Normal and Oncogenic Forms of the Receptor Tyrosine Kinase Kit - 《stemcells》2004-2005文献干细胞之家

干细胞之家 - 中国干细胞行业门户第一站 › 干细胞之家论坛 › 干细胞文献资源库 › 《stemcells》2004-2005文献 › Normal and Oncogenic Forms of the Receptor Tyrosine ...



免疫细胞治疗专区	欢迎关注干细胞微信公众号

12 3 4 5 6 7 8 9 10 ... 26 下一页

返回列表

查看: 329712\|回复: 258	go Normal and Oncogenic Forms of the Receptor Tyrosine Kinase Kit [复制链接]

江边孤钓

版主

Rank: 7 Rank: 7 Rank: 7

积分: 0
威望: 0
包包: 483

楼主

发表于 2009-3-5 10:52 |只看该作者 |倒序浏览 |打印

Normal and Oncogenic Forms of the Receptor Tyrosine Kinase Kit
  w' O7 E: F5 s# D- _0 O5 j! C! `
Johan Lennartsson, Tanya Jelacic, Diana Linnekin, R. Shivakrupa' [& R- I: K. O' E: X* r! p
, d" D5 I. R9 @7 v6 e5 s( y
Basic Research Laboratory, Center for Cancer Research, National Cancer Institute–Frederick, Frederick, Maryland, USA8 u5 U1 @3 G9 B

Key Words. Kit ? Signal transduction ? Hematopoiesis ? Oncogene ? Cancer
7 B& \+ K2 y& E
Correspondence: Johan Lennartsson, Ph.D., Ludwig Institute for Cancer Research, Box 595, Biomedical Center, SE-751 24 Uppsala, Sweden. Telephone: 46-18-160406; Fax: 46-18-160420; e-mail: Johan.Lennartsson@LICR.uu.se2 Z3 I7 c) Y* f7 D+ j( W
: ?2 `8 I6 d9 k' i! w' z
ABSTRACT

Kit is a receptor tyrosine kinase (RTK) that binds stem cell factor. This receptor ligand combination is important for normal hematopoiesis, as well as pigmentation, gut function, and reproduction. Structurally, Kit has both an extracellular and intracellular region. Theintra-cellular region is comprised of a juxtamembrane domain (JMD), a kinase domain, a kinase insert, and a carboxyl tail. Inappropriate expression or activation of Kit is associated with a variety of diseases in humans. Activating mutations in Kit have been identified primarily in the JMD and the second part of the kinase domain and have been associated with gastrointestinal stromal cell tumors and mastocytosis, respectively. There are also reports of activating mutations in some forms of germ cell tumors and core binding factor leukemias. Since the cloning of the Kit ligand in the early 1990s, there has been an explosion of information relating to the mechanism of action of normal forms of Kit as well as activated mutants. This is important because understanding this RTK at the biochemical level could assist in the development of therapeutics to treat primary and secondary defects in the tissues that require Kit. Furthermore, understanding the mechanisms mediating transformation of cells by activated Kit mutants will help in the design of interventions for human disease associated with these mutations. The objective of this review is to summarize what is known about normal and oncogenic forms of Kit. We will place particular emphasis on recent developments in understanding the mechanisms of action of normal and activated forms of this RTK and its association with human disease, particularly in hematopoietic cells.3 ^/ Z. j( b+ `( v9 n' z

INTRODUCTION  p6 {; |" n  W& `7 Q- q. o

Kit and Stem Cell Factor

The v-kit oncogene was identified in 1986 as the transforming gene in the Hardy-Zuckerman 4 feline sarcoma virus [1]. One year later, the cellular counterpart, c-kit, was identified based on sequence similarities [2]. The protein product of the c-kit gene will be referred to as Kit in this article. Kit is a receptor tyrosine kinase (RTK) belonging to subclass III, which also includes platelet-derived growth factor receptor  (PDGFR), PDGFR?, Flt3, and the CSF1R (c-Fms and M-CSFR). This subclass is characterized by an extracellular region consisting of five immunoglobulin (Ig)-like domains, a single transmembrane domain, and an intracellular tyrosine kinase domain split in two by an insert region (Fig. 1). Studies on the extracellular domain have shown that the first three N-terminal Ig-like domains of Kit are involved in ligand binding [3, 4]. Alternative splicing of Kit results in the presence or absence of four amino acids (GNNK) just outside the plasma membrane, and these isoforms exist in both mice and humans [5]. In humans, additional splice forms of Kit exist, which are characterized by the presence or absence of a serine residue in the kinase insert [6].

The ligand for Kit is called stem cell factor (SCF). SCF is expressed as a heavily glycosylated transmembrane protein. The biologically active form of SCF is a noncovalently associated dimer. Alternative splicing of the SCF transcript results in inclusion or exclusion of exon 6, which contains a proteolytic cleavage site [7]. The splice form that lacks the cleavage site remains associated with the cell surface (membrane-associated), whereas the other form is rapidly released through proteolysis (soluble form).4 i: ]0 W6 S5 t: j$ q
" k, l% C1 ^6 u7 j- h
The White Spotting and Steel Loci9 i" U0 k0 [$ x4 P. g

In 1988 it was established that c-kit is allelic with the White spotting (W) locus [8, 9]. Several naturally occurring loss-of-function mutations have been found in the c-kit gene. In mice with alleles that result in complete loss of Kit expression, the homozygotes die in utero or perinatally, likely because of anemia. In the heterozygous state, phenotypes include defects in pigmentation and reduced fertility. Mutations that affect kinase activity often result in death in utero or shortly after birth in the homozygous state, whereas heterozygous mice are anemic and have reduced fertility. In general, the severity of the phenotype correlates with the magnitude of the decrease in kinase activity of Kit [10].! q. H, K& _% C4 f

In 1990, the product of the Steel (Sl) locus was demonstrated to be identical to the ligand for Kit [11, 12]. Naturally occurring mutations in the Sl locus lead to complete or partial deletion of the gene as well as reduced SCF expression. The phenotypes of the Sl mutants are in most cases similar to those of W mutant mice; complete loss of SCF results in death in utero or perinatally, whereas heterozygous mice display less-severe phenotypes, with defects in fur color and mild anemia [10].
2 g# g4 R- O+ M* d
The phenotypes of W and Sl mice suggest that Kit signaling is important for normal hematopoiesis, gametogenesis, and melanogenesis, and studies of Kit function have indeed indicated roles for the receptor in these processes.

HEMATOPOIESIS; j* ~  ]3 }! L. I
) b* Z: k6 u( C& a6 L# H
During mammalian embryonic development, primitive hematopoiesis occurs in the yolk sac and in the aorta-gonad-mesonephros region [13]. Subsequently, hematopoietic stem cells (HSCs) migrate from these regions to the embryonic liver, thymus, and spleen. Kit is expressed by hematopoietic cells in the embryonic liver throughout its development. In contrast, expression of SCF is more restricted during this time [14]. Around the time of birth, the major site for hematopoiesis becomes the bone marrow, and this is maintained during adult life.: \0 \8 x$ P& K! u6 K

Most mature blood cells are short lived and must be continuously replenished through out life. HSCs are rare cells with the capacity to self-renew and give rise to all blood lineages, including lymphocytes, megakaryocytes, granulocytes, monocytes, and erythroid cells. CD34 is a transmembrane glycoprotein of unknown function that is expressed on hematopoietic stem/progenitor cells [15]. Most of the CD34  cells in the bone marrow also express Kit, suggesting an important role for Kit in primitive hematopoietic cells [16]. Most HSCs remain in a quiescent state, but they can be mobilized in response to stress on the hematopoietic system. Specific cytokines in combination induce HSC proliferation and differentiation. In vitro SCF in combination with Tpo has a central role in HSC proliferation [17]. In addition, HSC mobilization involves interactions between SCF and Kit [18]. Furthermore, G-CSF in combination with SCF is a particularly potent means to mobilize stem/progenitor cells in both humans and mice [19]. Physiological regulation of HSC growth, differentiation, and mobilization is a complicated process that is likely mediated by combinations of factors, including several cytokines and components of the extracellular matrix [20].# L/ ?8 K" W# O% o; |
  D" O1 k7 G8 `4 \2 G8 t: |7 B; p5 ^
HSCs have the capacity to differentiate into multipotential progenitor cells, thus initiating the process of lineage commitment. These latter cells have a more restricted ability to differentiate than stem cells and are functionally defined by their ability to form colonies in vitro. In the absence of Kit, other pathways can compensate and produce small numbers of HSCs as well as more differentiated hematopoietic cells. For example, HSCs can be maintained on stromal cells in the presence of an antibody that blocks SCF binding to Kit. This suggests that stromal cells can support HSCs in the absence of functional Kit signaling [21]. Indeed, Epo receptor (EpoR) signaling partially supported colony-forming unit–erythroid formation in W/W mice lacking functional Kit [22]. In addition, other growth factors may compensate for loss of Kit. Hepatocyte growth factor (HGF) promotes proliferation and differentiation of the HSCs and progenitor cells from W/W mice [23]. HGF in combination with GM-CSF, G-CSF, or M-CSF induces colony formation of CD34  cells from human cord blood [24]. Furthermore, insulin-like growth factor-I (IGF-I) in combination with G-CSF can maintain proliferation of CD34  cells isolated from human cord blood. The number of differentiated myelopoietic cells, as measured by increased expression of the granulocytic marker CD15 and the myelomonocytic marker CD64, increased in the presence of IGF-I in combination with G-CSF [25].
5 h6 Q; O4 M% Z5 t
Currently, no single marker specific for HSCs has been found. Consequently, these cells are often purified using combinations of markers. A common strategy for enrichment of murine HSCs has been to use lineage-negative cells that express Sca-1, Kit, and CD34 [26]. Progenitor cells differentiate into precursor cells, which are committed to a specific lineage and have no capacity for self-renewal. However, precursor cells have a high proliferative potential, leading to extensive amplification of cell numbers. The precursor cells differentiate further into fully mature blood cells. Although the mechanisms regulating these events are not fully understood, a variety of cytokines and growth factors play critical roles. Moreover, the hematopoietic microenvironment also plays an important role through structural support and adhesive interactions required for colocalization of primitive cells and regulatory factors. In fact, differentiation and proliferation of hematopoietic progenitor cells can be controlled by communication with stromal cells in the microenvironment [27]. Furthermore, it has been demonstrated that autocrine/paracrine loops, including Kit and SCF, are important for growth of primitive hematopoietic cells [28, 29]. Because these cells secrete a multitude of cytokines and growth factors, it is likely that synergistic interactions between these factors are important for survival and proliferation. For further information on autocrine/paracrine loops in normal and oncogenic hematopoiesis, please refer to the study by Janowska-Wieczorek et al. [30].% D/ n2 B& G% m0 [, y7 ?

Kit is also expressed on more committed progenitors, including myeloid, erythroid, megakaryocytic, natural killer, and dendritic progenitor cells, as well as pro-B and -T cells and mature mast cells [31, 32]. The expression of Kit is low on the earliest HSC and peaks in more mature progenitor cells. Interestingly, SCF is a potent chemoattractant for this population of cells [33]. As a single factor, SCF acts primarily to promote survival of hematopoietic progenitor and stem cells [34, 35]. However, it is insufficient to maintain self-renewal in vitro [36]. A recent study demonstrated that activation of JAK2 in combination with signals emanating from Kit or Flt3 promoted self-renewal of primary multipotential hematopoietic cells [37]. Interestingly, progenitor cells with different characteristics were obtained when Kit was activated instead of Flt3. Differentiation of HSCs to progenitor cell populations is, at least in some cases, linked to increased mitogenic activity of SCF [36].
; ]8 K$ J  |' i. U( |
Although Kit expression is generally lost during hematopoietic cell differentiation, some differentiated cell types, such as mast cells, retain this RTK (Fig. 2). SCF induces mast cell differentiation from primitive fetal liver hematopoietic cells [38]. Furthermore, SCF supports proliferation, survival, and final maturation of mast cells in vitro, as well as induces chemotaxis, adhesion, and survival of mature mast cells [39]. One striking phenotype of viable mice with W and Sl alleles is the deficiency in tissue mast cells [40]. Kit is not expressed on differentiated lymphocytes. Hence, in the lymphoid lineage, SCF acts primarily on lymphoid progenitors. Because mice with severe mutations in Kit or SCF die early in development, little is known about the effects these mutations have in adult animals. However, a recent study showed an age-dependent decline in the number of pro-T and pro-B cells, as well as reductions in common lymphoid progenitors in the bone marrow of the viable W/W mouse mutant Vickid [41]. The identity of the compensatory mutation that allows the Vickid mice to survive in the absence of Kit and the molecular mechanism by which this occurs are not presently known.
  J$ q; W2 c1 P
In conclusion, Kit can be detected on less than 0.1% of peripheral blood cells, indicating that most differentiated hematopoietic cells do not express this RTK [32, 42, 43]. Figure 2 summarizes expression of Kit on cells in the hematopoietic hierarchy. For detailed information on Kit in hematopoiesis, please refer to [19, 31, 44–46.]

