Surprisingly, phosphorylation of CRMP1 at Thr509 was dramatically lowered upon remedy of rat cortical neurons with purvalanol and in Cdk5/? and Cdk5?/? neurons compared with wild variety neurons, suggesting that this residue could be straight phosphorylated by Cdk5. This was supported by phosphorylation of CRMP1 at HER2 cancer Thr509 by Cdk5 in vitro. In summary, phosphorylation of Thr509 of human CRMP1 appears to be regulated by two mechanisms, direct phosphorylation by Cdk5, or by priming of Ser522 by Cdk5 followed by sequential phosphorylation of Ser518, Thr514, and Thr509 by GSK3. In rodents, phosphorylation of Thr509 cannot be accomplished by the latter mechanism, therefore Thr509 is phosphorylated straight by Cdk5. DYRK2 didn’t phosphorylate Ser522 of human or rodent CRMP1 and did not prime for subsequent phosphorylation by GSK3. Cdk5 Primes CRMP2, but Not CRMP4, for GSK3 mediated Phosphorylation in Vivo Main rat cortical neurons were treated with purvalanol, a extra potent inhibitor of Cdk5 and DYRK2 than roscovitine. Phosphorylation was monitored employing antibodies that specifically recognize CRMP2 when phosphorylated at Thr514/Thr509, or CRMP4 when phosphorylated at Thr509.
Loss of phosphorylation of Ser522 will avoid subsequent phosphorylation of Ser518/Thr514/Thr509 by GSK3. Incubation of neurons for 48 h with purvalanol caused significant, inhibition of CRMP2 phosphorylation. This therapy also lowered the phosphorylation of Thr509 of CRMP4.
Extended incubation of neurons with purvalanol was needed to observe CRMP dephosphorylation. The decrease in phosphorylation of kinase inhibitor both proteins was accompanied by a partial band shift to a lower relative molecular weight on SDS Page. The modification to CRMP2/4 causing this band shift is not but identified, while it really is unlikely to become due to dephosphorylation alone, for the reason that similar band shifts were not observed in other experiments that cut down CRMP phosphorylation. CRMP2 Thr509/514 phosphorylation was also considerably lowered in Cdk5 ?/? cortices compared with wild sort or Cdk5 /? heterozygous mice. In contrast, phosphorylation of CRMP4 was identical in wild kind, Cdk5/? Cdk5?/? cortices, suggesting that Cdk5 is not expected for Ser522 phosphorylation of CRMP4 in vivo. Nonetheless, treatment of key cortical neurons from Cdk5?/? mice with purvalanol reduced CRMP4 phosphorylation, implicating DYRK2 as a priming kinase for CRMP4. Since phosphorylation of CRMP2 was not completely inhibited in Cdk5?/? cortex, this suggested that a further Ser522 kinase exists that partially compensates for the loss of Cdk5. Alternatively, Thr514/Thr509 may possibly be directly phosphorylated by kinases apart from GSK3.