Absence of apparent phenotype in mice lacking Cdc25C protein phosphatase - PubMed (original) (raw)
Absence of apparent phenotype in mice lacking Cdc25C protein phosphatase
M S Chen et al. Mol Cell Biol. 2001 Jun.
Abstract
The Cdc25 family of protein phosphatases positively regulate the cell division cycle by activating cyclin-dependent protein kinases. In humans and rodents, three Cdc25 family members denoted Cdc25A, -B, and -C have been identified. The murine forms of Cdc25 exhibit distinct patterns of expression both during development and in adult mouse tissues. In order to determine unique contributions made by the Cdc25C protein phosphatase to embryonic and adult cell cycles, mice lacking Cdc25C were generated. We report that Cdc25C(-/-) mice are viable and do not display any obvious abnormalities. Among adult tissues in which Cdc25C is detected, its transcripts are most abundant in testis, followed by thymus, ovary, spleen, and intestine. Mice lacking Cdc25C were fertile, indicating that Cdc25C does not contribute an essential function during spermatogenesis or oogenesis in the mouse. T- and B-cell development was also found to be normal in Cdc25C(-/-) mice, and Cdc25C(-/-) mouse splenic T and B cells exhibited normal proliferative responses in vitro. Finally, the phosphorylation status of Cdc2, the timing of entry into mitosis, and the cellular response to DNA damage were unperturbed in mouse embryo fibroblasts lacking Cdc25C. These findings indicate that Cdc25A and/or Cdc25B may compensate for loss of Cdc25C in the mouse.
Figures
FIG. 1
Targeted disruption of mouse Cdc25C gene. (A) Structure of the targeting vector. The genomic organization of the mouse Cdc25C gene was disrupted by inserting into exon 3 the neomycin phosphotransferase gene (neo) driven by the thymidine kinase promoter (pTKneo) as a selectable marker. Restriction sites in the introns and exons flanking the targeted exon are indicated. Exons 1 to 4 are represented by black boxes. The bar under the targeted locus indicates the probe used for Southern blot analysis. _Eco_RI digestion fragments probed by Southern blotting are also indicated. (B) PCR analysis of mouse tail DNA; PCR strategy and expected sizes of the amplified DNA fragments for wild-type (top) and Cdc25C mutant (bottom) alleles. Small arrows depict the locations of PCR primers used for genotyping. Wild-type (WT) mice produced a 302-bp PCR fragment, null mice (KO) generated a 186-bp fragment, and heterozygous mice (HT) gave rise to both the 302- and 186-bp PCR products.
FIG. 2
Northern and Western blot analysis of _Cdc25C_−/− mouse tissues and MEFs. (A) RNA was isolated from the testes of Cdc25C+/+ (WT), Cdc25C+/−(HT), and _Cdc25C_−/− (KO) mice and processed for Northern blotting using probes specific for mCdc25A, mCdc25B, mCdc25C, neomycin phosphotransferase (neo), and GAPDH. (B) Lysates prepared from Sf9 insect cells infected with baculoviruses encoding GST-mCdc25A, GST-mCdc25B, and GST-mCdc25C were lysed and then resolved by SDS-PAGE. Proteins were analyzed by Western blotting using peptide antibody specific for 14 amino acids in the C terminus of mouse Cdc25C (Cdc25C) or with antibody specific for GST. (C) MEFs derived from Cdc25C+/+ (WT) and _Cdc25C_−/− (KO) mouse embryos were incubated with [35S]methionine-cysteine. Cells were lysed, and lysates were incubated with peptide antibody specific for Cdc25C in either the absence (−) or presence (+) of the immunizing peptide. Immunoprecipitates were boiled, and mCdc25C was reimmunoprecipitated prior to SDS-PAGE. Radiolabeled mCdc25C was visualized by fluorography.
FIG. 3
Growth curves. Cdc25C+/+(WT), Cdc25C+/−(HT), and _Cdc25C_−/−(KO) male (left) and female (right) mice were weighed beginning at birth and at weekly intervals up to 12 weeks. Weights (in grams) represent averages of at least 30 mice per genotype per time point.
FIG. 4
T-cell development is normal in Cdc25C−/− mice. Thymocytes (A and B) and splenocytes (C and D) harvested from 6-week-old mice were stained with FITC-anti-CD4 and PE-anti-CD8. Stained cells were analyzed by flow cytometry gated on lymphocytes, and results are shown as dot plots. The percentage of gated cells in each quadrant is indicated. These data are representative of four independent experiments.
FIG. 5
T- and B-cell proliferation assays. Splenic CD4+ T cells were stimulated to proliferate by incubation on plate-bound anti-CD3 (A) and B220+ splenocytes were induced to proliferate by incubation with anti-IgM Ab (20 μg/ml) (B) for the indicated times. Cell proliferation was measured by [3H]thymidine incorporation. Standard deviations for triplicate samples are shown as error bars along the y axis. These data are representative of six and eight independent T- and B-cell proliferation assays, respectively.
