Our long-term objective is to understand the relationship between cell proliferation and the initiation of terminal differentiation in the nervous system. In this study, we seek to understand the molecular mechanism of how a protein kinase that shares striking structural homology to kinases that are essential for cell division functions during neuronal differentiation. Cyclin-dependent kinase 5, a close homologue of cdk2 and cdc2, displays associated kinase activity only in the nervous system of adult mammals. During embryonic development, cdk5 kinase activity parallels the advance of neurogenesis. The unique temporal and spatial patterns of cdk5 activity are due in part, to the distribution of its regulatory partner p35. p35 is a novel cdk activator that associates with cdk5 in post-mitotic neurons. This association gives rise to active kinase in the absence of other protein molecules. Surprisingly, the primary sequence of p35 does not share any homology to known cyclins or other protein molecules in the database. During mouse embryonic development p35 mRNA appears immediately after neurons exit the cell cycle and is transiently expressed in post- mitotic neurons of the central nervous system (CNS). These observations indicate that the cdk5/p35 kinase is likely to play an important role in neurogenesis of the CNS during embryonic development. Indeed, we find that overexpression of a dominant negative form of cdk5 or antisense p35 inhibited neurite outgrowth in differentiating cultured cortical neurons. Conversely, co-expression of wildtype cdk5 and p35 enhanced neurite outgrowth. This research proposal has two aims; 1) To investigate the regulation of cdk5 kinase activity by p35. The minimal region(s) of p35 that is sufficient for association and activation of cdk5 will be determined. Also residues on cdk5 that allow its specificity to p35 will be defined. These experiments are likely to shed light on a novel activation mechanism of a cdk by its regulatory partner. 2) To further explore the biological function of the cdk5/p35 kinase in neurogenesis. Using the approach of overexpressing mutant and wildtype cdk5/p35 kinase in primary cortical cultures, we have shown that the cdk5/p35 kinase plays a role in neurite outgrowth. This system will be used to characterize the changes in neuronal differentiation that caused by the elevation or inactivation of cdk5/p35 kinase and to investigate a role of this kinase in the onset of neuronal differentiation. In a complementary approach, the consequence of loss of p35 function in mice will be analyzed. The phenotype displayed by animals homozygous for a deletion of the p35 gene will indicate stages of neurogenesis that require p35. The combination of in vitro and in vivo approaches will reveal the biological function of the cdk5/p35 kinase.
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