CRMP Family Protein: Novel Targets for Cdk5 That Regulates Axon Guidance, Synapse Maturation, and Cell Migration

Abstract

In the developing nervous system, post-mitotic neurons migrate and extend their neurites and form precise patterns of connections that emerge through the interaction between the growth cone and a myriad of environmental cues such as attractive or repulsive axon guidance molecules. Semaphorin3A (Sema3A) is the prototypical repulsive axon guidance molecule that potently induces growth cone collapse stalling neurite extension. Neuropilin-1 (NRP-1) and Plexin-As are ligand-binding and signal-transducing receptor components for Sema3A, respectively. Collapsin response mediator protein (CRMP) was identified as a signaling molecule of Sema3A. However, its molecular mechanisms have been ill-defined. CRMPs are now known to be composed of five homologous cytosolic proteins CRMP1–5; all of the family proteins are highly phosphorylated in developing brains. By screening pharmaceutical reagents and utilizing gene-deficient mice and through biochemical analysis, we found that Fyn and cyclin-dependent kinase 5 (Cdk5) mediate Sema3A-induced response in dorsal root ganglion (DRG) neurons. Cdk5 was associated with PlexA2 through the active state of Fyn. This raised the possibility that Sema3A induced growth cone collapse response through phosphorylation of CRMPs by Cdk5. The 2-D gel analysis of brain lysate from Cdk5-deficient mice revealed that CRMP2 was a substrate for Cdk5 in vivo. In vitro kinase assay revealed that Ser522 was the major site of CRMP1 and CRMP2 phosphorylation by Cdk5. Cdk5 primarily phosphorylated CRMP2 at Ser522, and GSK3β secondarily phosphorylates at Thr509. The dual-phosphorylated CRMP2 was recognized by the antibody 3F4, which is highly reactive with the neurofibrillary tangles of Alzheimer’s disease. In DRG neurons, Sema3A stimulation enhanced the levels of the phosphorylated form of CRMP2 detected by 3F4. Overexpression of CRMP2 mutant substituting either Ser522 or Thr509 with Ala attenuated Sema3A-induced growth cone collapse. Knockdown of CRMP1 and CRMP2 inhibited Sema3A-induced growth cone collapse. The phosphorylation of CRMP1 and/or CRMP2 is therefore an essential step for Sema3A signaling. CRMP1 and CRMP2 were also good substrates for Fyn. The phosphorylation of CRMP1 by Cdk5 and Fyn also appears to be involved in Sema3A and Reelin signaling, contributing to spine maturation and the regulation of cell migration during the development of the cerebral cortex.