Characterization of the roles of PAK5 in neuronal development

Abstract

P21-activated kinase 5 (PAK5) is a serine/threonine kinase downstream of the small GTPase Cdc42. PAK5 is predominantly detected in the brain and is involved in a number of vital cellular processes including cell survival and microtubules stabilization. It is also known that Pak5 promotes neuronal cell differentiation, but the molecular mechanism(s) by which Pak5 operates in this role is not entirely understood. To understand how PAK5 regulates neurite outgrowth, we have performed yeast two hybrid screening using PAK5 as the bait. After screening a yeast fetal brain library, we have identified a positive cDNA clone that matches to the sequence of a motor protein, called dynein cytoplasmic 1 intermediate chain 1 (DYNC1Il). To confirm the interaction between PAK5 and DYNC1Il, we have performed an affinity pull down assay. Purified glutathione-S-transferase (GST) tagged PAK5 was shown to interact with a purified his-tagged DYNC1Il, indicating that PAK5 can directly interact with DYNC1Il. This DYNC1Il isoform was isolated from the human fetal brain library and has 17 amino acids missing in the N-terminal region comparing with the longest DYNC1Il isoform. To characterize the binding between PAK5 and DYNC1Il, an in vitro reconstitution kinase assay was performed. A constitutively active form of purified GST-tagged PAK5 (mutation of histidine residues 19 and 22 to leucine) could phosphorylate the purified His-tagged Dync1i1 isoform. It has already been showed that cytoplasmic dynein participates in neuronal growth cone remodeling and neurite outgrowth. Taken together, the results suggest a possibility that PAK5 exert its functions, particularly neuronal cell differentiation, through phosphorylation of DYNC1Il.