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Unformatted text preview: Cyclin-Dependent Kinase 5 (cdk5) Activation Requires Interaction With Three Domains of p35 Niranjana D. Amin, Wayne Albers, and Harish C. Pant * Laboratory of Neurochemistry, National Institutes of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892 Cyclin-dependent kinase 5 (cdk5), in contrast to other members of the cyclin-dependent kinase family, is not activated by cyclins but instead is activated by complex- ing with neuron-specific activator molecules (p35, p39, and p67). The most effective activator of cdk5 both in vitro and in vivo is p35. We have taken a kinetic approach to study the interaction between p35, its various trun- cated forms, and cdk5 to understand better the mecha- nism of its activation. The cdk5 complexes formed with the truncated forms p25 and p21 produced similar max- imum active kinase, whereas the cdk5 complexed with full-length p35 and a further truncated form spanning amino acid residues from 138 to 291, with approximate molecular weight of 16 kDa (p16), produced slightly less (80%) activation than p25. P16 was the smallest frag- ment of p35 that produced activation equal to or greater than that of full-length p35. By examination of further truncations of p16, we found that a small number of residues, 11 and 4 at the N- and C-termini, respectively, of p16, are essential for cdk5 activation. Further truncation, removing both essential N- and C-terminal domains, pro- duces a peptide with markedly higher affinity for cdk5 com- pared with the peptides that retain either of these domains. Using these inactive truncated peptides as inhibitors, we examined the kinetics of activation. From these studies we conclude that activation involves at least three cdk5- interacting domains, one located at each end of p16 and at least one located in a central domain. The cdk5 activation process is slow: The second-order rate constant for p16 is about 1.2 M 1 hr 1 . On the basis of kinetic data, we suggest that cdk5 exists in two conformations. The inactive kinase conformation predominates in the absence of the activator. Activation occurs in two stages: a rapid and re- versible interaction of cdk5 with its activator, which involves only one or two binding domains, followed by a slow sta- bilization of the active conformation as interaction with all three domains is achieved. Published 2002 Wiley-Liss, Inc. Key words: cdk5; p35; p16; cyclin-dependent kinase Cdk5 is a member of the cyclin-dependent kinase family (Meyerson et al., 1992; Beaudette et al., 1993) of proline-directed kinases. Although it is found at low levels in proliferating cells, bound to cyclins D and E, it does not participate in the cell cycle (Hellmich et al., 1992; Lew et al., 1994; Tsai et al., 1994). Its activity is primarily re- stricted to neuronal tissues, because of the restricted ex- pression of the cdk5 specific activator molecules p39 and p35 or its truncated forms, p25 and p21 (Lew et al., 1994; Lee et al., 1996; Patrick et al., 1998). Another cdk5-Lee et al....
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