Sheela Akhila, Mahin Althaf, Ummar Samseera, Vattoth Nazah Naurah, Dcunha Leona, Gopalakrishnan Athira Perunelly, Raju Rajesh
Centre for Integrative Omics Data Science (CIODS), Yenepoya (Deemed to be University), Mangalore, Karnataka, India.
Cytoskeleton (Hoboken). 2025 Jun 16. doi: 10.1002/cm.70001.
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.
帽蛋白调节剂与肌球蛋白1连接蛋白1（CARMIL1）是肌动蛋白聚合、褶皱形成和片状伪足发育的多功能调节剂，对细胞铺展和迁移至关重要。虽然其蛋白质水平的功能已为人所知，但高度磷酸化的CARMIL1的磷酸信号传导仍未得到探索。本研究调查了CARMIL1的磷酸化及其调控机制。全球磷酸化蛋白质组数据集捕获了在不同条件下最常检测到且差异调节的CARMIL1磷酸化位点，位于CARMIL_C结构域（T916、S968和S1067）。采用基于共调节的方法来鉴定与磷酸化位点共调节的相互作用蛋白和上游激酶。这些位点呈现出一致的共出现模式，包括正向和负向共调节。复杂相互作用蛋白的磷酸化位点表现出正向和负向共调节，并参与细胞周期调控和细胞生长。AKT1、PAK2和MYLK被鉴定为CARMIL在S968位点的潜在上游激酶，而WNK1被预测为S1067位点的潜在上游激酶，表明这些磷酸化位点具有不同的调控机制。在人脑癌中，发现CDK1 S146、MAP4K2 S238、MINK1 S641和TNIK S678的磷酸化与CARMIL T916高度共调节。值得注意的是，大多数共调节蛋白与肌动蛋白细胞骨架途径的调控有关。我们的结果表明，CARMIL1 C末端结构域的磷酸化对肌动蛋白细胞骨架组织有高度影响。它为CARMIL1介导的细胞功能提供了新的见解，加深了我们对其参与细胞骨架动力学的理解。
