Critical role of PIP5KI87 in InsP3-mediated Ca2 signaling



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1 Department of Physiology and 2 Department of Cell Biology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 753903 d+ M( V+ c8 v- E
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Correspondence to Helen L. Yin: Helen.Yin@UTSouthwestern.edu

Abstract3 v/ v+ o! q# V' }, s) b- g
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Phosphatidylinositol 4,5-bisphosphate (PIP2) is the obligatory precursor of inositol 1,4,5-trisphosphate (InsP3 or IP3) and is therefore critical to intracellular Ca2  signaling. Using RNA interference (RNAi), we identified the short splice variant of type I phosphatidylinositol 4-phosphate 5-kinase  (PIP5KI87) as the major contributor of the PIP2 pool that supports G protein–coupled receptor (GPCR)-mediated IP3 generation. PIP5KI87 RNAi decreases the histamine-induced IP3 response and Ca2  flux by 70%. Strikingly, RNAi of other PIP5KI isoforms has minimal effect, even though some of these isoforms account for a larger percent of total PIP2 mass and have previously been implicated in receptor mediated endocytosis or focal adhesion formation. Therefore, PIP5KI87's PIP2 pool that supports GPCR-mediated Ca2  signaling is functionally compartmentalized from those generated by the other PIP5KIs., ~7 O0 k# r9 X* s0 J# S

Abbreviations used in this paper: i, intracellular Ca2  concentration; GPCR, G protein–coupled receptor; IP3, inositol 1,4,5-trisphosphate; PIP5KI, type I phosphatidylinositol 4-phosphate 5-kinase ; PIP2, phosphatidylinositol 4,5-bisphosphate; PM, plasma membrane; RNAi, RNA interference; siRNA, small interfering RNA.
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Introduction

Phosphatidylinositol 4,5-bisphosphate (PIP2) regulates multiple plasma membrane (PM) functions (Doughman et al., 2003; Yin and Janmey, 2003), and it is also a substrate for PLC-mediated inositol 1,4,5-trisphosphate (InsP3 or IP3) generation. In spite of PIP2's obligatory role as an IP3 precursor, and the importance of IP3 mediated Ca2  signaling, surprisingly little is known about the identity of the phosphoinositide kinases involved. Unlike yeast, which has a single type I phosphatidylinositol 4-phosphate 5-kinase (PIP5KI) that synthesizes PIP2 (Audhya and Emr, 2003), mammals have three major PIP5KI isoforms named , ? and  (Doughman et al., 2003).

PIP5KI has two splice variants (PIP5KI87 and 90) that are distinguished by a 28–amino acid extension at the COOH terminus of PIP5KI90 (Di Paolo et al., 2002; Ling et al., 2002; Fig. 1 A). PIP5KI90 is particularly enriched in neurons (Wenk et al., 2001); it is the major PIP2 synthesizing enzyme at the synapse, where it has been implicated in the regulation of clathrin coat recruitment, actin dynamics (Wenk et al., 2001) and focal adhesion formation (Di Paolo et al., 2002; Ling et al., 2002). In contrast, PIP5KI87 is not involved in focal adhesion formation or clathrin-mediated endocytosis (in HeLa cells; Padron et al., 2003).
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Figure 1. PIP5KI RNAi. (A) PIP5KI siRNA design. Pan siRNA is directed against both isoforms. (B) PIP5KI protein knockdown. Effect of PIP5K RNAi on protein expression of the targeted and nontargeted PIP5KIs. Western blots were probed with isoform specific antibodies. Additional data are provided in Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200408008/DC1. (C) Quantitative real-time PCR. PCR primers were used to quantitate PIP5KIpan and PIP5KI90 mRNA and PIP5KI87 mRNA was calculated from the difference. Numbers indicate the amounts of each isoform relative to PIP5KI90 in control cells. Data are the average of duplicate RNAi samples from a single experiment. Similar results were obtained from another experiment. (D) PIP5KI is enriched in the PM. Endogenous PIP5KI was detected with anti-PIP5KIpan antibody, and overexpressed HA-PIP5KI87 (in cDNA-transfected cells) were stained with anti-HA. Arrows indicate PM. Bars, 50 μm. (E) Differential PIP5KI membrane association. Fractions obtained after sequential sedimentation were loaded equivalently, except for the cytosol fraction (CYT), which was loaded 10 times less. Western blot band intensity was determined by quantitative densitometry, and expressed as a percent of total recovered, after correcting for differences in fraction of sample loaded.
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Here, we examined the role of PIP5KI87 in intracellular Ca2  signaling. Previous biochemical studies have shown that cells have agonist-sensitive and -insensitive PIP2 pools (Koreh and Monaco, 1986). Inhibitor studies suggest that the agonist-sensitive pool can be further classified as constitutive or de novo generated in response to agonists (Nakanishi et al., 1995). Some of these pools are enriched in cholesterol–sphingolipid raft domains (Pike and Casey, 1996) and the stringent spatial and temporal regulation of Ca2  may be specified by assembling key players into supramolecular signaling complexes (Delmas et al., 2004). We now report that PIP5KI87 is the major source of the agonist-sensitive PM PIP2 pool that fuels the initial Ca2  response to external stimuli.4 g; _" b; k  w! L# M4 W
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Results and discussion$ [* \$ Z2 C$ F' @
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Knockdown of PIP5KI87 by RNA interference
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We use small interfering RNA (siRNA) mediated RNA interference (RNAi) to knockdown each PIP5KI individually (Padron et al., 2003). Anti-PIP5KIpan antibody recognizes a sharp 87-kD band and a slower migrating diffuse band (Fig. 1 B) which is probably hyperphosphorylated PIP5KI87 (Park et al., 2001; Wenk et al., 2001). Anti-PIP5KIpan stains the PM, the perinuclear region and the nucleus (Fig. 1 D). Low level HA-PIP5KI87 overexpression confirms that PIP5KI is enriched in the PM and punctate internal structures, but rules out nuclear localization.* ]7 g+ I& o* t$ m4 J. ~# Z7 M

