Phosphoproteomic Data Integration Reveals Paralog-Specific Co-Regulatory Networks of Rho-Associated Kinases ROCK1 and ROCK2.

Rho-associated coiled-coil containing protein kinases 1 and 2 (ROCK1 and ROCK2) are serine/threonine kinases in the AGC family that regulate cytoskeletal dynamics, cell morphology, and mechanosignaling. Although their primary cellular functions are well established, their phosphoregulatory mechanisms remain insufficiently characterized at the system level. In this study, the analysis of 3825 human cell line phosphoproteomic datasets identified Class I phosphosites (localization probability ≥ 75% or A-score > 13) in both ROCK1 and ROCK2. The predominant phosphosites were ranked according to their frequency across the datasets. Co-regulation with phosphosites in other proteins (PsOPs) was assessed using Fisher's exact test with multi-criteria filtering. From 607 profiling and 153 differential datasets for ROCK1, and 667 profiling and 143 differential datasets for ROCK2, S1105 and S1341 for ROCK1, and S1137 and S1374 were considered the major ROCK2 phosphosites, all located outside the kinase domain. ROCK1_S1105 exhibited significant positive co-regulation with 141 PsOPs, whose annotated functions include cytoskeletal organization, cardiomyocyte survival, metabolism, and genomic stability, suggesting potential functional coordination. ROCK1_S1341 also displayed similar associations. In contrast, ROCK2_S1374 was associated with a broader network of 1349 PsOPs involved in cytoskeletal remodeling, cell cycle progression, hypertrophy, and fibrosis. Functional analyses identified both shared and paralog-specific regulatory networks, with C-terminal phosphosites as potential hubs associated with cytoskeletal regulation and the MAPK, PI3K/AKT/mTOR, and Wnt signaling pathways. ROCK2 exhibits co-regulation with a substantially larger network of phosphosites in proteins other than ROCK1, despite the positional conservation of the predominant sites, suggesting paralog-specific regulatory differences. This provides a hypothesis-generating resource for prioritizing ROCK phosphosites and co-regulated proteins as candidates for experimental validation in pathological contexts and for therapeutic target discovery.