Background: Cks proteins are small (9 kD), highly conserved proteins that associate with cyclin- dependent kinases (Cdks). Cyclin B1, Cdk1, and Cks protein form a hetero-trimer known as maturation- promoting factor (MPF) that regulates M phase in both mitotic and meiotic cell division cycles. Although the precise function(s) of Cks proteins is not understood, it is believed that they function as """"""""adaptors,"""""""" linking cyclin-Cdk complexes to a variety of cell division regulatory processes. We functionally inactivated the ortholog Cks2 in mice and found that it plays an essential role in mammalian meiosis. CKS2-/- male and female mice are sterile due to an arrest of spermatocytes and oocytes in metaphase of meiosis I (Ml). Objective/Hypothesis: Studies in lower eukaryotes have implicated Cks proteins in several important M phase regulatory processes including: 1) activation of MPF kinase activity;2) activation of the multi-subunit ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C);3) proteolysis of cyclin B1;and 4) transcriptional activation of APC/C activator protein Cdc20. Our hypothesis is that Cks2 performs analogous regulatory functions in mammalian Ml.
Specific Aims :
Aim1. Determine the role of Cks2 in regulating MPF and APC/C activity in Ml.
Aim2. Define the role of Cks2 in regulating 26S proteasome function in Ml.
Aim3. Investigate the ortholog-specific function(s) of Cks2 in mammalian Ml through generation of CKS2Cks1/Cks1 knock-in mice. Study Design:
Aim1) determine the role of Cks2 in regulating MPF activity, we will: 1) analyze Cdk1 kinase activity using H1 kinase assays;and 2) analyze Cdk1 phosphorylation status by Western blot analysis. The role of Cks2 in APC/C activation will be determined using in vitro ubiquitination assays on spermatocyte extracts.
Aim2) the role of Cks2 in mediating 26S proteasome functions will be evaluated by: 1) in vitro proteolysis assays using spermatocyte extracts and a recombinant cyclin B1 substrate;and 2) expression analysis and oocyte microinjection experiments to determine the effect of Cks2 on Cdc20 transcription.
Aim3) we will evaluate the ortholog-specific function(s) of Cks2 in mammalian meiosis I by generating CKS2(Cks1/Cks1) knock-in mice, which express Cks1 under regulation of the CKS2 promoter, by homologous targeting. Relevance to Public Health: Clarifying the molecular controls of meiosis I in mammals is highly relevant from both a basic science and clinical perspective. Very little is known regarding how meiosis is regulated at the molecular level and how dysregulation of this process leads to human infertility. The metaphase-to- anaphase transition of Ml is particularly relevant clinically because its dysregulation is associated with oocyte aneuploidy in women and infertility in both men and women. Aneuploidy (trisomy or monosomy) is the most common genetic abnormality in human pregnancies (5-25% of cases) and the predominant cause of mental retardation in humans. Uncovering the function(s) of Cks2 in mammalian meiosis could lead to a better understanding about how Ml is regulated, and help to define the underlying causes of infertility and birth defects in humans.
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