Biochemical fractionation shows that 60% of PIP5KI87 is sedimented by high speed centrifugation (Fig. 1 E), and approximately two thirds of this is associated with the PM enriched fraction. PIP5KI is much more membrane bound, whereas PIP5KI? is least membrane associated. Therefore, these PIP5KIs have different patterns of membrane association. Nevertheless, each can potentially generate PIP2 at the PM and internal membranes.# l" @3 i) N+ r* o7 D" x' h2 P/ y9 r
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Because it is not possible to knockdown PIP5KI87 exclusively, we compared the effects of siRNA directed against both PIP5KI isoforms (PIP5KIpan) to that of PIP5KI90 alone. PIP5KIpan siRNA decreases all PIP5KI bands in Western blots (Fig. 1 B) and reduces PM and cytoplasmic anti-PIP5KI immunofluorescence, but not the nonspecific nuclear staining (Fig. 1 D). PIP5KI90 siRNA has little effect on either PIP5KI bands (Fig. 1 B), even though quantitative real-time PCR established that PIP5KI90 mRNA is decreased by 70% (Fig. 1 C). We conclude that HeLa cells have very little PIP5KI90.
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Unexpectedly, PIP5KIpan siRNA preferentially knocks down PIP5K87 mRNA relative to PIP5KI90. Because PIP5K87 is more abundant than PIP5K90 in HeLa cells and PIP5Kpan siRNA generates a distinct phenotype (compared with that of PIP5K90 siRNA), the PIP5KIpan siRNA effects described here can be attributed primarily to PIP5KI87 knockdown. Importantly, PIP5KIpan siRNA has almost no effect on PIP5KI and ? protein expression (Fig. 1 B), establishing that the PIP5KI87 knockdown phenotype is not complicated by compensatory changes in the other PIP5KIs. This was originally a concern, because we have previously found that knockdown of one PIP5KI induces changes in the mRNA level of some other PIP5KIs (Padron et al., 2003).
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PIP5KI87 is the major supplier of the PIP2 pool that supports GPCR-mediated Ca2  signaling+ T/ a4 ]( F1 L8 I2 A7 `; Y
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Histamine binding to the H1 type G protein–coupled receptor (GPCR) in HeLa cells (Tilly et al., 1990) initiates a PLC? activation cascade that culminates in the hydrolysis of PIP2 to generate IP3 and diacylglycerol. When control cells were stimulated with a supramaximal histamine dose (100 μM), they generate a transient robust IP3 response (Fig. 2 A). PIP5KI87 RNAi decreases the initial IP3 peak by 69%  (Fig. 2, A and B). Neither PIP5KI90 nor PIP5KI? RNAi has any apparent effect, and PIP5KI RNAi only decreases the initial IP3 peak slightly  (Fig. 2 B). Although we cannot rule out a small contribution by PIP5KI? or  because each is less completely knocked down than PIP5KI, we can conclude that PIP5KI87 is the dominant regulator.+ Y" B7 u3 k' b# A# t3 ~6 n
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Figure 2. PIP5KIpan siRNA blocks histamine-stimulated IP3 generation. Cells were stimulated with 100 μM histamine. (A) Effect of PIP5KIpan siRNA on IP3 generation. Data shown are representative of five independent experiments. (B) Effect of PIP5KI RNAi on the initial IP3 peak response. Values are mean ± SEM of multiple independent experiments (n).
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Consistent with a decrease in IP3 production, PIP5KIpan RNAi also attenuates histamine-induced Ca2  signaling. In control cells, 100 μM histamine induces a rapid and transient rise in intracellular Ca2  concentration (i) in 95% of the cells examined (Fig. 3 A). PIP5KI87 RNAi reduces the percent of responding cells slightly (by 13%), decreases the amplitude of the first Ca2  peak more , and has the most impact on Ca2  flux  (Fig. 3 A). Because Ca2  flux correlates with the open probability of the IP3Rs and hence the rate of IP3 generation (Johenning et al., 2004), our results establish that PIP5KI87 RNAi depletes the PIP2 pool used for IP3 generation. A similarly large decrease in Ca2  flux was also observed when PIP2 was depleted by overexpressing the PIP2 phosphatase synaptojanin (Johenning et al., 2004).
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Figure 3. PIP5KIpan siRNA attenuates intracellular Ca2  signaling. Cells loaded with fura2-AM in randomly chosen microscopic fields were ratio imaged (F340/F380) to obtain baseline Ca2  values. Histamine was added in the absence of extracellular Ca2 , and the ratio image was recorded as a function of time. (A) Ca2  response to 100 μM histamine (indicated by the arrow). Representative tracings for each type of RNAi are shown. Values in bar graphs are expressed as percent (mean ± SEM) of control. (B) In vivo rescue of intracellular Ca2  signaling in PIP5KIpan siRNA-treated cells by Shuttle PIP2. Top panels are representative tracings. (Bottom) The Ca2  flux of the transients elicited by the second histamine addition was plotted and 10 cells were analyzed per condition.

