Analysis of Cdk5-related phosphoproteomics in growth cones

Neurons establish interactions with target cells via elongation and guidance of axons, and the growth cone plays pivotal roles in this process. Cyclin-dependent kinase 5 (Cdk5)is a key regulator of nervous system development. Cdk5 regulates several significant events by phosphorylating substrates that are involved in neurogenesis, and previous studies of Cdk5 have typically focused on single substrates. Here, we took anew approach to investigate Cdk5 substrates using mass spectrometry and bioinformatics analyses. Axonal growth cones were isolated and analyzed by HPLC-MALDI-MS/MS. In total, 178,617 MS/MS spectra were detected. Candidates were analyzed by GPS 2.1 and Scansite 3, which predicted that 2,664 and 275 sites, respectively, were potential phosphorylation sites of Cdk5. There were 190 overlapped phosphorylation sites, corresponding to 89 proteins. Those proteins correlated with axonal functions were classified, and two of them were verified using a classic site-specific mutation strategy. This is the first study in which the phosphoproteome of axonal growth cones was identified. The systematic examination of Cdk5 substrates could provide a reference for further study of molecular mechanisms of axonal growth cones, and new insights into treatments of neuronal disorders.