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

最近，我们克隆了一个可能的蛋白酪氨酸磷酸酶(PTP)，PTP36，它具有与Band 4.1蛋白的N-末端半部分同源的结构域。在附着于底物的小鼠成纤维细胞中，PTP36在丝氨酸残基上被磷酸化。PTP36被发现与丝氨酸/苏氨酸激肽(S)形成复合体，在体外使PTP36磷酸化。星形孢菌素在体外可抑制PTP36的磷酸化，而ML-9、Calphostin C、KN-62和W-7则不能抑制PTP36的磷酸化。星形孢子素还抑制体内的磷酸化。细胞-底物黏附的破坏导致PTP36迅速去磷酸化，并将PTP36移位到含有肌动蛋白的细胞骨架部分。丝氨酸/苏氨酸磷酸酶抑制剂冈田酸可完全阻断PTP36的去磷酸化和转位。因此，PTP36的磷酸化水平和细胞内定位受到细胞-底物黏附的调节，提示PTP36可能在细胞黏附的信号转导通路中发挥作用。为了深入了解PTP36的生物学功能，我们建立了一个稳定的HeLa细胞株(HtTA1-38)，其中PTP36的表达水平受到四环素或强力霉素(Dox)的调节。在野生型HeLa细胞和未诱导的HtTA1-38细胞中几乎检测不到PTP36蛋白。在这些细胞中没有发现任何表型的差异。只有在HtTA1-38中去除Dox后，才能观察到PTP36的强烈诱导。诱导PTP36过表达后，细胞形态、肌动蛋白细胞骨架、细胞生长和Fas抗体诱导的细胞凋亡均发生改变。我们的结果表明，PTP36参与了细胞黏附、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.