(Received for publication, August 21, 1995; and in revised form, September 15, 1995)
From the
It has been demonstrated that Ras is involved in interleukin 3 (IL-3)-stimulated signal transduction in various hematopoietic cultured cells (Satoh, T., Nakafuku, M., Miyajima, A., and Kaziro, Y.(1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3314-3318; Duronio, V., Welham, M. J., Abraham, S., Dryden, P., and Schrader, J. W.(1992) Proc. Natl. Acad. Sci. U. S. A. 89, 1587-1591). However, it has not been fully understood which of IL-3-promoted cellular responses, i.e. proliferation, survival, and differentiation, requires Ras function. We employed a system of inducible expression of the dominant-negative (S17N) or dominant-active (G12V) mutant of Ras in BaF3 mouse pro-B cell line to analyze the role of Ras in IL-3-stimulated signal transduction. Induction of the dominant-negative Ras(S17N) effectively inhibited the IL-3induced activation of c-Raf-1 and mitogen-activated protein kinase (MAPK). 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 Ras-independent 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), ()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 (Fantl et
al., 1993; 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 Ras-GAPs (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 signal-regulated kinase (ERK) kinase (MEK) kinase (MEKK) (Lange-Carter 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/threonine kinase activity of Raf is enhanced by interaction with membrane components (Leevers et al., 1994; Stokoe et al., 1994; Dent et al., 1995; Marais et al., 1995), and then the activated Raf stimulates MEK and MAPK. In addition to the Raf/MAPK pathway, it has been proposed that Rac-mediated pathways function downstream of Ras and are essential for the induction of transformed phenotypes by oncogenic Ras in Rat1 and NIH3T3 cells (Qiu et al., 1995). Furthermore, sequential activation of Rac and Rho upon cell stimulation to trigger the assembly of focal complexes has been reported (Nobes and Hall, 1995), suggesting a cascade of small GTPases.
In fibroblast cells, Ras is implicated in the signal transduction of normal growth as well as malignant transformation. On the other hand, in PC12 pheochromocytoma and 3T3-L1 cells, Ras stimulates the differentiation to neuronal cells and adipocytes, respectively. It has also been revealed that Ras plays a crucial role in T cell activation signaling. Furthermore, in factor-dependent hematopoietic cell lines, accumulation of the active GTP-bound Ras was detected in response to the stimulation with various cytokines including IL-2, IL-3, granulocyte/macrophage colony-stimulating factor (GM-CSF), and Steel factor, which induce both proliferation and differentiation (see Satoh et al. (1992b), Boguski and McCormick(1993), and Lowy and Willumsen(1993) for reviews).
IL-3 is
indispensable for survival and proliferation of a mouse pro-B cell
line, BaF3. Upon binding of IL-3, the IL-3 receptor, consisting of a
heterodimer of and
subunits, triggers multiple signals, for
instance, Ras-dependent, Janus kinase (JAK)/signal transducers
and activators of transcription (STAT) protein-mediated, and
Myc-related pathways. It is likely that these signaling pathways
cooperatively regulate cellular proliferation, survival, and
differentiation although the role and relationship of each pathway
remain unclear (see Darnell et al.(1994), Ihle et
al.(1994), and Sato and Miyajima(1994) for reviews).
In this study, we investigated the roles of Ras-dependent signal transduction pathways in IL-3-induced cell growth of BaF3 cells by utilizing a system for inducible expression of dominant-negative (S17N) as well as dominant-active (G12V) mutants of Ras protein. The dominant-negative Ras(S17N) almost completely blocked the signal transduction downstream of Ras including the activation of c-Raf-1 protein and subsequent hyperphosphorylation of MAPK. Under these conditions, the cell growth was not interfered with at all, indicating that the Ras pathway may be dispensable for IL-3-promoted proliferation. It was also found that a constitutively active Ras(G12V) by itself was able to prevent the cells from apoptotic cell death caused by IL-3 deprivation.
To clarify the role of Ras in IL-3-induced signal transduction for cell growth, we established cell lines in which the dominant-negative (S17N) or dominant-active (G12V) form of Ras protein is expressed in an inducible manner. Mouse IL-3-dependent BaF3 pro-B cells were used as the parental cell line. The cells were transfected with two expression plasmids; one for Ras and the other for the repressor of E. coli lactose operon (termed pOPRSVI-Ras and p3`SS, respectively). The repressor binds to the operator sequence that was inserted into the Rous sarcoma virus (RSV)-derived promoter of pOPRSVI-Ras to suppress the expression. When IPTG is added to the medium, the repressor is released from the lactose operator sequence, and transcription of the exogenously introduced cDNA is initiated. Two stable transfectants designated N6 (for Ras(S17N)) and V2 (for Ras(G12V)) were selected and characterized in detail. Fig. 1shows the expression of the mutant Ras proteins in N6 and V2 cells upon stimulation with IPTG. In both cases, expression of the mutant Ras was detected after 4 h of treatment with IPTG and reached a plateau after 16 h. Faint bands of endogenous Ras were detected above Ras(S17N) in A, and below Ras(G12V) in B. It seems likely that a band of unmodified Ras(S17N) appears at the same position as the endogenous Ras after 24-h induction. We consider that it does not interfere with the action of modified Ras(S17N) because the population of the unmodified form seems relatively small.
