THE RECEPTOR-LIKE PROTEIN-TYROSINE-PHOSPHATASE, RPTP-ALPHA, IS PHOSPHORYLATED BY PROTEIN-KINASE-C ON 2 SERINES CLOSE TO THE INNER FACE OF THE PLASMA-MEMBRANE

To determine whether the receptor-like protein-tyrosine phosphatase, R PTP alpha, 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 S-35-labeled NIH3T3 cells. Using this antiserum, we sho wed that endogenous RPTP alpha is constitutively phosphorylated in NIH 3T3 cells, predominantly on two serines, which we identified as Ser-18 0 and Ser-204, lying in the juxtamembrane domain. 12-O-tetradecanoylph orbol-13 acetate (TPA) stimulation of quiescent NIH3T3 cells rapidly i ncreased phosphorylation of Ser-180 and Ser-204. Purified protein kina se C (PKC) phosphorylated bacterially expressed RPTP alpha at Ser-180 and Ser-204. When wild type and S180A/S204A double mutant RPTP alpha w ere transiently expressed in 293 human embryonic kidney cells. TPA sti mulated phosphorylation of wild type but not of double mutant RPTP alp ha. PKC down-regulation following prolonged exposure to TPA diminished TPA-stimulated RPTP alpha phosphorylation. Taken together, these resu lts indicate that RPTP alpha is a direct substrate for (PKC). Examinat ion of 293 cells expressing exogenous RPTP alpha using immunofluoresce nce confocal microscopy showed that RPTP alpha exists predominantly in two subcellular compartments: in dense intracellular granules or disp ersed within the plasma membrane, TPA treatment caused redistribution of some intracellular RPTP alpha to the cell surface, but this did not require direct phosphorylation of RPTP alpha at Ser-180/Ser-204. Our results suggest that activation of PKC by cytokines modulates RPTP alp ha function in several different ways.