Regulation of p190 Rho-GAP by v-Src is linked to cytoskeletal disruption during transformation.
The v-Src oncoprotein perturbs the dynamic regulation of the cellular cytoskeletal and adhesion network by a mechanism that is poorly understood. Here, we have examined in detail the effects of a temperature-dependent v-Src protein on the regulation of p190 RhoGAP, a GTPase activating protein (GAP) that has been implicated in disruption of the organised actin cytoskeleton, and addressed the dependence of v-Src-induced stress fibre loss on inhibition of Rho activity. We found that activation of v-Src induced association of tyrosine phosphorylated p190 with p120(RasGAP) and stimulation of p120(RasGAP)-associated RhoGAP activity, although p120(RasGAP) itself was not a target for phosphorylation by v-Src in chicken embryo cells. These events required the catalytic activity of v-Src and were linked to loss of actin stress fibres during morphological transformation and not mitogenic signalling. Furthermore, these effects were rapidly reversible since switching off v-Src led to dissociation of the p190/p120(RasGAP) complex, inactivation of p120(RasGAP)-associated RhoGAP activity and re-induction of actin stress fibres. In addition, transient transfection of Val14-RhoA, a constitutively active Rho protein that is insensitive to RhoGAPs, suppressed v-Src-induced stress fibre loss and cell transformation. Thus, we show here for the first time that an activated Src kinase requires the inactivation of Rho-mediated actin stress fibre assembly to induce its effects on actin disorganisation. Moreover, our work supports p190 as a strong candidate effector of v-Src-induced cytoskeletal disruption, most likely mediated by antagonism of the cellular function of Rho.
The Beatson Institute for Cancer Research, CRC Beatson Laboratories, Garscube Estate, Switchback Road, Bearsden, Glasgow G61 1BD, UK.
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Fincham
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Chudleigh
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D000199:Actins / Q000502:physiology; D000818:Animals; D048429:Cell Size; D002471:Cell Transformation, Neoplastic; D002642:Chick Embryo; D003599:Cytoskeleton / Q000502:physiology* / Q000648:ultrastructure*; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D019204:GTP-Binding Proteins / Q000378:metabolism; D020690:GTPase-Activating Proteins; D016391:Genes, src; D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins / Q000378:metabolism*; D015688:Oncogene Protein pp60(v-src) / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D020742:rhoA GTP-Binding Protein
D000199:Actins; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011506:Proteins; D011994:Recombinant Proteins; D015688:Oncogene Protein pp60(v-src); D019204:GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein