Involvement of cdc2-mediated phosphorylation in the cell cycle-dependent regulation of p185^neu

Abstract

We previously reported cell cycle-dependent negative regulation of p185^neu (decreased tyrosine phosphorylation and kinase activity, with electrophoretic mobility retarded by serine/threonine phosphorylation) in M phase and the escape of mutation-activated p185^neu* from this regulation. Our present results showed that retardation of electrophoretic mobility occurs independently of the cells' transformed status. We found that normal p185^neu lost its ability to dimerize in the M phase. We demonstrated a physical association between cdc2 (a serine/threonine kinase, active in M phase) and p185^neu. We showed that the carboxy terminal portion of p185^neu is phosphorylated in vitro by cdc2. Many phosphopeptides (at least three phosphoserine residues) unique to the M phase were identified, and the in vivo and in vitro phosphopeptide patterns were superimposable. In contrast, mutation-activated p185^neu* dimerized in the M phase with no changes in electrophoretic mobility, failed to associate with cdc2 and no unique phosphoserine residues could be identified in the M phase (data not shown), consistent with the escape of p185^neu* from cell cycle-dependent regulation. Our results suggest that this escape is an intrinsic property of the mutation-activated p185^neu* independent of its ability to transform cells. Our results also suggest the involvement of serine/threonine kinases such as cdc2 in the cell cycle-dependent negative regulation of p185^neu.