UV-induced downregulation of the CDC25B protein in human cells.

The CDC25B phosphatase regulates the activation of CDK1-Cyclin B at the onset of mitosis, being a key target of the checkpoint pathways activated by cellular stress and DNA damage. Previous work has reported that checkpoint activation induces the sequestration of CDC25B in the cytoplasm. Here we show that in response to UV irradiation, the levels of CDC25B protein can be downregulated independently of classical checkpoints pathways such as p53, ATM/ATR and p38 MAPK. We also show that translational repression mediated by eIF2alpha phosphorylation regulates CDC25B expression levels. Taken together, our results illustrate a new mechanism of CDC25B regulation in response to stress.

NanoLC-MS/MS analysis provides new insights into the phosphorylation pattern of Cdc25B in vivo: full overlap with sites of phosphorylation by Chk1 and Cdk1/cycB kinases in vitro.

NanoLC-MS/MS analysis was used to characterize the phosphorylation pattern in vivo of CDC25B3 (phosphatase splice variant 1) expressed in a human cell line and to compare it to the phosphorylation of CDC25B3 by Cdk1/cyclin B and Chk1 in vitro. Cellular CDC25B3 was purified from U2OS cells conditionally overexpressing the phosphatase. Eighteen sites were detectably phosphorylated in vivo. Nearly all existing (S/T)P sites were phosphorylated in vivo and in vitro. Eight non(S/T)P sites were phosphorylated in vivo. All these sites could be phosphorylated by kinase Chk1, which phosphorylated a total of 11 sites in vitro, with consensus sequence (R/K) X(2-3) (S/P)-non P. Nearly half of the sites identified in this study were not previously described and were not homologous to sites reported to be phosphorylated in other CDC25 species. We also show that in vivo a significant part of CDC25B molecules can be hyperphosphorylated, with up to 13 phosphates per phosphatase molecule.

CDC25B phosphorylation by p38 and MK-2.

CDC25B is one of the three human phosphatases that are involved in the control of the activation of cyclin-dependent kinases. CDC25B participates in regulating entry into mitosis and appears to play a key role in the checkpoint response to DNA injury. CDC25B has been reported to be regulated by a number of kinases and controversial evidence suggests that it is phosphorylated by p38SAPK and/or MAPKAP kinase-2. In this report, we clarify this issue using an approach combining mass spectrometry and the use of specific antibodies against phosphorylated CDC25B residues. We report that MAPKAP kinase-2 phosphorylates CDC25B on multiple sites including S169, S323, S353 and S375, while p38SAPK phosphorylates CDC25B on S249. We show that the S323-phosphorylated form of CDC25B is detected at the centrosome during a normal cell cycle. Since most of these sites are also phosphorylated by several other kinases, our observations highlight the difficulty in characterizing and understanding in vivo phosphorylation patterns.

Ability of human CDC25B phosphatase splice variants to replace the function of the fission yeast Cdc25 cell cycle regulator.

CDC25 phosphatases are essential and evolutionary-conserved actors of the eukaryotic cell cycle control. To examine and compare the properties of three splicing variants of human CDC25B, recombinant fission yeast strains expressing the human proteins in place of the endogenous Cdc25 were generated and characterized. We report, that the three CDC25B variants: (i) efficiently replace the yeast counterpart in vegetative growth, (ii) partly restore the gamma and UV radiation DNA damage-activated checkpoint, (iii) fail to restore the DNA replication checkpoint activated by hydroxyurea. Although these yeast strains do not reveal the specific functions of the human CDC25B variants, they should provide useful screening tools for the identification of new cell cycle regulators and pharmacological inhibitors of CDC25 phosphatase.

Evolutionary conservation of a novel splice variant of the Cds1/CHK2 checkpoint kinase restricted to its regulatory domain.

The Cds1/CHK2 kinase plays a key role in the activation of the G(2) checkpoint after DNA damage. Here we report the existence in fission yeast of a short variant (Sv) of Cds1 that is produced through an alternative splicing mechanism leading to a frame shift and premature termination. This SvCds1 protein consists solely of the regulatory region and lacks the catalytic domain. Expression of SvCds1 increases sensitivity to ionizing radiation and, to a lesser extent, to hydroxyurea, but not to UV radiation. We also report that in the human orthologue of Cds1, CHK2, differential splicing of a cryptic exon leads to a frame shift and premature termination producing a short variant (SvCHK2). Thus, we have discovered the existence of an evolutionary conserved mechanism ensuring the production of a catalytically inactive variant Cds1/CHK2 that is restricted to SQTQ and FHA domains and that can act as a dominant negative. The role that this short variant of Cds1/CHK2 might play in the response to DNA damage and the physiopathological consequences are discussed.

A fission yeast strain expressing human CDC25A phosphatase: a tool for selectivity studies of pharmacological inhibitors of CDC25.

Fission yeast is a simple eukaryotic model organism in which many aspects of cell cycle control can be explored. We examined by homologous recombination whether the human CDC25A phosphatase could substitute for the function of the fission yeast Cdc25. We first show: (a). that CDC25A efficiently replaces the endogenous Cdc25 mitotic inducer for vegetative growth and (b). that CDC25A is able to partially restore a functional checkpoint in response to both ionising and UV irradiation, but not a DNA replication checkpoint. We then describe a simple assay in which we demonstrate that growth of the humanised CDC25A strain is strongly repressed in a CDC25-dependent manner by BN2003, a potent chemical inhibitor of CDC25 belonging to the benzothiazoledione family. The ease of manipulation of fission yeast humanised CDC25 cells and the simplicity of the above assay offer a powerful tool with which to investigate the specificity of pharmacological inhibitors of CDC25.

Mesangial IgG glomerulonephritis: a distinct type of primary glomerulonephritis.

Fourteen cases of mesangial IgG glomerulonephritis characterized by exclusive or predominant mesangial IgG deposits are reported. The median age at onset was 19 yr (range, 13 to 47 yr). No patient exhibited evidence of systemic lupus erythematous or other systemic diseases. Proteinuria was present in all cases (median, 2.4 g/d; range, 1 to 13 g/d), microscopic hematuria in 12 cases, and macroscopic hematuria in two cases. Five patients were hypertensive at the time of referral. In all cases, renal biopsies revealed mesangial IgG deposits and varying degrees of mesangial matrix expansion, in the absence of significant mesangial cell proliferation. Complement component (mainly C3) deposits were present in virtually all cases. Subepithelial deposits were also noted in nine cases. IgG deposits were polyclonal and consisted mainly of IgG1 and IgG3 subclasses. In electron-microscopic analyses, deposits were electron dense and granular. Treatment was purely supportive. After a mean follow-up period of 11 yr, seven patients had experienced progression to chronic renal failure, including four who had reached end-stage renal failure. Three patients exhibited persistently normal renal function. For one patient, a symptomatic recurrence of mesangial IgG deposits in the renal graft was diagnosed 4 yr after renal transplantation. Such a recurrence highlights the specificity of this type of glomerulonephritis. Mesangial IgG glomerulonephritis is a distinct, albeit rare, type of glomerulonephritis that exhibits far from benign outcome and may recur in renal transplants.