Opposing effects of Rho-associated coiled-coil kinase 1 (ROCK1) and ROCK2 in TGF-β-SMAD signaling.

BACKGROUND: Transforming growth factor-β (TGF-β) signals through type I and type II kinase-associated transmembrane receptors to activate both SMAD-dependent and SMAD-independent pathways, including the Rho/ROCK axis with its effectors ROCK1 and ROCK2. However, the role of ROCK isoforms in TGF-β-SMAD signaling in breast cancer cells, has not been elucidated. METHODS: CAGA12-luciferase reporter assays were utilized to measure SMAD3/4-dependent transcription. Small-molecule kinase inhibitors and RNA interference-mediated gene silencing were provided for loss-of-function analyses. Immunoblotting, immunofluorescence staining, subcellular fractionation, in vitro kinase activity assay and mRNA expression assays were performed to gain mechanistic evidence. Mass spectrometry analysis coupled with co-immunoprecipitation assays examined the interaction between ROCK isoforms and other members of the TGF-ꞵ signaling pathway. RESULTS: ROCK2 knockdown or treatment with the selective ROCK2 kinase inhibitor KD025 suppressed TGF-β-SMAD signaling, whereas ROCK1 knockdown produced the opposite effect. Overexpression studies revealed that ROCK1 inhibited, while ROCK2 enhanced, TGF-β-induced SMAD3/4-dependent transcriptional activity. These effects were reversed by expression of kinase-dead ROCK1 (K105R) or ROCK2 (K121D) mutants, and by treatment with ROCK inhibitors, demonstrating that the kinase activities of the two isoforms are required for their opposing functions. We further showed that ROCK1 and ROCK2 exhibit distinct subcellular localizations, and that SMAD3 interacts with ROCK1, but not with ROCK2. ROCK2 inhibition reduced phosphorylation at serine residues in the C-terminal (Ser423/425) and linker region (Ser204 and Ser208) of SMAD3, decreased nuclear accumulation of SMAD3 and SMAD4, and suppressed TGF-β-SMAD target gene expression. Functionally, ROCK1 depletion promoted cell proliferation and invasion in MDA-MB-231 cells, whereas ROCK2 depletion reduced proliferation and invasion. CONCLUSIONS: ROCK1 and ROCK2 exert opposing, kinase-dependent effects on TGF-β-SMAD signaling and differentially affect breast cancer cell behaviours.