SIGNAL TRANSDUCTION
1 G: H2 U. J, U3 m! s- Y
The focus of this section is on biochemical pathways activated by wild-type Kit, with special consideration given to mechanisms in hematopoietic cells. Table 1 and Figure 3 are summaries of signaling proteins that interact with Kit.  g$ g& |/ G* t4 |+ s' E- ^
* w. C3 ~' Q- e( m1 u* [
Receptor Dimerization and Autophosphorylation
& [! y9 A: [( j+ A* f
Stimulation of Kit with SCF results in dimerization and subsequent activation of its intrinsic tyrosine kinase activity [47]. The dimerization process is believed to be induced by simultaneous binding of dimeric SCF to two receptor monomers [48, 49]. Ligand binding also induces a conformational change in the receptor, leading to receptor–receptor interactions that further stabilize the dimeric complex [50]. Ig-like domain 4 in the extracellular domain of Kit has been implicated in the receptor–receptor interaction. The crystal structure of human SCF has been determined and suggests that the receptor-binding site in SCF contains charged regions that could provide specificity and a hydrophobic region that could provide binding energy [51, 52]. In a fluorescence resonance energy transfer study of Kit dimerization in hemato poietic cells, SCF induced detectable dimers within minutes after stimulation [53]. Internalization of Kit is a consequence of its activation, and cholesterol depletion experiments have suggested that membrane lipid rafts are essential for this process [54]. After initiation of kinase activity, tyrosine residues, primarily outside the kinase domain, become phosphorylated and function as docking sites for signal transduction molecules with phosphotyrosine binding or Src homology 2 (SH2) domains [55]. Recently, the crystal structure of the active Kit kinase domain was solved [56]. The structure indicated that the conformation of the kinase domain of monomeric, unphosphorylated Kit is very similar to that of the activated receptor. Thus, physical separation of receptor monomers is the likely mechanism of preventing autophosphorylation in the absence of ligand.% C7 o$ F$ ]/ J, G
% V2 z; b! y# F- S& f
The soluble and membrane-associated forms of SCF have different effects on Kit autophosphorylation. Stimulation with the soluble form results in rapid but transient auto-phosphorylation of Kit, whereas stimulation with the membrane-bound form results in more sustained phosphorylation [57]. Interestingly, soluble and membrane-associated SCFs have overlapping but distinct functions in vivo. This was clearly demonstrated in a mutant mouse strain that expresses only the soluble form of SCF (Sld) [58]. The homozygous Sld mice are viable but have severe anemia, are sterile, and lack pigmentation, and their skin is deficient of mast cells. In mice with a disrupted SCF gene (Sl), the homozygotes die in utero or perinatally [10]. Moreover, in an erythroid progenitor cell line, stimulation with membrane-associated SCF results in higher proliferation compared with soluble SCF [59]. Biochemical analysis showed that this correlated with more persistent phosphorylation of Erk1/2 and p38 mitogen-activated protein kinase (MAPK) after stimulation with membrane-associated SCF compared with soluble ligand [60].) y5 _0 ~6 I* k2 Z6 V9 k$ ], l6 {
' @8 G: ^- q+ a6 V( w
In summary, ligand-induced dimerization of Kit monomers increases the local concentration of kinase domains, thus facilitating autophosphorylation. Most of the tyrosine phosphorylation sites are located outside the kinase domain, and these function as docking sites for intracellular signal transduction proteins." {5 N: {2 c- ?2 G9 v; @  T5 w
$ p, K4 d) x0 S9 q
The Phosphoinositide 3'-Kinase Pathway
8 `& l. `- O  Z2 l
Phosphoinositide 3'-kinase (PI3K) phosphorylates the 3'-hydroxyl group in the inositol ring of phosphati-dylinositol (PtdIns), phosphatidylinositol-4-phosphate [PtdIns(4)P], or phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] in vitro. However, in vivo, the preferred substrate is PtdIns(4,5)P2, and this subsequently generates PtdIns(3,4,5)P3 [61]. PI3K is a heterodimeric complex with a regulatory (p85) and a catalytic (p110) subunit [62]. PI3K has been implicated in many aspects of cellular signaling, such as DNA synthesis, cell survival, membrane ruffling, and chemotaxis, as well as receptor and vesicular trafficking. Activation of PI3K involves association of the SH2 domain of the regulatory subunit, with phosphorylated receptors leading to translocation of PI3K to the plasma membrane, where its sub-strates are located. Downstream targets for 3'-phospholipids generated by PI3K include the serine/threonine kinases Akt and protein kinase C (PKC). Akt is important for antiapoptotic signaling. The PH (Pleckstrin homology) domain of Akt interacts with phospholipids generated by PI3K, resulting in its localization to the plasma membrane, where it is activated by serine and threonine phosphorylation.6 p0 _+ ]0 m+ i' Y

SCF induces association of the SH2 domain of the 85-kDa regulatory subunit of PI3K with tyrosine residue 721 in the kinase insert of human Kit (Y719 of murine Kit). The role of this interaction in SCF signaling in hematopoietic cells has been examined by several groups, and many of the earlier studies were reviewed previously [63]. In brief, infection of murine mast cells lacking endogenous Kit with KitY719F demonstrated that interaction with PI3K was required for optimal membrane ruffling, adhesion, actin assembly, and proliferation in response to SCF [64]. In contrast, a recent study showed that restoration of the PI3K binding site in a Kit receptor where all autophosphorylation sites were mutated to phenylalanine led to only minor increases in cell survival and migration and had no effect on proliferation [65]. A human KitY721F mutant was unable to protect against Bad-induced apoptosis in U2-OS cells [66]. Similar results were obtained with bone marrow mast cells (BMMCs) expressing the KitY719F mutant. On the cellular level, there were reductions in SCF-induced proliferation and protection from apoptosis. Biochemically, this resulted in reductions in SCF-induced activation of Akt, as well as c-Jun NH2-terminal kinase (JNK) in BMMCs [67]. These data, in conjunction with earlier work, demonstrate that direct association of PI3K and Kit contribute to, but are not required for, most SCF-mediated responses. Studies from Besmer and colleagues [68] suggest that the Kit binding sites for both PI3K and Src family kinases (SFKs) are required for most SCF-mediated responses. Consistent with this notion, Ueda et al. [69] demonstrated that PI3K and SFK binding sites were the only tyrosines in the Kit cytoplasmic domain sufficient for SCF-induced chemotaxis and calcium mobilization.

In nonhematopoietic tissue, studies with tyrosine 719 knock-in mice demonstrated that direct interaction of PI3K and Kit is vital for mouse fertility [67, 70]. Sterility in males was attributable to a block in spermatogenesis with decreased proliferation and extensive apoptosis. Reductions in fertility in females were caused by impaired follicle development. A recent study supports an important role for Akt in survival and growth of primordial germ cells [71].

Data with p85PI3K-deficient mice support an important role for PI3K in SCF-mediated responses beyond that mediated by direct interaction of Kit with p85. SCF-induced proliferation of BMMCs from p85-deficient mice was dramatically reduced compared with wild-type cells. This decrease in proliferation correlated with a defect in the SCF-induced activation of JNK in the p85-deficient BMMCs. This is consistent with previous studies demonstrating that activation of the JNK pathway in BMMCs is required for SCF-induced proliferation [68, 72]. Surprisingly, SCF-induced activation of Akt was only partially affected in p85-deficient BMMCs, and no defects in SCF-mediated survival were observed in this study. Similar results were obtained in fetal liver mast cells from p85-deficient mice [73]. Thus, other pathways must be contributing to survival mediated through Kit. Kapur and colleagues [74] demonstrated using mast cells from p85 or Rac2-deficient mice that SCF-induced chemotaxis is dependent on signal transduction involving these proteins. Furthermore, they showed that the 4 integrin and Kit cooperate in promoting directed migration of mast cells.
$ U5 c. F( D% w1 i* C2 ^
The differences in results using p85-deficient cells and cells expressing the KitY719F receptor mutant are of note. In the knock-in studies, other proteins that potentially bind to tyrosine 719 are unable to interact. In addition, p85 can associate indirectly with other signaling components, independent of interaction with the PI3K docking site on Kit. Thus, further study using both these important models will be required to define biochemical mechanisms underlying the contribution of PI3K to SCF-mediated responses.' H( e  K6 D" v

The JAK-STAT Pathway
1 |* ^0 @  E! b$ q
The Janus kinases (JAKs) are cytoplasmic tyrosine kinases that are rapidly activated by ligand binding to cytokine receptors or RTKs. Important targets for activated JAKs are the signal transducers and activators of transcription (STAT) [75]. STATs are a group of transcription factors that have a central DNA binding domain followed by a SH2 domain and a C-terminal transactivation domain. Activated JAKs or RTKs phosphorylate the cytoplasmic STATs, which then undergo a SH2 domain–dependent dimerization and subsequent translocation to the nucleus. Nuclear STATs regulate expression of responsive genes by binding to their promotors. STATs (at least 1, 3, and 5) are also serine phosphorylated at the C-terminus, which regulates their transcriptional activity./ g' }5 M. G- }/ e$ p

SCF stimulation results in activation of the JAK-STAT pathway [76, 77]. JAK2 constitutively associates with Kit and becomes transiently activated in response to SCF [78]. Studies using antisense oligonucleotides specific for the JAK2 sequence suggest that it is important for maximal SCF-induced proliferation [77]. Recently, we showed that partially purified populations of fetal liver hemato-poietic progenitor cells are dependent on JAK2 for proliferation and differentiation in response to SCF [79]. After SCF stimulation, STAT1, STAT5A, and STAT5B associate with Kit and become tyrosine phosphorylated [80]. SCF-induced STAT tyrosine phosphorylation has been associated with increased DNA binding [81, 82]. In addition to tyrosine phosphorylation, SCF also induces serine phosphorylation of STAT3 [83]. Interestingly, different regions of Kit are involved in the activation of STAT1 and STAT5A/B [80]. Complete activation of STAT5, but not STAT1, requires the C-terminal region of Kit.% c/ ]0 ]- i; g+ b) M' F

In contrast to the above findings, several studies have not observed activation of JAK-STAT pathway by SCF [84–86]. One potential explanation for these differences is lineage-specific differences in JAK-STAT activation. Consistent with this possibility, we recently showed that JAK2 is important for SCF-induced growth of Kit-positive progenitor cells but not critical for mast cell response to SCF [79]. A second potential explanation is differences in the experimental procedures. For example, many studies looked at only one time point after stimulation with SCF. In contrast to interleukin 3 (IL-3), SCF-induced activation of this kinase is extremely rapid and transient [77, 78]. In addition, JAK2 is rapidly dephosphorylated in the absence of high levels of phosphatase inhibitors [78]. Thus, differences in the components of the lysis buffer can have a dramatic effect on the ability to detect phosphorylation of JAK2.
) q  I: w2 t' g- Z: m0 {0 D5 E
The Ras-Erk Pathway
9 ^5 ]/ o: }. R1 U6 I5 W9 j
Activation of the Ras-Erk pathway is of critical importance for several cellular responses such as cell division and survival [75]. Ras has two conformations, an active guanine triphosphate (GTP)-associated form and an inactive guanosine diphosphate (GDP)-associated form. RTKs activate Ras by associating with Sos, a guanine nucleotide exchange factor that favors the guanosine diphosphate–bound state. Sos associates constitutively with the adaptor protein Grb2, which in turn associates with activated RTKs via its SH2 domain either directly or indirectly via other molecules such as SHP2 and ShcA. The active receptor recruits the Grb2/Sos complex, bringing it close to the plasma membrane where Ras is located. Activated Ras can interact with and initiate the activation of the serine/threonine kinase Raf-1. Active Raf-1 can phosphorylate and activate the dual-specificity kinases Mek1 and 2. However, Raf-1–deficient cells are still able to activate Erk1/2, suggesting a redundancy with other kinases such as A-Raf and B-Raf. The mitogen-activated protein kinases Erk1 and Erk2 are serine/threonine kinases that are activated through phosphorylation by Mek1/2. Targets for Erks in the cytoplasm include the serine/threonine kinases RSK and Mnk1/2, as well as phospholipase A2. A fraction of the activated Erk and Ribosomal S6 kinase(RSK) proteins translocate to the nucleus, where they phosphorylate various transcription factors, thereby influencing gene transcription. A recent study showed that disruption of the Erk2 gene is embryonic lethal despite the presence of Erk1 [87]. Thus, Erk1 and Erk2 have unique nonredundant functions during mouse development.0 N9 V+ Y+ G1 P7 P" H1 B/ I, j

It has been well established that SCF activates the Ras-Erk pathway through multiple signaling components. The adaptor protein Grb2 can interact directly with tyrosine residues 703 and 936 of human Kit or indirectly via proteins that associate with Kit such as ShcA or SHP2 [88–90]. Some studies, but not all, have demonstrated that SCF-induced activation of SFKs is important for activation of the Ras-Erk cascade [68, 69, 91, 92]. In a recent study, Kapur and colleagues [65] showed that activation of the Ras-Erk cascade requires only an intact SFK binding site in Kit. Consistent with these findings, knock-in mice expressing a Kit receptor lacking the SFK interaction site are unable to activate the Erk cascade [93]. Variability relating to the role of SFKs in activation of the Ras-Erk pathway may relate to differences in experimental procedures, lineage specificity, and which GNNK splice form of Kit that is predominantly expressed. In addition, Ras can be activated by the guanine nucleotide exchange factor Vav, which is phosphorylated in response to SCF in hematopoietic cells [94, 95]. The adaptor protein Gab2 can link to the Ras-Erk pathway through association with SHP2 [96]. Indeed, Gab2 was demonstrated to be involved in the activation of the Ras-Erk pathway after SCF stimulation of mast cells [97].1 m8 @+ j% ^5 G; F# E4 l5 z1 ^  _/ L
4 R. J( h5 F+ t0 k( G
Activation of Erk1/2 is important for hematopoietic differentiation [98]. This was demonstrated in the UT-7/GM cell line, which can differentiate into erythroid or mega-karyocytic lineages. Transient activation of Erk1/2 by Epo results in erythroid differentiation. In contrast, Erk1/2 activation by Tpo is sustained and induces megakaryocytic differentiation. Furthermore, pharmacological inhibition of the Ras-Erk pathway with PD98059 promotes Epo-induced erythroid differentiation and suppresses Tpo-induced mega-karyocytic differentiation in UT-7/GM cells. Hence, this study shows that the duration of Erk1/2 activity is important for the biological outcome. Furthermore, inhibition of the Ras-Erk pathway with PD98059 suppresses megakaryocytic differentiation from CD34  hematopoietic progenitor cells from human cord blood [99].
+ G4 G. p; W0 f% p5 Z
The SFK Pathway, q0 b2 z6 ^+ f6 C
. h* Q, R5 A" l: X: m
SFKs are involved in a wide range of cellular functions, including cell cycle progression, chemotaxis, adhesion, survival, and protein trafficking [100]. Gene disruption studies have demonstrated that SFKs are essential for normal development and that there is a great deal of functional redundancy between the different family members.1 F) ]% Z: j5 }2 y2 E2 T
  u- n: P! e0 ^& B6 Q
Activation of Kit results in a rapid increase in SFK activity [101, 102]. Furthermore, SFKs associate with tyrosine 568 and to some extent with tyrosine 570 in the juxta-membrane domain (JMD) of human Kit (Y567 and Y569 in murine Kit) through their SH2 domain [68, 91, 103].4 e& `9 r5 }) p7 v* ]- M

