Collapsin response mediating protein‐2 (CRMP2) has been identified as an intracellular protein mediating Semaphorin3A (Sema3A), a repulsive guidance molecule. In this study, we demonstrate that cyclin‐dependent kinase 5 (Cdk5) and glycogen synthase kinase 3β (GSK3β) plays a critical role in Sema3A signalling. In In vitro kinase assay, Cdk5 phosphorylated CRMP2 at Ser522, while GSK3β did not induce any phosphorylation of CRMP2. Phosphorylation by GSK3β was exclusively observed in Cdk5‐phosphorylated CRMP2, but barely in CRMP2T509A. These results indicate that Cdk5 primarily phosphorylates CRMP2 at Ser522 and GSK3β secondarily phosphorylates at Thr509. The dual‐phosphorylated CRMP2, but not non‐phosphorylated or single‐phosphorylated CRMP2, is recognized with the antibody 3F4, which is highly reactive with the neurofibrillary tangles of Alzheimer's disease. 3F4 recognized the CRMP2 in the wild‐type but not cdk5^−/− mouse embryonic brain lysates. The phosphorylation of CRMP2 at Ser522 caused reduction of its affinity to tubulin. In dorsal root ganglion neurones, Sema3A stimulation enhanced the levels of the phosphorylated form of CRMP2 detected by 3F4. Over‐expression of CRMP2 mutant substituting either Ser522 or Thr509 to Ala attenuates Sema3A‐induced growth cone collapse response. These results suggest that the sequential phosphorylation of CRMP is an important process of Sema3A signalling and the same mechanism may have some relevance to the pathological aggregation of the microtubule‐associated proteins.

A member of semaphorin family, semaphorin3A (Sema3A), acts as a chemorepellent or chemoattractant on a wide variety of axons and dendrites in the development of the nervous systems. We here show that Sema3A induces clustering of both postsynaptic density-95 (PSD-95) and presynaptic synapsin I in cultured cortical neurons without changing the density of spines or filopodia. Neuropilin-1 (NRP-1), a receptor for Sema3A, is present on both axons and dendrites. When the cultured neurons are exposed to Sema3A, the cluster size of PSD-95 is markedly enhanced, and an extensive colocalization of PSD-95 and NRP-1 or actin-rich protrusion is seen. The effects of Sema3A on spine morphology are blocked by PP2, an Src type tyrosine kinase inhibitor, but not by the PP3, the inactive-related compound. In the cultured cortical neurons fromfyn^−/−mice, dendrites bear few spines, and Sema3A does not induce PSD-95 cluster formation on the dendrites. Sema3A and its receptor genes are highly expressed during the synaptogenic period of postnatal days 10 and 15. The cortical neurons in layer V, but not layer III, show a lowered density of synaptic bouton-like structure on dendrites insema3A- andfyn-deficient mice. The neurons of the double-heterozygous mice show the lowered spine density, whereas those of single heterozygous mice show similar levels of the spine density as the wild type. These findings suggest that the Sema3A signaling pathway plays an important role in the regulation of dendritic spine maturation in the cerebral cortex neurons.

Collapsin response mediator proteins (CRMPs) are intracellular proteins that mediate signals for several extracellular molecules, such as Semaphorin3A and neurotrophins. The phosphorylation of CRMP1 and CRMP2 by Cdk5 at Ser522 is involved in axonal guidance and spine development. Here, we found that the Ser522-phosphorylated CRMP1 and/or CRMP2 are enriched in the dendrites of cultured cortical neurons and P7 cortical section. To determine the physiological role of CRMPs in dendritic development, we generated CRMP2 knock-in mutant mice (crmp2^ki/ki) in which the Ser residue at 522 was replaced with Ala. Strikingly, the cortical basal dendrites of double mutantcrmp2^ki/kiandcrmp1^−/−mice exhibited severe abnormal dendritic patterning, which we defined as “curling phenotype.” These findings demonstrate that the function of CRMP1 and CRMP2 synergistically control dendritic projection, and the phosphorylation of CRMP2 at Ser522 is essential for proper dendritic field organizationin vivo.

Collapsin response mediator protein 1 (CRMP1) is one of the CRMP family members that mediates signal transduction of axonal guidance and neuronal migration. We show here evidence that CRMP1 is involved in semaphorin3A (Sema3A)-induced spine development in the cerebral cortex. In the cultured cortical neurons fromcrmp1^+/−mice, Sema3A increased the density of clusters of synapsin I and postsynaptic density-95, but this increase was markedly attenuated incrmp1^−/−mice. This attenuation was also seen incyclin-dependent kinase 5(cdk5)^−/−neurons. Furthermore, the introduction of wild-type CRMP1 but not CRMP1-T509A/S522A, (Thr 509 and Ser 522 were replaced by Ala), a mutant that cannot be phosphorylated by Cdk5, intocrmp1^−/−neurons rescued the defect in Sema3A responsiveness. The Golgi-impregnation method showed that thecrmp1^−/−layer V cortical neurons showed a lower density of synaptic bouton-like structures and that this phenotype had genetic interaction withsema3A. These findings suggest that Sema3A-induced spine development is regulated by phosphorylation of CRMP1 by Cdk5.