The weakened Ca2  response by PIP5KI87 RNAi cells is not due to depletion of Ca2  stores, because thapsigargin releases similar amounts of Ca2  into the cytosol of control and siRNA cells (unpublished data). The i response to UTP, which binds a different GPCR than histamine (P2Y and H1, respectively), is blunted as well (unpublished data). i increase in the presence of 1.3 mM of extracellular Ca2  (Table S2, available at http://www.jcb.org/cgi/content/full/jcb.200408008/DC1) is also attenuated, confirming that the lack of PIP2 impacts the entire Ca2  signaling cascade that starts with Ca2  release from internal stores and ends with capacitative Ca2  entry (that usually follows the depletion of intracellular Ca2  stores).

As expected, in control cells, 1 μM histamine induces a slower Ca2  flux than 100 μM histamine . Paradoxically, PIP5KI87 RNAi cells have the same low Ca2  flux  at both histamine doses. We cannot explain why this is the case. Perhaps because PIP2 is already limiting at submaximal stimulation, increasing the intensity of the stimulus does not significantly increase the amount of IP3 generated due to lack of substrate." X, D% J& `3 E( E# ^+ j$ V

Unlike PIP5KI87 RNAi, PIP5KI90,  or ? RNAi has much less effects (Fig. 3 A), paralleling the trend observed with IP3 production (Fig. 2 B). Thus, the PIP5KIpan siRNA phenotype can be most simply explained by a decrease in the amount of PLC? accessible PM PIP2 and that this pool is generated primarily by PIP5KI87.