SFKs are important for ligand-induced internalization of Kit [54, 104]. This was demonstrated by the use of point mutants of the receptor and pharmacological inhibitors of SFKs and in Lyn-deficient cells. Moreover, overexpression of c-Src results in an increased rate of epidermal growth factor receptor (EGFR) internalization, suggesting a conserved role for SFKs in internalization of members of the RTK super family [105].6 q/ r/ j7 _. h1 ]+ j  m4 L, g

The involvement of SFK in Kit-induced mitogenicity is not clear. In some studies, SFKs have no or only a partial effect on proliferation, whereas other studies have shown them to be essential [68, 91, 101, 106, 107]. Tan et al. [107] used chimeric Kit receptor mutants to show that inactivation of the SFK pathway had the most severe effect on proliferation compared with other signaling pathways. The same study also showed that a Kit mutant that only had the SFK binding site intact restored proliferation to 50% of that induced by wild-type Kit. SFKs have been shown to phosphorylate Kit on tyrosine residue 900 within the second part of the kinase domain [108]. Mutating tyrosine 900 to phenylalanine significantly inhibited SCF-induced DNA synthesis compared with wild-type Kit in transfected NIH3T3 cells. Tan et al. [107] could also demonstrate a reduction in c-Myc expression using a chimeric Kit receptor in which the SFK binding sites had been mutated compared with the chimeric receptor with the wild-type intracellular domain of Kit. In contrast, no defect could be seen in the mutated receptor’s ability to induce STAT5 phosphorylation or Bcl-xL expression. In addition, this study also demonstrated that activation of Src family kinases plays a crucial role in the proliferative cooperation between Kit and the EpoR.  x& \' F2 e0 R! L! `

The SFK member Lyn has an important role in SCF-induced chemotaxis in primary hematopoietic cells [106]. Furthermore, a recent study in which all individual tyrosine residues in the intracellular part of murine Kit were mutated to phenylalanine showed that tyrosine residues 567 and 719 were important for SCF-induced chemotaxis and Ca2  mobilization [69]. Restoring individual tyrosine residues in a receptor in which all tyrosines had been mutated to phenylalanine demonstrated that the SFK binding site in Kit is critical for survival and migration [65]. However, this receptor mutant only partially restored proliferation compared with wild-type Kit.
3 Z! f$ I" E6 D. Q5 w+ |1 F% ^
Besmer et al. [109] recently generated KitY567F knock-in mice. Homozygous KitY567F mice displayed normal hema-topoiesis but defective lymphocyte development in older animals with a block at the pro-B and pro-T stage. These results are in agreement with studies on Vickid mice, in which loss of Kit signaling led to an age-dependent reduction in pro-B and pro-T cells [41].( n' K6 T  G% |- a% K4 j% F

Two splice forms of Kit differ only by the presence or absence of four amino acids in the extracellular juxtamem-brane region. However, these splice forms display major differences in kinetics of autophosphorylation and signaling capabilities [110]. Recently, the shorter splice form was reported to bind and activate SFKs more efficiently [111]. This led to stronger phosphorylation of ShcA, Erk1/2, and Cbl compared with the longer splice form. The difference in kinetics of receptor autophosphorylation was also dependent on SFK activation.  a7 w4 C! e! K0 x# j  m

Knock-in of a Kit receptor lacking the SFK binding site in mice resulted in decrease in receptor phosphorylation and a selective inhibition of the Erk phosphorylation [93]. Further analysis demonstrated these mice have defects in the development of mast cells and in the melanogenesis but normal development of erythroid blood cells, germ cells, and interstitial cells of Cajal (ICC).

Recent studies with knock-in mice have demonstrated the importance of cellular context on signaling pathways activated downstream of Kit [93, 109]. For example, loss of SFK binding(KitY567F)leads to defects primarily in lymphopoiesis, whereas mice harboring KitY567F/Y569Falso had major defects in pigmentation, mast cell development, and splenomegaly [93, 109]. Defects in pigmentation and reduced numbers of mast cells were also observed in mice with single tyrosine mutations but were more severe in the double tyrosine mutant [93]. This indicates that these two tyrosines in the JMD of Kit cooperate to activate certain signaling pathways. It is interesting to note that in the hematopoietic system, Kimura et al. [93] report age-dependent defects of B-cell development and megakaryopoiesis, whereas the study by Agosti et al. [109] found defects in B and T cells. This could be due to differences in the genetic background, because Kimura et al. studied the phenotype on mixed 129/SvCP and C57 BL/6 background, whereas the work from Agosti et al. used C57 BL/6J. Another potential explanation is difference in the age of mice when they were analyzed. In contrast, loss of PI3K binding (KitY719F) resulted in defects in gametogenesis but not lymphopoiesis, as seen for loss of SFK binding. Thus, signaling pathways have distinct roles in different cell lineages.

Phospholipase C# ]" s' n) z  ?3 z

Two isoforms of phospholipase C (PLC) have been identified: PLC1, which is expressed ubiquitously, and PLC2, which is mainly expressed in hematopoietic cells [112]. PLC hydrolyzes phosphatidylinositol-4,5-bisphosphate (PIP2), generating diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP3). DAG binds and activates conventional and novel PKCs, whereas IP3 induces release of Ca2  from intracellular stores. A variety of cellular functions are regulated by Ca2 , including membrane localization of conventional PKCs. The PLC association with Kit is dependent on tyrosine residues 730 and 936 of the human receptor [113, 114]. Studies using soluble SCF have in most cases failed to detect an increase in activity or phosphorylation of PLC [115, 116]. These studies have instead suggested activation of phospholipase D after SCF stimulation. In contrast, studies using the membrane-bound form of SCF have indicated a role for PLC in SCF-induced proliferation and survival [114, 117]. This suggests a differential effect on signal transduction by soluble and membrane-bound SCF. How this occurs is not clear, but one possibility is that the membrane-bound SCF results in slower internalization of active Kit, whereas soluble SCF induces rapid internalization [57, 118, 119]. The difference in localization of the active receptor could explain the differences in coupling to PLC and potentially to other molecules. It is well established that SCF protects Kit-expressing cells against apoptosis [66, 120–122]. Studies have demonstrated that PLC1 has a central role in SCF-induced protection against apoptosis induced by irradiation as well as the cytotoxic agent daunorubicin [123, 124].
! N4 F% Y* k/ d+ N( |
Adaptor Proteins

Adaptor proteins can be defined as proteins lacking enzymatic activity but with the ability to simultaneously interact with at least two proteins. The capability of linking proteins together via specific and in many cases inducible protein–protein interactions offers multiple flexible mechanisms to regulate signal transduction.0 f0 f' y4 n, g! H3 L' ~
( s* M9 N6 U. t" j, j, @* ^
Grb2 is a widely expressed adaptor protein that couples RTKs to the Ras-Erk pathway. This occurs through association with the Ras guanine nucleotide exchange factor Sos. Activated Kit has been shown to bind Grb2 [88]. The interaction sites of the Grb2 SH2 domain have been mapped in vitro to tyrosine residues 703 and 936 in human Kit [90]. However, the contributions to Ras-Erk pathway activation in vivo by direct binding of Grb2 to Kit compared with indirect contributions through other adaptors, such as ShcA or SHP2, remains to be defined. Grap is an adaptor protein related to Grb2. Studies on Grap have suggested a role analogous to Grb2, i.e., coupling Sos to activated receptors, including Kit [125].4 {8 h7 g6 ~0 z/ w2 S7 W

ShcA is an adaptor protein that interacts with Grb2 after phosphorylation. Thus, ShcA can mediate recruitment of Grb2 to receptors that lack direct Grb2 binding sites. Activation of Kit results in increased tyrosine phosphorylation of ShcA, which correlates with the activation of the Ras-Erk pathway [91, 126]. However, ShcA likely serves additional functions in signal transduction through association with proteins such as Gab1 and the hematopoietic protein Gads [127, 128]. In vitro studies have suggested that ShcA interacts with the JMD of Kit [103].
  Y) v1 k8 o7 s$ K
Grb10 association with activated Kit requires SH2 domains [129]. Furthermore, this adaptor protein forms a constitutive complex with Akt and is a cofactor in SCF-induced activation. Grb10 belongs to the Grb7 family of adaptor proteins. In the case of Grb7, the interaction site with Kit has been mapped in vitro to tyrosine residue 936 in the C-terminal tail of the receptor [90].
$ s8 y" ~8 ]( i) z0 C
Adaptor protein containing PH and SH2 domains (APS) is an adaptor protein cloned in a yeast two-hybrid screen using the activated mutant KitD816V as bait [130]. APS becomes phosphorylated in response to SCF but also by stimulation with growth factor and cytokines such as PDGF-BB, IGF-I, IGF-II, and GM-CSF [131]. The SH2 domain of APS interacts with tyrosine residues 568 and 936 in human Kit [132]. Phosphorylation of APS leads to its association with the ubiquitin ligase Cbl. Recruitment of Cbl through APS to the PDGFR? leads to inhibition of PDGF-BB–induced proliferation [131]. Lnk is a protein structurally related to APS but expressed mainly in hematopoietic cells. Lnk–/– mice have increased numbers of hematopoietic progenitor cells in part because of increased signaling from Kit [133].; m; S/ S9 [  s

The adaptor protein Dok-1 was originally discovered as a tyrosine-phosphorylated protein associated with RasGAP [134]. Several growth factors, including SCF, induce Dok-1 tyrosine phosphorylation. Recently, it was demonstrated that SCF-induced Dok-1 phosphorylation was dependent on PI3K as well as the SFK Lyn [135, 136]. Liang et al. [136] showed that Dok-1 associated readily with the JMD as well as the C-terminal tail of Kit. Phosphorylated Dok-1 forms a complex with the kinases Lyn and Tec as well as other as-yet unidentified molecules and hence functions as an adaptor protein [135]. Studies on Dok-1–/– mice have suggested that Dok-1 is a negative regulator of proliferation [137, 138].

SCF stimulation of MO7e cells leads to tyrosine phosphorylation of CrkL [139]. Sattler et al. [139] showed CrkL associates with Kit indirectly through the p85 subunit of PI3K. Furthermore, CrkL recruits Cbl to the Kit complex, likely downregulating its activity by increasing ubiquitination and degradation of this RTK. In NIH3T3 cells transfected with Kit, SCF induces tyrosine phosphorylation of Crk-II, a protein related to CrkL [108]. Moreover, SCF-induced Crk-II phosphorylation was dependent on tyrosine residue 900 in Kit.
7 w8 v  P0 V& z+ G& i* y
The adaptor protein Gab2 becomes tyrosine phosphorylated in response to SCF [140]. Nishida et al. [140] showed that Gab2 forms a complex with SHP2 and PI3K and that in vitro Gab2 is a substrate for SHP2 [140]. A study using BMMCs derived from Gab2–/– mice demonstrated that Gab2 is important for SCF-induced activation of Erk1/2 and Akt [97]. BMMCs from Gab2–/– mice grow poorly in response to SCF, indicating an important role for Gab2 in mast cells proliferation.+ G2 p) @% r# Q* I. e( g- R
1 t" [! r- W9 A# A3 i) L, k8 l% S  i
These data highlight the importance of adaptor proteins in amplification and integration of signals downstream of Kit. They contribute to activation of proteins that cannot directly associate with the receptor as well as provide multiple means to activate a particular pathway. For example, the Ras-Erk pathway can be activated by association of Grb2 directly with the receptor or through proteins such as ShcA and SHP2.
; Z- Z, v7 O/ G& S
Synergistic Signaling& O- {% b" k% j+ `# `% _

An important feature of SCF is its strong capability to synergize with ligands for the cytokine receptor super family members [141]. This includes GM-CSF, IL-3, and Epo. Although the subject of rather intense study, the molecular mechanisms mediating synergy of SCF in combination with these cytokines remain to be determined. Below is a summary of some of the salient features of published work on this intriguing phenomenon.

Most studies indicate it is unlikely that synergistic growth of SCF in combination with other growth factors is mediated by alterations in expression or affinity of either population of receptors. Several studies suggest that SCF does not alter the number or affinity of GM-CSF or IL-3 receptors in either human or murine cell lines [142–144]. Furthermore, neither IL-3 nor GM-CSF influences Kit mRNA production [145]. However, GM-CSF has been reported to downregulate Kit expression in FDC-P1 cells [146]. Although receptor expression is not altered by SCF, Kit does interact with some cytokine receptors. This includes both the IL-7 receptor as well as the Epo receptor. Furthermore, our preliminary findings suggest that Kit also interacts with the ?-chain subunit of the GM-CSF receptor (unpublished data).% A! }4 X1 _3 A/ C
1 ]! L$ [8 `# \5 p
Several groups have examined tyrosine phosphorylation of receptors after costimulation with two growth factors. In general, GM-CSF and SCF costimulation does not induce synergistic increases in Kit tyrosine phosphorylation, although one group did report an increase in tyrosine phosphorylation of Kit in HML-2 cells stimulated with both SCF and GM-CSF compared with SCF alone [143, 147, 148]. Although one report indicates that SCF induces serine and threonine phosphorylation of the ?-chain common for GM-CSF, IL-3, and IL-5 receptor complexes in a PKC-dependent manner, the significance of this finding is not understood [144].
5 X+ i; Z) Y% m* ~; y, e0 x  R
Downstream of Kit and the GM-CSF receptor, most early signaling pathways are not synergistically activated by SCF and GM-CSF. This includes JAK family members, PLC, Akt, Jnks, and p38 MAPK [84, 143, 149, 150]. However, several studies have implicated activation of Erk1/2 in synergistic signaling induced by SCF in combination with either GM-CSF or IL-3 [84, 85, 142, 143, 151, 152]. Broxmeyer et al. [149] showed that SCF and GM-CSF synergistically activate Erk but not the stress activated protein kinase (SAPK)/JNK and p38 MAPK pathways in MO7e cells. Similar findings have been reported in normal erythroid progenitors stimulated with SCF in combination with Epo [152].& {1 Q( o1 T) |
4 e2 S/ X% r; P5 q' ]' Z3 ~
Downstream of early signaling pathways, which are activated within minutes of costimulation, are proteins involved in cell cycle progression. Concurrent stimulation with SCF and GM-CSF synergistically induces phosphorylation of the Rb protein as well as increasing the cyclin-dependent kinase inhibitor Cip-1 and decreasing the Kip-1 expression [153]. SCF in combination with IL-3 induces the expression of the 68-kDa calmodulin-binding protein (CaM-BP68) [154]. Both Rb phosphorylation and expression of CaM-BP68 are required for growth factor–induced proliferation.

