Transformation mediated by RhoA requires activity of ROCK kinases

Background: The Ras-related GTPase RhoA controls signalling processes requi red for cytoskeletal reorganisation, transcriptional regulation, and transf ormation. The ability of RhoA mutants to transform cells correlates not wit h transcription but with their ability to bind ROCK-I, an effector kinase i nvolved in cytoskeletal reorganisation. We used a recently developed specif ic ROCK inhibitor, Y-27632, and ROCK truncation mutants to investigate the role of ROCK kinases in transcriptional activation and transformation. Results: In NIH3T3 cells, Y-27632 did not prevent the activation of serum r esponse factor, transcription of c-fos or cell cycle re-entry following ser um stimulation. Repeated treatment of NIH3T3 cells with Y-27632, however, s ubstantially disrupted their actin fibre network but did not affect their g rowth rate. Y-27632 blocked focus formation by RhoA and its guanine-nucleot ide exchange factors Dbl and mNET1. It did not affect the growth rate of ce lls transformed by Dbl and mNET1, but restored normal growth control at con fluence and prevented their growth in soft agar. Y-27632 also significantly inhibited focus formation by Ras, but had no effect on the establishment o r maintenance of transformation by Src. Furthermore, it significantly inhib ited anchorage-independent growth of two out of four colorectal tumour cell lines. Consistent with these data, a truncated ROCK derivative exhibited w eak ability to cooperate with activated Raf in focus formation assays. Conclusions: ROCK signalling is required for both the establishment and mai ntenance of transformation by constitutive activation of RhoA, and contribu tes to the Ras-transformed phenotype. These observations provide a potentia l explanation for the requirement for Rho in Ras-mediated transformation. M oreover, the inhibition of ROCK kinases may be of therapeutic use.