Chew T L, Masaracchia R A, Goeckeler Z M, Wysolmerski R B
Department of Pathology, St Louis University School of Medicine, Missouri 63104-1028, USA.
J Muscle Res Cell Motil. 1998 Nov;19(8):839-54. doi: 10.1023/a:1005417926585.
Myosin regulatory light chain (RLC) phosphorylation has been implicated in Rho-mediated stress fibre formation. The recent observation that Rho kinase phosphorylates RLC in vitro suggests that serine/threonine kinases other than those in the myosin light chain kinase (MLCK) family have the potential to activate myosin II. In this study we report that gamma-PAK, which is activated by the GTP-binding proteins Cdc42 and Rac, catalyses phosphorylation of intact non-muscle myosin II and isolated recombinant RLC. gamma-PAK phosphorylated endothelial cell myosin II to 0.85 +/- 0.02 mol PO4 per mol RLC. Phosphorylation is Ca2+/calmodulin-independent and the enzyme has a K(m) and Vmax for myosin II regulatory light chain of 12 microM and 180 nmol/min/mg respectively. No myosin II heavy chain phosphorylation was detected. Phosphopeptide maps and phosphoamino acid analysis revealed that gamma-PAK phosphorylates Ser-19 but does not phosphorylate Thr-18. A panel of recombinant RLC mutants was used to confirm that Ser-19 is the only phosphorylation site modified by gamma-PAK. On substitution of both Ser-19 and Thr-18 with Ala or Glu, no phosphorylation of other Ser/Thr residues in the RLC was detected. Similar to MLCK, Arg-16 is required for interaction of gamma-PAK with the substrate, since converting Arg-16 to Ala significantly reduced RLC phosphorylation. Endothelial cell monolayers permeabilized with saponin retract upon exposure to either Cdc42 or trypsin-activated gamma-PAK and ATP. Activation of gamma-PAK is required to initiate Ca2+/calmodulin-independent cell retraction and actin rearrangement. Taken together, these data suggest that myosin II activation by the p21-activated family of kinases may be physiologically important in regulating cytoskeletal organization.
肌球蛋白调节轻链（RLC）磷酸化与Rho介导的应力纤维形成有关。最近有观察表明，Rho激酶可在体外使RLC磷酸化，这表明除肌球蛋白轻链激酶（MLCK）家族中的激酶外，丝氨酸/苏氨酸激酶也有激活肌球蛋白II的潜力。在本研究中，我们报告称，由GTP结合蛋白Cdc42和Rac激活的γ-PAK可催化完整的非肌肉型肌球蛋白II和分离的重组RLC的磷酸化。γ-PAK可将内皮细胞肌球蛋白II磷酸化至每摩尔RLC含0.85±0.02摩尔磷酸根。磷酸化不依赖于Ca2+/钙调蛋白，该酶对肌球蛋白II调节轻链的米氏常数（Km）和最大反应速度（Vmax）分别为12微摩尔和180纳摩尔/分钟/毫克。未检测到肌球蛋白II重链的磷酸化。磷酸肽图谱和磷酸氨基酸分析表明，γ-PAK使Ser-19磷酸化，但不使Thr-18磷酸化。一组重组RLC突变体被用于证实Ser-19是γ-PAK修饰的唯一磷酸化位点。当Ser-19和Thr-18都被丙氨酸或谷氨酸取代时，未检测到RLC中其他丝氨酸/苏氨酸残基的磷酸化。与MLCK相似，Arg-16是γ-PAK与底物相互作用所必需的，因为将Arg-16转换为丙氨酸会显著降低RLC磷酸化。用皂素通透的内皮细胞单层在暴露于Cdc42或胰蛋白酶激活的γ-PAK和ATP后会收缩。γ-PAK的激活是启动不依赖于Ca2+/钙调蛋白的细胞收缩和肌动蛋白重排所必需的。综上所述，这些数据表明，p21激活的激酶家族对肌球蛋白II的激活在调节细胞骨架组织方面可能具有重要的生理意义。
