SERINE-16 OF ONCOPROTEIN-18 IS A MAJOR CYTOSOLIC TARGET FOR THE CA2+ CALMODULIN-DEPENDENT KINASE-GR/

Oncoprotein 18 (Op18) is a cytosolic protein that was initially identi fied due to its up-regulated expression in acute leukemia and its comp lex pattern of phosphorylation in response to diverse extracellular si gnals. We have previously identified in vivo phosphorylation sites and some of the protein kinase systems involved. Two distinct proline-dir ected kinase families phosphorylate Ser25 and Ser38 of Op18 with overl apping but distinct site preference. These two kinase families, mitoge n-activated protein (MAP) kinases and cyclin-dependent cdc2 kinases, a re involved in receptor-regulated and cell-cycle-regulated phosphoryla tion events, respectively. During analysis of Op18 phosphorylation in the Jurkat T-cell line, we also found that Serl6 of Op18 is phosphoryl ated in response to a Ca2+ signal generated by T-cell receptor stimula tion or the Ca2+ ionophore ionomycin. As suggested by a previous study , T-cell-receptor-induced phosphorylation events may be mediated by th e Ca2+/CaM-dependent protein kinase type Gr (CaM kinase-Gr). The prese nt study shows that activation of this protein kinase correlates with phosphorylation of Serl6 of Op18, and in vitro experiments reveal effi cient and selective phosphorylation of this residue. The CaM kinase-Gr is only expressed in certain lymphoid cell lines, and the present stu dy shows that ionomycin-induced phosphorylation of Op18 Serl6 is restr icted to cells expressing this protein kinase. Finally, CaM kinase-Gr- dependent in vitro phosphorylation of a crude cellular extract reveals a striking preference of this protein kinase for Op18 compared to oth er cellular substrates. In conclusion, the results suggest that Serl6 of Op18 is a major cytosolic target for activated CaM kinase-Gr.