CDK4 maintains cytoskeletal homeostasis by restraining actomyosin contractility

Cyclin-dependent kinase 4 (CDK4) is best known for its canonical role in cell-cycle control, yet how CDK4 activity intersects with cellular architecture remains poorly defined. Here, we uncover a non-canonical function of CDK4 in maintaining cytoskeletal mechanical homeostasis in triple-negative breast cancer (TNBC) cells. We show that CDK4 phosphorylates the RhoA-targeting RhoGAP Myo9b at serine 1935, enhancing its GAP activity and restraining RhoA–ROCK–pMLC-dependent actomyosin contractility. Loss or inhibition of CDK4 alters cell morphology, disrupts actin organization, and increases contractility. Elevated contractility in CDK4-deficient cells induces collapse of the intermediate filament network, characterized by Vimentin reorganization and mitochondrial redistribution, indicating a higher-order defect in cytoskeletal architecture. Furthermore, CDK4 expression negatively correlates with pMLC levels in both mouse TNBC tumors and human breast cancer samples, supporting the physiological relevance of this pathway. Together, these findings identify CDK4 as a regulator of cellular mechanical stability, integrating actin dynamics, intermediate filament integrity, and organelle positioning, and reveal an unexpected role for CDK4 beyond cell-cycle regulation.