Src and FAK kinases cooperate to phosphorylate paxillin kinase linker, stimulate its focal adhesion localization, and regulate cell spreading and protrusiveness
Src and FAK kinases cooperate to phosphorylate paxillin kinase linker, stimulate its focal adhesion localization, and regulate cell spreading and protrusiveness
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DOI：
10.1091/mbc.e05-02-0131
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发表时间：
2005-09-01
影响因子：
3.3
通讯作者：
Turner, CE
中科院分区：
文献类型：
--
作者：
Brown, MC;Cary, LA;Turner, CE
ArfGAP桩蛋白激酶连接体（PKL）/G蛋白偶联受体激酶相互作用蛋白（GIT）2通过瞬时募集p21激活的激酶（PAK）至粘着斑而参与调节细胞铺展和运动。Nck-PAK-PIX-PKL蛋白复合物在整合素接合和Rac活化后被桩蛋白募集至粘着斑。在这份报告中，我们确定酪氨酸磷酸化PKL作为一种蛋白质，与SH 3-SH 2适配器NCK，在Src依赖的方式，细胞粘附到纤连蛋白后。细胞粘附和Rac激活均刺激PKL酪氨酸磷酸化。PKL在酪氨酸残基286/392/592上被Src和/或FAK磷酸化，并且这些位点是PKL定位于粘着斑和桩蛋白结合所需的。FAK或Src家族激酶的缺乏阻止PKL磷酸化并抑制PKL而不是GIT 1在Rac激活后局部粘连的定位。活化的FAK突变体的表达在Src家族激酶的情况下部分恢复PKL的本地化，这表明Src激活FAK是PKL磷酸化和本地化所需的。非磷酸化的GFP-PKL三YF突变体的过表达刺激细胞铺展和伸展性，类似于不结合PKL的桩蛋白突变体的过表达，这表明未能招募PKL至局灶性粘附干扰正常的细胞铺展和运动。
The ArfGAP paxillin kinase linker (PKL)/G protein-coupled receptor kinase-interacting protein (GIT)2 has been implicated in regulating cell spreading and motility through its transient recruitment of the p21-activated kinase (PAK) to focal adhesions. The Nck-PAK-PIX-PKL protein complex is recruited to focal adhesions by paxillin upon integrin engagement and Rac activation. In this report, we identify tyrosine-phosphorylated PKL as a protein that associates with the SH3-SH2 adaptor Nck, in a Src-dependent manner, after cell adhesion to fibronectin. Both cell adhesion and Rac activation stimulated PKL tyrosine phosphorylation. PKL is phosphorylated on tyrosine residues 286/392/592 by Src and/or FAK and these sites are required for PKL localization to focal adhesions and for paxillin binding. The absence of either FAK or Src-family kinases prevents PKL phosphorylation and suppresses localization of PKL but not GIT1 to focal adhesions after Rac activation. Expression of an activated FAK mutant in the absence of Src-family kinases partially restores PKL localization, suggesting that Src activation of FAK is required for PKL phosphorylation and localization. Overexpression of the nonphosphorylated GFP-PKL Triple YF mutant stimulates cell spreading and protrusiveness, similar to overexpression of a paxillin mutant that does not bind PKL, suggesting that failure to recruit PKL, to focal adhesions interferes with normal cell spreading and motility.