Paxillin is tyrosine-phosphorylated by and preferentially associates with the calcium-dependent tyrosine kinase in rat liver epithelial cells
Paxillin is tyrosine-phosphorylated by and preferentially associates with the calcium-dependent tyrosine kinase in rat liver epithelial cells
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DOI：
10.1074/jbc.272.22.14341
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发表时间：
1997-05-30
影响因子：
4.8
通讯作者：
Earp, HS
中科院分区：
文献类型：
--
作者：
Li, X;Earp, HS
我们和其他人最近克隆了一种非受体的钙依赖性酪氨酸激酶（CADTK;也称为PYK 2、CAK β和RAFTK），其与p125（FAK）共享整体结构域结构和45%氨基酸同一性。我们已经研究了这些相关酶在表达CADTK和p125（FAK）水平大致相似的GN 4大鼠肝上皮细胞中的信号传导、活化和潜在功能，p125（FAK）在静息GN 4细胞中几乎完全酪氨酸磷酸化。相反，虽然CADTK在未处理的细胞中没有酪氨酸自磷酸化，但血管紧张素II使CADTK Tyr（P）增加5-10倍。CADTK活化与c-Jun N-末端激酶和p70（S6 K）途径的刺激相关，但与促分裂原活化蛋白激酶或p90（RSK）的刺激无关，在本报告中，我们评估了CADTK和p125（FAK）对局部接触蛋白酪氨酸磷酸化的贡献。在粘附的GN 4细胞中，p125（FAK）的组成性活性与基础桩蛋白、张力蛋白和p130（CAS）酪氨酸磷酸化相关。桩蛋白酪氨酸磷酸化的延长的3-4倍增加是最显著的变化。在表达比GN 4细胞系少3倍的CADTK的WE细胞系中，激动剂依赖性桩蛋白酪氨酸磷酸化类似地降低。来自GN 4细胞的CADTK免疫沉淀显示CADTK桩蛋白复合物在500 mM NaCl中持续存在，但在0.1%SDS细胞裂解缓冲液中不存在。该复合物在很大程度上不依赖于任一蛋白质的酪氨酸磷酸化状态。令人惊讶的是，我们没有检测到p125（FAK）使用两种p125（FAK）抗体中的任何一种在免疫沉淀物中检测到桩蛋白复合物，当CADTK和p125（FAK）在293（T）细胞中瞬时过表达时，两种酶都与桩蛋白相关，但CADTK的亲合力似乎更大。此外，在转染的293（T）细胞中，检测到CADTK和另一种潜在底物p130（CAS）之间的复合物。在GN 4大鼠肝上皮细胞中，CADTK的刺激与桩蛋白酪氨酸磷酸化高度相关;此外，CADTK而不是p125（FAK）以可检测的水平与桩蛋白复合。这表明上皮细胞中激动剂依赖性细胞骨架变化可能部分通过CADTK依赖性机制进行。
We and others have recently cloned a non-receptor, calcium-dependent tyrosine kinase (CADTK; also known as PYK2, CAK beta, and RAFTK) that shares both overall domain structure and 45% amino acid identity with p125(FAK), We have studied the signaling, activation, and potential function of these related enzymes in GN4 rat liver epithelial cells that express CADTK and p125(FAK) at roughly similar levels, p125(FAK) is nearly fully tyrosine-phosphorylated in resting GN4 cells, In contrast, while CADTK is not tyrosine-autophosphorylated in untreated cells, angiotensin II increases CADTK Tyr(P) by 5-10-fold, With regard to signaling, CADTK activation is correlated with stimulation of c-Jun N-terminal kinase and p70(S6K) pathways but not with the stimulation of mitogen-activated protein kinase or p90(RSK), In this report we assessed the contribution of CADTK and p125(FAK) to tyrosine phosphorylation of focal contact proteins, In adherent GN4 cells, the constitutive activity of p125(FAK) was correlated with basal paxillin, tensin, and p130(CAS) tyrosine phosphorylation, A rapid increase in the tyrosine phosphorylation of each protein was detected after treatment with angiotensin II or other agonists that stimulate CADTK; the prolonged 3-4-fold increase in paxillin tyrosine phosphorylation was the most substantial change, In the WE cell line that expresses 3-fold less CADTK than GN4 cell line agonist-dependent paxillin tyrosine phosphorylation is similarly reduced. Immunoprecipitation of CADTK from GN4 cells revealed CADTK paxillin complexes that persisted in 500 mM NaCl but not in 0.1% SDS cell lysis buffer, The complexes were largely independent of the tyrosine phosphorylation state of either protein, Surprisingly, we did not detect pl25(FAK) paxillin complexes in immunoprecipitates using either of two p125(FAK) antibodies, When CADTK and p125(FAK) were transiently overexpressed in 293(T) cells, both enzymes associated with paxillin, but the avidity of CADTK appeared to be greater, In addition, in transfected 293(T) cells, complexes between CADTK and another potential substrate, p130(CAS), were detected, In summary, in GN4 rat liver epithelial cells stimulation of CADTK was highly correlated with paxillin tyrosine phosphorylation; in addition, CADTK but not p125(FAK) was complexed to paxillin at detectable levels, This suggests that agonist-dependent cytoskeletal changes in epithelial cells might proceed, in part, by CADTK-dependent mechanisms.