9507109 Tsai The cell division cycle is mainly regulated by a cascade of protein phosphorylation and dephosphorylation events. In higher eukaryotes, a family of cyclin-dependent kinases (cdks) are known to participate in the protein phosphorylation cascade that is required for the progression of cells through the cell division cycle. A common feature of these kinases is their requirement for a positive regulatory subunit named cyclin. One of these kinases, cdk5, shares a high degree of sequence homology to cdc2 and cdk2, however, its putative cell cycle function has yet to be determined. In search for a function of cdk5 we and others have isolated a regulatory partner of cdk5 known as p35. The primary sequence of p35 shares no homology to other isolated proteins including cyclins. During embryonic development, p35 is only expressed in post-mitotic neuronal cells of the central nervous system (CNS) but not in proliferating cells. Thus, p35 represents a novel cdk regulator that renders cdk5 active only in post-mitotic neurons, Appearance of the cdk5/p35 kinase activity correlates with the advancing differentiated phenotype of neurons in developing rodent neocortex and primary cell cultures derived from the embryonic forebrain. In adult animals, p35 is expressed at a high level only in certain regions of the forebrain including CA1 and CA3 of the hippocampus, the dentate gyrus and amygdala. These observations suggest a role of the cdk5/p35 kinase in neuronal differentiation/development and possibly for normal functioning of neurons in certain regions of the adult brain. The aim of this research proposal is to understand the regulation of the cdk5/p35 kinase and to get better insight into the biology of the p35 molecule. It has been shown that p35 is the only element required for activation of cdk5 kinase activity, therefore, the cdk5/p35 kinase activity may be regulated by the synthesis and degradation of p35. Alternatively, a negative regulator may be required to monitor the activity of the cdk5/p35 kinase. Preliminary results indicated the presence of an inhibitory activity in cell lysates. Thus, conventional protein purification procedures will be used to identify this activity. Primary cultures from embryonic as well as newborn rat cerebral cortices will be used to study the regulation of the p35 subunit in vivo. These cultures have been shown to express p35 and display active cdk5/p35 kinase. Thus, the half-life and the subcellular localization of p35 will be analyzed in these cells. Immunoprecipitations from the cortical cultures using p35 antibodies indicated the existence of multiple cellular proteins associated with p35. These proteins will be purified via their affinity for p35 and protein microsequencing will be used to determine their identify. This will help elucidating the in vivo function of p35. While the regulation of cell division has been explored extensively at the molecular level, the mechanism of cell differentiation is relatively less understood. To date, the cdk protein kinase family is known for their roles in cell cycle regulation, this research proposal will add another dimension to our understanding of the role of the cdks in a different biological aspect, namely cell differentiation. Studying the biology of the cdk5/p35 kinase might also help learning the differentiation process of neurons and possibly, how the cell fate between cell proliferation and differentiation might be determined by the interplay among different cdks and their regulators. %%% This project examines a protein which appears only in differentiating nerve cells. The protein interacts with a cell cycle regulatory protein which normally stimulates cell division. However, this protein appears in large quantities only in forming nerve cells. Its appearance suggests that it may play an important role inhibiting the cell cycle as nerve cells form. Investigation of this novel protein is likely to shed light on the function of this unique cell cycle protein. * **
