Abstract
Increasing evidence implicates cyclin-dependent kinase 5 (Cdk5) in neuronal synaptic function. We searched for Cdk5 substrates in synaptosomal fractions prepared from mouse brains. Mass spectrometric analysis after two-dimensional SDS-PAGE identified several synaptic proteins phosphorylated by Cdk5-p35; one protein identified was Sept5 (CDCrel-1). Although septins were isolated originally as cell division-related proteins in yeast, Sept5 is expressed predominantly in neurons and is implicated in exocytosis. We confirmed that Sept5 is phosphorylated by Cdk5-p35 in vitro and identified Ser17 of adult type Sept5 (Sept5_v1) as a major phosphorylation site. We found that Ser17 of Sept5_v1 is phosphorylated in mouse brains. Coimmunoprecipitation from synaptosomal fractions and glutathione S-transferase-syntaxin-1A pulldown assays of Sept5_v1 expressed in COS-7 cells showed that phosphorylation of Sept5_v1 by Cdk5-p35 decreases the binding to syntaxin-1. These results indicate that the interaction of Sept5 with syntaxin-1 is regulated by the phosphorylation of Sept5_v1 at Ser17 by Cdk5-p35.
Highlights
Cyclin-dependent kinase 5 (Cdk5)3 is a proline-directed Ser/ Thr kinase that is activated by binding to a neuron-specific activator, p35 or p39 [1,2,3]
In contrast to other members of the Cdk family, which are known as cell cycle promoting factors, Cdk5 plays a role in neuronal activities unrelated to cell cycle progression
Successful fractionation of synaptosomes was confirmed by the enrichment of synaptic proteins such as VAMP-2, dynamin 1, and NR2A (Fig. 1A). Both Cdk5 and p35 were detected in the isolated synaptosomal fractions, as reported previously (Fig. 1A) [13, 41]. p35 was more concentrated in synaptosomes than was Cdk5, which was distributed more evenly in other fractions
Summary
Cyclin-dependent kinase 5 (Cdk5)3 is a proline-directed Ser/ Thr kinase that is activated by binding to a neuron-specific activator, p35 or p39 [1,2,3]. Coimmunoprecipitation from synaptosomal fractions and glutathione S-transferase-syntaxin-1A pulldown assays of Sept5_v1 expressed in COS-7 cells showed that phosphorylation of Sept5_v1 by Cdk5-p35 decreases the binding to syntaxin-1. These results indicate that the interaction of Sept5 with syntaxin-1 is regulated by the phosphorylation of Sept5_v1 at Ser17 by Cdk5-p35.
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