FIG. 6
Cell cycle and checkpoint analysis of cells lacking Cdc25C. MEFs prepared from Cdc25C+/+ and _Cdc25C_−/− mice were pulse labeled with BrdU for 1 h and then treated with 0 (−IR) or 4 Gy (+IR) of γ-irradiation. Cells were harvested at the indicated times (hours) and stained with PI and for BrdU. Cellular DNA content of BrdU-positive cells was analyzed by flow cytometry.
FIG. 7
Phosphorylation status of Cdc2 in cells lacking Cdc25C. Early-passage MEFs prepared from Cdc25C+/+ and _Cdc25C_−/− mice were synchronized in early S phase. Cells were harvested prior to release (time 0) or at 3, 6, 9, or 12 h after release. Cell lysates were prepared and incubated with p13_suc1_-agarose to precipitate Cdc2. Precipitates were resolved on a 10% SDS gel, and Cdc2 was detected by Western blotting. The arrows indicate the three electrophoretic forms of Cdc2.
Similar articles
- Normal cell cycle and checkpoint responses in mice and cells lacking Cdc25B and Cdc25C protein phosphatases.
Ferguson AM, White LS, Donovan PJ, Piwnica-Worms H. Ferguson AM, et al. Mol Cell Biol. 2005 Apr;25(7):2853-60. doi: 10.1128/MCB.25.7.2853-2860.2005. Mol Cell Biol. 2005. PMID: 15767688 Free PMC article. - Cell cycle regulation by the Cdc25 phosphatase family.
Nilsson I, Hoffmann I. Nilsson I, et al. Prog Cell Cycle Res. 2000;4:107-14. doi: 10.1007/978-1-4615-4253-7_10. Prog Cell Cycle Res. 2000. PMID: 10740819 Review. - A maternal form of the phosphatase Cdc25A regulates early embryonic cell cycles in Xenopus laevis.
Kim SH, Li C, Maller JL. Kim SH, et al. Dev Biol. 1999 Aug 15;212(2):381-91. doi: 10.1006/dbio.1999.9361. Dev Biol. 1999. PMID: 10433828 - In vivo roles of CDC25 phosphatases: biological insight into the anti-cancer therapeutic targets.
Kiyokawa H, Ray D. Kiyokawa H, et al. Anticancer Agents Med Chem. 2008 Dec;8(8):832-6. doi: 10.2174/187152008786847693. Anticancer Agents Med Chem. 2008. PMID: 19075565 Free PMC article. Review.
Cited by
- The subcortical maternal complex modulates the cell cycle during early mammalian embryogenesis via 14-3-3.
Han Z, Wang R, Chi P, Zhang Z, Min L, Jiao H, Ou G, Zhou D, Qin D, Xu C, Gao Z, Qi Q, Li J, Lu Y, Wang X, Chen J, Yu X, Hu H, Li L, Deng D. Han Z, et al. Nat Commun. 2024 Oct 15;15(1):8887. doi: 10.1038/s41467-024-53277-3. Nat Commun. 2024. PMID: 39406751 Free PMC article. - CHK1-CDC25A-CDK1 regulate cell cycle progression and protect genome integrity in early mouse embryos.
Knoblochova L, Duricek T, Vaskovicova M, Zorzompokou C, Rayova D, Ferencova I, Baran V, Schultz RM, Hoffmann ER, Drutovic D. Knoblochova L, et al. EMBO Rep. 2023 Oct 9;24(10):e56530. doi: 10.15252/embr.202256530. Epub 2023 Sep 11. EMBO Rep. 2023. PMID: 37694680 Free PMC article. - The p38/MK2 Pathway Functions as Chk1-Backup Downstream of ATM/ATR in G2-Checkpoint Activation in Cells Exposed to Ionizing Radiation.
Luo D, Mladenov E, Soni A, Stuschke M, Iliakis G. Luo D, et al. Cells. 2023 May 14;12(10):1387. doi: 10.3390/cells12101387. Cells. 2023. PMID: 37408221 Free PMC article. - Downregulation of CDC25C in NPCs Disturbed Cortical Neurogenesis.
Zhou X, Lu D, Yi W, Xu D. Zhou X, et al. Int J Mol Sci. 2023 Jan 12;24(2):1505. doi: 10.3390/ijms24021505. Int J Mol Sci. 2023. PMID: 36675024 Free PMC article. - Mechanisms of Oocyte Maturation and Related Epigenetic Regulation.
He M, Zhang T, Yang Y, Wang C. He M, et al. Front Cell Dev Biol. 2021 Mar 19;9:654028. doi: 10.3389/fcell.2021.654028. eCollection 2021. Front Cell Dev Biol. 2021. PMID: 33842483 Free PMC article. Review.
References
- Chamberlain J P. Fluorographic detection of radioactivity in polyacrylamide gels with the water soluble fluor sodium salicylate. Anal Biochem. 1979;98:132–135. - PubMed
- Chauhan S, Gottesman M. Construction of a new universal vector for insertional mutagenesis by homologous recombination. Gene. 1992;120:281–286. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Molecular Biology Databases
Research Materials
Miscellaneous