Abstract
Cytoskeletal proteins, such as actin and myosin, are essential for regulating cell morphology and motility. Rho-associated kinase (ROCK; herein referring collectively to ROCK1 and ROCK2) is a key regulator of actomyosin dynamics, including stress fibre formation and cell migration. Our previous study revealed that cyclin G-associated kinase (GAK) antagonises ROCK signalling during autophagic flux regulation; however, the underlying molecular mechanisms remained unclear. In this study, we investigated the role of GAK in cytoskeletal dynamics and cell motility by genetically disrupting GAK. GAK-knockout cells exhibited enhanced stress fibre formation and cell migration, accompanied by increased phosphorylation of myosin light chain (MLC). Notably, the inhibition of stress fibre formation and MLC phosphorylation was more dependent on the intrinsically disordered region (IDR) of GAK than on its kinase activity. GAK IDR interacted with ARHGEF2, and ARHGEF2 knockdown suppressed stress fibre formation in GAK-knockout cells. Furthermore, the GAK IDR contributes to the regulation of MLC expression. Together, these findings indicate that GAK IDR is a crucial regulator of actomyosin dynamics and cell motility, and suggest that GAK antagonises ROCK-dependent cytoskeletal regulation through the coordinated control of ARHGEF2 activity and MLC expression.