Functional cdc25C Dual-Specificity Phosphatase Is Required for S-Phase Entry in Human Cells

Abstract

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.

Histone demethylase Kdm4b functions as a co-factor of C/EBPβ to promote mitotic clonal expansion during differentiation of 3T3-L1 preadipocytes

“…30 A study indicates that Cdc25c not only has a role in mitosis, but also contributes to S-phase entry. 31 Our results verify that the four cell cycle genes are downstream target genes of C/EBPb during MCE (Figures 4, 5; Supplementary Figure S5-S6). Knockdown of the four genes impairs MCE, followed by the inhibition of terminal adipocyte differentiation (Supplementary Figure S5).…”

A caspase-dependent cleavage of CDC25A generates an active fragment activating cyclin-dependent kinase 2 during apoptosis

“…Indeed, full-size CDC25A is almost totally downregulated in these cells, and the other members of the CDC25 family were not described as CDK2 regulators, although CDC25C was reported to induce S-phase entry in one study. 23 Furthermore, the fact that ectopic expression of the truncation mutant reduced Tyr15 phosphorylation of CDK2 further reinforced this conclusion. Importantly, CDK2 activation was recently found necessary for Myc-induced apoptosis in murine and human fibroblasts.…”

Linking PCNA-dependent replication and ATR by human Claspin

“…Rabbit polyclonal Anti PRK was from BD (UK). For Chk1 activation and p21 expression, cells were fixed as previously described [24] stained with anti-phosphoser317 of Chk1 (Upstate, UK) and anti HA monoclonal antibody (12CA5) and revealed with anti rabbit alexa555 and anti mouse alexa488 (Molecular probe, Neth) respectively. Chk1 monoclonal antibodies (8408 from Santa Cruz, USA), anti phosphoSer345 Chk1 (Upstate, UK) and anti HA (12CA5) were used for western-blotting 50 µg of DLD1 extracts.…”