Figure 1:
IPTG induction of Ras mutants in
BaF3-derived transfectants. A, expression of Ras(S17N) in N6
cells. Ras(S17N) was induced by treatment of the cells with IPTG (5
mM) for the indicated periods. Then, the cells were dissolved
in SDS-PAGE sample buffer and applied to SDS-PAGE (15% acrylamide, 5
10
cells per lane). Ras proteins were detected by
immunoblotting using a specific antibody (Has 6). B,
expression of Ras(G12V) in V2 cells was analyzed as described in A.
Evidence for the specific interaction between Ras and c-Raf-1 proteins was provided in various types of mammalian cells using several different methods, such as co-immunoprecipitation, affinity chromatography, and yeast two-hybrid systems (Koide et al., 1993; Avruch et al., 1994; Daum et al., 1994). Furthermore, it has been demonstrated that the blockade of c-Raf-1 function resulted in the failure of Ras to stimulate proliferation (Kolch et al., 1991), indicating that c-Raf-1 acts downstream from Ras. Hence, we examined whether the dominant-negative Ras(S17N) sufficiently inhibited the Ras-mediated pathway by comparing the activation of c-Raf-1 protein in response to IL-3 stimulation between IPTG-treated and untreated N6 cells. Endogenous c-Raf-1 protein was immunoprecipitated with a specific antibody, and the kinase activity of the protein was assayed using recombinant MAPK kinase (MAPKK or MEK) as a substrate and by measuring the incorporation of radioactive phosphate into recombinant kinase-negative MAPK added to the reaction mixture. As illustrated in Fig. 2A, IL-3 rapidly triggered the activation of c-Raf-1 protein in N6 cells in the absence of IPTG, indicating that Ras/Raf pathway functions downstream of the IL-3 receptor. When the cells were treated with IPTG for 16 h, the induction of c-Raf-1 activation was completely diminished, suggesting that the induced Ras(S17N) interferes with the function of endogenous Ras. Then, we investigated the phosphorylation and subsequent activation of one of the endogenous MAPKs, ERK2, following IL-3 stimulation, which can be detected by immunoblotting as the appearance of mobility-shifted bands corresponding to phosphorylated ERK2. As shown in Fig. 2B, IL-3 induced the phosphorylation of ERK2 in a similar time course as c-Raf-1 activation in N6 cells without IPTG pretreatment, whereas, in cells treated with IPTG for 16 h prior to the addition of IL-3, ERK2 was no longer activated.
Figure 2:
Inhibition of IL-3-induced activation of
c-Raf-1 and ERK2 by Ras(S17N). A, inhibition of IL-3-induced
activation of c-Raf-1. N6 cells were treated with or without IPTG (5
mM) for 16 h. During the last 3 h, the cells were starved in
RPMI 1640 containing bovine serum albumin (1 mg/ml). Then, the cells
were stimulated with mouse IL-3 (50 ng/ml) for the indicated periods,
and cell lysates were prepared. c-Raf-1 protein was immunoprecipitated
with a specific antibody (sc-227) from the cell lysates (3
10
cells per lane), and the precipitates were subjected to
MEK kinase assay. Phosphorylated GST-MAPK(K57D) was detected by
SDS-PAGE (9% acrylamide) and the following autoradiography. B,
inhibition of IL-3-induced hyperphosphorylation of ERK2. N6 cells were
treated as described in A. The cellular proteins were
separated by SDS-PAGE (10% acrylamide, 5
10
cells
per lane), and ERK2 was detected by immunoblotting with a specific
antibody (05-157).
Analyses of Ras-bound guanine nucleotide in N6 cells were carried out to confirm that Ras(S17N) actually inhibits the formation of an active form of Ras within the cell since it is well known that only a GTP-bound conformation can transduce the signal to downstream targets including c-Raf-1. The molar ratios of the GTP-bound form were: 2.6% in control and 18.5% in IL-3-stimulated N6 cells without IPTG pretreatment, and 1.8% in control and 5.4% in IL-3-stimulated cells after the induction of Ras(S17N) by IPTG, respectively.
We next examined the effect of Ras(S17N) on IL-3-enhanced transcription from c-fos promoter using c-fos-luciferase as a reporter plasmid. As shown in Fig. 3A, IL-3 stimulated the luciferase activity in control N6 cells, which was significantly reduced when Ras(S17N) was induced by IPTG treatment of the cell. Co-transfection of increasing amounts of a plasmid that expresses Ras(S17N) constitutively, designated pCMV5-Ras(S17N), also diminished IL-3-promoted transcription from c-fos promoter (Fig. 3B). The results indicate that c-fos induction in response to IL-3 stimulation is Ras-dependent, and the induction of Ras(S17N) with IPTG in N6 cells is sufficient for interfering with this signaling pathway.
