Separation of v-Src-induced mitogenesis and morphological transformation by inhibition of AP-1.
v-Src activity results in both morphological transformation and reentry of quiescent chick embryo fibroblasts (CEF) into cell cycle. We have previously used temperature-sensitive v-Src mutants to show that enhanced activity of cellular AP-1 in the first few hours after activation of v-Src invariably precedes the biological consequences. Here we have investigated whether the early activation of AP-1 is essential for any or all of the v-Src responses by using a mutant c-Fos that comprises the leucine zipper and a disrupted basic region. Expression of the c-Fos mutant partially reduced cellular AP-1 activity in exponentially growing cells. However, in CEF that had been made quiescent by serum deprivation, v-Src-induced stimulation of AP-1 DNA binding activity was substantially reduced. In addition, quiescent CEF stably transfected with this mutant show an impaired mitogenic response to v-Src, indicating that the AP-1 stimulation is a necessary prerequisite for cell-cycle reentry. The ability of v-Src to morphologically transform quiescent CEF was not impaired by the inhibition of AP-1 stimulation, indicating that the mitogenic and morphological consequences of v-Src have distinguishable biochemical mediators. Focal adhesion kinase, a recently identified determinant of cell morphology, undergoes a gel mobility shift, characteristic of its hyperphosphorylated state, in response to v-Src activation in cells expressing the inhibitory AP-1 protein. This provides further evidence that the pathways that regulate morphological transformation are independent of AP-1.
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Beatson Institute for Cancer Research, Beatson Laboratories, Bearsden, Glasgow, United Kingdom.
Simpson
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K
Fincham
V J
VJ
Crouch
D H
DH
D000818:Animals; D002453:Cell Cycle / Q000235:genetics / Q000502:physiology; D048429:Cell Size / Q000235:genetics; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D002478:Cells, Cultured; D002642:Chick Embryo; D004268:DNA-Binding Proteins / Q000378:metabolism; D005786:Gene Expression Regulation / Q000235:genetics*; D016762:Genes, fos / Q000235:genetics; D016391:Genes, src / Q000235:genetics; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014162:Transfection / Q000235:genetics
D004268:DNA-Binding Proteins; D016760:Proto-Oncogene Proteins c-fos; D018808:Transcription Factor AP-1