Regulation of Human Cdc25A Stability by Serine 75 Phosphorylation Is Not Sufficient to Activate a S-phase Checkpoint

Abstract

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.

Human Embryonic Stem Cells Are Capable of Executing G1/S Checkpoint Activation

“…Two types of kinases are currently known to perform this task in somatic cells: p38 MAP kinase [15] and the checkpoint kinases Chk1 and Chk2 [5]. We found that all three Cdc25A-phosphorylating kinases are activated by phosphorylation in hESCs, and at least in the case of p38, this is detectable by an anti-phosphoresidue antibody.…”

“…Abbreviation: hESC, human embryonic stem cell; UVC, ultraviolet radiation-C range. Although Chk1 and Chk2 can functionally substitute for one another, since the degradation of Cdc25A is prevented and UVC-induced cell cycle block is abrogated only when the activity of both is eliminated, two other kinases that are capable of phosphorylating Cdc25A, p38 and GSK-3b [15,34], do not phosphorylate Cdc25A in UVC-treated hESCs (inhibition of GSK-3b by lithium chloride, data not shown). In this context, the recent finding that GSK-3b is responsible for Cdc25A phosphorylation in c-irradiated mouse ESCs is of note [17].…”

Inactivation of the Wip1 phosphatase inhibits mammary tumorigenesis through p38 MAPK–mediated activation of the p16Ink4a-p19Arf pathway

“…This result indicates that even after elimination of p16 expression as the primary block to tumor development, secondary mechanisms that contribute to tumor resistance in Ppm1d -/-mice still operated. Thus, Ppm1d deficiency through activation of p38 MAPK could target other molecules that may negatively contribute to proliferation, such as inhibition of Cdc25 phosphatases 9,10,28 or induction of p19. Thus, we propose that p38 MAPK-dependent modulation of cell cycle regulators including expression of the Cdkn2a locus, through inactivation or depletion of Wip1, will be a powerful approach to treating human primary breast cancers as well as other human malignancies.…”

“…In turn, the p38 MAPK pathway has been implicated as a negative regulator of cell cycle progression by several mechanisms, which implies that Wip1 is also an important regulator of cell cycle progression. p38 MAP kinase inhibits expression of D-type cyclins at transcriptional and post-translational levels 7,8 , phosphorylates and induces degradation of the Cdc25A phosphatase 9 , inhibits the Cdc25B phosphatase through phosphorylation of 14-3-3 sites 10 and phosphorylates the p53 tumor suppressor on two activating sites (Ser33 and Ser46) in the N-terminal region, which contribute to p53-mediated apoptosis 2,11 . Together, these p38 MAPK targets cooperate to activate cell cycle checkpoints, which implies that defects in p38 MAPK regulation or function may perturb cell cycle progression leading to increased tumorigenesis.…”

Cdc25A phosphatase: combinatorial phosphorylation, ubiquitylation and proteolysis

“…The same group demonstrated that Ser 76 mediates Cdc25A stabilization in response to UV-induced DNA damage. Moreover, Goloudina et al (2003), using phosphositespecific antibodies, showed that Chk1 and p38 kinase can phosphorylate both Ser 76 and 124, upon UVmediated DNA damage and osmotic stress, respectively ( Figure 1 and Table 1). However, mutations at these sites did not result in an RDS phenotype, raising the question as to whether other mechanisms acting on Cdc25A or related pathways contribute to the S-phase checkpoint.…”