A rate limiting function of cdc25A for S phase entry inversely correlates with tyrosine dephosphorylation of Cdk2

The cdc25A phosphatase removes inhibitory phosphates from threonine-14 and tyrosine-15 of cyclin dependent kinase-2 (cdk2) in vitro, and it is therefo re widely assumed that cdc25A positively regulates cyclin E- and A-associat ed cdk2 activity at the G1 to S phase transition of the mammalian cell divi sion cycle. Human cdc25A was introduced into mouse NIH3T3 fibroblasts co-ex pressing a form of the colony-stimulating factor-1 (CSF-1) receptor that is partially defective in transducing mitogenic signals. Cdc25A enabled these cells to form colonies in semisolid medium containing serum plus human rec ombinant CSF-1 in a manner reminiscent of cells rescued by c-myc. However, cdc25A-rescued cells could not proliferate in chemically defined medium con taining CSF-1 and continued to require c-myc function for S phase entry. Wh en contact-inhibited cells overexpressing cdc25A were dispersed and stimula ted to synchronously enter the cell division cycle, they entered S phase 2- 3 h earlier than their parental untransfected counterparts. Shortening of G 1 phase temporally correlated with more rapid degradation of the cdk inhibi tor p27(Kip1) and with premature activation of cyclin A-dependent cdk2. Par adoxically, tyrosine phosphorylation of cdk2 increased considerably as cell s entered S phase, and cdc25A overexpression potentiated rather than dimini shed this effect. At face value, these results are inconsistent with the hy pothesis that cdc25A acts directly on cdk2 to activate its S phase promotin g function.