Lammers R, Lerch M M, Ullrich A
Max-Planck-Institut für Biochemie, Am Klopferspitz 18A, D-82152 Martinsried, Germany.
J Biol Chem. 2000 Feb 4;275(5):3391-6. doi: 10.1074/jbc.275.5.3391.
The receptor protein-tyrosine phosphatase alpha (PTPalpha) is involved in the activation of c-Src kinase as well as in down-regulation of the insulin signal. To investigate the role of PTPalpha in activation of the Src kinase in more detail we tried to overexpress this phosphatase in NIH3T3 fibroblasts. Although PTPalpha has been overexpressed in rat embryonic fibroblasts and in embryonic carcinoma cells and should increase mitogenic responses we were not able to achieve a detectable overexpression. In contrast, expression of partially (C442S) or completely inactive (C442S,C732S) PTPalpha or of phosphatase active PTPalpha containing mutation Y781F or Y798F was possible. The level of expression, however, was reduced to background after several passages of lines expressing PTPalphaC442S,C732S and PTPalphaY781F. When employed in a focus formation assay, only infection with virus encoding PTPalphaY798F induced Src-dependent formation of foci. In immunofluorescence studies, PTPalphaC442S and PTPalphaY781F but not PTPalphaY798F colocalized with proteins found in focal adhesion plaques. Treatment of PTPalphaC442S-overexpressing cells with vanadate abolished this colocalization and led to proteolytic processing of the phosphatase. We conclude that tyrosine 798 in PTPalpha is important for localization at focal adhesion plaques. Inhibition of phosphatases by vanadate treatment releases PTPalpha from focal adhesions.
受体蛋白酪氨酸磷酸酶α（PTPα）参与c-Src激酶的激活以及胰岛素信号的下调。为了更详细地研究PTPα在Src激酶激活中的作用，我们试图在NIH3T3成纤维细胞中过表达这种磷酸酶。尽管PTPα已在大鼠胚胎成纤维细胞和胚胎癌细胞中过表达，且应该会增加促有丝分裂反应，但我们未能实现可检测到的过表达。相反，部分失活（C4C42S）或完全失活（C442S，C732S）的PTPα或含有Y781F或Y798F突变的磷酸酶活性PTPα的表达是可能的。然而，表达PTPαC442S、C732S和PTPαY781F的细胞系传代几次后，表达水平降至背景值。在焦点形成试验中，只有感染编码PTPαY798F的病毒才能诱导Src依赖性的焦点形成。在免疫荧光研究中，PTPαC442S和PTPαY781F而非PTPαY798F与粘着斑中发现的蛋白质共定位。用钒酸盐处理过表达PTPαC442S的细胞消除了这种共定位，并导致磷酸酶的蛋白水解加工。我们得出结论，PTPα中的酪氨酸798对于在粘着斑中的定位很重要。钒酸盐处理抑制磷酸酶会使PTPα从粘着斑中释放出来。
