Dual phosphorylation controls Cdc25 phosphatases and mitotic entry

Negative regulation of the Cdc25C protein phosphatase by phosphorylation on Ser 216, the 14-3-3-binding site, is an important regulatory mechanism used by cells to block mitotic entry under normal conditions and after DNA damage. During mitosis, Cdc25C is not phosphorylated on Ser 216 and ionizing radiation (IR) does not induce either phosphorylation of Ser 216, or binding to 14-3-3. Here, we show that Cdc25C is phosphorylated on Ser 214 during mitosis, which in turn prevents phosphorylation of Ser 216. Mutation of Ser 214 to Ala reconstitutes Ser 216 phosphorylation and 14-3-3 binding during mitosis. Introduction of exogenous Cdc25CS214A into HeLa cells depleted of endogenous Cdc25C results in a substantial delay to mitotic entry. This effect was fully reversed in a S214A/S216A double-mutant, implying that the inhibitory effect of S214A mutant was entirely dependent on Ser 216 phosphorylation. A similar regulatory mechanism may also apply to another mitotic phosphatase, Cdc25B, as well as mitotic phosphatases of other species, including Xenopus laevis. We propose that this pathway ensures that Cdc2 remains active once mitosis is initiated and is a key control mechanism for maintaining the proper order of cell-cycle transitions.

[1]  C. Peng,et al.  C-TAK1 protein kinase phosphorylates human Cdc25C on serine 216 and promotes 14-3-3 protein binding. , 1998, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[2]  J. Maller,et al.  Elimination of cdc2 phosphorylation sites in the cdc25 phosphatase blocks initiation of M-phase. , 1993, Molecular biology of the cell.

[3]  Catherine G. Takizawa,et al.  Control of mitosis by changes in the subcellular location of cyclin-B1-Cdk1 and Cdc25C. , 2000, Current opinion in cell biology.

[4]  E. Appella,et al.  Initiation of a G2/M checkpoint after ultraviolet radiation requires p38 kinase , 2001, Nature.

[5]  Edward A. Sausville,et al.  The Chk1 Protein Kinase and the Cdc25C Regulatory Pathways Are Targets of the Anticancer Agent UCN-01* , 2000, The Journal of Biological Chemistry.

[6]  K. Sakaguchi,et al.  Phosphorylation of human p53 by p38 kinase coordinates N‐terminal phosphorylation and apoptosis in response to UV radiation , 1999, The EMBO journal.

[7]  Ingrid Hoffmann,et al.  Cdc25b and Cdc25c Differ Markedly in Their Properties as Initiators of Mitosis , 1999, The Journal of cell biology.

[8]  C. Peng,et al.  Mitotic and G2 checkpoint control: regulation of 14-3-3 protein binding by phosphorylation of Cdc25C on serine-216. , 1997, Science.

[9]  A. Murray,et al.  Cell cycle extracts. , 1991, Methods in cell biology.

[10]  P. Graves,et al.  Localization of human Cdc25C is regulated both by nuclear export and 14-3-3 protein binding , 2001, Oncogene.

[11]  Hui Zhao,et al.  Disruption of the checkpoint kinase 1/cell division cycle 25A pathway abrogates ionizing radiation-induced S and G2 checkpoints , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[12]  H. Piwnica-Worms,et al.  DNA Damage and Replication Checkpoints in Fission Yeast Require Nuclear Exclusion of the Cdc25 Phosphatase via 14-3-3 Binding , 1999, Molecular and Cellular Biology.

[13]  A. Fornace,et al.  p38 and Chk1 kinases: different conductors for the G(2)/M checkpoint symphony. , 2002, Current opinion in genetics & development.

[14]  E. Karsenti,et al.  Phosphorylation and activation of human cdc25‐C by cdc2‐‐cyclin B and its involvement in the self‐amplification of MPF at mitosis. , 1993, The EMBO journal.

[15]  Andrew W. Murray,et al.  Chapter 30 Cell Cycle Extracts , 1991 .

[16]  S. Kornbluth,et al.  Maintenance of G2 arrest in the Xenopus oocyte: a role for 14‐3‐3‐mediated inhibition of Cdc25 nuclear import , 1999, The EMBO journal.

[17]  A. Kumagai,et al.  Binding of 14-3-3 proteins and nuclear export control the intracellular localization of the mitotic inducer Cdc25. , 1999, Genes & development.

[18]  U. Strausfeld,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. , 1994, The Journal of biological chemistry.