Int J Biochem Cell Biol. 2014 Jan;46:19-31.
Protein tyrosine phosphatse interacting protein (PTPIP51) is involved in the modulation of the mitogen activated protein kinase (MAPK) signaling pathway. Up to now, less is known about the regulation of this modulation. A recent study hinted to the phosphorylation status of PTPIP51 being essential for correct regulation of PTPIP51 function.In this study we investigate the phosphorylation status of PTPIP51 under the inhibition of the main interacting kinases and phosphatases of PTPIP51. c-Src was inhibited by Dasatinib, EGF receptor by Gefitinib, protein kinase C by staurosporine, protein kinase A by RpcAMPs and PTP1B by its specific inhibitor. Furthermore, a combination of PP2 with Gefitinib and RpcAMPs was used, respectively. The data were acquired for non-EGF and EGF treated HaCaT cells.All cells were analyzed relative to the subcellular distribution and change in the amount of tyrosine 176 phosphorylated PTPIP51. Furthermore, the protein interactions were assayed by duolink proximity ligation assay.HaCaT cells submitted to the respective inhibitor displayed a subcellular redistribution of tyrosine 176 phosphorylated PTPIP51 depending on the applied inhibitor. Yet, the amount of tyrosine 176 phosphorylated PTPIP51 remained unchanged by inhibitor treatment except for Gefitinib and simultaneous PP2 and Gefitnib treatment in non EGF-stimulated cells, but was elevated if cells were also EGF stimulated, in control and inhibitor treated cells. Interestingly, the interaction with EGFR, 14-3-3, Raf-1, c-Src, PTP1B, PKA and PKC was influenced by the application of inhibitors. Also EGF application resulted in a sharp drop of the PTPIP51 interaction with the MAPK pathway (e.g. Raf-1) in the control group.Summarizing these new findings, we postulate that PTPIP51 is regulated by its phosphorylation status combined with a thereby induced subcellular redistribution. In addition, the EGF receptor regulates PTPIP51 interaction with Raf-1 by its phosphorylation, thus preventing an overshooting activation of the MAPK pathway.
蛋白酪氨酸磷酸酶相互作用蛋白（PTPIP51）参与丝裂原活化蛋白激酶（MAPK）信号通路的调节。到目前为止，对于这种调节的调控知之甚少。最近的一项研究表明，PTPIP51 的磷酸化状态对于正确调节 PTPIP51 的功能至关重要。在这项研究中，我们研究了在 PTPIP51 的主要相互作用激酶和磷酸酶抑制下 PTPIP51 的磷酸化状态。通过达沙替尼抑制 c-Src，通过吉非替尼抑制表皮生长因子受体，通过 staurosporine 抑制蛋白激酶 C，通过 RpcAMPs 抑制蛋白激酶 A，通过其特异性抑制剂抑制 PTP1B。此外，还分别使用了 PP2 与吉非替尼和 RpcAMPs 的组合。对未用 EGF 处理和用 EGF 处理的 HaCaT 细胞获取数据。所有细胞均相对于酪氨酸 176 磷酸化 PTPIP51 的亚细胞分布和数量变化进行分析。此外，通过 duolink 邻近连接测定法测定蛋白质相互作用。用各自的抑制剂处理的 HaCaT 细胞显示出酪氨酸 176 磷酸化 PTPIP51 的亚细胞重新分布，这取决于所应用的抑制剂。然而，除了非 EGF 刺激细胞中的吉非替尼和同时的 PP2 和吉非替尼处理外，抑制剂处理对酪氨酸 176 磷酸化 PTPIP51 的量没有影响，但如果细胞也接受 EGF 刺激，则在对照和抑制剂处理的细胞中升高。有趣的是，与 EGFR、14-3-3、Raf-1、c-Src、PTP1B、PKA 和 PKC 的相互作用受抑制剂的应用影响。此外，EGF 的应用导致对照组成员中 PTPIP51 与 MAPK 途径（例如 Raf-1）的相互作用急剧下降。总结这些新发现，我们假设 PTPIP51 通过其磷酸化状态与由此诱导的亚细胞重新分布相结合进行调节。此外，表皮生长因子受体通过其磷酸化调节 PTPIP51 与 Raf-1 的相互作用，从而防止 MAPK 途径的过度激活。
