Lyons P D, Dunty J M, Schaefer E M, Schaller M D
Department of Cell and Developmental Biology and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
J Biol Chem. 2001 Jun 29;276(26):24422-31. doi: 10.1074/jbc.M011080200. Epub 2001 May 3.
Protein-tyrosine phosphatase (PTP)-PEST is a cytoplasmic tyrosine phosphatase that can bind and dephosphorylate the focal adhesion-associated proteins p130(CAS) and paxillin. Focal adhesion kinase (FAK) and cell adhesion kinase beta (CAKbeta)/PYK2/CADTK/RAFTK are protein-tyrosine kinases that can colocalize with, bind to, and induce tyrosine phosphorylation of p130(CAS) and paxillin. Thus, we considered the possibility that these kinases might be substrates for PTP-PEST. Using a combination of substrate-trapping assays and overexpression of PTP-PEST in mammalian cells, CAKbeta was found to be a substrate for PTP-PEST. Both the major autophosphorylation site of CAKbeta (Tyr(402)) and activation loop tyrosine residues, Tyr(579) and Tyr(580), were targeted for dephosphorylation by PTP-PEST. Dephosphorylation of CAKbeta by PTP-PEST dramatically inhibited CAKbeta kinase activity. In contrast, FAK was a poor substrate for PTP-PEST, and treatment with PTP-PEST had no effect on FAK kinase activity. Tyrosine phosphorylation of paxillin, which is greatly enhanced by CAKbeta overexpression, was dramatically reduced upon coexpression of PTP-PEST. Finally, endogenous PTP-PEST and endogenous CAKbeta were found to localize to similar cellular compartments in epithelial and smooth muscle cells. These results suggest that CAKbeta is a substrate of PTP-PEST and that FAK is a poor PTP-PEST substrate. Further, PTP-PEST can negatively regulate CAKbeta signaling by inhibiting the catalytic activity of the kinase.
蛋白酪氨酸磷酸酶（PTP）-PEST是一种细胞质酪氨酸磷酸酶，它能够结合粘着斑相关蛋白p130（CAS）和桩蛋白并使其去磷酸化。粘着斑激酶（FAK）和细胞粘着激酶β（CAKβ）/PYK2/CADTK/RAFTK是蛋白酪氨酸激酶，它们能够与p130（CAS）和桩蛋白共定位、结合并诱导其酪氨酸磷酸化。因此，我们推测这些激酶可能是PTP-PEST的底物。通过在哺乳动物细胞中结合使用底物捕获分析和PTP-PEST的过表达，发现CAKβ是PTP-PEST的底物。CAKβ的主要自磷酸化位点（Tyr(402)）以及激活环酪氨酸残基Tyr(579)和Tyr(580)均被PTP-PEST靶向去磷酸化。PTP-PEST对CAKβ的去磷酸化显著抑制了CAKβ激酶活性。相比之下，FAK是PTP-PEST的不良底物，用PTP-PEST处理对FAK激酶活性没有影响。CAKβ过表达可显著增强的桩蛋白酪氨酸磷酸化，在与PTP-PEST共表达时显著降低。最后，发现内源性PTP-PEST和内源性CAKβ定位于上皮细胞和平滑肌细胞中的相似细胞区室。这些结果表明CAKβ是PTP-PEST的底物，而FAK是PTP-PEST的不良底物。此外，PTP-PEST可通过抑制激酶的催化活性来负向调节CAKβ信号传导。
