Regulation of human Cdc25A stability by Serine 75 phosphorylation is not sufficient to activate a S phase checkpoint.
Degradation of Cdc25A phosphatase is an ubiquitous feature of stress. There are some discrepancies in the reported roles for different phosphorylation sites in the regulation of Cdc25A stability. Using a panel of doxycycline-inducible phosphorylation mutants we show that the stability of human Cdc25A protein is dependent upon phosphorylation at S75. In non-stressed conditions and in non-mitotic cells, Cdc25A is unstable and its stability is regulated in a Chk1-dependent manner. During mitosis, Cdc25A becomes stable and does not undergo degradation after DNA damage. We further show that Chk1 kinase regulates Cdc25A stability after UV irradiation. Similar to Chk1 kinase, p38 MAPK controls Cdc25A protein level after osmotic stress. Using phospho-specific antibodies, we find that both kinases can phosphorylate S75 and S123 in vitro. Inactivation of either Chk1 after UV-irradiation or p38 MAPK after osmotic stress prevents activation of a S phase checkpoint and S75 and S123 phosphorylation. However, introduction of stable Cdc25A (S75A or S75/123A) proteins is not sufficient to overcome this checkpoint. We propose that regulation of human Cdc25A stability by its phosphorylation at S75 may contribute to S phase checkpoint activation only in cooperation with other regulatory mechanisms.
Goloudina
Anastasia
A
Gene Response Section, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Yamaguchi
Hiroshi
H
Chervyakova
Daria B
DB
Appella
Ettore
E
Fornace
Albert J
AJ
Bulavin
Dmitry V
DV
D000071877:Checkpoint Kinase 1; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D016193:G1 Phase / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008938:Mitosis / Q000502:physiology; D009097:Multienzyme Complexes / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex; D025941:Protein Interaction Mapping; D011494:Protein Kinases / Q000378:metabolism; D016196:S Phase / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology; D014466:Ultraviolet Rays; D020687:cdc25 Phosphatases / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases
D009097:Multienzyme Complexes; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D018844:Cyclin-Dependent Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C517004:CDC25A protein, human; D020687:cdc25 Phosphatases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex