Expression of constitutively active alpha-PAK reveals effects of the kinase on actin and focal complexes
Expression of constitutively active alpha-PAK reveals effects of the kinase on actin and focal complexes
复制标题
DOI：
10.1128/mcb.17.3.1129
复制
发表时间：
1997-03-01
影响因子：
5.3
通讯作者：
Lim, L
中科院分区：
文献类型：
--
作者：
Manser, E;Huang, HY;Lim, L
P21激活蛋白激酶(PAK)家族似乎存在于所有具有CDC42样GTP酶的生物体中，在哺乳动物细胞中，PAK参与了丝裂原激活的蛋白激酶级联反应的激活，但这些蛋白激酶对细胞骨架的影响尚未见报道。最近，我们发现果蝇PAK在经历背部闭合的胚胎上皮细胞的前沿富含(N.Harden，J.Lee，H.Y.Loh，Y.-Mng，I.Tan，T.Leung，E.Manser，L.Lim，Mel)。细胞，Biol，16：1896-1908,1996)，在那里它共定位，其结构类似焦点复合体，在这里，我们通过转染显示，在上皮HeLa细胞中，α-PAK从细胞质中招募到不同的焦点复合体，它们本身共同定位到这些位置，通过缺失分析，PAK的N端包含焦点粘连的靶向序列，这表明这些复合体是体内的激酶功能部位。CDC42和Rad引起α-PAK的自磷酸化和激酶激活，定位α-PAK的自磷酸化位点允许产生一个结构性活性的激酶突变体，通过将CDC42的区域融合到PAK的C末端，也获得了激活的嵌合体，当这些不同的结构性活性的α-PAK被导入HeLa细胞和成纤维细胞时，编码这些不同的结构性活性的α-Pak的质粒导致应力纤维的丢失，这与导入Cdc42(G12V)或rac1(G12V)的效果相似。还观察到显著的局灶性粘连丢失，这些联合作用导致了微注射后细胞周边的回缩。这些数据支持我们之前提出的PAK在CDC42和Rad下游的作用，并表明PAK的功能包括溶解应力纤维和重组焦点复合体。
The family of p21-activated protein kinases (PAKs) appear to be present in all organisms that have Cdc42-like GTPases, In mammalian cells, PAKs have been implicated in the activation of mitogen-activated protein kinase cascades, but there are no reported effects of these kinases on the cytoskeleton. Recently we have shown that a Drosophila PAK is enriched in the leading edge of embryonic epithelial cells undergoing dorsal closure (N. Harden, J. Lee, H.-Y. Loh, Y.-M. Ong, I. Tan, T. Leung, E. Manser, and L. Lim, Mel. Cell, Biol, 16:1896-1908, 1996), where it colocalizes,vith structures resembling focal complexes, We show here by transfection that in epithelial HeLa cells alpha-PAK is recruited from the cytoplasm to distinct focal complexes by both Cdc42(G12V) and Rac1(G12V), which themselves colocalize to these sites, By deletion analysis, the N terminus of PAK is shown to contain targeting sequences for focal adhesions which indicate that these complexes are the site of kinase function in vivo. Cdc42 and Rad cause alpha-PAK autophosphorylation and kinase activation, Mapping alpha-PAK autophosphorylation sites has allowed generation of a constitutively active kinase mutant, By fusing regions of Cdc42 to the C terminus of PAK, activated chimeras were also obtained, Plasmids encoding these different constitutively active alpha-PAKs caused loss of stress fibers when introduced into both HeLa cells and fibroblasts, which was similar to the effect of introducing CdC42(G12V) or Rac1(G12V). Significantly dramatic losses of focal adhesions were also observed, These combined effects resulted in retraction of the cell periphery after plasmid microinjection. These data support our previous suggestions of a role for PAK downstream of both Cdc42 and Rad and indicate that PAK functions include the dissolution of stress fibers and reorganization of focal complexes.