Regulatory properties of neuronal Cdc2-like kinase

Neuronal Cdc2-like kinase (NCLK) is a heterodimer of Cdk5 and a neuron-specific protein, p35. Three p35-derived fragments, p25, p23 and p21, were expressed in and purified from E. coli as glutathione S-transferase recombinant proteins. p21 was used to precipitate an active Cdk5 from bovine brain extract. When recombinant p35 fragments were incubated with bacterially expressed Cdk5, all three p35 fragments activated Cdk5 with equal efficacy. NCLK has been reconstituted from bacterially expressed Cdk5 and the shortest form or p35, p21. After purification, reconstituted NCLK displays similar specific enzyme activity and substrate specificity to NCLK isolated from bovine brain.; Activation of Cdk5 by p35-derivatives has been characterized in detail with p21. Several observations suggest that, unlike other well characterized Cdc2-like kinases whose activities depend on phosphorylation of the catalytic subunits by a distinct kinase, activation of Cdk5 by p35-derivatives is independent of Cdk5 phosphorylation. Cdk5 is highly activated by p35-derivatives without requiring any other protein in the reconstitution reaction. The rate and extent of Cdk5 activation by reconstitution were not significantly affected by inclusion of ATP-Mg{dollar}sp{lcub}2+{rcub}{dollar} in the reaction. Potential autophosphorylation of Cdk5 has been ruled out, as no phosphorylation occurred on Cdk5 during Cdk5-catalyzed phosphorylation. Protein phosphatase 2A, which dephosphorylates and inactivates Cdk2/cyclin A, has no discernible effect on reconstituted NCLK activity.; Cdk5/p35 may exist in a complex with several proteins in brain. Seven different sequences encoding p35-interacting proteins were isolated by the yeast two-hybrid system from a human brain cDNA library. They are fragments of glial fibrillary acidic protein (GFAP), {dollar}n(alphasb2{dollar})-chimaerin, clusterin, and four novel peptides displaying no significant homology to any clearly defined protein in the current database, {dollar}n(alphasb2{dollar})-chimaerin and clusterin are expressed in neurons. GFAP is a glial-specific protein. However, neurofilaments are neuronal homologues of GFAP, and contain a region of homology to the isolated p35-interacting region in GFAP. Further study is required to verify interactions between p35 and these isolated binding proteins and to determine the nature of these interactions.