If PIP5KI87 RNAi suppresses Ca2  signaling by depleting PM PIP2, restoring membrane PIP2 should rescue the Ca2  response. We used a membrane permeant polyamine shuttle carrier to deliver exogenous PIP2 into intact cells (Ozaki et al., 2000; Wang et al., 2003). Control HeLa cells respond to sequential histamine challenges identically, and Shuttle PIP2 does not change the Ca2  response significantly (Fig. 3 B). PIP5KI87 RNAi cells, which are already less responsive to the first stimulus than control cells, have an even more blunted response to the second stimulus in the absence of Shuttle PIP2 (Fig. 3 B, left). This is consistent with depletion of the already small PIP2 pool by the first stimulus, and inadequate refilling before the second stimulus. Significantly, Shuttle PIP2 restores the Ca2  response of PIP5KI87 RNAi cells to 74% of that observed in Ctrl RNAi cells (Fig. 3 B). Therefore, the Ca2  signaling defect is due to PIP2 depletion by PIP5KI RNAi.

Together, our results demonstrate that PIP5KI87 has a critical role in GPCR-mediated IP3 signaling in HeLa cells. Interestingly, overexpressed mouse PIP5KI? (equivalent to human PIP5KI described in this paper) stimulates tyrosine kinase receptor activated-IP3 generation in B lymphocytes (Saito et al., 2003). Together, these results raise the intriguing possibility that the GPCR- and tyrosine kinase receptor-coupled PIP2 pools may be governed by different PIP5KIs. We plan to determine if this is the case in future studies.
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Effects of PIP5KI87 RNAi on PIP2 content and distribution8 E3 z  @3 A& ]9 {: I7 ^3 w  g$ K: I% B

To understand how PIP5KI87 uniquely contributes to GPCR mediated IP3 signaling, we estimated the size and location of its PIP2 pool relative to those of other PIP5KIs. PIP5KIpan siRNA reduces PIP2 mass, determined by HPLC (Nasuhoglu et al., 2002), by 14%  and 32P-incorporation into PIP2, determined by TLC, to a similar extent (Fig. 4 A). PIP5KI90 siRNA has no statistically significant effect. PIP5KI? RNAi decreases PIP2 mass by 34% (Fig. 4 A), which is consistent with the large decrease in PIP2 reported previously (Padron et al., 2003). Paradoxically, although PIP5KI RNAi does not decrease PIP2 (Padron et al., 2003), it decreases PIP2 mass by 33%. The difference between the TLC and HPLC estimates may be because they measure different parameters. 32P-labeling/TLC detects PIP2 that turns over during the labeling period, whereas the HPLC method does not involve radiolabeling (Nasuhoglu et al., 2002) and measures PIP2 mass. It is possible that the 4-h labeling interval we used was not long enough to completely equilibrate a particularly stable PIP2 pool, and therefore underestimates its size.

