Characterization and function of cks2-a cyclin dependent kinase regulatory subunit-during spermatogenesis

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Title: Characterization and function of cks2-a cyclin dependent kinase regulatory subunit-during spermatogenesis
Author: Attaya, Ebtesam Nabil
Abstract: Cell division is controlled by cyclin dependent kinases (CDKs ) that are activated to phosphorylate various downstream targets . My goal was to study the CDKs and interacting proteins that control meiosis . CDK2beta , the alternatively spliced isoform of cyclin dependent kinase 2 (CDK2 ) , may be a key meiotic regulator due to its expression at prophase of meiosis I in spermatogenesis . Therefore , we used the yeast two -hybrid system to identify binding partners of CDK2beta . The mouse homolog of CKS2 (CDC28 Kinase Subunit 2 ) was identified as a CDK2beta interactor after screening an adult mouse testis cDNA library . CKS2 was confirmed to bind to CDK2beta . Cks2 was more abundant in day 17 after birth and adult mice , and was only weakly detected in mutants that lack germ cells . Further , Cks2 mRNA levels were significantly higher in pachytene spermatocytes than early and late spermatids , while Cks1 was nearly undetectable . CKS2 protein was detected with both CDK2beta and cyclin A1 in germ cells during the first meiotic division . The Cks2 gene has three exons . Cks2 - / - mice were generated by another group by deletion of exon 1 . Cks2 - / - mice are sterile due to a block in spermatogenesis which arrests at metaphase I of meiosis . I show here that the major biochemical effect in the absence of Cks2 was an increase of CCNA1 and CDK2 -associated kinase activity . Surprisingly , reverse transcription -PCR analysis showed that a mutant form of Cks2 was being transcribed in testes of these mice . We also found that Cks2 - / - mice produced a protein that was immunoreactive with the CKS antibody , suggesting that a translatable mRNA was present in testes of Cks2 - / - mice . Cloning of the mutant Cks2 cDNA indicated that it consisted of a portion of the Cks2 coding sequence (exons 2 and 3 ) fused to portions of introns 1 from the Cks2 gene and the plasmid used to generate the Cks2 - / - ES cells . We postulate that this cDNA is derived from a mRNA transcribed from a cryptic promoter active in the testes of Cks2 - / - mice . Interestingly , the aberrant protein did not possess all of the function attributed to the wild type protein , as Cks2 - / - mice were sterile . However , these data indicate that the interpretation that this phenotype is due to complete loss of Cks2 must be interpreted with care . While not definitive , these data suggest that a truncated form of Cks2 was being expressed in testes of these mice , and suggest a more complicated explanation for the observed infertility phenotype . These data indicate that CKS2 appears to act as a CDK2beta /CCNA1 repressor and that Cks2 - / - mice generate a Cks2 mutant that is being transcribed and possibly translated in the testis .
URI: http : / /hdl .handle .net /2346 /1212
Date: 2005-05

Citation

Characterization and function of cks2-a cyclin dependent kinase regulatory subunit-during spermatogenesis. Doctoral dissertation, Texas Tech University. Available electronically from http : / /hdl .handle .net /2346 /1212 .

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