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Myosin light chain kinase (MLCK) is a busy protein〞 it phosphorylates myosin's regulatory light chain (and thus activates myosin) during nonmuscle cell contraction, cytokinesis, stress fiber formation, and motility. In general, MLCK is known to be present at the cellular sites implicated in these events, but the results of Chew et al. (page 543) are some of the first hints at the dynamics of that localization and the in situ activity of the kinase.
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; O" P. O, t' a: G0 qChew et al. track MLCK by adding a module with a calcium–calmodulin-binding domain flanked by BFP and GFP. In the absence of calcium, the BFP and GFP are close enough to each other to allow fluorescence resonance energy transfer (FRET) between them. When calcium–calmodulin binds, however, this disrupts FRET. Chew et al. suspect that when there is sufficient calmodulin to bind to the added cassette, there will also be calmodulin binding to a domain in MLCK, an event that activates the kinase. Thus, the absence of FRET is interpreted as a sign of an activated kinase.
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0 K2 E* ]4 y; `& I) E. u! T0 n7 _* m' eThe authors measure both the absolute abundance of MLCK (by exciting GFP directly), and the kinase activity (by measuring FRET from BFP to GFP). In motile cells, they find that active MLCK is prominent in the leading lamella, where myosin is present only in small clumps. Activation of myosin may help pull it into larger aggregates, and drag the nucleus toward the front of the cell. At the back of the cell, however, there is less active MLCK, suggesting that other myosin activators, such as Rho kinase, may help in pulling up the back of the cell.Meanwhile, MLCK can be seen transiently associating with and activating on stress fibers, and localizing at the equator of dividing cells before it gets activated. Chew et al. suspect that a similar technique for monitoring both abundance and activity may be useful for other calmodulin-regulated kinases.(Abundance (peak height) and activity (bl) |
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发表于 2009-3-6 00:09
发表于 2015-6-1 15:43
