Abstract
Neuronal Cdc2-like kinase (Nclk) plays an important role in a variety of cellular processes, including neuronal cell differentiation, apoptosis, neuron migration, and formation of neuromuscular junction. The active kinase consists of a catalytic subunit, Cdk5, and an essential regulatory subunit, neuronal Cdk5 activator (p35(nck5a) or p25(nck5a)), which is expressed primarily in neurons of central nervous tissue. In our previous study using the yeast two-hybrid screening method, three novel p35(nck5a)-associated proteins were isolated. Here we show that one of these proteins, called C42, specifically inhibits the activation of Cdk5 by Nck5a. Co-immunoprecipitation data suggested that C42 and p35(nck5a) could form a complex within cultured mammalian cells. Deletion analysis has mapped the inhibitory domain of C42 to a region of 135 amino acids, which is conserved in Pho81, a yeast protein that inhibits the yeast cyclin-dependent protein kinase Pho85. The Pho85.Pho80 kinase complex has been shown to be the yeast functional homologue of the mammalian Cdk5/p35(nck5a) kinase.
Highlights
From the ‡Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China and the ¶Department of Medical Biochemistry, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 1N4, Canada
Inhibition of Nck5a Activating Activity of Cdk5 Kinase by Full-length C42 Protein—The C42 clone obtained from the yeast two-hybrid screen encodes a polypeptide of 112 amino acid residues corresponding to Glu475–Thr586
The C42-FL and C42-BF were bacterially expressed in glutathione S-transferase (GST) fusion forms and tested for their effect on Cdk5 kinase activity
Summary
From the ‡Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China and the ¶Department of Medical Biochemistry, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 1N4, Canada. The Pho851⁄7Pho kinase complex has been shown to be the yeast functional homologue of the mammalian Cdk5/p35nck5a kinase. While Nck5a and Nck5ai are homologous proteins, they show little or no sequence similarity to cyclins Members of both Cdk inhibitor families have been tested and shown to have no activity toward Cdk. In Saccharomyces cerevisiae, there is a cyclin-dependent protein kinase, Pho, and its cyclin partner Pho, which have been shown to be the functional homologues of mammalian Cdk and Nck5a, respectively [14]. We show that one of the novel proteins, C42, displays an inhibitory effect on Cdk kinase activity While both the full-length C42 and the C42 fragment obtained from the yeast two-hybrid screen can bind Nck5a, only the full-length protein has Cdk inhibitory activity. We have mapped the inhibitory domain of C42 to a 135-amino acid region, which shows a significant homology to the inhibitory region of Pho
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