||Cyclin-dependent kinase 5 (Cdk5) is a member of Cdk family. While other members of Cdk family associate with cyclins, Cdk5 interacts with its neuronal specific activators, p35 or p39. Unlike other Cdks, Cdk5 is not involved in the control of cell cycle, but is important during central nervous system (CNS) development. We have previously demonstrated the involvement of Cdk5 in the regulation of acetylcholine receptor expression at the neuromuscular junction, suggesting a novel functional role of Cdk5 at the synapse. Here we report the identification of Pctaire1, a member of Cdk-related kinase family, as a p35-interacting protein in muscle. Binding of Pctaire1 to p35 can be demonstrated by in vitro binding assay and co-immunoprecipitation experiments. In addition, both p35 and Pctaire1 are concentrated at the neuromuscular junction. Furthermore, Pctaire1 can be phosphorylated by active Cdk5, both in vitro and in vivo, and Ser95 is the major phosphorylation site. In brain and muscle of Cdk5 null mice, Pcatire1 activity is significantly reduced. Moreover, Pctatire1 activity is increased following pre-incubation with adult rat brain extracts and phosphorylation by Cdk5/p25 complex. Our recent findings reveal prominent expression of Pctaire1 at the growth cone regions in primary cortical neurons. Moreover, Pctaire1 protein is concentrated in the synaptosomal membrane and synaptic vesicle enriched fractions in adult rat brain and associates with synapsin. These observations may shed light on Pctaire1 and Cdk5 functions in synapse. Taken together, our findings demonstrate that Pctaire1 interacts with p35, both in vitro and in vivo, and that phosphorylation of Pctaire1 by Cdk5 enhances its kinase activity, although the physiological significance of this interaction and phosphorylation remains to be elucidated.