Regulation of a cytoplasmic protein tyrosine phosphatase through cell-substrate adhesion

最近，我们已经克隆了一个假定的蛋白酪氨酸磷酸酶（PTP），PTP 36，它具有一个结构域同源的N-末端的一半带4.1蛋白。在小鼠成纤维细胞粘附到基板上，磷酸化的丝氨酸残基上的PTP 36。发现PTP 36与丝氨酸/苏氨酸激酶形成复合物，其在体外磷酸化PTP 36。Staurosporine，而不是ML-9，Calphostin C，KN-62，和W-7，抑制PTP 36的磷酸化在体外。Staurosporine也抑制了体内的磷酸化，破坏细胞与底物的粘附可使PTP 36快速去磷酸化，并使PTP 36移位到含有肌动蛋白的细胞骨架中。丝氨酸/苏氨酸磷酸酶抑制剂冈田酸可完全阻断PTP 36的去磷酸化和易位。为了深入研究PTP 36的生物学功能，我们建立了一个稳定的转染HeLa细胞的转染子（HtTA 1 -38），其中PTP 36的表达水平受四环素或多西环素（Dox）的调控。PTP 36蛋白在野生型HeLa细胞以及未诱导的HtTA 1 -38中几乎检测不到。这些细胞的表型存在差异。当从培养物中去除Dox时，仅在HtTA 1 -38中观察到PTP 36的强诱导。在诱导PTP 36过表达后，Fas抗体诱导的形态学、肌动蛋白细胞骨架、细胞生长和凋亡的变化。我们的研究结果表明，PTP 36参与细胞粘附，细胞生长和细胞死亡的调节过程。

Recently we have cloned a putative protein tyrosine phosphatase (PTP), PTP36, which possesses a domain homologous to the N-terminal half of band 4.1 protein. In mouse fibroblast adhered to substrate, PTP36 was phosphorylated on serine residues. PTP36 was found to make a complex with serine/threonine kinae (s), which phosphorylated PTP36 in vitro. Staurosporine but not ML-9, Calphostin C,KN-62, and W-7, inhibited the phosphorylation of PTP36 in vitro. Staurosporine also inhibited the in vivo phosphorylation.Disruption of cell-substrate adhesion induced rapid dephosphorylation of PTP36 and translocation of PTP36 into cytoskeletal fraction containing actin. Both dephosphorylation and translocation of PTP36 was completely blocked by okadaic acid, an inhibitor of serine/threonine phosphatase. Thus, the phosphorylation level and intracellular localization of PTP36 is regulated by cell-substrate adhesion, suggesting that PTP36 may play roles in the signal transduction pathway of cell adhesion.To gain insight into the biological function of PTP36, we established a stable transfectant (HtTA1-38) of HeLa cells in which the expression level of PTP36 was regulated by tetracycline or doxycycline (Dox). PTP36 protein was hardly detectable in wild type HeLa cells as well as in uninduced HtTA1-38. Any difference of Phenotype was found in these cells. Strong induction of PTP36 was observed only in HtTA1-38 when Dox was removed from the culture. Upon induction of PTP36 overexpression, changes in morphology, actin cytoskeleton, cell growth, and apoptosis by Fas antibody were induced. Our results suggest that PTP36 is involved in the regulatory processes of cell adhesion, cell growth and cell death.