Figure 4. Effect of PIP5KI RNAi on PIP2. (A) PIP2 mass (HPLC) and 32P-incorporation (TLC). Means ± SEM of independent experiments are shown. (B) PIP2 distribution as detected with anti-PIP2 and overexpressed GFP-PLC-PH. Cross-sectional plots of fluorescence intensity are shown next to the image. Bars, 50 μm. (C) Analysis of PIP2 quantitation. (Left) The average intensities of anti-PIP2 at the PM and inside the cell are expressed in arbitrary units (mean ± SEM). 10 cells were analyzed per RNAi condition. (Right) PM/cytoplasmic intensity ratios of anti-PIP2 and GFP-PLC-PH were shown. 10 cells were analyzed per label per RNAi condition.
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PIP5KI87 RNAi decreases PM PIP2 significantly. PIP2 was detected by single cell fluorescence imaging using overexpressed GFP-PLC-PH (Varnai and Balla, 1998; Watt et al., 2002) and anti-PIP2 (Laux et al., 2000; Matsuda et al., 2001; Fig. 4 B). Although it is generally accepted that the PM is particularly enriched in PIP2 and that GFP-PLC-PH labels the PM intensely, internal GFP-PLC-PH labeling has also been reported in at least some types of cells (Matsuda et al., 2001). However, because GFP-PLC-PH was overexpressed and also binds IP3 (Hirose et al., 1999), it is difficult to determine if the internal GFP-PLC-PH is bound to PIP2/IP3 or represents unliganded PH. This issue is clarified somewhat by a quantitative immuno-electron microscopic study which shows that the PM accounts for 40% of total GST-PLC-PH labeling, and internal organelles account for the remainder (Watt et al., 2002).
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Our anti-PIP2 staining results clearly shows that PIP2 is present in internal membranes as well as PM in HeLa cells. Using image quantitation (Fig. 4, B and C), we estimate that anti-PIP2 fluorescence in the vicinity of the PM accounts for 12.3 ± 1.2% (n = 10) of total, and its intensity is 1.54 ± 0.13 times (n = 10) higher than in internal sites (PM/cytosol ratio; Fig. 4 C). Although these cross-sectional analyses underestimate the size of the PM pool (compared with morphometric analysis by electron microscopy, as described by Watt et al., 2002), it can be used to compare the effects of different PIP5KI RNAi.- B" a7 Q1 W  g/ ^5 e
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PIP5KIpan RNAi decreases the PM anti-PIP2 intensity from 226 ± 7 to 134 ± 8 arbitrary units (59% of control) and internal PIP2 from 157 ± 12 to 130 ± 12 arbitrary units (84% of control; Fig. 4 C). Therefore, PM PIP2 is preferentially depleted. PIP5KI? RNAi decreases anti-PIP2 intensity at the PM to the same extent (to 58% of control; Fig. 4 B) but has a greater effect on internal PIP2 (71% of control). In contrast, PIP5KI RNAi has no detectable effect on PM PIP2 (98% remains after RNAi), but decreases internal staining (76% remaining; Fig. 4, B and C). The differential responses of PM versus internal PIP2 is evident when the average intensity of PM PIP2 is expressed as a ratio to that in the cytosol (Fig. 4 C, right). Qualitatively similar, but not identical, changes are also observed with GFP-PLC-PH (Fig. 4 C). Thus, these two independent methods both show that PIP5KI87 and PIP5KI? RNAi decrease PM PIP2, even though only the former suppresses GPCR-mediated IP3/Ca2  signaling." D. G% ?+ i, W$ b- T+ X
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In conclusion, PIP5KI87 is the major source of the GPCR mobilized PIP2 pool. This specialized PIP2 accounts for a small fraction of total PIP2, a significant fraction of PM PIP2 and most of the histamine induced IP3 response. The exquisitely selective effect of PIP5KI87 RNAi on Ca2  signaling suggests that the cell's PIP2 is functionally compartmentalized in a PIP5KI-dependent manner. This study provides a mechanistic understanding of how PIP2 can regulate multiple PM functions independently. Additional studies will determine if PIP5KI87 is part of the supramolecular PLC? signaling scaffold that specifies rapid local Ca2  generation and propagation (Delmas et al., 2004), and whether the functionally compartmentalized PIP2 is physically segregated in the PM., C. a/ n! H" B5 Z
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Materials and methods

Antibodies
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Anti-PIP5KI was purchased from Santa Cruz Biotechnology, Inc. Anti-PIP5KI? and PIP5KIpan were gifts from C. Carpenter (Harvard Medical School, Boston, MA) and P. De Camilli (Yale University, New Haven, CT; Wenk et al., 2001), respectively. Monoclonal anti-PIP2 (Fukami et al., 1988) was a gift from K. Fukami (University of Tokyo, Tokyo, Japan).! D% {8 `1 z2 p# K! ~& X# ?: ?7 i
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RNAi
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We used the human PIP5KI isoforms designation, which is different from the mouse designation. siRNA oligonucleotides were performed as described previously (Padron et al., 2003). HeLa cells were transfected with the siRNA and used 48–72 h later.* @" o) k2 G( j8 u$ X

Quantitative real-time PCR( W( K+ Q4 q: Q) ^3 ^$ p

RNA extracted from HeLa cells transfected with siRNA were reverse transcribed and used for PCR in a sequence detection system (Prism 7000; Applied Biosystems). Primers directed at nucleotides 414–478 (pan) and 1993–2048 (unique to PIP5KI90) were used (Padron et al., 2003).
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PIP2 measurements( f5 V/ N8 R$ f( f! y0 S
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PIP2 mass was determined by a nonradioactive HPLC detection system (Nasuhoglu et al., 2002). 32P-incorporation into PIP2 was determined by labeling cells for 4 h with 32P-PO4 (NEN Life Science Products), resolving lipids by TLC, and quantitation by phosphorimager analysis (Wang et al., 2003). PM PIP2 was determined by image analysis of cells overexpressing low amounts of GFP-PLC-PH (Varnai and Balla, 1998), or labeling with anti-PIP2 (Laux et al., 2000; Matsuda et al., 2001). Fluorescence images were captured by confocal microscopy (model LSM5; Carl Zeiss MicroImaging, Inc.) and intensity plots were analyzed by MetaMorph Offline software (Varnai and Balla, 1998). The average fluorescence intensity of the two cell edges and between the cell edges were defined as PM and cytoplasmic PIP2, respectively, and are expressed in arbitrary units.  W: z$ d" q; b6 N+ ]2 f
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Immunofluorescence microscopy" k. J: O+ b+ n
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For most purposes, 0.4% formaldehyde fixed cells were permeabilized with Triton X-100 and processed for confocal microscopy as described previously (Wang et al., 2003). Anti-PIP2 staining was detected by permeabilizing fixed cells with 10 μg/ml digitonin, which preserves the lipid signal better than Triton X-100.; h; p# X! T7 Q) N# n- `

