Ras Is Not Required for the Interleukin 3-induced Proliferation of a Mouse Pro-B Cell Line, BaF3

Abstract

It has been demonstrated that Ras is involved in in-. Furthermore, the activation of fos gene promoter following IL-3 stimulation was almost completely abolished when Ras(S17N) was induced. Under these conditions, Ras(S17N) exhibited no inhibitory effect on IL-3-dependent proliferation assessed by the increase of cell numbers and a mitochondrial enzyme activity. The results indicate that Ras-dependent pathways, including the Raf/MAPK/Fos pathway, are dispensable for IL-3-induced growth stimulation. When BaF3 cells were treated with a tyrosine kinase inhibitor, herbimycin A, IL-3-dependent proliferation of the cells was impaired, suggesting that tyrosine kinase-mediated pathways are critical for growth promotion. On the other hand, apoptotic cell death caused by deprivation of IL-3 was prevented by the induction of the activated mutant Ras(G12V), although the rate of cell number increase was markedly reduced. Thus, it is likely that Rasindependent pathways play important roles to facilitate the proliferation although they may not be essential for IL-3-stimulated antiapoptotic signal transduction.In various types of cells, Ras functions as a molecular switch that regulates intracellular signaling pathways for proliferation, differentiation, and other physiological responses. Tyrosine kinase receptors, such as epidermal growth factor receptor and platelet-derived growth factor receptor, when stimulated by their specific ligands, form a complex with a variety of signal-transducing molecules including phosphatidylinositol 3-kinase, Ras-GTPase activating protein (Ras-GAP), 1 phospholipase C-␥1, and adaptor proteins (e.g. Grb-2, Nck, and Shc) through the interaction between specific phosphotyrosines on the receptor and Src homology 2 (SH2) domains of the binding molecules Schlessinger, 1993). Among the above components of the signal-transducing complex, Shc and Grb-2 are well characterized as elements that link the receptor and a Ras-guanine nucleotide exchange factors (Ras-GEFs), such as mSos-1. Adaptors and Ras-GEFs are known to participate also in Ras regulation through non-tyrosine kinase-type receptors including interleukin 2 (IL-2), IL-3, and T cell antigen receptors (Burns et al., 1993;Cutler et al., 1993;Ravichandran et al., 1993;Sato et al., 1993;Buday et al., 1994;Reif et al., 1994;Ravichandran and Burakoff, 1994;Welham et al., 1994). The active GTP-bound form of Ras specifically binds RasGAPs (Boguski and McCormick, 1993), c-Raf-1 (Avruch et al., 1994), B-Raf (Moodie et al., 1994;Vaillancourt et al., 1994), phosphatidylinositol 3-kinase (Rodriguez-Viciana et al., 1994), Ral-guanine nucleotide dissociation stimulator (Hofer et al., 1994;Kikuchi et al., 1994;Spaargaren and Bischoff, 1994), mitogen-activated protein kinase (MAPK)/extracellular signalregulated kinase (ERK) kinase (MEK) kinase (MEKK) (LangeCarter and Johnson, 1994), and Rin1 (Han and Colicelli, 1995), among which Raf proteins are best characterized as direct targets of Ras (Daum et al., 1994;Marshall, 1995). After the binding of Ras, the serine/t...

Anti-apoptotic function of Rac in hematopoietic cells

“…Of these, the Ras pathway has been well characterized by the use of an array of the receptor mutants and dominant-negative as well as activated mutants of Ras (Kinoshita et al, 1995;Terada et al, 1995). In BaF3 cells, overexpression of dominant-negative Ras did not impair IL-3-dependent growth under conditions where the Raf/ extracellular signal-regulated kinase (ERK) cascade was diminished, raising the notion that Ras is not crucial for cell proliferation .…”

“…In BaF3 cells, overexpression of dominant-negative Ras did not impair IL-3-dependent growth under conditions where the Raf/ extracellular signal-regulated kinase (ERK) cascade was diminished, raising the notion that Ras is not crucial for cell proliferation . Instead, Ras is implicated in the prevention of cell death as evidenced by an anti-apoptotic e ect of an ectopically expressed active Ras mutant (Kinoshita et al, 1995;Terada et al, 1995). For this function, PI3-K and ERK were identi®ed as important players downstream of Ras .…”

Small contribution of G1 checkpoint control manipulation to modulation of p53-mediated apoptosis

“…These results are supported by the ®ndings that expression of constitutively activated Ras or Raf protect from apoptosis induced by growth factor deprivation and p53 (Canman et al, 1995;Lin et al, 1995a;Kinoshita et al, 1995;Cleveland et al, 1994;Terada et al, 1995). Paradoxically, introduction of a dominant-negative Ras protein failed to promote apoptosis in BaF3 cells cultured in IL-3, arguing against Ras-dependent pathways playing a role in anti-apoptosis signaling in these cells (Terada et al, 1995). Recently, the Janus kinase (Jak) family of kinases, which regulate the Stat (Signal transducers and activators of transcription) family of transcription factors, have received attention as important mediators of cytokine signal transduction.…”

“…Activation of the Ras/Raf/MAP kinase pathway has also been implicated in survival signaling mediated by several di erent receptor systems, including those activated by cytokines (Kinoshita et al, 1995;Xia et al, 1995). These results are supported by the ®ndings that expression of constitutively activated Ras or Raf protect from apoptosis induced by growth factor deprivation and p53 (Canman et al, 1995;Lin et al, 1995a;Kinoshita et al, 1995;Cleveland et al, 1994;Terada et al, 1995). Paradoxically, introduction of a dominant-negative Ras protein failed to promote apoptosis in BaF3 cells cultured in IL-3, arguing against Ras-dependent pathways playing a role in anti-apoptosis signaling in these cells (Terada et al, 1995).…”

Analysis of mechanisms involved in the prevention of γ irradiation-induced apoptosis by hGM-CSF

“…Further analysis using the tyrosine kinase inhibitor genistein and the MEK1 inhibitor, PD98059, indicated that activation of either the genistein sensitive signaling or the PD98059 sensitive signaling pathways of bc may be su cient to suppress apoptosis (Liu et al, 1999c). Consistent with this notion, it was reported that constitutive active Ras prevents DNA fragmentation induced by factor depletion but that dominant negative Ras failed to block the anti-DNA fragmentation activity or the cell viability induced by IL-3 (Okuda et al, 1994;Terada et al, 1995). These observations indicate that activation of the Ras/MAPK pathway by IL-3/GM-CSF is dispensable for the anti-apoptosis activity of GM-CSF while forced activation of this pathway can rescue cells from factor depletion induced apoptosis.…”