CDK5-related Phosphoproteomic Analysis Of Mice Neuronal Axon Growth Cone

Background and ObjectiveNeuron axonal growth cone, which is the core part of neuron axon, plays a pivotal role ofestablishing links with the target cells. The proline-directed serine/threonine kinase CDK5is akey regulator in the developing nervous system. Studies have confirmed that CDK5mayregulate important developmental events of neuron system by phosphorylating the proteinsthat involved in brain development, such as axon differentiation and axon guidance. It alsoplays a critical role in the degenerative disease of the nervous system, such as DNA damageand nervous system tumors. The degeneration and atrophy of neuron axon is one of theearliest changes in the nervous system of many neuronal diseases, however, its mechanism isnot clear. Therefore, it is essential to intensively studying phosphorylation proteomics ofaxonal growth cones and predicting phosphorylation sites of kinase CDK5by bioinformaticssoftwares and revealing the molecular mechanism of CDK5in regulation of axon. It canprovide a comprehensive understanding of the axonal guidance molecular mechanisms andnew ideas to solve the nerve injury and neurodegenerative diseases.MethodsThe axonal growth cone particles were isolated from mouse brains as previously described byKarl H. Pfenningers. Cerebral cortexes were dissected from mice embryos (E16-18). Dicedcortexes were homogenized in6volumes of homogenization buffer, The homogenate wasfirst centrifuged at1660×g for15min at4°C, The supernatant was saved for furtherultracentrifugation. Discontinues gradient of sucrose density were0.83M,1.0M and2.66M.After ultracentrifugation, the axonal growth cone proteins were located between the layer of0.32M and0.83M. The protein was digested into peptides after extracting. Then TiO2enrichment was taken to enrich the peptides that containing phosphate. The purified peptides were detected by ESI-QUAD-TOF mass spectrometry. The data were finally analyzed by GPSand Scansite to predict the possible phosphorylation sites of CDK5. Peptides of11aminoacids with the phosphorylation site were synthesized in vitro, and inserted into PGEX-6P-1vector. The recombination plasmids were transformed into BL21competent cells toduplicating and translating. Finally, we use Western blots and in vitro kinase reaction to verifythe results.ResultsA total of120fetal mice were obtained from10pregnant mice. The wet weight of corticalprotein was about5g. The low speed supernatant was loaded on discontinuous sucrose densitygradient in four tubes. After ultracentrifugation, three layers can clearly be seen from eachtube. Totally2.5mg axonal growth cone proteins were obtained by Bradford proteinquantitative method.In our study，totally about178617MS/MS spectra were detected. The proteins identified inuniprot-mouse database were3837. After Mascot analysis,11881unique peptides wereobtained. The number of non-redundant phosphorylation sites was36587, of which20833were phosphorylated serine sites,13162were threonine sites and2592were tyrosine sites,with proportions of57%,36%and7%respectively.The proteins were analyzed with GPS2.1and Scansite3with high threshold to predictpossible sites of CDK5. A total of2664sites were found to math with GPS2.1predictionresults; however; only275sites were consistent with the prediction results of Scansite. Thesites that coincide with the GPS2.1and Scansite3software were190.By document retrieve and classification analysis, we classified these sites one by one. Wefound that most of the sites we predicted were new. Only10sites were determined by site-specific methods,3of which were phosphorylated by CDK5. It is type of protein kinase,cytoskelet proteins and proteins unknown function that take the most proportion. Proteins ofneuronal axon related were selected for verification. We chose two of these to verify in vitro.In our study, the target DNA fragment was inserted into PGEX-6P-1vector by DNAsequencing technique. The protein was successfully expressed by Coomassie blue G-250stain.These proteins were verified to be new substrates of CDK5by in vitro kinase reaction andwest blot.ConclusionIn our study，we combine MS/MS and Bioinformatics technique predict a number of newsubstrates of CDK5in axonal growth cones, and it may provide a reference for further studyof the molecular mechanism of axon growth cone proteins and new ideas to solve the nerveinjury and neurodegenerative diseases.