The receptor-like protein-tyrosine phosphatase, RPTPα, is phosphorylated by protein kinase C on two serines close to the inner face of the plasma membrane

Abstract

To determine whether the receptor-like protein-tyrosine phosphatase, RPTPα, which is widely expressed in both the developing and adult mouse, is regulated by phosphorylation, we raised antiserum against a C-terminal peptide. This antiserum precipitated a 140-kDa protein from metabolically 35S-labeled NIH3T3 cells. Using this antiserum, we showed that endogenous RPTPα is constitutively phosphorylated in NIH3T3 cells, predominantly on two serines, which we identified as Ser-180 and Ser-204, lying in the juxtamembrane domain. 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulation of quiescent NIH3T3 cells rapidly increased phosphorylation of Ser-180 and Ser-204. Purified protein kinase C (PKC) phosphorylated bacterially expressed RPTPα at Ser-180 and Ser-204. When wild type and S180A/S204A double mutant RPTPαs were transiently expressed in 293 human embryonic kidney cells, TPA stimulated phosphorylation of wild type but not of double mutant RPTPα. PKC down-regulation following prolonged exposure to TPA diminished TPA-stimulated RPTPα phosphorylation. Taken together, these results indicate that RPTPα is a direct substrate for (PKC). Examination of 293 cells expressing exogenous RPTPα using immunofluorescence confocal microscopy showed that RPTPα exists predominantly in two subcellular compartments: in dense intracellular granules or dispersed within the plasma membrane. TPA treatment caused redistribution of some intracellular RPTPα to the cell surface, but this did not require direct phosphorylation of RPTPα at Ser-180/Ser-204. Our results suggest that activation of PKC by cytokines modulates RPTPα function in several different ways.