Translocation of Src kinase to the cell periphery is mediated by the actin cytoskeleton under the control of the Rho family of small G proteins.
We have isolated Swiss 3T3 subclones that are resistant to the mitogenic and morphological transforming effects of v-Src as a consequence of aberrant translocation of the oncoprotein under low serum conditions. In chicken embryo and NIH 3T3 fibroblasts under similar conditions, v-Src rapidly translocates from the perinuclear region to the focal adhesions upon activation of the tyrosine kinase, resulting in downstream activation of activator protein-1 and mitogen-activated protein kinase, which are required for the mitogenic and transforming activity of the oncoprotein. Since serum deprivation induces cytoskeletal disorganization in Swiss 3T3, we examined whether regulators of the cytoskeleton play a role in the translocation of v-Src, and also c-Src, in response to biological stimuli. Actin stress fibers and translocation of active v-Src to focal adhesions in quiescent Swiss 3T3 cells were restored by microinjection of activated Rho A and by serum. Double labeling with anti-Src and phalloidin demonstrated that v-Src localized along the reformed actin filaments in a pattern that would be consistent with trafficking in complexes along the stress fibers to focal adhesions. Furthermore, treatment with the actin-disrupting drug cytochalasin D, but not the microtubule-disrupting drug nocodazole, prevented v-Src translocation. In addition to v-Src, we observed that PDGF-induced, Rac-mediated membrane ruffling was accompanied by translocation of c-Src from the cytoplasm to the plasma membrane, an effect that was also blocked by cytochalasin D. Thus, we conclude that translocation of Src from its site of synthesis to its site of action at the cell membrane requires an intact cytoskeletal network and that the small G proteins of the Rho family may specify the peripheral localization in focal adhesions or along the membrane, mediated by their effects on the cytoskeleton.
Fincham
V J
VJ
Beatson Institute for Cancer Research, CRC Beatson Laboratories, Glasgow, Scotland.
Unlu
M
M
Brunton
V G
VG
Pitts
J D
JD
Wyke
J A
JA
Frame
M C
MC
D016475:3T3 Cells; D000199:Actins / Q000502:physiology*; D000818:Animals; D001692:Biological Transport; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002455:Cell Division; D002462:Cell Membrane / Q000378:metabolism; D002642:Chick Embryo; D003599:Cytoskeleton / Q000502:physiology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D051379:Mice; D008870:Microtubules / Q000502:physiology; D015688:Oncogene Protein pp60(v-src) / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D020741:rho GTP-Binding Proteins
D000199:Actins; D018808:Transcription Factor AP-1; D015688:Oncogene Protein pp60(v-src); D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019204:GTP-Binding Proteins; D020741:rho GTP-Binding Proteins