The studies above have focused primarily on SCF in combination with GM-CSF or IL-3 with regards to mechanisms mediating synergy. However, Epo in combination with SCF has also been examined in some detail. As mentioned above, Kit physically interacts with the EpoR. Kit also induces phosphorylation of the EpoR and vice versa [155]. It was recently shown that a Kit mutant unable to activate SFKs could not efficiently mediate phosphorylation of the EpoR [107]. Studies on the EpoR have demonstrated that a truncated recept or that only retains the STAT5 binding site (tyrosine residue 343) can support Epo-induced proliferation and erythroid development as efficiently as the wild-type EpoR [156]. In addition, the truncated receptor still synergizes with Kit. Studies on the molecular mechanism mediating synergy in FDCER cells have identified both EpoR tyrosine phosphorylation–dependent and –independent contributions [157]. A mutated EpoR with no intracellular tyrosine phosphorylation sites could still synergize with Kit in proliferation. In contrast, the same study also showed that SCF-induced Bcl-xL gene expression was dependent on tyrosine residue 343 in the EpoR. Furthermore, it was also demonstrated that activation of the EpoR initiates a negative feedback loop involving SOCS-3 (suppressor of cytokine signaling-3) that inhibits both Kit and EpoR-induced proliferation [157]. Studies using G1E-ER2 cells revealed that activation of Kit is critical for maintenance of EpoR and STAT5 protein expression [158]. Subsequently, the EpoR activates STAT5 that in turn induces Bcl-xL expression, ultimately leading to cell survival. A recent study in AS-E2 cells demonstrated that SCF can increase Epo-induced STAT5 transactivation through a mechanism that does not involve serine or tyrosine phosphorylation of STAT5 [159]. However, the increase in STAT5 transactivation was dependent on SCF-induced protein kinase A activity and CREB phosphorylation. Thus, the synergistic interaction between Kit and the EpoR occurs at the level of transcription, as well as through alterations in protein interaction and phosphorylation.
2 h2 w% |( n( L6 |" ]; ~8 l5 E+ {
Another important ligand that synergizes with SCF is IL-7. The IL-7 receptor (IL-7R) consists of an  and a  subunit. Comparison of the IL-7–/– and IL-7R –/– mice has shown that the phenotype is more severe in IL-7R –/– mice. This suggests the existence of an IL-7–independent way to activate the IL-7R. Interestingly, Kit interacts with and phosphorylates the IL-7R in 293T cells, and this led to activation of the JAK-STAT pathway in the absence of IL-7 [160].
  b* W+ t3 t, O8 G) Z: ?8 g. X
G-CSF is another important hematopoietic growth factor that synergizes with SCF. Although G-CSF induces tyrosine and partial serine phosphorylation of STAT1 and STAT3, SCF only induces serine phosphorylation of STAT3 [161]. Stimulation with the combination of SCF and G-CSF results in complete serine phosphorylation of STAT3, which is necessary for maximal DNA binding. Furthermore, SCF and G-CSF induce STAT3 serine phosphorylation through different pathways, which to some extent are dependent on PI3K and Erk activity.. l( v3 L, k9 B7 |! F
: P, X# u& _. Q# D* }5 {" K
In total, these data support the possibility that synergistic growth induced by SCF in combination with other growth factors is mediated by multiple mechanisms that could include receptor interaction, amplification of activation of the Ras-Erk cascade, and activation of STAT family members, as well as synergistic expression of components of the cell cycle. Much remains to be learned about this interesting and important biological phenomenon.
7 i% d$ u& n3 V6 c" X4 u8 a
Negative Regulators of Kit Signaling

Because Kit activates multiple signaling pathways, it is not surprising that negative regulation of this potent receptor also occurs through a variety of mechanisms.* T) s% R7 s1 p- E' h! ~" n) v
2 U8 G! s' {8 b* a1 h/ X# `+ }- k
PKC is a large family of structurally related serine/threonine kinases. PKCs are important in downregulating several RTKs, including the EGFR and Kit [162, 163]. In cells transfected with Kit, activation of DAG-responsive PKCs by soluble SCF occurs through a mechanism involving a PI3K-dependent activation of phospholipase D [116]. The down-regulation of Kit by PKC occurs in two ways: Activation of PKC induces proteolytic release of the extracellular ligand binding domain of Kit [119, 164], and PKC phosphorylates serine residues 741 and 746 in the kinase insert, which inhibits Kit kinase activity by an unknown mechanism [165].
/ H: u( C% s- r2 |; ?
The tyrosine phosphatase SHP1 (PTP1C, HCP, SH-PTP1) interacts with Kit in the JMD [88, 166]. One report suggestst hat SHP1 binds to tyrosine 569 of murine Kit [166]. Expression of KitY569F in BaF3 cells results in increased SCF-induced proliferation compared with wild-type Kit. Furthermore, genetic experiments support a negative role for SHP1 in Kit signaling. Motheaten (me) mice have a loss-of-function mutation in SHP1, and these mice have hyper-proliferative hematopoietic progenitor cells [167]. Crossing me mice with mice expressing a mutated Kit receptor (W v) to produce mice harboring both mutations showed that this combination had a milder phenotype than me or W v mice [168, 169]. This suggests a functional relationship in which SHP1 negatively regulates Kit. Interestingly, loss of SHP1 had no effect on SCF-induced bone marrow–derived mast cell proliferation, suggesting a cell type–specific role of SHP1 in Kit signaling [169].
$ W+ m* E& d+ X
SOCS-1 protein levels increase after SCF stimulation of mast cells, and this leads to inhibition of proliferation [170]. Although SOCS-1 inhibits JAK and Tec by blocking kinase activity [171–173], it is unlikely that this mechanism inhibits Kit signaling. De Sepulveda et al. [170] demonstrated that SOCS-1 binds to Kit as well as several downstream signal transduction proteins, including Grb2 and the guanine nucleotide exchange factor Vav. This suggests that the inhibitory effect of SOCS-1 on Kit could be attributable to interference with coupling and function of downstream molecules. Interestingly, a recent study using BMMCs from SOCS-1–deficient mice demonstrated that these cells had a reduced proliferative response to both IL-3 and SCF compared with wild-type cells [174]. This unexpected finding was explained by accumulation of proteases in the absence of SOCS-1, resulting in degradation of essential signal transduction molecules. Hence, it appears that besides its function in negative feedback loops, SOCS-1 is also important for maintenance of certain protein levels.- @* F' C  S* t, R
! \7 f* [" q7 I# a: @1 Y
SHIP is a 5'-inositol phosphatase hydrolyzing phosphatidylinositol-3,4,5-trisphosphate (PIP3) to PIP2. SCF stimulation of mast cells leads to SHIP phosphorylation [175]. Disruption of the SHIP gene has revealed that it is a negative regulator of SCF-induced mast cell degranulation [176]. Furthermore, SHIP –/–mice have granulocytes and macrophages with increased responsiveness to IL-3, GM-CSF, and SCF compared with wild-type cells, supporting a role for SHIP as a negative regulator of cytokine signaling [177]./ n2 }5 ?; b' P% I' }

The GTPase activating protein neurofibromin-1 (NF-1) has been suggested to be a negative regulator of Kit function [178]. In NF-1–/– progenitor cells, SCF induces a stronger Erk activation and increases colony formation compared with normal cells. The NF-1 protein is a tumor suppressor commonly lost in juvenile myelomonocytic leukemia (JMML) [179]. It is not clear if Kit has a role in the development of JMML. Interestingly, it has been suggested that JMML originates from a pluripotent hematopoietic stem cell, indicating a potential role for increased Kit signaling in tumorigenesis [180].

Ubiquitin molecules are covalently attached to lysine residues on target proteins, and this labels them for degradation via the 26S proteosome complex. In RTK signaling, the ubiquitin ligase Cbl has an important role, because it can bind to activated receptors, or other tyrosine phosphorylated proteins, with its SH2 domain [181]. Activation of Kit leads to its poly-ubiquitination [118]. Cbl can associate with Kit through CrkL or APS and becomes phosphorylated in response to SCF [132, 139, 182]. Hence, it is likely that Cbl has an important role in Kit ubiquitination and subsequent downregulation.6 Z4 d/ T8 c* [; z
# u+ q/ L$ T8 S3 C7 ]* u
Signaling and Cellular Context8 D0 ~$ ?" @* c2 J4 H  x

When reading the literature on Kit signaling, it is important to note that studies have been done with a variety of experimental designs. This includes the use of primary cells from different species, primary cells from genetically engineered mice, cell lines expressing endogenous Kit, and cell lines that do not express endogenous Kit but are transfected with wild-type or mutant Kit. Although each of these approaches generates valuable information, the data must be interpreted in the context of the experimental approach. For example, in primary cells, there are differences in pathways activated by SCF in different lineages [67, 93, 109]. There are also differences in the outcome of disrupting the same pathway in different lineages [93, 109]. Thus, stimulus-response coupling mechanisms of Kit vary in mast cells, hematopoietic progenitors, lymphoid cells, melanocytes, and germ cells. This is also a consideration in work done with cell lines, in which lineage as well as extent of differentiation is a factor. In addition, as mentioned above, cell lines expressing endogenous Kit, as well as cell lines transfected with wild-type Kit or mutants, have been used extensively. An advantage of transfected cell lines is comparison of wild-type Kit and Kit mutants in a specific cell context. However, ectopic expression of Kit may generate an inappropriate biochemical or biological readout due to absence of certain signaling proteins, an inability to couple with Kit, or activation of pathways not physiologically relevant. In summary, consideration of the strengths and weaknesses of each of these experimental approaches is critical in integrating the vast amount of data available in this field.( U$ A1 v8 W) r; K

KIT AND HUMAN DISEASE4 Z  R/ k3 |+ A+ g; H5 W

Abnormalities in the expression and function of Kit are associated with several human diseases. Although the primary focus of this article is Kit and hematopoiesis, recent work indicates that targeting Kit holds promise in the treatment of diseases in nonhematopoietic tissues also. Consequently, studies of both hematopoieitc and nonhematopoietic tissues will be included in this section.
5 H! U9 C- J3 U& L8 p7 y
Loss-of-Function Mutations

Autosomal-dominant piebaldism is associated with loss-of-function mutations in Kit [183]. This syndrome results in deafness, megacolon, and abnormalities in pigmentation of skin and hair. The pigmentation defect in persons with piebaldism is remarkably similar to that seen in heterozygous W mice. Study of megacolon in patients with piebaldism helped define the critical role for Kit in development of the ICC. Kit mutations found in patients with piebaldism include point mutations and deletions.: W7 w) D( S" M7 P0 g% w* _
$ b0 B( F, C3 `
Inappropriate Expression of Kit or SCF

One mechanism leading to inappropriate activation of Kit is autocrine secretion of SCF. Tumors reported to have Kit/SCF autocrine loops include small cell lung carcinomas (SCLC), colorectal carcinoma, breast carcinoma, gynecological tumors, and neuroblastomas [184]. These data suggest that these diseases could respond to drugs that inhibit wild-type Kit. Indeed, in vitro, the tyrosine kinase inhibitor STI571 (imatinib mesylate/gleevec/glivec; Novartis Pharmaceuticals, St. Louis) does inhibit proliferation of SCLC cell lines [185, 186]. Furthermore, Krystal et al. [187] have identified two other Kit inhibitors, SU5416 and SU6597, that can inhibit proliferation of SCLC cell lines. A similar inhibitory effect of STI571 on proliferation of neuroblastoma cells was also recently demonstrated [188]. Thus, inhibition of Kit kinase activity may be one approach for treatment of diseases associated with autocrine activation of Kit.
# o% p7 n; h6 [3 j. x* y3 Q
Mast cells and hematopoietic progenitor cells express Kit on their surface, whereas most differentiated hematopoietic cells lack Kit expression. However, certain human leukemia cells, especially blasts from patients with acute myelogenous leukemia (AML) [43, 189], and certain non-hematopoietic tumors, such as gastrointestinal stromal cell tumors (GIST) [190] and seminoma germ cell tumors [191], also express surface Kit.

Kit is also expressed in cells such as cutaneous and choroidal melanocytes. Melanomas are variable with regard to chromosomal abnormalities, but loss of Kit expression seems to be associated with progression of certain forms of melanoma. Approximately 70% of metastatic lesions and human melanoma cell lines do not express detectable levels of Kit receptor [192]. Interestingly, transient expression of Kit in melanoma cell lines significantly inhibited tumor growth and metastasis [193]. Exposure of Kit-positive mela-noma cells to SCF, in vitro and in vivo, triggered apoptosis of these cells but not of normal melanocytes. These results suggested that loss of Kit may allow malignant melanoma cells to escape Kit-mediated apoptosis, thus allowing tumor growth and eventually metastasis. Further support for this possibility is a study demonstrating that the absence of Kit expression directly correlated with the metastatic potential of human melanoma cells in nude mice [194]. Although the exact mechanism for the loss of Kit gene expression during melanoma progression is unknown, Bar-Eli and colleagues [194] recently demonstrated that the transcription factor AP-2 is downregulated in melanomas. The Kit promoter has three AP-2 binding sites, suggesting loss of function of AP-2 plays a role in regulation of Kit expression and subsequently melanoma progression [195]. In contrast to these findings, Kit is expressed on choroidal melanoma tumors [196].! }: L. \5 m: b0 z  S3 O$ u$ ]/ E
# f, V) A- s7 r9 T+ H2 j% N
Gain-of-Function Mutations

In 1993, Furistu et al. [197] identified two gain-of-function mutations in Kit (V560G and D816V) in the human mast cell line HMC-1. These mutations map to the JMD and the second part of the kinase domain of Kit, respectively. A variety of activating mutations in the JMD have been identified, including deletions, point mutations, tandem duplications, and combinations of deletions and point mutations. These mutations are frequently associated with GISTs [198]. Similar JMD mutations have also been identified in RTKs closely related to Kit such as Flt3 (internal tandem repeats in the JMD of AML patients) and Met (JMD missense mutations or insertional mutations in SCLC) [199, 200].

