SERINE-25 OF ONCOPROTEIN-18 IS A MAJOR CYTOSOLIC TARGET FOR THE MITOGEN-ACTIVATED PROTEIN-KINASE

Oncoprotein 18 (Op18) is an 18-19-kDa cytoplasmic phosphoprotein, of u nknown function, that is frequently up-regulated in transformed cells. Stimulation of various cell-surface receptors results in extensive ph osphorylation of Op18 and this protein has, therefore, previously been implicated in intracellular signaling. In the present study, by expre ssion of specific Op18 cDNA mutant constructs and phosphopeptide mappi ng, we have identified in vivo phosphorylation sites. In conjunction w ith in vitro phosphorylation experiments, using purified wild-type and mutant Op18 proteins in combination with a series of kinases, these r esults have identified two distinct proline-directed kinase families t hat phosphorylate Op 1 8 with overlapping but distinct site preference . These two kinase families, mitogen activated protein (MAP) kinases a nd cyclin dependent cdc2 kinases, are involved in receptor and cell cy cle-regulated phosphorylation events, respectively. Therefore, Op18 ma y reside at a junction where receptor and cell cycle-regulated kinase families interact with the same substrate. The present study shows tha t the MAP kinase has a 20-fold preference for Ser25 as opposed to Ser3 8 of Op18, while cdc2 kinases have a 5-fold preference for the Ser38 r esidue. Only a minor fraction of the 4.5 x 10(6)Op18 molecules/cell in a leukemic T-cell line are normally in their Ser25 phosphorylated for m. However, antigen receptor stimulation of this cell line is shown to result in a rapid conversion of 35-45% of all Op18 molecules to the S er25 phosphorylated form. These results suggest that Ser25 of Op18 may be a major cytoplasmic target for the MAP kinase in cells with high e xpression of Op18.