G2 M TRANSITION REQUIRES MULTISITE PHOSPHORYLATION OF ONCOPROTEIN-18 BY 2 DISTINCT PROTEIN-KINASE SYSTEMS/

Oncoprotein 18 (Op18) is a conserved cytosolic protein that is a targe t for both cell cycle and cell surface receptor-regulated phosphorylat ion events, The four residues Ser(16), Ser(25), Ser(38), and Ser(63) a re all subject to cell cycle-regulated phosphorylation, Ser(25) and Se r(38) are targets for cyclin dependent kinases (CDKs), while Ser(16) a nd Ser(63) are phosphorylated by an unidentified protein kinase, We ha ve recently shown that induced expression of a CDK target site deficie nt mutant, Op18-S25A,S38A, blocks human cell lines during G2/M transit ion, In the present report we show that mitosis is associated with com plete phosphorylation of the two Op18 CDK target sites Ser(25) and Ser (38) and that Ser(16) and Ser(63) are also phosphorylated to a high st oichiometry, To evaluate the function of multisite phosphorylation of Op18, we expressed and analyzed the cell cycle phenotype of different kinase target site-deficient mutants. The data showed that induced exp ression of the S16A,S63A, S25A,S38A, and S16A,S25A,S38A,S63A mutants a ll resulted in an indistinguishable phenotype, i.e. immediate G2/M blo ck and subsequent endoreduplication, a given fraction of G2 versus M-p hase blocked cells, and a characteristic nuclear morphology of M-block ed cells, This result was unexpected; however, a likely explanation wa s provided by analysis of Op18 phosphoisomers, which revealed that mut ations of the CDK sites interfere with phosphorylation of Ser(16) and Ser(63). The simplest interpretation of our results is that phosphoryl ation of Ser(16) and Ser(63) is essential during G2/M transition and t hat the phenotype of the S25A,S38A mutant is mediated by the observed block of Ser(16)/Ser(63) phosphorylation.