Mas oncogene signaling and transformation require the small GTP-binding protein Rac - PubMed (original) (raw)

Mas oncogene signaling and transformation require the small GTP-binding protein Rac

I E Zohn et al. Mol Cell Biol. 1998 Mar.

Abstract

The Mas oncogene encodes a novel G-protein-coupled receptor that was identified originally as a transforming protein when overexpressed in NIH 3T3 cells. The mechanism and signaling pathways that mediate Mas transformation have not been determined. We observed that the foci of transformed NIH 3T3 cells caused by Mas were similar to those caused by activated Rho and Rac proteins. Therefore, we determined if Mas signaling and transformation are mediated through activation of a specific Rho family protein. First, we observed that, like activated Rac1, Mas cooperated with activated Raf and caused synergistic transformation of NIH 3T3 cells. Second, both Mas- and Rac1-transformed NIH 3T3 cells retained actin stress fibers and showed enhanced membrane ruffling. Third, like Rac, Mas induced lamellipodium formation in porcine aortic endothelial cells. Fourth, Mas and Rac1 strongly activated the JNK and p38, but not ERK, mitogen-activated protein kinases. Fifth, Mas and Rac1 stimulated transcription from common DNA promoter elements: NF-kappaB, serum response factor (SRF), Jun/ATF-2, and the cyclin D1 promoter. Finally, Mas transformation and some of Mas signaling (SRF and cyclin D1 but not NF-kappaB activation) were blocked by dominant negative Rac1. Taken together, these observations suggest that Mas transformation is mediated in part by activation of Rac-dependent signaling pathways. Thus, Rho family proteins are common mediators of transformation by a diverse variety of oncogene proteins that include Ras, Dbl family, and G-protein-coupled oncogene proteins.

PubMed Disclaimer

Figures

FIG. 1

FIG. 1

Mas-transformed cells exhibit a transformed phenotype similar to those of Rac- and Rho-transformed cells. (A) Transformed foci from NIH 3T3 cultures transfected with pZIP-ras(61L), pZIP-rhoA(63L), and pZIP-mas. (B) Morphology of NIH 3T3 cells stably transfected with pZIP-NeoSV(x)1, pZIP-ras(61L), pZIP-rac1(115I), and pZIP-mas. Multiple (>100) G418-resistant colonies were pooled to establish the cell lines used for these analyses.

FIG. 2

FIG. 2

Like Rac1, Mas cooperates with Raf(340D) and causes synergistic focus-forming activity. NIH 3T3 cells were cotransfected with pZIP expression plasmids encoding the indicated proteins. One hundred nanograms of pZIP-mas cDNA and 1 μg of all other DNAs were transfected per 60-mm-diameter dish. The data are shown as mean ± standard error for triplicate plates and are representative of at least three separate experiments.

FIG. 3

FIG. 3

Mas and Rac1 cause similar changes in the organization of the actin cytoskeleton. (A) Mas- and Rac-transformed cells retain stress fibers and show enhanced membrane ruffling. Stably transfected NIH 3T3 cell lines expressing the indicated proteins were subject to immunofluorescence analysis as described in Materials and Methods. Shown are actin stress fibers and focal adhesions stained with RITC-phalloidin and FITC-antivinculin antibodies, respectively. Membrane ruffles are indicated by arrowheads. (B) Like Rac1(12V), Mas induces membrane ruffles in PAE cells. PAE cells were microinjected with expression constructs encoding either Mas along with GFP (a and b) or Myc epitope-tagged Rac1(12V) (c and d) as described in Materials and Methods. Cells were serum starved following injection, and actin was stained with RITC-phalloidin (a and c). Microinjected cells were identified by expression of GFP (for Mas) (b) or anti-Myc antiserum (for Rac1) (d).

