A transcriptional cross-talk between RhoA and c-Myc inhibits the RhoA/Rock-dependent cytoskeleton.

The GTPase RhoA participates in a number of cellular processes, including cytoskeletal organization, mitogenesis and tumorigenesis. We have previously shown that the transforming activity of an oncogenic version of RhoA (Q63L mutant) was highly dependent on the transcriptional factor c-Myc. In contrast to these positive effects in the RhoA route, we show here that c-Myc affects negatively the F-actin cytoskeleton induced by RhoA(Q63L) and its downstream effector, the serine/threonine kinase Rock. This effect entails the activation of a transcriptional program that requires synergistic interactions with RhoA-derived signals and that includes the upregulation of the GTPase Cdc42 and its downstream element Pak1 as well as the repression of specific integrin subunits. The negative effects of c-Myc in the F-actin cytoskeleton are eliminated by the establishment of cell-to-cell contacts, an effect associated with the rescue of Pak1 and integrin levels at the post-transcriptional and transcriptional levels, respectively. These results reveal the presence of a hitherto unknown signaling feed-back loop between RhoA and c--Myc oncogenes that can contribute to maintain fluid cytoskeletal dynamics in cancer cells.

Identification of the Rock-dependent transcriptome in rodent fibroblasts

Rock proteins are Rho GTPase-dependent serine/ threonine kinases with crucial roles in F-actin dynamics and cell transformation. By analogy with other protein kinase families, it can be assumed that Rock proteins act, at least in part, through the regulation of gene expression events. However, with the exception of some singular transcriptional targets recently identified, the actual impact of these kinases on the overall cell transcriptome remains unknown. To address this issue, we have used a microarray approach to compare the transcriptomes of exponentially growing NIH3T3 cells that had been untreated or treated with Y27632, a well known specific inhibitor for Rock kinase activity. We show here that the Rock pathway promotes a weak impact on the fibroblast transcriptome, since its inhibition only results in changes in the expression of 2.3% of all the genes surveyed in the microarrays. Most Y27632-dependent genes are downregulated at moderate levels, indicating that the Rock pathway predominantly induces the upregulation of transcriptionally active genes. Although functionally diverse, a common functional leitmotiv of Y27632-dependent genes is the implication of their protein products in cytoskeletal-dependent processes. Taken together, these results indicate that Rock proteins can modify cytoskeletal dynamics by acting at post-transcriptional and transcriptional levels. In addition, they suggest that the main target of these serine/threonine kinases is the phosphoproteome and not the transcriptome (AU)

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Transcriptomal profiling of the cellular transformation induced by Rho subfamily GTPases.

We have used microarray technology to identify the transcriptional targets of Rho subfamily guanosine 5'-triphosphate (GTP)ases in NIH3T3 cells. This analysis indicated that murine fibroblasts transformed by these proteins show similar transcriptomal profiles. Functional annotation of the regulated genes indicate that Rho subfamily GTPases target a wide spectrum of functions, although loci encoding proteins linked to proliferation and DNA synthesis/transcription are upregulated preferentially. Rho proteins promote four main networks of interacting proteins nucleated around E2F, c-Jun, c-Myc and p53. Of those, E2F, c-Jun and c-Myc are essential for the maintenance of cell transformation. Inhibition of Rock, one of the main Rho GTPase targets, leads to small changes in the transcriptome of Rho-transformed cells. Rock inhibition decreases c-myc gene expression without affecting the E2F and c-Jun pathways. Loss-of-function studies demonstrate that c-Myc is important for the blockage of cell-contact inhibition rather than for promoting the proliferation of Rho-transformed cells. However, c-Myc overexpression does not bypass the inhibition of cell transformation induced by Rock blockage, indicating that c-Myc is essential, but not sufficient, for Rock-dependent transformation. These results reveal the complexity of the genetic program orchestrated by the Rho subfamily and pinpoint protein networks that mediate different aspects of the malignant phenotype of Rho-transformed cells.

Involvement of the Rho/Rac family member RhoG in caveolar endocytosis.

We show here that the GTPase RhoG is involved in caveolar trafficking. Wild-type RhoG moves sequentially to the plasma membrane, intracellular vesicles, and the Golgi apparatus along markers of this endocytic pathway. Such translocation is associated with changes in RhoG GDP/GTP levels and is highly dependent on lipid raft integrity and on the function of the GTPase dynamin2. In addition, the constitutively active RhoG(Q61L) mutant is preferentially located in endocytic vesicles that can be decorated with markers of the caveola-derived endocytic pathway. RhoG(Q61L), but not the analogous Rac1 mutant protein, affects caveola internalization and the subsequent delivery of endocytic vesicles to the Golgi apparatus. The expression of RhoG/Rac1 chimeric proteins and RhoG(Q61L) effector mutants in cells induces alterations in the internalization of caveolae and severe changes in vesicle structure, respectively. However, the knockdown of endogenous rhoG transcripts using small interfering RNAs does not affect significantly the trafficking of caveola-derived vesicles, suggesting that RhoG function is dispensable for this endocytic process or, alternatively, that its function is compensated by other molecules. Taken together, these observations assign a novel function to RhoG and suggest that caveolar trafficking, as previously shown for other endocytic routes, is modulated by GTPases of the Ras superfamily.