Szczepanowska Joanna, Korn Edward D, Brzeska Hanna
Laboratory of Cell Biology, National Heart, Lung and Blood Institute, Bethesda, MD 20892, USA.
Cell Motil Cytoskeleton. 2006 Jun;63(6):356-74. doi: 10.1002/cm.20125.
Activation of actomyosin II by phosphorylation of its regulatory light chain is one of the main factors involved in the regulation of cytoskeletal dynamics. Phosphorylation of myosin regulatory light chain may be mediated directly and indirectly by several kinases including myosin light chain kinase (MLCK) and kinases activated by small GTP-binding proteins. Most of the myosin kinases, including PAK, can also interact with other proteins through binding sites located outside of their catalytic domains. In an attempt to study the effects due only to phosphorylation of myosin light chain, we expressed the constitutively active catalytic domain of ameba PAK in HeLa cells. The catalytic domain phosphorylates myosin light chain in vitro with high specific activity but has none of the sequences that target mammalian PAK to other proteins and membranes. Expression of the catalytic domain caused disassembly of focal adhesions and stress fibers in the cell center and accumulation of focal adhesions and F-actin at the cell periphery. There was a twofold increase in the phosphorylation level of endogenous myosin light chain and changes in cell shape consistent with enhanced cell contractility. The phenotype was independent of MLCK, ROCK, MEK, Rac, and Rho activities but was abolished by blebbistatin, a specific inhibitor of myosin II activity. Our data are consistent with myosin being directly phosphorylated by the expressed catalytic domain of ameba PAK with the induced phenotype resulting from cell retraction driven by contraction of peripheral actomyosin. The phenotype induced by expression of the catalytic domain is reminiscent of that caused by expression of active mammalian PAK, suggesting that myosin phosphorylation may play an important role in PAK-induced cytoskeletal changes. The catalytic domain of ameba PAK may be a useful tool for studying the effects of myosin light chain phosphorylation in other cells.
肌动球蛋白II通过其调节性轻链的磷酸化而激活，是参与细胞骨架动力学调节的主要因素之一。肌球蛋白调节性轻链的磷酸化可能由包括肌球蛋白轻链激酶（MLCK）和由小GTP结合蛋白激活的激酶在内的几种激酶直接或间接介导。大多数肌球蛋白激酶，包括PAK，也可以通过位于其催化结构域之外的结合位点与其他蛋白质相互作用。为了仅研究肌球蛋白轻链磷酸化的影响，我们在HeLa细胞中表达了变形虫PAK的组成型活性催化结构域。该催化结构域在体外以高比活性磷酸化肌球蛋白轻链，但没有将哺乳动物PAK靶向其他蛋白质和膜的序列。催化结构域的表达导致细胞中心的粘着斑和应力纤维解体，以及粘着斑和F-肌动蛋白在细胞周边积累。内源性肌球蛋白轻链的磷酸化水平增加了两倍，并且细胞形状发生改变，这与细胞收缩性增强一致。该表型与MLCK、ROCK、MEK、Rac和Rho的活性无关，但被肌球蛋白II活性的特异性抑制剂blebbistatin消除。我们的数据表明，肌球蛋白被变形虫PAK的表达催化结构域直接磷酸化，诱导的表型是由外周肌动球蛋白收缩驱动的细胞回缩引起的。催化结构域表达诱导的表型让人联想到由活性哺乳动物PAK表达引起的表型，表明肌球蛋白磷酸化可能在PAK诱导的细胞骨架变化中起重要作用。变形虫PAK的催化结构域可能是研究其他细胞中肌球蛋白轻链磷酸化影响的有用工具。
