MULTISITE PHOSPHORYLATION OF THE PROTEIN-TYROSINE PHOSPHATASE, PTP1B - IDENTIFICATION OF CELL-CYCLE REGULATED AND PHORBOL ESTER STIMULATED SITES OF PHOSPHORYLATION

The non-transmembrane protein tyrosine phosphatase, PTP1B, comprises 4 35 amino acids, of which the C-terminal 114 residues have been implica ted in controlling both localization and function of this enzyme. Insp ection of the sequence of the C-terminal segment reveals a number of p otential sites of phosphorylation. We show that PTP1B is phosphorylate d on seryl residues in vivo. Increased phosphorylation of PTP1B is see n to accompany the transition from G2 to M phase of the cell cycle. Tw o major tryptic phosphopeptides appear in two-dimensional maps of PTP1 B from mitotic cells. One of these comigrates with the peptide generat ed following phosphorylation of PTP1B in vitro at Ser386 by the mitoti c protein Ser/Thr kinase p34cdc2:cyclin B. The site of phosphorylation that is responsible for the pronounced retardation in the electrophor etic mobility of PTP1B from mitotic cells has been identified by site directed mutagenesis as Ser352. The identity of the kinase responsible for this modification is presently unknown. We also show that stimula tion of HeLa cells with the phorbol ester TPA enhances phosphorylation of PTP1B. Two dimensional phosphopeptide mapping reveals that the bul k of the phosphate is in a single tryptic peptide. The site, identifie d as Ser378, is also the site of phosphorylation by protein kinase C ( PKC) in vitro. Thus the TPA-stimulated phosphorylation of PTP1B in viv o appears to result directly from phosphorylation by PKC. The effect o f phosphorylation on the activity of PTP1B has been examined in immuno precipitates from TPA-treated and nocodazole-arrested cells. TPA treat ment does not appear to affect activity directly, whereas the activity of PTP1B from nocodazole-arrested cells is only 70% of that from asyn chronous populations.