Multistep membrane fractionation
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Cells were homogenized by 25 strokes in a prechilled steel homogenizer and homogenates were centrifuged sequentially to obtain the crude organelle/membrane fractions as described previously (Wei et al., 2002). LSP is enriched for Golgi membranes and early endosomes, and HSP is enriched for lysosomes and late endosomes. The PM fraction was obtained by placing the 19,000 g pellet on top of a sucrose cushion, and collecting the membranes at the top after centrifugation at 100,000 g.. D6 p3 U) J- B) s! H; b& F
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IP3 measurement$ t- M5 \; Q/ p" Q7 v; ]

Cell monolayers incubated in Ca2 -free Hank's buffer supplemented with 0.1% BSA were stimulated with 100 μM histamine (Sigma-Aldrich) for 0–25 s at RT and the reaction was stopped with PCA. IP3 content was assayed by competition with exogenous IP3 to bind calf cerebellar microsomes (Sun et al., 1995).
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Single cell Ca2  imaging4 J! z6 `8 S" d& O3 o' F) Y& J
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Cells plated on glass-bottom culture dishes (Mat Tek) were loaded with fura2/AM, washed and incubated for 30 min at RT to allow de-esterification of the loaded dye. The dish was mounted on the stage of an inverted fluorescence microscope (Axiovert 200; Carl Zeiss MicroImaging, Inc.) with a 40x objective. Cells were excited at 340 and 380 nm and the change in fluorescence ratio values (F340/F380) as a function of time in individual cells within a field was recorded simultaneously. The percent of responding cells was obtained by dividing those with a Ca2  signal to total cells recorded. The ratio of maximal F340/F380 induced by histamine to basal F340/F380 is defined as the Ca2  peak. Ca2  flux is defined as the slope of a line between the initiation of a persistent increase in F340/F380 and the maximal F340/F380.
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Intracellular delivery of PIP2 by Shuttle PIP2  `0 W, Y: N. o- e% ^( \

siRNA-treated cells were stimulated with 1 μM histamine in the absence of extracellular Ca2  and ratio imaged. Histamine was washed out and cells were loaded with a mixture of 1 μM diC16-PIP2 and 1 μM of carrier 2 (Shuttle PIP2; Echelon Biosciences, Inc.) diluted in the Hank's buffer (PIP2) or buffer only (Mock; Wang et al., 2003) for 10–15 min on the microscope stage. Cells were then reexposed to 1 μM histamine and imaged again.
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Online supplemental material
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Table S1 illustrates the effect of PIP5KI RNAi on PIP5KI protein expression. HeLa cells transfected with siRNA for each PIP5KI was lysed and subjected to Western blotting with isoform specific antibodies. Table S2 illustrates the effect of PIP5KI RNAi on histamine induced Ca2  transients. HeLa cells transfected with siRNA were stimulated with histamine, and Ca2  transients were recorded on single cells loaded with fura2. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200408008/DC1.& T$ z/ {% x% |* k

Acknowledgments; O1 f) }4 Y+ B0 i

We thank D.W. Hilgemann, S. Muallem, and I. Bezprozvanny for stimulating discussions, and K.M. Lin for help with initial Ca2  measurements.

This work is supported by National Institutes of Health GM21681 and the Robert A. Welch Foundation to H.L. Yin, the Leukemia and Lymphoma Society Career Development Fellowship to Y.J. Wang and the Welch Foundation and the American Diabetes Association Career Development Award to W.H. Li.( V- w2 S' n1 h
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