Phosphoproteomic analysis reveals CDK5-Mediated phosphorylation of MTDH inhibits protein synthesis in microglia

CDK5 plays a crucial role in maintaining normal central nervous system (CNS) development and synaptic function, while microglia are the primary immune cells present in the CNS and play vital physiological roles in CNS development, immune surveillance, and regulation of synaptic plasticity. Despite this, our understanding of both the substrate proteins and functional mechanisms of CDK5 in microglia remains limited. To address this, we utilized CRISPR-Cas9 knockout of Cdk5 in BV2 cells and conducted quantitative phosphoproteomics analysis to systematically screen potential CDK5 substrates in microglia. Our findings identified 335 phosphorylation sites on 234 proteins as potential CDK5 substrates in microglia based on the reported sequence motif. Through in vitro kinase assay and intracellular inhibition and knockout of CDK5 experiments, we confirmed that ER proteins MTDH (protein LYRIC) and Calnexin are novel substrate proteins of CDK5. Moreover, we demonstrated for the first time a critical mechanism for regulating protein synthesis in microglia, that the phosphorylation of S565 site on MTDH, a key protein mediating cell growth, by CDK5 inhibits protein synthesis. Our data provide valuable insights for the discovery of new substrate proteins of CDK5 and the in-depth investigation of the function and mechanism of CDK5 in microglia.