Rosenberg Michael, Ravid Shoshana
Department of Biochemistry, Institute of Medical Sciences, Faculty of Medicine, The Hebrew University, Jerusalem 91120, Israel.
Mol Biol Cell. 2006 Mar;17(3):1364-74. doi: 10.1091/mbc.e05-07-0597. Epub 2006 Jan 4.
Nonmuscle myosin II is an important component of the cytoskeleton, playing a major role in cell motility and chemotaxis. We have previously demonstrated that, on stimulation with epidermal growth factor (EGF), nonmuscle myosin heavy chain II-B (NMHC-IIB) undergoes a transient phosphorylation correlating with its cellular localization. We also showed that members of the PKC family are involved in this phosphorylation. Here we demonstrate that of the two conventional PKC isoforms expressed by prostate cancer cells, PKCbetaII and PKCgamma, PKCgamma directly phosphorylates NMHC-IIB. Overexpression of wild-type and kinase dead dominant negative PKCgamma result in both altered NMHC-IIB phosphorylation and subcellular localization. We have also mapped the phosphorylation sites of PKCgamma on NMHC-IIB. Conversion of the PKCgamma phosphorylation sites to alanine residues, reduces the EGF-dependent NMHC-IIB phosphorylation. Aspartate substitution of these sites reduces NMHC-IIB localization into cytoskeleton. These results indicate that PKCgamma regulates NMHC-IIB phosphorylation and cellular localization in response to EGF stimulation.
非肌肉肌球蛋白II是细胞骨架的重要组成部分，在细胞运动和趋化作用中起主要作用。我们之前已经证明，在用表皮生长因子（EGF）刺激时，非肌肉肌球蛋白重链II-B（NMHC-IIB）会发生瞬时磷酸化，这与其细胞定位相关。我们还表明PKC家族成员参与了这种磷酸化。在此我们证明，在前列腺癌细胞表达的两种传统PKC亚型PKCβII和PKCγ中，PKCγ直接使NMHC-IIB磷酸化。野生型和激酶失活的显性负性PKCγ的过表达都会导致NMHC-IIB磷酸化和亚细胞定位的改变。我们还确定了PKCγ在NMHC-IIB上的磷酸化位点。将PKCγ磷酸化位点转换为丙氨酸残基会降低EGF依赖性的NMHC-IIB磷酸化。这些位点的天冬氨酸替代会减少NMHC-IIB在细胞骨架中的定位。这些结果表明，PKCγ在EGF刺激下调节NMHC-IIB的磷酸化和细胞定位。
