The small GTPases Cdc42Hs, Rac1 and RhoG delineate Raf-independent pathways that cooperate to transform NIH3T3 cells.
BACKGROUND
Ras-mediated transformation of mammalian cells has been shown to activate multiple signalling pathways, including those involving mitogen-activated protein kinases and the small GTPase Rho. Members of the Rho family affect cell morphology by controlling the formation of actin-dependent structures: specifically, filopodia are induced by Cdc42Hs, lamellipodia and ruffles by Rac, and stress fibers by RhoA. In addition, Rho GTPases are involved in progression through the G1 phase of the cell cycle, and Rac1 and RhoA have recently been directly implicated in the morphogenic and mitogenic responses to transformation by oncogenic Ras. In order to examine the cross-talk between Ras and Rho proteins, we investigated the effects on focus-forming activity and cell growth of the Rho-family members Cdc42Hs, Rac1 and RhoG by expressing constitutively active or dominant-negative forms in NIH3T3 cells.


RESULTS
Expression of Rac1 or RhoG modulated the saturation density to which the cells grew, probably by affecting the level of contact inhibition. Although all three GTPases were required for cell transformation mediated by Ras but not by constitutively active Raf, the selective activation of each GTPase was not sufficient to induce the formation of foci. The coordinated activation of Cdc42Hs, RhoG and Rac1, however, elicited a high focus-forming activity, independent of the mitogen-activated ERK and JNK protein kinase pathways.


CONCLUSIONS
Ras-mediated transformation induces extensive changes in cell morphology which require the activity of members of the Rho family of GTPases. Our data show that the pattern of coordinated Rho family activation that elicits a focus-forming activity in NIH3T3 cells is distinct from the regulatory cascade that has been proposed for the control of actin-dependent structures in Swiss 3T3 cells.
Roux
P
P
Institut de Génétique Moléculaire, C.N.R.S. UMR5535, 1919 route de Mende, F-34293 Montpellier cedex 5, France. roux@igm.cnrs-mop.fr
Gauthier-Rouvière
C
C
Doucet-Brutin
S
S
Fort
P
P
D016475:3T3 Cells; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002452:Cell Count; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002471:Cell Transformation, Neoplastic; D004789:Enzyme Activation; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D051379:Mice; D011506:Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000502:physiology*; D015398:Signal Transduction; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection; D020764:cdc42 GTP-Binding Protein; D020703:ras GTPase-Activating Proteins; D020741:rho GTP-Binding Proteins
D018797:Cell Cycle Proteins; D020690:GTPase-Activating Proteins; D011506:Proteins; C489577:Rhog protein, mouse; D014157:Transcription Factors; D020703:ras GTPase-Activating Proteins; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020764:cdc42 GTP-Binding Protein; D020741:rho GTP-Binding Proteins