Activating mutations in the second part of the kinase domain have been identified as point mutations at codon 816 of human Kit, which corresponds to codon 814 in murine Kit. Aspartic acid 816/814 is located in the activation loop and can be mutated to tyrosine, histidine, asparagine, or valine with similar consequences [201]. Activating mutations in the Kit kinase domain are associated with human malignancies such as mast cell leukemia, masto-cytosis [202, 203], acute myeloid leukemia (D816Y) [204, 205], and germ cell tumors (D816H) [206].
/ B. c, J$ h. Y9 ?# q/ [7 X
In a recent review, Longley et al. [207] classified activating mutations of Kit as either regulatory or enzymatic. The JMD mutations are considered regulatory in nature. These likely change the conformation of Kit and lead to constitutive dimerization and activation. This may also impact the binding of Kit to secondary signaling molecules. Mutations of the kinase domain are considered the enzymatic type. Interestingly, these two classes of mutations differ in their sensitivity to inhibitors of wild-type Kit, including STI571 and SU9529. JMD mutants are more sensitive to these inhibitors than the kinase domain mutants [208, 209]. In vitro studies have identified four small molecule inhibitors to be effective against the activated form of Kit mutant found in many patients with mastocytosis. These drugs, SU6577, SU11652, SU11654, and SU11655, all indolinone derivatives, are active against Kit JMD as well as kinase domain mutants [210, 211]. As of now there are no drugs identified that can specifically target the kinase domain mutants. Our laboratory is currently involved in screening for novel drugs to inhibit the kinase domain mutant of Kit.

In addition, a considerable number of patients with sinonasal natural killer/T-cell lymphomas have mutations in codon 825 of Kit [212]. Last, small subsets of patients with myeloproliferative disease contain point mutations in the extracellular domain of Kit, resulting in a D52N substitution [213, 214]. It is as yet unclear if gain of function of Kit is involved in disease progression in either of these cases.5 E: m: @% _5 x) b. z  Y

The following sections will highlight the salient features of three of the most prevalent diseases associated with gain-of-function mutations in Kit, as well as summarize available therapeutic approaches.7 r+ Y6 X7 G. y  g( @7 V3 I

Gastrointestinal Stromal Cell Tumors( _# [. [/ ~! w  n/ n0 `  M

Although GISTs constitute less than 1% of all tumors of the gastrointestinal (GI) tract, they are the most common form of mesenchymal tumors of the digestive tract [198]. GISTs occur in the GI tract from the esophagus to the rectum (5%) but are most commonly found in the gastric (6%–70%) and small intestine (25%–30%) regions. These tumors are believed to arise from the ICC or their mesenchymal stem cell precursors. On average, 10 to 20 GIST cases per million people are recorded annually. Of these, 20%–30% are malignant. Expression of Kit, readily detectable with immunohistochemistry, is often associated with this disease. Ninety percent of GIST patients express Kit, and many of them contain activating mutations in the JMD. Specifically, 57%–71% of GIST patients have mutations in exon 11, and 4%–17% of patients have mutations in exons 9 or 13. In certain rare cases, mutation in the kinase domain of Kit is also observed in GIST patients. The presence of these mutations is associated with poor prognosis. The percentage of patients surviving 5 years with GIST without mutations in Kit is 68%, compared with 49% for GIST patients with mutation in Kit.0 n, G8 n1 C: ^. I" P! V  C
; a" i2 x6 w8 \& ]/ L. M  C
As mentioned earlier, mutations in the JMD are considered regulatory and are believed to work through disruption of an inhibitory alpha helical structure [215]. This alpha helix is implicated in suppression of kinase activity and phosphorylation of Kit in the absence of ligand. The presence of mutations in this region is believed to relieve this inhibitory configuration, rendering Kit constitutively active.2 l' Z8 j6 O0 J& x5 S
( |* d4 {; |6 W( C6 r$ K: z
Although most GISTs have activating mutations in Kit, a certain population of GIST patients lacks Kit mutations [216, 217]. These findings suggest that there are other mechanisms that could potentially result in GIST. A recent study by Heinrich et al. [218] has demonstrated that 35% of GIST patients (14 of 40) did not carry a Kit mutation but instead had mutations in PDGFR, a related RTK [218]. They also demonstrated that the tumors with either Kit or PDGFR mutations did not differ in their potential to activate downstream signaling molecules such as Akt, STAT1, STAT3, and Erk1/2. These tumors, irrespective of a mutation in Kit or PDGFR, have deletions in chromosome 1p and monosomies of chromosomes 14 and 22. These results imply that Kit and PDGFR could be alternative and mutually excusive oncogenic mechanisms leading to GISTs.
3 \! \& G  z# k5 B' D* r2 q/ r
GISTs represent a wide spectrum of disease, ranging from benign to highly malignant forms. This makes prediction of clinical responses difficult. The malignant potential of GIST relies on tumor size, mitotic activity, and anatomical origin. Certain conventional treatment strategies are available to treat GISTs. Surgical resection is currently the only option for treatment of primary GIST. Unfortunately, these tumors are fragile and often require secondary removal. Adjuvant therapy is useful after surgical resection in patients with rectal GISTs with close or positive microscopic margins [219]. Metastatic GISTs are treated with conventional chemotherapeutic agents such as doxorubicin and ifosfamide together with surgical resection. However, conventional chemotherapy has limited efficacy. For patients with liver metastasis, hepatic artery embolization (i.e., cutting off the primary blood supply to liver) is an alternative that may be used in combination with traditional chemotherapy [220]. In certain rare cases, radiotherapy is also used mostly for palliation or to stop bleeding from peritoneal recurrence [221].

More recent, the tyrosine kinase inhibitor STI571, which is active against BCR-ABL fusion protein in chronic myelogenous leukemia (CML) as well as PDGFRs and Kit, has been shown to be effective in the treatment of GIST [222]. STI571 has recently been cocrystallized with Kit [223]. STI571 binds near the active site and stabilizes a conformation that is incompatible with kinase activity. Because of its ability to directly target the JMD mutant of Kit and its low toxicity in CML patients, use of STI571 as a treatment for GIST is increasing. In 2001, Joensuu et al. [224] reported promising results of treating a patient with metastatic GIST with STI571. Data obtained in follow-upstudies indicated STI571 had minimal toxicity and substantial activity against GIST. There was a partial response rate of 59%, with 23% showing disease stabilization [225]. Only approximately 13% of patients experienced progression of disease. STI571 is now considered a prime adjuvant therapy for primary GISTs after resection. Most patients are at high risk of recurrence after resection and do not respond to conventional therapies. Thus, the adjuvant therapy is expected at least to delay recurrence and hence prolong survival. However, because STI571 does not elicit a complete response, it may be best used in combination with conventional therapies. Despite the effectiveness of STI571, there are concerns regarding the development of resistance. In certain patients with CML, gene amplification and point mutations in the kinase domain in response to STI571 have already been demonstrated [226–228].

Mastocytosis4 ]+ e; E" z1 E) r6 w' y
: `. @, P* t0 U8 c! Z
Mastocytosis is characterized by accumulation of mast cells in various organs and release of mast cell mediators. Mastocytosis is reported in 1,000 to 8,000 patients in the U.S. every year belonging to all races and both sexes [229]. Peak incidence is during infancy and early childhood, with a second peak occurring in middle age. It usually occurs as a transient and limited disease in children, whereas it is persistent and progressive in adults. Because of its heterogeneous clinical presentation, mastocytosis is classified into four categories. Indolent mastocytosis is the most common form and involves the skin, bone marrow, and GI tract. Clinical features range from a single cutaneous nodule to multiple pigmented macules resulting from increased epidermal melanin and papules (urticaria pigmentosa) or diffuse cutaneous involvement; bullae, vesicles, and abnormal telangiectasia may be seen. GI involvement leads to symptoms such as nausea, vomiting, and abdominal pain. Mastocytosis with an associated hematologic disorder refers to cases in which urticaria pigmentosa symptoms are accompanied by a variety of hematological findings due to mast cell infiltration of the bone marrow, spleen, liver, and lymph nodes. Mast cell leukemia is characterized by proliferation and infiltration of immature mast cells in bone marrow, peripheral blood, and various extramedullary tissues. Aggressive mastocytosis is characterized by severe involvement of bone marrow, liver, spleen, and lymphatic system.2 P4 _) {, C) |3 |7 Z0 M9 E: e6 W9 j
) j8 p7 Y) z0 @  B$ Y
The prognosis of mastocytosis is highly dependent on the form. Although indolent mastocytosis, the most common form, is not generally fatal, the other three forms can be.
! N, |! d/ E. |* i1 l$ V9 O: |
Interaction of SCF and Kit is critical for mast cell development, survival, and function. Although abnormalities in this receptor ligand combination have been implicated in mastocytosis, mechanisms mediating the diverse clinical patterns have not been elucidated. As previously discussed, activating mutations in Kit have been reported in a variety of patients with mastocytosis. Most of these mutations map to the kinase domain of Kit. In contrast, familial mastocytosis may occur in the absence of Kit mutations [230]. These mutations are summarized in Table 2. Direct and indirect evidence supports the contention that these mutations promote ligand-independent autophosphorylation of the mutant receptor. This will be discussed in greater detail in the following section dealing with oncogenic Kit signaling.5 g$ [% L& W3 T! @) R# _

Interestingly, activating mutations are more common in adult than pediatric patients. Pediatric mastocytosis falls into three categories. The first class of patients has an activating mutation in codon 816 of Kit. This is very rare. The second class of patients, identified by Longley et al. [230], have a novel inactivating mutation at codon 839 of Kit, resulting in substitution of a lysine with glutamic acid in three out of six patients. The third class of patients has no known mutations. However, mutations found during childhood disease are associated with a higher risk for extensive or persistent disease. Notably, most studies on pediatric mastocytosis have only analyzed the exons encoding the JMD and the region containing codon 816. Hence, there is a possibility of mutations elsewhere in the receptor.
% _6 E4 R( |% F6 f
Altered expression of SCF may also be involved in mastocytosis. The soluble form of SCF is prevalent in the dermis and extracellular spaces between kertinocytes in skin from mastocytosis patients [231]. Altered distribution of SCF in the skin of patients with cutaneous mastocytosis, a disease characterized by dermal accumulations of mast cells and increased epidermal melanin, is consistent with abnormal production of the soluble form of SCF. This likely results from an increase in cleavage of the membrane-bound form of SCF [232]. This suggests cutaneous mastocytosis could be the result of a hyperplastic response to a local increase in soluble SCF in the tissue.: H3 _: S6 n9 y  B+ S$ ^

To date, there is no cure for mastocytosis. Patients are asked to avoid classic mast cell triggers, as well as extreme temperatures, alcohol, and certain drugs. Pharmacological treatment is aimed at reducing discomfort associated with symptoms and includes stabilizing mast cell membranes to decrease the extent of degranulation in conjunction with blocking the action of inflammatory mediators. Surgical removal of isolated mastocytomas, topical corti-costeroid therapy, and psoralen plus ultraviolet A therapy to fade multiple pigmentation lesions are other available approaches for treatment.& R, r6 W/ r4 n. N: ^
; k6 {" M$ G- {/ U
Acute Myelogenous Leukemia) m5 X9 \8 L7 ~+ B- Q6 Z& c$ r. u

Wild-type Kit is expressed in most cases (80%–90%) of human AML and in some cases during CML blast crisis [184]. The presence of Kit has been attributed to the immature stage of differentiation of AML cells [233]. In contrast, Kit is rarely expressed in leukemia cells of lymphoid origin. In addition, Kit-positive AML specimens show constitutive phosphorylation of Kit [189, 234, 235]. Kit is also activated in some AML blasts in the absence of SCF. Furthermore, SCF can stimulate proliferation of certain AML blasts in vitro [189, 234, 235]. Autocrine and paracrine stimulation of Kit by SCF may thus play a role in the pathobiology of AML. Indeed, it has been shown that approximately 30% of AML blasts coexpress Kit and SCF [236]. In addition, Gewirtz et al. [44] reported that 25% of AML blasts showed decreased growth in the presence of Kit antisense oligonucleotides. The same study also found this to be the case for 33% of ALL and 50% of CML blasts. For a detailed review on autocrine/paracrine loops in normal and oncogenic hematopoiesis, please refer to the study by Janowska-Wieczorek et al. [30].
9 d5 Y4 s8 r! U/ |: p8 r
One third of the cases of AML with inv (16) karyotype also have deletion and insertion in exon 8 that code for the Kit extracellular domain [237]. Mutations in codon 416 are involved in all of these cases, and this is presumed to be a gain-of-function mutation. However, this mutation has not been linked with increased activation of Kit [237]. The other site for Kit mutations in patients with AML is codon 816 (D816Y or D816V). This mutation was observed in 7 of 15 patients with core binding factor leukemia [t(8;21) or inv(16)] [238, 239]. The heterodimeric core binding transcription complex is crucial for normal hematopoiesis [240]. Interestingly, mutations and rearrangements of the core binding complex have often been found in leukemia. However, the functional significance of the correlation between the translocations of core binding factors and Kit mutations is as-yet unclear." O/ s6 A9 D/ C
; E" z- {3 A6 W2 _6 r! x: r
Although a significant number of Kit-positive AMLs have been identified, there is still insufficient evidence to use Kit as a prognostic marker. Overall, Kit expression does not correlate well with cytogenetic analysis. Despite accepted prognostic indicators, approximately 40% of patients with newly diagnosed AML do not achieve complete remission after conventional therapy. The response rate can be improved to 50%–75% with the use of sibling bone marrow transplant therapy. Recent studies have shown that the small molecule inhibitors SU5416 and SU6668 effectively block kinase activity of wild-type Kit in cultured AML blasts from patients [241, 242]. Complex biological defects in hematopoietic progenitor cell differentiation, proliferation, and survival are critical in the pathogenesis of AML and pose significant challenges in development of treatment strategies.
9 N' I. l/ d' Y" A# g" [
AML is also associated with mutations in Flt3, a RTK closely related to Kit. These occur as either internal tandem duplications of the JMD (25%–30%) or point mutations in the kinase domain region (7%–8%) [243].

