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 therefore widely assumed that cdc25A positively regulates cyclin E- and A-associated cdk2 activity at the G1 to S phase transition of the mammalian cell division cycle. Human cdc25A was introduced into mouse NIH3T3 fibroblasts co-expressing 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 recombinant CSF-1 in a manner reminiscent of cells rescued by c-myc. However, cdc25A-rescued cells could not proliferate in chemically defined medium containing CSF-1 and continued to require c-myc function for S phase entry. When contact-inhibited cells overexpressing cdc25A were dispersed and stimulated to synchronously enter the cell division cycle, they entered S phase 2-3 h earlier than their parental untransfected counterparts. Shortening of G1 phase temporally correlated with more rapid degradation of the cdk inhibitor p27Kip1 and with premature activation of cyclin A-dependent cdk2. Paradoxically, tyrosine phosphorylation of cdk2 increased considerably as cells entered S phase, and cdc25A overexpression potentiated rather than diminished this effect. At face value, these results are inconsistent with the hypothesis that cdc25A acts directly on cdk2 to activate its S phase promoting function.
Sexl
V
V
Department of Tumor Cell Biology, St Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
Diehl
J A
JA
Sherr
C J
CJ
Ashmun
R
R
Beach
D
D
Roussel
M F
MF
D016475:3T3 Cells; D000818:Animals; D042846:CDC2-CDC28 Kinases; D002461:Cell Line, Transformed; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D051379:Mice; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000378:metabolism*; D016196:S Phase; D014443:Tyrosine / Q000378:metabolism*; D020687:cdc25 Phosphatases