Regulation of alpha2 chimaerin and associated phosphorylation pathways in neuronal signalling and morphogenesis.

The generation of neuronal dendrites and axons is a highly dynamic process. These extensive structural changes are regulated by molecules such as GTPases, as well as kinases/phosphatases, which provide cyclical control mechanisms. Rho family GTPases are regulated, in part, by GAPs such as neurone-enriched chimaerin, and act through downstream kinase effectors, including cyclin dependant kinase 5 (Cdk5). ?2 chimaerin is a Rac GAP and phorbol ester receptor, which also displays Cdc42/Rac effector functions. From this study it appears ?2 chimaerin exists in an auto- inhibited state in the cytosol and that DAG/phorbol ester association, or selected mutations translocates ?2 chimaerin to the membrane, leading to GAP activation. ?2 chimaerin GAP activity was implicated in PMA induced neurite collapse of N1E-115 neuroblastoma cells. PMA also promoted the association of ?2 chimaerin with target proteins Collapsin Response Mediator Protein-2 (CRMP-2) and p35/Cdk5, possibly as a result of a conformational change. The Rac effector, Cdk5 is a proline directed, Ser/Thr kinase, whose activity contributes to neurite outgrowth, growth cone collapse and neuronal migration. Deregulation of Cdk5 in neurodegenerative diseases is implicated in pathological changes. ?2-chimaerin GAP domain was shown to interact in vivo both with p35, the neuronal activator of Cdk5 and with Cdk5 itself. CRMP-2 interacted in vivo with ?2 chimaerin SH2 domain, for which tyrosine phosphorylation was not essential. The functional correlates of these protein interactions of ?2 chimaerin were investigated in vivo. CRMP-2, which is hyperphosphorylated in Alzheimer's disease was identified as a novel Cdk5 substrate in vitro with CRMP-2 Ser522 identified as the target site. Phosphorylation of CRMP-2 Ser522, potentially primes for GSK-3? phosphorylation of CRMP-2 Ser518 and Thr 514 which alters CRMP-2 conformation. ?2 chimaerin was not a Cdk5 substrate but may be tyrosine phosphorylated, which could potentially regulate its activity or associations in neuronal cells