ONCOGENIC KIT SIGNALING
0 s: A' I) s- x" O! R( J6 P! `( f
Oncogenic Kit Mutations2 A6 Y. D3 Q+ Y2 r" d; K& g
5 ^, N" c. P9 b9 W) d9 v6 C
As discussed above, gain-of-function mutations in Kit associated with human disease occur most frequently in the JMD or in the kinase domain. These activating mutations have both common and unique features. Both types of mutations result in constitutive phosphorylation of Kit on tyrosine, although the magnitude of phosphorylation of the kinase domain mutant is generally greater than that of the JMD mutants [244]. A comparison of the JMD mutant KitV559G with the kinase domain mutant KitD814V in 293T cells indicated that the kinase domain mutant has enhanced enzymatic activity compared with wild-type or KitV559G [197]. Similar results were observed in later studies comparing the two types of mutants with wild-type Kit [245, 246]. Comparison of wild-type and mutant kinase domains indicated that the kinase domain mutant (D814V) has an enhanced affinity for ATP. In contrast, the JMD deletion mutant and wild-type Kit bound ATP with similar affinity [245, 246].% D* a$ K# _. B" B! P7 L
( r( ^- w2 f7 r
A shift in substrate specificity has also been observed with oncogenic mutants of Kit. In vitro kinase assays using optimal peptide substrates for EGF receptor and Abl kinase showed a shift in substrate specificity with the D814Y mutation [247]. Both wild-type and KitD814Y phosphorylated an EGFR peptide, but KitD814Y phosphorylated an Abl peptide much more efficiently. A similar shift in specificity was reported with a recently identified JMD deletion mutation designated K27 (in frame deletion at codons 547–555) compared with wild-type Kit [248]. This shift in substrate specificity could contribute to differential activation of transforming pathways by mutant forms of Kit.
% s7 q$ {: ?2 _) t  `' y
Factor-dependent hematopoietic cell lines expressing Kit with mutations in the JMD or the kinase domain become factor independent and tumorigenic in mice [238, 244, 249–253]. However, hematopoietic cells expressing JMD or kinase domain Kit mutants do not behave identically. Although both types of Kit mutants can promote transformation in hematopoietic cells, kinase domain mutants have a more potent effect on hematopoietic lineages. Bone marrow mast cells from knock-in mice expressing the Kit JMD deletionmutation, V558, do not rely on SCF for survival but do require it for cell-cycle progression [254]. These cells are also resistant to growth factor deprivation–induced apoptosis. Surprisingly, lineage-negative hematopoieitc cells and T-cell development were unaffected in these knock-in mice. In contrast, murine bone marrow cells retrovirally infected with the kinase domain mutant D814V differentiated into larger, more numerous granulocyte-macrophage and mast cell colonies in the absence of growth factors compared with bone marrow cells infected with the JMD mutant V559G [255]. In addition, bone marrow infected with the D814V mutant differentiated into mixed erythroid/myeloid colonies. Six out of 10 mice transplanted with D814V-infected bone marrow developed lymphoid leukemia, whereas only 1 out of 10 mice transplanted with V559G-infected bone marrow did [255]. Enhanced SCF-dependent chemotaxis has been observed in Jurkat cells transfected with KitD816V and in circulating mast cell precursors from mastocytosis patients with the D816V mutation [256]. Recently, Besmer and colleagues [254] developed a mouse model for GIST in which they knocked in an activating Kit mutation derived from a mutation identified in human familial GIST syndrome (V558). This study also shows that activating mutations in the Kit JMD is predisposing for GIST development.
: K; X: [- v1 y& g6 U0 N
The Hardy-Zuckerman-4 strain of feline sarcoma virus contains a transforming version of Kit, v-Kit, that lacks the extracellular, transmembrane domains and has a deletion of two amino acids in the JMD and the C-terminal region. Herbst and colleagues [257] showed that the deletion of tyrosine 569 and valine 570 in the JMD in combination with loss of the C-terminal region were crucial for the transforming ability of v-Kit. More recent work suggests this would lead to loss of SFK and SHP1 binding. In addition, APS binds to both these regions in Kit [132]. APS is connected to receptor downregulation by linking to Cbl and subsequently protein degradation. Thus, the binding sites for two negative regulatory signaling components are deleted in v-Kit. This could, at least in part, contribute to the transforming activity of v-Kit. The importance of the inability of v-Kit to interact with SFKs has not been thoroughly investigated.* T9 V8 y4 z9 v4 ]6 T- ^2 n
0 s' n2 G* o. u% B
Dimerization of Oncogenic Kit Mutants" l9 W3 Q- g7 U  j9 [+ C

Wild-type Kit dimerizes and autophosphorylates after stimulation with SCF. JMD gain-of-function mutants form stable dimers in the absence of SCF. Similar to wild-type Kit, JMD mutants dimerize through the extracellular domain. In contrast, the kinase domain mutants are not constitutively dimerized via the extracellular domain but do dimerize in the presence of SCF [249]. Indeed, dimerization of the Kit kinase domain mutants likely occurs by mechanisms that differ from wild-type [249, 251, 258].- T$ i- H$ w+ \! o7 a3 B8 J

Deletion constructs of KitD814Y, lacking the extracellular ligand binding and dimerization domains, result in truncated Kit receptors that are still constitutively phosphorylated on tyrosine [258]. Studies on phosphorylation kinetics using purified recombinant wild-type Kit kinase domains demonstrated a nonlinear concentration dependence, supporting activation by transphosphorylation and oligomerization. In contrast, purified KitD814Y kinase domain had a linear relationship with concentration. This suggests that the Kit kinase domain mutant could be activated by cis rather than transphosphorylation or that it exists as preformed dimers through regions of the kinase domain [245]. Gel filtration analysis of the purified kinase domains supported the hypothesis that KitD814Y spontaneously forms stable ligand-independent dimers, whereas the wild-type receptor forms transient ligand-dependent dimers. These studies suggested the D814Y mutant dimerizes via a unique intracellular mechanism. However, there are conflicting reports regarding constitutive dimerization of the kinase domain mutant [245, 249, 258]. Whereas the aforementioned groups used mutant Kit constructs that lacked the extracellular domains, a third group used the entire mutant protein and did not detect constitutive dimers [249]. The presence or absence of the extracellular domain could impart conformational changes in the mutant protein that could alter dimerization properties and substrate specificities. This hypothesis is additionally substantiated by studies using the GNNK JMD splice variants. Voytyuk et al. [111] demonstrated that the presence or absence of these four amino acids in the JMD greatly influenced receptor phosphorylation as well as downstream signaling events.; j+ \" U( f4 S
& i! \% ^" P9 ~  ^8 M
Regulation of Kit Kinase Activity( \/ Z: Y8 Z' p; n* F1 E: w
" {5 T5 i; g+ p' @- |7 A7 C& |
Regulation of Kit kinase activity is a multistep process. Studies with constitutively active Kit mutants have shed light on some of these regulatory mechanisms. Mutations in the JMD of Kit play a critical role in regulating kinase activity through modulation of dimerization as well as potentially altering interaction with negative regulatory molecules. For example, SHP1 negatively regulates Kit activity by interacting with the Kit JMD [166]. Another potential negative regulator of Kit is the adapter protein APS. APS interacts with the Kit JMD, and this interaction facilitates recruitment of Cbl, a negative regulator of tyrosine kinases, into the complex [131, 132]. Lnk, a homologue of APS, is tyrosine phosphorylated and negatively regulates Kit-mediated proliferation [133]. Another potential negative regulator of Kit that may interact indirectly with the JMD is Dok-1 [136]. Mutations in the Kit JMD could alter the interaction with one or more of these negative regulators and thus result in cellular transformation.

Exon 11 of the cytosolic region of the JMD plays a critical role in negative regulation of Kit dimerization. Residues 553–663 of Exon 11 form a putative alpha helix that inhibits activation of ligand unoccupied receptor [215]. A similar autoinhibitory region was identified in the RTK EphB2, which functions by direct interaction with the kinase domain [259]. Thus, this seems to be a general mechanism of kinase regulation. Therefore, mutations in the helix region (substitutions, deletions, or insertions) result in major alterations in the kinase activity.

The effect of the JMD on dimerization became evident from studies using peptides corresponding to the intracellular JMD. The highly organized secondary structure of this peptide was lost after tyrosine phosphorylation [246]. Although unphosphorylated peptide bound the ATP-binding region of Kit and suppressed autophosphorylation, the tyrosine-phosphorylated peptides or peptides with mutations corresponding to activating Kit mutations did not. This suggested that phosphorylation of wild-type Kit, or mutation of the JMD, relieves the negative effect of the JMD on Kit dimerization. This was confirmed when coexpression of wild-type unphosphorylated peptide with a JMD deletion mutant of Kit decreased cell growth. Thus, the oncogenic potential of the JMD mutations may be attributable to the loss of JMD inhibitory function.3 D4 K( c. f, V
+ k- m$ t5 B) c5 l  b* b; C
Mutations in the kinase domain mutation have unpredictable effects. Heinrich et al. [184] have speculated that codon 816 mutations alter the conformation of the mobile activation loop. A recent structural study of the active form of wild-type Kit suggested that Y823 of the activation loop is one of the last residues to be autophosphorylated [56]. This further led to the conclusion that the monomeric Kit kinase domain is close to the active conformation but positioned in a way that prevents transactivation. Dimerization alters the conformation and orientation of the kinase domains into one that allows efficient autophosphorylation. This may explain the differences in substrate specificities of the JMD mutants, which can dimerize like wild-type Kit, and the kinase domain mutant that may dimerize through a different mechanism. Furthermore, this could also lead to differences in the sensitivities of these two mutants toward STI571.

Downstream Signaling by Oncogenic Kit Mutants

Several groups have compared signaling of wild-type Kit with different JMD mutants. Frost et al. [209] have demonstrated that the JMD mutation G560V constitutively activated Akt as well as STAT3. Furthermore, STAT3 was constitutively activated in GIST cells with mutations in exon 11 of the JMD [260]. Activation of JMD mutants and subsequent activation of Erk1/2 and Akt can be effectively blocked by using STI571 [261–263]. Thus, this mutant activates similar signaling components as wild-type Kit, including Akt and Erks.

Signaling through the Kit kinase domain mutant has also been studied. Similarities with wild-type Kit include activation of the PI3K pathway. The p85 subunit of PI3K was constitutively associated with the Kit kinase domain mutant (human KitD816V and murine KitD814Y) and was constitutively phosphorylated. Our group has recently demonstrated that this association is likely mediated by constitutive phosphorylation of tyrosine 721 of human Kit (719 of murine Kit), the docking site for PI3K on wild-type Kit. In contrast, both phosphorylation of p85PI3K and association with wild-type Kit required stimulation with SCF [66, 253, 264]. Inhibition of PI3K activity with wortmannin or LY249002 or by inhibition of p85 association with KitD816V by mutation of tyrosine Y719/721 led to dramatic decreases in growth and tumorigenicity of cells expressing the Kit kinase domain mutant both in vitro and in vivo [253, 264, 265]. Together, these data demonstrate the importance of the PI3K pathway in transformation by the Kit kinase domain mutant. However, consistent with previous findings, it is unlikely that PI3K is sufficient for transformation mediated by the Kit kinase domain mutant.

Akt is one of the best known targets downstream of PI3K. Interestingly, Akt was not constitutively phosphorylated in cells stably expressing KitD816V but was in cells transiently transfected with the Kit kinase domain mutant [253]. The absence of Akt phosphorylation in cells stably transfected with the Kit kinase domain mutant could be attributable to rapid turnover or degradation of activated Akt in these cells as a secondary adaptation due to stable expression of the oncoprotein. Indeed, stable expression of KitD814Y results in rapid degradation of SHP1, a negative regulator of wild-type Kit activity [247]. One implication of these data is that PI3K is contributing to transformation of cells expressing the Kit kinase domain mutant through a signaling component other that Akt.8 C0 P4 G' T% h1 p# q7 }
- }+ H  L3 K0 z" x0 c7 U. g3 A
Another signaling component activated in cells expressing the Kit kinase domain mutant is STAT3. Arceci and colleagues [205] showed constitutive STAT3 tyrosine phosphorylation and DNA binding activity in MO7e cells expressing human KitD816H/N. In contrast, MO7e cells expressing wild-type Kit exhibited only weak activation of STAT3 in response to SCF stimulation. Expression of potential downstream effectors of STAT3, Bcl-xL, and c-Myc was also increased in MO7e cells expressing KitD816H. Transfection of a dominant-negative STAT3 construct in MO7e cells expressing KitD816H led to formation of fewer, smaller colonies in semisolid media, indicating the importance of STAT3 to the oncogenic potential of the kinase-domain mutant. Another study comparing 293 cells stably transfected with wild-type human Kit or KitD816H had similar results [266]. Thus, similar to PI3K, STAT3 is necessary but not sufficient for factor-independent growth mediated by a kinase domain Kit mutant. These data also suggest that STAT3 is more important in signaling through the Kit kinase domain mutant than wild-type Kit.* C6 L+ y8 M, m" U; Q

Another signaling pathway examined by multiple investigators in cells expressing the Kit kinase domain mutant is the MAPK cascade. Whereas SCF induces activation of Erk1/2 in cells expressing wild-type Kit, these MAPK family members were not phosphorylated in MIHC cells that expressed KitD816V or in the megakaryoblastic cell line MO7e cells infected with KitD816H/N [205, 266]. In contrast, one report suggests Erks may be activated by the Kit kinase domain mutant in an erythroid lineage cell line [267]. These data raise the interesting possibility that the Kit kinase domain mutant either preferentially activates or preferentially maintains the activity of some, but not all, signaling pathways stimulated by wild-type Kit.

