Mitogenic activation of the Ras guanine nucleotide exchange factor in NIH 3T3 cells involves protein tyrosine phosphorylation.

Abstract

We report biochemical evidence that epidermal growth factor and platelet-derived growth factor stimulate the Ras gua nucleotide exchange factor activity in quiescent NIH 3T3 cells. Moreover, the exchange activity is costutively enhanced in NIH 3T3 cells transformed by Src and ErbB2 oncogenic tyrosine protein kinases (TPKs), whereas transformation by oncogenic Mos and Raf does not alter the activity. GTPase-activating protein activity was not affected under these conditions. Overexpression of pp6O"src mutants contining activated and suppressor TPK mutations resulted in stimulation and inhibition ofthe exchange factor activity, respectively. A TPK inhibitor, genistein, prevented the activation of the exchange factor in epidermal growth factor/platelet-derived growth factor-treated cells and src-transformed cels. Furthermore, the exchange factor activity bound to an antiphosphotyrosine antibody immunalnity column. These findings suggest that the ganine nucleotide exchange factor, but not GTPase-activating protein, plays a major role in the Ras activation in cell proliferation initiated by growth factor receptor TPKs and malignant transformation by oncogenic TPKs and that tyrosine phosphorylation of either the exchange factor or a tightly bound protein(s) may mediate the activation of the exchange factr by these TPKs.

Isoform-specific insertion near the Grb2-binding domain modulates the intrinsic guanine nucleotide exchange activity of hSos1

“…In that regard, it is interesting to note recent reports (Li et al, , 1993a(Li et al, , 1995(Li et al, , 1996 suggesting that, in contrast to the currently prevailing translocation model (reviewed in Schlessinger 1993;Downward 1994), an increase in the intrinsic catalytic activity of Sos1 may suce to account for the activation of Ras by some tyrosine kinase receptors including NGF, EGF, PDGF and macrophage-stimulating protein receptor. Indeed, our direct comparison of the Ras guanine nucleotide exchange (GEF) activity detectable in lysates of transfected cells overexpressing Isf I and Isf II con®rmed that view.…”

“…In support of this notion, constitutive or conditional membrane targeting of these exchange factors has been shown to potentiate Ras activation in transfected cells (Aronheim et al, 1994;Quilliam et al, 1994;Holsinger et al, 1995). However, some recent reports suggest that, irrespective of subcellular location, the intrinsic guanine nucleotide releasing activity of Sos may be dierent before and after stimulation of surface tyrosine kinase receptors (Li et al, 1993a(Li et al, , 1995(Li et al, , 1996. Such a notion would be consistent with other reports suggesting that the Cterminal portion of Sos may exert negative regulation over the activity of the whole Sos1 protein (Aronheim et al, 1994;Quilliam et al, 1994;Karlovich et al, 1995;McCollam et al, 1995;Wang et al, 1995;Byrne et al, 1996;Corbalan-Garcia et al, 1998).…”

“…3 H]GDP.Ras (0.4 mg) binary complexes in 100 ml of the reaction mixture for 25 min at 378C by constant agitation as described (Li et al, 1993a(Li et al, , 1996. […”

“…Lysates were spun at 100 000 g for 10 min, and the supernatant were immunoprecipitated with rabbit anti-SOS-1 antibody (1 mg; Santa Cruz Biotechnologies) for 90 min at 48C. The immune complexes retained onto protein A-Sepharose (Pharmacia) were washed four times with 1 ml of buer B [20 mM Tris HCl, pH 7.5, 1 mM MgCl 2 , 10 mM NaCl, 1 mM dithiothreitol, bovine serum albumin (50 mg/ml), leupeptin (10 mg/ml), and pepstatin (10 mg/ml)], as described (Li et al, 1993a(Li et al, , 1996. Then, the immunoprecipitates were reacted with […”

“…Lysates from unstimulated and stimulated 31-1 (overexpressing Isf I) and 33-2 (overexpressing Isf II) cells were immunoprecipitated with anti-Sos1 antibodies, and the resulting Isf I or Isf II immunoprecipitates were then analysed for Ras guanine nucleotide exchange activity assay (Li et al, 1993a(Li et al, , 1996. We consistently observed (Figure 7) that the Isf II immunoprecipitates displayed about threefold higher speci®c activity than Isf I immunoprecipitates when comparing their ability to promote the release of GDP bound to Ras under basal, unstimulated conditions.…”