| Budget Amount *help |
¥12,300,000 (Direct Cost: ¥12,300,000)
Fiscal Year 2000: ¥4,900,000 (Direct Cost: ¥4,900,000)
Fiscal Year 1999: ¥7,400,000 (Direct Cost: ¥7,400,000)
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| Research Abstract |
Dormant ERM (ezrin/radixin/moesin) proteins in the cytoplasm are activated with concomitant threonine-phosphorylation in a Rho-dependent manner, and recruited to the plasma membranes, where they function as actin filament/plasma membrane cross-linkers to form microvilli. Rho-associated kinase, a direct effector of Rho, stoichiometrically threonine-phosphorylates ERM proteins in vitro, which keeps ERM proteins at the active state as cross-linkers. PI4P5K, another direct effector of Rho, produces phosphatidylinositol 4,5-bisphosphate (PIP_2), which activates ERM proteins in vitro. We then have examined whether the Rho-dependent activation of ERM proteins is mediated by Rho-associated kinases and/or PI4P5K in vivo. Immunofluorescence microscopy of serum-starved NIH3T3 cells with a mAb specific for threonine-phosphorylated ERM proteins (CPERMs) was performed. Transfection of a constitutively active RhoA (V14RhoA), but not Rac1 or Cdc42, increased the level of CPERMs, which were exclusively concentrated in microvilli. This V14RhoA-induced activation of ERM proteins was not mimicked by transfection of constitutively active Rho-associated kinase, and was not suppressed by Y-27632, a specific inhibitor or ROCK kinases including Rho-associated kinase. In contrast, overexpression of PI4P5K, but not its kinase-dead mutant, activated ERM proteins as V14RhoA did. These PI4P5K-induced phenotypes were not affected by Y-27632 or dominant-negative RhoA (N19RhoA) These findings indicate that PI4P5K but not ROCK kinases are involved in the Rho-dependent activation of ERM proteins in vivo. We speculate that PI4P5K-mediated production of PIP2 primarily activates ERM proteins, and that the active state of ERM proteins is maintained through their threonine-phosphorylation by not-yet-determined kinases. This activation process would result in the microvilli formation.
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