In summary, the Kit JMD mutant likely acts through similar signaling pathways as wild-type Kit. In contrast, the kinase domain mutants of Kit may maintain the activity of some (PI3K and STAT3) but not all (Erks) pathways normally used by wild-type Kit.

CONCLUSION

When the older literature on the White spotting and Steel mice is combined with more recent work on the gene products of these loci, an impressive research story spanning close to eight decades emerges. Mice with mutations in either the Kit (White spotting mice) or SCF (Steel mice) gene were first identified in 1927 and 1956, respectively. In 1986, the v-kit oncogene was isolated, and the corresponding proto-oncogene c-kit was described in 1987. In 1990, we learned that SCF is the ligand for Kit. Fueled with the ability to transfect/infect primary cells and cell lines, as well as to generate transgenic/knockout mice, the research community has made impressive advances in understanding some of the structure-function relationships of the Kit RTK. There has also been some progress in understanding the biochemical and molecular mechanisms mediating the capacity of SCF to synergize with other growth factors, although much remains to be learned. Since the mid-1990s, activating mutations in Kit have been identified in patients with a variety of diseases. Indeed, targeted therapy to inhibit one class of constitutively active Kit mutant has shown great promise in treatment of patients with GIST. Work to identify additional compounds that inhibit activated Kit mutants found in mastocytosis and other diseases is ongoing in several laboratories. It is encouraging to see the rate of discoveries increasing our understanding of the basic mechanisms of this important RTK accelerating over time, as well as advances that directly benefit patients. As we learn about fundamental issues of cell biology through this interesting and important RTK, we trust that this will lead to further success in the treatment of cancer and other diseases.3 W" ?8 z3 ~! Y/ ^1 t3 Y3 \

ACKNOWLEDGMENTS
; W! K, C  C, m4 t& L6 ^
The authors wish to thank Dr. Reuben Kapur, Dr. Jonathan Keller, and Dr. Lars R?nnstrand for reviewing this manuscript.

The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. government." D) {# I, N0 b& N' }
; |% t& H# p& f0 z
REFERENCES" Z+ [; {( Q$ L9 q6 ?

Besmer P, Murphy JE, George PC et al. A new acute transforming feline retrovirus and relationship of its oncogene v-kit with the protein kinase gene family. Nature 1986;320:415–421.
3 c7 @8 ^. s7 b. G# H! O0 a7 c
Yarden Y, Kuang WJ, Yang-Feng T et al. Human protooncogene c-kit: a new cell surface recept or tyrosine kinase for an unidentified ligand. EMBO J 1987;6:3341–3351

Blechman JM, Lev S, Brizzi MF et al. Soluble c-kit proteins and antireceptor monoclonal antibodies confine the binding site of the stem cell factor. J Biol Chem 1993;268:4399–4406.% ?4 y8 I5 b0 z2 V
' i' M8 {/ z7 g
Lev S, Blechman J, Nishikawa S et al. Interspecies molecular chimeras of kit help define the binding site of the stem cell factor. Mol Cell Biol 1993;13:2224–2234.. t$ K# x1 ^3 b

Reith AD, Ellis C, Lyman SD et al. Signal transduction by normal isoforms and W mutant variants of the Kit receptor tyrosine kinase. EMBO J 1991;10:2451–2459.5 N! j8 F* ]) C

Crosier PS, Ricciardi ST, Hall LR et al. Expression of isoforms of the human receptor tyrosine kinase c-kit in leukemic cell lines and acute myeloid leukemia. Blood 1993;82:1151–1158.2 q- e1 `7 x% D. W: J+ a

Huang EJ, Nocka KH, Buck J et al. Differential expression and processing of two cell associated forms of the kit-ligand: KL-1 and KL-2. Mol Biol Cell 1992;3:349–362.

Chabot B, Stephenson DA, Chapman VM et al. The proto-oncogene c-kit encoding a transmembrane tyrosine kinase receptor maps to the mouse W locus. Nature 1988;335:88–89.6 G2 q3 C0 M5 b) K! w

Geissler EN, Ryan MA, Housman DE. The dominant-white spotting (W) locus of the mouse encodes the c-kit proto-oncogene. Cell 1988;55:185–192.

Kissel H, Timokhina I, Hardy MP et al. Point mutation in kit receptor tyrosine kinase reveals essential roles for kit signaling in spermatogenesis and oogenesis without affecting other kit responses. EMBO J 2000;19:1312–1326.# H  n7 ?# Q; j0 X( y  H0 A

Timokhina I, Kissel H, Stella G et al. Kit signaling through PI 3-kinase and Src kinase pathways: an essential role for Rac1 and JNK activation in mast cell proliferation. EMBO J 1998;17:6250–6262.
+ l6 L. ^) i/ K
Ueda S, Mizuki M, Ikeda H et al. Critical roles of c-kit tyrosine residues 567 and 719 in stem cell factor-induced chemotaxis: contribution of src family kinase and PI3-kinase on calcium mobilization and cell migration. Blood 2002;99:3342–3349.+ D  h5 S8 R6 o% W& A
& ~% n" j7 L; F* k- S: m5 |$ V
Blume-Jensen P, Jiang G, Hyman R et al. Kit/stem cell factor receptor-induced activation of phosphatidylinositol 3'-kinase is essential for male fertility. Nat Genet 2000;24:157–162" ]2 N& I3 J  {" f4 f6 G* L+ W5 H
; n: W, I, ]# y: x$ ~* D6 l/ S3 X
De Miguel MP, Cheng L, Holland EC et al. Dissection of the c-Kit signaling pathway in mouse primordial germ cells by retroviral-mediated gene transfer. Proc Natl Acad Sci U S A 2002;99:10458–10463.+ O* N( s- o$ }" _% L7 \2 v

Fukao T, Yamada T, Tanabe M et al. Selective loss of gastrointestinal mast cells and impaired immunity in PI3K-deficient mice. Nat Immunol 2002;3:295–304.3 v5 V% S, f6 L! u0 ?+ e. _4 |! T

Lu-Kuo JM, Fruman DA, Joyal DM et al. Impaired kit- but not FcepsilonRI-initiated mast cell activation in the absence of phosphoinositide 3-kinase p85alpha gene products. J Biol Chem 2000;275:6022–6029.

Tan BL, Yazicioglu MN, Ingram D et al. Genetic evidence for convergence of c-Kit- and 4 integrin-mediated signals on class Ia PI-3kinase and the rac pathway in regulating integrin-directed migration in mast cells. Blood 2003;101:4725–4732.
' R7 L5 i3 G5 l
Steelman L, Pohnert S, Shelton J et al. JAK/STAT, Raf/MEK/ERK, PI3K/Akt and BCR-ABL in cell cycle progression and leukemogenesis. Leukemia 2004;18:189–218." ^0 {5 q( J* D

Brizzi MF, Zini MG, Aronica MG et al. Convergence of signaling by interleukin-3, granulocyte-macrophage colony-stimulating factor, and mast cell growth factor on JAK2 tyrosine kinase. J Biol Chem 1994;269:31680–31684.! Q" w- q- M* g! E6 ~

Weiler SR, Mou S, DeBerry CS et al. JAK2 is associated with the c-kit proto-oncogene product and is phosphorylated in response to stem cell factor. Blood 1996;87:3688–3693.

Linnekin D, Weiler SR, Mou S et al. JAK2 is constitutively associated with c-Kit and is phosphorylated in response to stem cell factor. Acta Haematol 1996;95:224–228.5 ?* D' d/ g# Y' A" Y
: h9 S' Y4 i- s- f! G
Radosevic N, Winterstein D, Keller JR et al. JAK2 Contributes to the intrinsic capacity of primary hematopoietic cells to respond to SCF. Exp Hematol 2004;32:149–156.
( s4 V, E1 {( p, H. [
Voytyuk O, Lennartsson J, Mogi A et al. Src family kinases are involved in the differential signaling from two splice forms of c-Kit. J Biol Chem 2003;278:9159–9166.! @0 B, d' ~' m0 c8 W
) B* D- U- ?8 Z4 u$ v
Wilde JI, Watson SP. Regulation of phospholipase C gamma isoforms in haematopoietic cells: why one, not the other? Cell Signal 2001;13:691–701.

Herbst R, Shearman MS, Jallal B et al. Formation of signal transfer complexes between stem cell and platelet-derived growth factor receptors and SH2 domain proteins in vitro. Biochemistry 1995;34:5971–5979.

Gommerman JL, Sittaro D, Klebasz NZ et al. Differential stimulation of c-Kit mutants by membrane-bound and soluble Steel Factor correlates with leukemic potential. Blood 2000;96:3734–3742.# `8 Q  x# g, u/ c4 Y

Koike T, Hirai K, Morita Y et al. Stem cell factor-induced signal transduction in rat mast cells: activation of phospholipase D but not phosphoinositide-specific phospholipase C in c-kit receptor stimulation. J Immunol 1993;151:359–366.+ O+ t9 o, e; |5 w" e" h$ g
& F& e1 g  w4 Y$ O' i8 Z
Kozawa O, Blume-Jensen P, Heldin CH et al. Involvement of phosphatidylinositol 3'-kinase in stem-cell-factor- induced phospholipase D activation and arachidonic acid release. Eur J Biochem 1997;248:149–155.
6 v  F, \9 x1 j# S( q
Trieselmann NZ, Soboloff J, Berger SA. Mast cells stimulated by membrane-bound, but not soluble, steel factor are dependent on phospholipase C activation. Cell Mol Life Sci 2003;60:759–766.

Miyazawa K, Toyama K, Gotoh A et al. Ligand-dependent polyubiquitination of c-kit gene product: a possible mechanism of receptor down modulation in M07e cells. Blood 1994;83:137–145.
6 W" @  C5 N/ W" _/ Z5 m' s. a* X
Yee NS, Hsiau CW, Serve H et al. Mechanism of down-regulation of c-kit receptor: roles of receptor tyrosine kinase, phosphatidylinositol 3'-kinase, and protein kinase C. J Biol Chem 1994;269:31991–31998.2 Q% H) ~" A8 v. e- B
# A0 Y* `/ d) \
Zsebo KM, Smith KA, Hartley CA et al. Radioprotection of mice by recombinant rat stem cell factor. Proc Natl Acad Sci U S A 1992;89:9464–9468.
0 o) ?, O9 P9 T& V; E; [
Neta R, Williams D, Selzer F et al. Inhibition of c-kit ligand/steel factor by antibodies reduces survival of lethally irradiated mice. Blood 1993;81:324–327.
1 a" s, o- q" Z* I
Liebmann J, DeLuca AM, Epstein A et al. Protection from lethal irradiation by the combination of stem cell factor and tempol. Radiat Res 1994;137:400–404.
7 @0 ^) }( R% k1 \4 r6 n
Plo I, Lautier D, Casteran N et al. Kit signaling and negative regulation of daunorubicin-induced apoptosis: role of phospholipase Cgamma. Oncogene 2001;20:6752–6763.$ L5 [1 e  q7 a; W& r8 N/ |+ Z
; q. `8 n/ M$ L1 a3 s/ d
Maddens S, Charruyer A, Plo I et al. Kit signaling inhibits the sphingomyelin-ceramide pathway through PLC gamma 1: implication in stem cell factor radioprotective effect. Blood 2002;100:1294–1301.6 m5 e3 O. Y2 p5 b

Feng GS, Ouyang YB, Hu DP et al. Grap is a novel SH3-SH2-SH3 adaptor protein that couples tyrosine kinases to the Ras pathway. J Biol Chem 1996;271:12129–12132.9 `, I5 e) h; m5 f
5 _! x5 U8 U( Q) G. M; n1 _" h7 ~
Cutler RL, Liu L, Damen JE et al. Multiple cytokines induce the tyrosine phosphorylation of Shc and its association with Grb2 in hemopoieticcells. J Biol Chem 1993;268:21463–21465.

Liu SK, McGlade CJ. Gads is a novel SH2 and SH3 domain-containing adaptor protein that binds to tyrosine-phosphorylated Shc. Oncogene 1998;17:3073–3082.( v2 I* J% d* t7 A, n& T# S
# d# ~( T8 ~2 D) g  U
Holgado-Madruga M, Wong AJ. Gab1 is an integrator of cell death versus cell survival signals in oxidative stress. Mol Cell Biol 2003;23:4471–4484.8 m& Q6 C: g" N3 f- i$ U

Jahn T, Seipel P, Urschel S et al. Role for the adaptor protein Grb10 in the activation of Akt. Mol Cell Biol 2002;22:979–991.
  J6 X. C  \# {/ w1 @7 S
Yokouchi M, Suzuki R, Masuhara M et al. Cloning and characterization of APS, an adaptor molecule containing PH and SH2 domains that is tyrosine phosphorylated upon B-cell receptor stimulation. Oncogene 1997;15:7–15.$ {+ C( m/ W$ J* U: q4 {7 c( r

Yokouchi M, Wakioka T, Sakamoto H et al. APS, an adaptor protein containing PH and SH2 domains, is associated with the PDGF receptor and c-Cbl and inhibits PDGF-induced mitogenesis. Oncogene 1999;18:759–767./ _' r- ]0 }' @5 Y+ [$ G
* ~$ O; ?6 `& A8 k7 {: t
Wollberg P, Lennartsson J, Gottfridsson E et al. The adapter protein APS associates to the multifunctional docking sites Tyr568 and Tyr936 in c-Kit: possible role in v-Kit transformation. Biochem J 2003;370:1033–1038.