FIG. 4

FIG. 4

Mas and Rac are strong activators of p38 and JNK but not ERK. (A) Activation of ERK2 by Mas. Cos-7 cells were transfected with either pCGN-hyg (vector), pCGN-ras(61L), pCGN-_rac_1(61L), or pCGN-mas along with an HA epitope-tagged ERK2 expression vector. Immunocomplex kinase assays with myelin basic protein (MBP) as a substrate were performed following immunoprecipitation of HA-ERK2 (top panel). Fold activation (Act) (middle panel) of ERK was determined by PhosphorImager analysis and expressed relative to phosphorylation levels in vector-transfected cells. Twenty-five micrograms of lysate was resolved by SDS-PAGE, transferred to an Immobilon membrane, and subsequently probed with anti-HA antibody to ensure equivalent expression levels of HA-ERK2 (bottom panel). (B) Activation of JNK1 by Mas. Cos-7 cells were transfected as for panel A but with FLAG epitope-tagged JNK1. JNK1 kinase activity was determined with GST–c-Jun(1-79) as a substrate (top panel). Fold activation (middle panel) and JNK1 levels (bottom panel) were determined as described for panel A. Data in panels A and B are representative of at least three independent experiments in Cos-7 and NIH 3T3 cells, using pCGN and pCDNA3 expression plasmids. (C) Activation of p38 by Mas. Cos-7 cells were transfected as described for panels A and B but with FLAG epitope-tagged p38. p38 kinase activity was determined with GST–ATF-2(1-254) as a substrate (top panel); fold activation (middle panel) and p38 expression levels (bottom panel) were determined as described above. Data are representative of two separate experiments.

FIG. 5

FIG. 5

Mas and Rac stimulate transcription from common promoter elements. NIH 3T3 cells were transfected with pAX142 (vector), pAX142-ras(61L), pAX142-rac1(61L), pAX142-rhoA(63L), or pAX142-mas along with luciferase gene reporter constructs for SRF transcriptional activity (A), NF-κB transcriptional activity (B), c-Jun/ATF-2 transcriptional activity (C), and cyclin D1 expression (D). Data shown are representative of at least three independent experiments using both pAX142 and pCDNA3 mammalian expression constructs.

FIG. 6

FIG. 6

Mas, but not Rac1, caused activation of Elk-1. NIH 3T3 cells were transfected with pAX142 (vector), pAX142-ras(61L), pAX142-rac1(61L), pAX142-rhoA(63L), or pAX142-mas along with Gal4–Elk-1 and the Gal4-responsive 5XGal4-Luc construct. Data shown are representative of at least three independent experiments using both pAX142 and pCDNA3 mammalian expression constructs.

FIG. 7

FIG. 7

Dominant negative Rac1 blocks Mas signaling. NIH 3T3 cells were cotransfected with pAX142-mas and either pAX142-rac1(17N) or pAX142-rac1(WT) along with luciferase gene reporter constructs for SRF (A), cyclin D1 (B), or NF-κB (C) expression. Data are expressed as the mean of the percentage of the activation in the Mas-plus-vector samples ± standard deviation of duplicate samples and are representative of at least two independent experiments.

FIG. 8

FIG. 8

Dominant negative Rac, RhoA, and Ras block Mas transformation. NIH 3T3 cells were transfected with pZIP-mas and the indicated wild-type (WT) and dominant negative Rho family proteins, and the focus formation assay was performed as described in Materials and Methods. Data are expressed as the mean of the percentage of the total number of foci in the Mas-plus-vector dishes ± standard error and are the average of six separate experiments performed in duplicate or triplicate.

Similar articles

Cited by

References

    1. Albanese C, Johnson J, Watanabe G, Eklund N, Vu D, Arnold A, Pestell R G. Transforming p21ras mutants and c-Ets-2 activate the cyclin D1 promoter through distinguishable regions. J Biol Chem. 1995;270:23589–23597. - PubMed
    1. Allen L F, Lefkowitz R J, Caron M G, Cotecchia S. G-protein-coupled receptor genes as protooncogenes: constitutively activating mutation of the α1B-adrenergic receptor enhances mitogenesis and tumorigenicity. Proc Natl Acad Sci USA. 1991;88:11354–11358. - PMC - PubMed
    1. Andrawis N S, Dzau V J, Pratt R E. Autocrine stimulation of mas oncogene leads to altered growth control. Cell Biol Int Rep. 1992;16:547–556. - PubMed
    1. Angel P, Hattori K, Smeal T, Karin M. The jun proto-oncogene is positively autoregulated by its product, Jun/AP-1. Cell. 1988;55:875–885. - PubMed
    1. Baichwal V R, Baeuerle P A. Apoptosis: activate NF-KB or die? Curr Biol. 1997;7:R94–R96. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources