Ras-independent transformation by v-Src

Abstract

Signaling by a variety of receptor and nonreceptor tyrosine kinases is mediated by Ras, a membrane-associated GTPase. Expression of v-Src, a transforming nonreceptor tyrosine kinase, results in Ras activation, and inhibition of Ras function in NIH 3T3 cells suppresses transformation by v-Src, indicating that in these cells Ras-dependent signaling pathways are required for v-Src to exert its biological effects. However, we show here that Ras was not activated in Rat-2 fibroblasts transformed by wild-type v-Src, or in chicken embryo fibroblasts transformed by SRX5, a v-Src mutant with a linker insertion at the major site of autophosphorylation. Expression of a dominant-negative mutant of Ras completely inhibited the ability of v-Src to activate the mitogen-activated protein kinase ERK2, which is downstream of Ras. However, dominant-negative Ras did not suppress transformation by v-Src as judged by a variety of criteria. Thus, v-Src can transform at least some cell types in the absence of Ras activation or Ras-stimulated ERK2 activity, and in these cells activation of Ras-independent signaling pathways must therefore be sufficient for transformation.

Figure 1

Activation of Ras in CEF and Rat-2 cells expressing v-Src or SRX5. CEF (open bars) were infected with vector control, v-Src, or SRX5 virus 2 days before serum starvation. The Rat-2 cell lines (solid bars) fpGV1-1-1 (vector control), v-Src-A4, and SRX5-C20 were plated 24 hr before serum starvation. The activation state (% GTP-bound) of endogenous Ras was assayed as described and is expressed relative to the level observed in vector control cells. The data are expressed as the mean ± SEM of three independent experiments.

Figure 2

Expression of dominant-negative Ras in CEF and Rat-2 cells. (A) CEF were infected with an envelope subgroup B empty vector virus or a similar virus encoding N17ras, and 2 days later were superinfected with an envelope subgroup A empty vector virus or a similar virus encoding v-src. Lysates were prepared 3 days after infection with the subgroup A virus and were subjected to immunoblotting for Src and Ras as described. (B) Rat-2 cell lines stably transfected with a vector encoding N17ras under the control of a metal-inducible promoter were infected with a retrovirus encoding v-src. Infected cell lines (Rat-2/pM2NSrc and Rat-2/N17RasSrc) and uninfected control cell lines (Rat-2/pM2N-1 and Rat-2/N17Ras-2) were grown in serum-containing medium in the absence or presence of 100 μM ZnCl₂ and 2 μM CdCl₂ for 24 hr, and then lysed and subjected to SDS/PAGE and immunoblotting as described.

Figure 3

Effect of N17Ras expression on morphological transformation of CEF and Rat-2 cells. (A–C) Morphology of CEF that were infected with empty vector viruses (A), empty vector subgroup B and v-src subgroup A viruses (B), or N17ras subgroup B and v-src subgroup A viruses (C). (D–F) Morphology of Rat-2 cell lines that were grown in serum-containing medium in the presence of 100 μM ZnCl₂ and 2 μM CdCl₂ for 24 hr: (D) Rat-2/pM2N-1, (E) Rat-2/pM2NSrc, and (F) Rat-2/N17RasSrc (see Fig. 2B legend for description of cell lines). Images were acquired using a Zeiss Axiovert microscope with a ×20 objective.

Figure 4

Effect of dominant-negative Ras on transformation by a threshold dose of v-Src. (A) Expression of Src and Ras in untransfected Rat-2 and MS1 cells, MS1 cells stably transfected with the empty expression vector (MS1/pcDNA3), and three independent clones transfected with the N17Ras expression construct (MS1/N17Ras-1, -2, and -3). (B) Soft agar colony assays were performed with the cell lines described in A. Colonies were stained with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) 16 days after plating.

Figure 5

Effect of dominant-negative Ras on the activation of the MAP kinase ERK2 by v-Src. (A) CEF were infected as in Fig. 2A, and the activity of endogenous ERK2 was determined as described. (B) CEF were infected as in Fig. 2A, except that a temperature-sensitive mutant of v-Src (tsUP1) was used. Cells were maintained at the nonpermissive temperature (41°C) during the entire incubation period (Temp. Shift, −) or were shifted to the permissive temperature (36°C) 30 min prior to lysis (Temp. Shift, +). The activity of endogenous ERK2 was then determined as described. (C) Rat-2-derived cell lines carrying the empty metal-inducible expression vector (pM2N-1), metal-inducible N17Ras (N17Ras-2), or clones of these cells that also express v-Src (pM2NSrc and N17RasSrc) were assayed for the activity of endogenous ERK2 as described. Numbers below the autoradiograms refer to the radioactivity incorporated into the substrate (myelin basic protein, MBP) as determined by PhosphorImager analysis, and are expressed as percentages relative to the vector control lanes.