Takaki S, Morita H, Tezuka Y et al. Enhanced hematopoiesis by hematopoietic progenitor cells lacking intracellular adaptor protein, Lnk. J Exp Med 2002;195:151–160.; |  R- ]1 L  T4 w

Carpino N, Wisniewski D, Strife A et al. p62(dok): a constitutively tyrosine-phosphorylated, GAP-associated protein in chronic myelogenous leukemia progenitor cells. Cell 1997;88:197–204.! N7 N; w' q! B* B; G9 T

van Dijk TB, van Den Akker E, Amelsvoort MP et al. Stem cell factor induces phosphatidylinositol 3'-kinase-dependent Lyn/Tec/Dok-1 complex formation in hematopoietic cells. Blood 2000;96:3406–3413.3 L! G. J0 x1 @4 b  G6 d3 i, i0 Q

Liang X, Wisniewski D, Strife A et al. Phosphatidylinositol 3-kinase and Src family kinases are required for phosphorylation and membrane recruitment of Dok-1 in c-Kit signaling. J Biol Chem 2002;277:13732–13738./ I7 P6 c# w/ d/ K
( L5 G0 _: \$ E! c: `* y: F
Yamanashi Y, Tamura T, Kanamori T et al. Role of the rasGAP-associated docking protein p62(dok) in negative regulation of B cell receptor-mediated signaling. Genes Dev 2000;14:11–16.
, M6 G0 S0 w! i, @
Di Cristofano A, Niki M, Zhao M et al. p62(dok), a negative regulator of Ras and mitogen-activated protein kinase (MAPK) activity, opposes leukemogenesis by p210(bcr-abl). J Exp Med 2001;194:275–284.! ~6 i$ q9 D6 r% x  R) j7 P, c

Sattler M, Salgia R, Shrikhande G et al. Steel factor induces tyrosine phosphorylation of CRKL and binding of CRKL to a complex containing c-kit, phosphatidylinositol 3-kinase, and p120(CBL). J Biol Chem 1997;272:10248–10253.) a) a( F; G5 T

Duarte RF, Frank DA. SCF and G-CSF lead to the synergistic induction of proliferation and gene expression through complementary signaling pathways. Blood 2000;96:3422–3430.

Cochet C, Gill GN, Meisenhelder J et al. C-kinase phos-phorylates the epidermal growth factor receptor and reduces its epidermal growth factor-stimulated tyrosine protein kinase activity. J Biol Chem 1984;259:2553–2558.

Blume-Jensen P, Siegbahn A, Stabel S et al. Increased kit/SCF receptor induced mitogenicity but abolished cell motility after inhibition of protein kinase C. EMBO J 1993;12:4199–4209.
, H5 W( }$ N2 x' x
Yee NS, Langen H, Besmer P. Mechanism of kit ligand, phorbol ester, and calcium-induced down-regulation of c-kit receptors in mast cells. J Biol Chem 1993;268:14189–14201.* X' X* E! O& [7 A; ?3 z% P

Blume-Jensen P, Wernstedt C, Heldin CH et al. Identification of the major phosphorylation sites for protein kinase C in kit/stem cell factor receptor in vitro and in intact cells. J Biol Chem 1995;270:14192–14200.

Kozlowski M, Larose L, Lee F et al. SHP-1 binds and negatively modulates the c-Kit receptor by interaction with tyrosine 569 in the c-Kit juxtamembrane domain. Mol Cell Biol 1998;18:2089–2099.  o, T' y  f) J3 k+ {3 w

Shultz LD, Schweitzer PA, Rajan TV et al. Mutations at the murine motheaten locus are within the hematopoietic cell protein-tyrosine phosphatase (Hcph) gene. Cell 1993;73:1445–1454.0 \' x" s% \) C" B3 N. s

Paulson RF, Vesely S, Siminovitch KA et al. Signalling by the W/Kit receptor tyrosine kinase is negatively regulated in vivo by the protein tyrosine phosphatase Shp1. Nat Genet 1996;13:309–315.! y! ~% \3 ~% I  h' b2 {
" x' W1 f3 @- {8 F$ X
Lorenz U, Bergemann AD, Steinberg HN et al. Genetic analysis reveals cell type-specific regulation of receptor tyrosine kinase c-Kit by the protein tyrosine phosphatase SHP1. J Exp Med 1996;184:1111–1126.

De Sepulveda P, Okkenhaug K, Rose JL et al. Socs1 binds to multiple signalling proteins and suppresses steel factor-dependent proliferation. EMBO J 1999;18:904–915.. F; E! m6 y) V6 S- t- r
8 @. R6 t( F3 d  j( e: @! y# ]: w
Endo TA, Masuhara M, Yokouchi M et al. A new protein containing an SH2 domain that inhibits JAK kinases. Nature 1997;387:921–924.- D' u, l, v9 ^# o9 ^8 B8 n

Naka T, Narazaki M, Hirata M et al. Structure and function of a new STAT-induced STAT inhibitor. Nature 1997;387:924–929.  b; I1 T4 H0 @, Q
7 p/ L& @& Z3 a) N3 _/ y
Ohya K, Kajigaya S, Yamashita Y et al. SOCS-1/JAB/SSI-1 can bind to and suppress Tec protein-tyrosine kinase. J Biol Chem 1997;272:27178–27182.0 `9 u% \- J: L0 F7 B5 Y
7 _" G: w0 Y8 Y
Ilangumaran S, Finan D, Raine J et al. Suppressor of cyto-kine signaling 1 regulates an endogenous inhibitor of a mast cell proteaseo. J Biol Chem 2003;278:41871–41880.
* ]/ S! Z* c, R1 f3 N; l# \6 {
Longley BJ, Tyrrell L, Lu SZ et al. Somatic c-KIT activating mutation in urticaria pigmentosa and aggressive mastocytosis: establishment of clonality in a human mast cell neoplasm. Nat Genet 1996;12:312–314." r0 t. k( l3 _* E; k1 m

Ashman LK, Ferrao P, Cole SR et al. Effects of mutant c-kit in early myeloid cells. Leuk Lymphoma 1999;34:451–461.% J7 i: K: T3 v- S' A1 R& y7 j

Ning ZQ, Li J, Arceci RJ. Signal transducer and activator of transcription 3 activation is required for Asp(816) mutant c-Kit-mediated cytokine-independent survival and proliferation in human leukemia cells. Blood 2001;97:3559–3567.) z( T+ M1 \' r$ w
4 r/ _: U: \4 {7 a) Y5 i
Tian Q, Frierson HF Jr, Krystal GW et al. Activating c-kit gene mutations in human germ cell tumors. Am J Pathol 1999;154:1643–1647.
6 W4 o* W  k9 C; X) a, \) `+ A" S
Longley BJ, Reguera MJ, Ma Y. Classes of c-KIT activating mutations: proposed mechanisms of action and implications for disease classification and therapy. Leuk Res 2001;25:571–576.  T7 k6 n, K6 n) x8 e9 n. z: S
1 G- }2 J* Q" D. _& U& `8 k5 L
Ma Y, Zeng S, Metcalfe DD et al. The c-KIT mutation causing human mastocytosis is resistant to STI571 and other KIT kinase inhibitors; kinases with enzymatic site mutations show different inhibitor sensitivity profiles than wild-type kinases and those with regulatory-type mutations. Blood 2002;99:1741–1744.

Frost MJ, Ferrao PT, Hughes TP et al. Juxtamembrane mutant V560GKit is more sensitive to Imatinib (STI571) compared with wild-type c-kit whereas the kinase domain mutant D816VKit is resistant. Mol Cancer Ther 2002;1:1115–1124.

Ma Y, Carter E, Wang X et al. Indolinone derivatives inhibit constitutively activated KIT mutants and kill neoplastic mast cells. J Invest Dermatol 2000;114:392–394.2 F- l: @# O! \) n1 a2 u

Liao AT, Chien MB, Shenoy N et al. Inhibition of constitutively active forms of mutant kit by multitargeted indolinone tyrosine kinase inhibitors. Blood 2002;100:585–593.! U$ z& p) m, Q
% g$ b" d+ Z- h: S
Hongyo T, Li T, Syaifudin M et al. Specific c-kit mutations in sinonasal natural killer/T-cell lymphoma in China and Japan. Cancer Res 2000;60:2345–2347.$ f4 [. Q, V4 j( |. e7 Z8 R+ l, f

Nakata Y, Kimura A, Katoh O et al. c-kit point mutation of extracellular domain in patients with myeloproliferative disorders. Br J Haematol 1995;91:661–663

Kimura A, Nakata Y, Katoh O et al. c-kit point mutation in patients with myeloproliferative disorders. Leuk Lymphoma 1997;25:281–287.

Ma Y, Cunningham ME, Wang X et al. Inhibition of spontaneous receptor phosphorylation by residues in a putative a-helix in the Kit intracellular juxtamembrane region. J Biol Chem 1999;274:13399–13402.

Joensuu H, Roberts PJ, Sarlomo-Rikala M et al. Effect of the tyrosine kinase inhibitor STI571 in a patient with a metastatic gastrointestinal stromal tumor. N Engl J Med 2001;344:1052–1056.' a2 ?: k' Z# L: s( Z

Dematteo RP, Heinrich MC, El-Rifai WM et al. Clinical management of gastrointestinal stromal tumors: before and after STI-571. Hum Pathol 2002;33:466–477.$ K/ G* b- ?0 f# r' J6 l! n( W
5 W9 |2 ^3 B, B( H" q: Z2 n, W
Gorre ME, Mohammed M, Ellwood K et al. Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification. Science 2001;293:876–880.
  p. l0 s& K2 G7 u1 T. D
Weisberg E, Griffin JD. Mechanisms of resistance ima-tinib (STI571) in preclinical models and in leukemia patients. Drug Resist Updat 2001;4:22–28.
) U$ T) T8 K2 a! m: x3 X
Shah N, Sawyers C. Mechanisms of resistance to STI571 in Philadelphia chromosome-associated leukemias. Oncogene 2003;22:7389–7395.
$ l1 K. k2 q2 E4 Z" X$ ]
Fine J. Mastocytosis. Int J Dermatol 1980;19:117–123.

Longley BJJ, Metcalfe DD, Tharp M et al. Activating and dominant inactivating c-Kit catalytic domain mutations in distinct clinical forms of human mastocytosis. Proc Natl Acad Sci U S A 1999;96:1609–1614.! G) }6 p/ |: Q& O# ?9 q

de Paulis A, Minopoli G, Arbustini E et al. Stem cell factor is localized in, released from, and cleaved by human mast cells. J Immunol 1999;163:2799–2808.

Longley BJ Jr, Morganroth GS, Tyrrell L et al. Altered metabolism of mast-cell growth factor (c-kit ligand) incutaneous mastocytosis. N Engl J Med 1993;328:1302–1307.8 m0 c; T0 S& R( d

Schwartz S, Heinecke A, Zimmermann M et al. Expression of the C-kit receptor (CD117) is a feature of almost all subtypes of de novo acute myeloblastic leukemia (AML), including cytogenetically good-risk AML, and lacks prognostic significance. Leuk Lymphoma 1999;34:85–94.# `5 ~# _: C9 d: q1 U4 ~
. n+ b$ e5 Y0 x3 B
Kanakura Y, Ikeda H, Kitayama H et al. Expression, function and activation of the proto-oncogene c-kit product in human leukemia cells. Leuk Lymphoma 1993;10:35–41.

Ikeda H, Kanakura Y, Furitsu T et al. Changes in phenotype and proliferative potential of human acute myeloblastic leukemia cells in culture with stem cell factor. Exp Hematol 1993;21:1686–1694.3 r$ m+ r$ w, H- [3 V: {2 }

Pietsch T. Paracrine and autocrine growth mechanisms of human stem cell factor (c-kit ligand) in myeloid leukemia. Nouv Rev Fr Hematol 1993;35:285–286., a4 p- q2 s) B2 k8 t- r
- K# [% k% ]3 `; `
Gari M, Goodeve A, Wilson G et al. c-kit proto-oncogene exon 8 in-frame deletion plus insertion mutations in acute myeloid leukaemia. Br J Haematol 1999;105:894–900." h4 W8 q7 T0 B& Q

Ferrao P, Gonda TJ, Ashman LK. Expression of constitutively activated human c-Kit in Myb transformed early myeloid cells leads to factor independence, histiocytic differentiation, and tumorigenicity. Blood 1997;90:4539–4552.0 c9 ^1 Z7 `$ O  D1 W& b& y

Beghini A, Peterlongo P, Ripamonti CB et al. C-kit mutations in core binding factor leukemias. Blood 2000;95:726–727.
7 K. a* N2 q4 L* R- p2 R
Hart SM, Foroni L. Core binding factor genes and human leukemia. Haematologica 2002;87:1307–1323.
7 {% q# b6 u. j- S
Giles FJ, Stopeck AT, Silverman LR et al. SU5416, a small molecule tyrosine kinase receptor inhibitor, has biologic activity in patients with refractory acute myeloid leukemia or myelodysplastic syndromes. Blood 2003;102:795–801.
: {1 u' h  F+ B; E6 q. b
Giles FJ, Cooper MA, Silverman L et al. Phase II study of SU5416〞a small-molecule, vascular endothelial growth factor tyrosine-kinase receptor inhibitor〞in patients with refractory myeloproliferative diseases. Cancer 2003;97:1920–1928./ c3 {# ^( g* o$ E/ j

O’Farrell AM, Abrams TJ, Yuen HA et al. SU11248 is a novel FLT3 tyrosine kinase inhibitor with potent activity in vitro and in vivo. Blood 2003;101:3597–3605.! d. t  g8 H0 z4 _

Hirota S, Isozaki K, Moriyama Y et al. Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors. Science 1998;279:577–580.
0 Y; r% U* h, H& J. g/ h
Lam LPY, Chow RYK, Berger SA. A transforming mutation enhances the activity of the c-kit soluble tyrosine kinase domain. Biochem J 1999;338:131–1386 B$ V8 |) R- A. D
+ x' v  R) D& p, c
Chan PM, Ilangumaran S, La Rose J et al. Autoinhibition of the kit receptor tyrosine kinase by the cytosolic juxta-membrane region. Mol Cell Biol 2003;23:3067–3078.(Basic Research Laboratory)

收藏0 分享0 顶0 踩0