Cyclin‐dependent kinase 5 (cdk5) activation requires interaction with three domains of p35

Abstract

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 complexing 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 truncated forms, and cdk5 to understand better the mechanism of its activation. The cdk5 complexes formed with the truncated forms p25 and p21 produced similar maximum 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 fragment 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, produces a peptide with markedly higher affinity for cdk5 compared 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 microM(-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 reversible interaction of cdk5 with its activator, which involves only one or two binding domains, followed by a slow stabilization of the active conformation as interaction with all three domains is achieved.

Cyclin-Dependent Kinase 5 Phosphorylation of Human Septin SEPT5 (hCDCrel-1) Modulates Exocytosis

“…Expression and purification of the glutathione S-transferase (GST) and GST-SEPT5 full-length and fragment fusion proteins were performed essentially as described previously (Amin et al, 2002). GST-pull-down assays were performed as described previously (Kesavapany et al, 2001).…”

“…Active Cdk5 was immunoprecipitated from 250 g of rat brain lysates by using a well characterized Cdk5 antibody (C8) as described previously (Veeranna et al, 1998). The bacterially expressed GST-SEPT5 was phosphorylated essentially as described previously (Amin et al, 2002). Briefly, 1-2 g of GST-SEPT5 protein was incubated with 2 U of active Cdk5 (or Cdk5 immunoprecipitated from 500 g of rat brain lysates) for 30 min at 30°C in a kinase reaction buffer as described previously (Veeranna et al, 1998).…”

Study of interaction of the cyclin-dependent kinase 5 with its activator, p25 and with the p25-derived inhibitor, CIP

“…Therefore the flexibility hypothesis shows that p25 truncation that creates the inhibitory characteristics of CIP does so by increasing its flexibility relative to that of p25, such that the cdk5/CIP complex does not align the substrate protein or peptide with the catalytic site adequately to produce a significant rate of phosphorylation. This follows directly from the experiments described in [Ami02], which show that CIP inhibits cdk5 activity with high affinity and competitively with p16 or p25 activation.…”

“…Experimental studies, which are described in [Ami02], provided evidence that small truncations of p35 produce a high-affinity inhibitor protein called CIP. These results are of fundamental importance for neurodegenerative diseases such as Alzheimer's, which are known to be associated with hyperphosphorylation of specific neuronal proteins.…”

Peptides Derived from Neuronal Cell Cycle like Kinase 5 Activator p35, in Neurodegeneration; Pathology and Therapy

“…Although roscovitine and related compounds have been proposed and evaluated, their effects are nonspecific as they bind the common ATP site shared by other Cdks and most other kinases. Our lab has taken a different approach based on a study of truncated fragments of the p35 regulator [28]. Two peptides were identified, CIP (126 a.a) and a smaller peptide p5 (24 a.a.), derived from the p25 domain of the parent sequence, exhibited vigorous inhibition of Cdk5/p35 and Cdk5/p25 activities in test-tube experiments [29][30][31].…”