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.