Differential phosphorylation of Cdc25C phosphatase in mitosis
Section snippets
Materials and methods
Cell culture, synchronization, and siRNA transfection. HeLa cells were cultured in DMEM supplemented with 10% FCS, at 37 °C in an atmosphere containing 5% CO2. Cell culture media, serum, and antibiotics were purchased from Invitrogen. Cells were synchronized in mitosis for 12 h with 250 μg/ml Nocodazole, and collected by mitotic shakeoff. siRNA smart-pools™ targeting Cdc25C and control siRNA (non-targeting) were obtained from Dharmacon. 50 nM siRNA were transfected twice at a 24 h interval with
Characterization of human Cdc25C variants in interphasic and mitotic cells
In order to characterize the different species of Cdc25C present in interphasic and mitotic HeLa cells, we probed cell extracts by Western blotting with a generic antibody directed against the C-terminus of the phosphatase (C-20). We identified several forms of Cdc25C, which we classified into three categories, according to their electrophoretic mobility (Fig. 1A). In asynchronous HeLa cell extracts, two major species of Cdc25C were detected: a doublet in the 60 kDa range (molecular weight range
Discussion
Mitotic hyperphosphorylation of Cdc25C is associated with an increase in its phosphatase activity and is readily detected through a significant shift in its electrophoretic mobility [3], [15], [16], [17], [18], [19], [20], [26]. Although it is commonly assumed that Cdc25C oscillates between an hypophosphorylated (Ser216 phosphorylated) inactive state, and an hyperphosphorylated active mitotic form, growing evidence suggests that intermediate phosphorylation variants are likely to occur [18],
Acknowledgments
This work was supported by the CNRS (Centre National de la Recherche Scientifique) and grants from the Association de Recherche contre le Cancer (ARC) and the French National Research Agency (ANR) to MCM. J.B. was supported by fellowships from the French Ministry of Research and La Ligue Nationale Contre le Cancer. We thank J.M. Donnay and J.-C. Mazur from the CRBM animal core facility for immunizations. We thank B. Ducommun, V. Baldin, P. Coopman, D. Fesquet, V. Dulic, and all members of the
References (34)
Self-perpetuating states in signal transduction: positive feedback, double-negative feedback and bistability
Curr. Opin. Cell Biol.
(2002)- et al.
The when and wheres of CDC25 phosphatases
Curr. Opin. Cell Biol.
(2006) - et al.
Activation of p34cdc2 protein kinase by microinjection of human cdc25C into mammalian cells. Requirement for prior phosphorylation of cdc25C by p34cdc2 on sites phosphorylated at mitosis
J. Biol. Chem.
(1994) - et al.
The human polo-like kinase, PLK, regulates cdc2/cyclin B through phosphorylation and activation of the cdc25C phosphatase
Cell Signal.
(2000) - et al.
Hyperphosphorylation of the N-terminal domain of Cdc25 regulates activity toward Cyclin B1/cdc2 but not cyclin A/cdk2
J. Biol. Chem.
(1997) - et al.
Calcium/calmodulin-dependent phosphorylation and activation of human Cdc25-C at the G2/M phase transition in HeLa cells
J. Biol. Chem.
(1999) - et al.
Alternative splicing in the regulatory region of the human phosphatases CDC25A and CDC25C
Eur. J. Cell Biol.
(2000) - et al.
Differential expression of cdc25 cell-cycle-activating phosphatases in human colorectal carcinoma
Lab. Invest.
(2001) - et al.
Multiple splicing variants of cdc25B regulate G2/M progression
Biochem. Biophys. Res. Commun.
(1999) Principles of CDK regulation
Nature
(1995)
Re-staging mitosis: a contemporary view of mitotic progression
Nat. Cell Biol.
Phosphorylation and activation of human cdc25-C by cdc2-cyclin B and its involvement in the self-amplification of MPF at mitosis
EMBO J.
Building a cell cycle oscillator: hysteresis and bistability in the activation of Cdc2
Nat. Cell Biol.
Active cyclin B1-Cdk1 first appears on centrosomes in prophase
Nat. Cell Biol.
Cdc25 and Wee1: analogous opposites?
Cell Div.
The cdc25B phosphatase is essential for the G2/M phase transition in human cells
J. Cell Sci.
Cdc25B and Cdc25C differ markedly in their properties as initiators of mitosis
J. Cell Biol.
Cited by (21)
Nuclear-cytoplasmic compartmentalization of cyclin B1-Cdk1 promotes robust timing of mitotic events
2022, Cell ReportsCitation Excerpt :To design a new Cdk1 sensor with an improved signal-to-noise ratio, we modified a substrate sequence of an existing ERK FRET biosensor known as EKAREV.19,20 The resulting sensor (hereafter named Cdk1-EV) includes a donor-acceptor fluorescent protein pair with optimized energy transfer efficiency (SECFP and YPet), sequence from Cdc25C as a consensus phosphorylation site for Cdk1 in the mitotic phase21,22,23 (sensor domain), the WW domain known as the phosphopeptide binding domain (ligand domain), and a flexible 116 amino acid EV linker (Figure 1A). ERK-specific binding sequence FQFP peptide is further removed to improve the specificity for Cdk1 (Figure 1B).
The Catalytic Subunit of DNA-Dependent Protein Kinase Coordinates with Polo-Like Kinase 1 to Facilitate Mitotic Entry
2015, Neoplasia (United States)Citation Excerpt :The timely expression of Cdk1 pY15 is regulated through the balance between Wee1-type kinases [7,8] and Cdc25C phosphatase [9]. Consistent with the elevation of Cdk1 pY15 in DNA-PKcs–defective cells, we observed that activation of Cdc25C phosphatase, as indicated by its hyperphosphorylation [28], was significantly attenuated in DNA-PKcs–depleted HeLa cells (Figure 2A). Additionally, we observed that DNA-PKcs is involved in the timely inactivation of Wee1 and Myt1 kinases.
Suppression of tumor proliferation and angiogenesis of hepatocellular carcinoma by HS-104, a novel phosphoinositide 3-kinase inhibitor
2013, Cancer LettersCitation Excerpt :In the present study, cell cycle analyses showed a clear arrest of cells by the accumulation of cells in the G2/M phase after HS-104 treatments, a result which indicated delay of their entry into mitosis and lead to the delay of cell division. A crucial step in G2/M phase arrest is the dephosphorylation of cdc2, which was phosphorylated by cdc25c [33,34]. We found that treatment by HS-104 resulted in upregulation of p-cdc2 and p-cdc25, an indication that mitosis was arrested.
Spatholobus suberectus inhibits cancer cell growth by inducing apoptosis and arresting cell cycle at G2/M checkpoint
2011, Journal of EthnopharmacologyCitation Excerpt :A crucial step in G2/M phase arrest was reported as dephosphorylation of Cdc2 at Thr14 and Tyr15 by Cdc25c phosphorylation at Ser216. This was activated by Chk1/2 in response to DNA damage (Bonnet et al., 2008; Reinhardt and Yaffe, 2009; Zegerman and Diffley, 2009). P53 can also regulate the G2/M transition either through the induction of P21 or of 14-3-3σ, (Roos and Kaina, 2006; Watson and Irwin, 2006).
Positive-feedback loops in cell cycle progression
2009, FEBS Letters