Functional cdc25C dual-specificity phosphatase is required for S-phase entry in human cells.
In view of the common regulatory mechanism that induces transcription of the mitotic phosphatase cdc25C and cyclin A at the beginning of S-phase, we investigated whether cdc25C was required for S-phase transit. Here, we show that in both nontransformed human fibroblasts and HeLa cells, cdc25C protein levels significantly increased concomitant with S-phase onset and cyclin A synthesis. Activity measurements on immunoprecipitates from synchronized HeLa cells revealed a sharp rise in cdc25C-associated phosphatase activity that coincided with S-phase. Microinjection of various antisense-cdc25C molecules led to inhibition of DNA synthesis in both HeLa cells and human fibroblasts. Furthermore, transfection of small interfering RNA directed against cdc25C specifically depleted cdc25C in HeLa cells without affecting cdc25A or cdc25B levels. Cdc25C RNA interference was also accompanied by S-phase inhibition. In cells depleted of cdc25C by antisense or siRNA, normal cell cycle progression could be re-established through microinjection of wild-type cdc25C protein but not inactive C377S mutant protein. Taken together, these results show that cdc25C not only plays a role at the G2/M transition but also in the modulation of DNA replication where its function is distinct from that of cdc25A.
Turowski
Patric
P
Cell Biology Unit, Institut de Genetique Humaine, Centre National de la Recherche Scientifique Unité Mixte de Recherche 1142, F-34396 Montpellier, France.
Franckhauser
Celine
C
Morris
May C
MC
Vaglio
Philippe
P
Fernandez
Anne
A
Lamb
Ned J C
NJ
D002453:Cell Cycle / Q000502:physiology; D002478:Cells, Cultured; D019925:Cyclin A / Q000096:biosynthesis*; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D019384:Nucleic Acid Synthesis Inhibitors / Q000378:metabolism; D010766:Phosphorylation; D034622:RNA Interference / Q000502:physiology; D016372:RNA, Antisense / Q000502:physiology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D016196:S Phase / Q000502:physiology*; D015398:Signal Transduction; D020687:cdc25 Phosphatases / Q000096:biosynthesis / Q000378:metabolism*
D019925:Cyclin A; D019384:Nucleic Acid Synthesis Inhibitors; D016372:RNA, Antisense; D011994:Recombinant Proteins; C517004:CDC25A protein, human; D020687:cdc25 Phosphatases