Phosphoproteomic Analysis of CARMIL1 Reveals Novel Regulatory Mechanisms and Upstream Kinases Involved in Actin Dynamics and Cell Migration.

Capping protein regulator and myosin 1 Linker 1 (CARMIL1) is a multifunctional regulator of actin polymerization, ruffle formation, and lamellipodia development, making it essential for cell spreading and migration. While its protein-level functions are perceived, phospho-signaling of highly phosphorylated CARMIL1 remains unexplored. This study investigates CARMIL1 phosphorylation and its regulatory mechanisms. Global phosphoproteome datasets captured the most frequently detected and differentially regulated CARMIL1 phosphosites under different conditions to be in the CARMIL_C domain (T916, S968, and S1067). A coregulation-based method was employed to identify interactors and upstream kinases that are coregulated with the phosphorylation sites. These sites exhibited a consistent co-occurrence pattern including both positive and negative coregulation. The phosphosites of complex interactors showed positive and negative coregulation and were involved in cell cycle regulation and cell growth. AKT1, PAK2, and MYLK were identified as potential upstream kinases for CARMIL at S968, while WNK1 was predicted as a potential upstream kinase for S1067, suggesting distinct regulatory mechanisms for these phosphorylation sites. Phosphorylation at CDK1 S146, MAP4K2 S238, MINK1 S641, and TNIK S678 was found coregulated high with CARMIL T916 in human brain cancer. Notably, most coregulated proteins were associated with regulation of the actin cytoskeleton pathway. Our results show that phosphorylation of CARMIL1 in the C-terminal domain highly influences actin cytoskeletal organization. It offers new insights on CARMIL1-mediated cellular functions, deepening our comprehension of its involvement in cytoskeletal dynamics.