IDENTIFICATION OF IN-VIVO PHOSPHORYLATION SITES OF CD45 PROTEIN-TYROSINE-PHOSPHATASE IN 70Z 3.12 CELLS/

Phosphorylation of CD45, a transmembrane protein-tyrosine phosphatase (PTPase), has been proposed to mediate docking of signaling proteins a nd to modulate PTPase activity, To study the role of phosphorylation i n CD45, in vivo phosphorylation sites of CD45 from 70Z/3.12 cells were identified using P-32 labeling, trypsin digestion, two-dimensional pe ptide mapping, high performance liquid chromatography, phosphoamino ac id analysis, matrix-assisted laser desorption/ionization mass spectrom etry, and specific enzymatic degradation, Eight phosphopeptides, a thr ough h, were isolated and four phosphorylation sites were identified, All four phosphorylation sites were in the membrane-distal PTPase doma in (D2) and the C-terminal tail and none were in the membrane-proximal PTPase domain (D1), One site, Ser(P)(939) peptide h, was in the D2 do main and, by comparison to the three-dimensional structure of PTP1B, i s predicted to lie at the apex of the substrate binding loop, Ser(939) was the only in vitro phosphorylation site for protein kinase C among the phosphorylation sites identified, Four of the C-terminal peptides identified (d, e, f, and g) spanned the same sequence and were derive d from the same phosphorylation site in the C-terminal tail, Ser(1204) , Peptide a was derived from the intact C terminus and comprised a mix ture of monophosphorylated peptides containing either Ser(P)(1248) or. Thr(P)(1246). Knowledge of the precise phosphorylation sites of CD45 will lead to the design of experiments to define the role of phosphory lation in PTPase activity and in signaling.