Figure 3:
Effects of Ras(S17N) on IL-3-stimulated
transcription from c-fos promoter. A, inhibition of
IL-3-stimulated transcription from c-fos promoter in N6 cells.
c-fos-luciferase (30 µg) was introduced into N6 cells
(2-3 10
cells) by electroporation followed by
a 12-h incubation in the culture medium without IL-3 in the presence or
absence of IPTG (5 mM). Then, mouse IL-3 (approximately 1
nM) or control buffer was added to the cells, which were
subjected to further incubation for 12 h. Following IL-3 stimulation,
cell lysates were prepared, and the luciferase activity (10
cells for each assay) was measured. Relative luciferase
activities as mean ± S.E. (n = 3) were shown. B, inhibition of IL-3-stimulated transcription from c-fos promoter in BaF3 cells. c-fos-luciferase (15 µg) and
indicated amounts of pCMV5-Ras(S17N) were introduced into BaF3 cells
(1-2
10
cells) by electroporation. Then, the
cells were incubated in the culture medium with or without mouse IL-3
(approximately 1 nM) for 20 h. Luciferase activities were
measured as in A, and relative activities as mean ±
S.E. (n = 3) were shown.
Then, we analyzed the effects of dominant-negative Ras(S17N) on IL-3-dependent cell survival and long-term proliferation (Fig. 4). N6 cells showed IL-3-dependent characteristics: they were not able to survive and proliferate in the absence of IL-3, while, in the presence of increasing concentrations of IL-3, MTT-reducing activity was detected, and the cells continuously proliferated. Interestingly, even when the dominant-negative Ras(S17N) was induced with IPTG, the growth properties did not change at all in spite of the almost complete abrogation of IL-3-dependent stimulation in the Raf/MAPK/Fos pathway as described in Fig. 2and Fig. 3. The results indicate that the Ras pathway is not essential for IL-3-induced growth stimulation in BaF3 cells.
Figure 4:
Effects of Ras(S17N) on proliferation of
N6 cells. A, cell number increase of N6 cells in the culture
medium with () or without (
) IPTG (5 mM). B, MTT assay of N6 cells. N6 cells were cultivated for 48 h in
the culture medium containing various concentrations of mouse IL-3 with
(
) or without (
) IPTG (5 mM). Then, the
colorimetric assay using MTT was performed.
Various adaptor proteins and Ras-GEFs are involved in tyrosine kinase receptor-mediated activation of Ras (Schlessinger, 1993). Similarly, lymphokine receptors, for example IL-2 and IL-3 receptors, activate Ras through the interaction with tyrosine phosphorylated proteins, adaptors, and Ras-GEFs (Burns et al., 1993; Cutler et al., 1993; Sato et al., 1993; Ravichandran and Burakoff; 1994; Welham et al., 1994). To test whether Ras(S17N) or Ras(G12V) might affect IL-3 receptor-stimulated responses of Ras regulators, we compared tyrosine phosphorylation of Shc and its interaction with Grb-2 in BaF3, N6, and V2 cells. As shown in Fig. 5, when stimulated with IL-3 for 7 min, tyrosine phosphorylation of both species of Shc (p46 and p52) and Shc/Grb-2 association were observed in all of the above cell lines. A tyrosine-phosphorylated protein with a molecular weight of 150,000 that was co-immunoprecipitated with Shc (Buday et al., 1994) was also detected in these cell lines (data not shown). Even though Ras(S17N) or Ras(G12V) was induced by IPTG treatment for 16 h, these responses were unaffected, suggesting that the mutant Ras proteins do not interfere with the signal transduction pathways upstream of Ras. To clarify whether IL-3-induced activation of tyrosine kinases and their interaction with downstream signaling molecules are required for cell growth, we next analyzed the effect of a tyrosine kinase-specific inhibitor, herbimycin A. As illustrated in Fig. 6A, treatment of BaF3 cells with herbimycin A prior to the stimulation completely blocked both IL-3-induced tyrosine phosphorylation of Shc and Shc/Grb-2 association. Moreover, herbimycin A-treated BaF3 cells were unable to survive and proliferate even in the presence of IL-3 (Fig. 6B). The results suggest that tyrosine kinase-mediated pathways are critical for the induction of cell growth in response to IL-3.
Figure 5:
Tyrosine phosphorylation and association
of Ras-regulating molecules. A, tyrosine phosphorylation of
p46 and p52
in response
to IL-3. V2, N6, and BaF3 cells were treated with or without IPTG (5
mM) for 16 h. During the last 3 h, the cells were starved in
RPMI 1640 containing bovine serum albumin (1 mg/ml). Then, the cells
were stimulated with mouse IL-3 (50 ng/ml) for 7 min, and the lysate
was prepared. Shc proteins were immunoprecipitated with a specific
antibody (06-203) from the cell lysate (3
10
cells per lane), and the precipitate was subjected to SDS-PAGE
(10% acrylamide) and subsequent immunoblotting using
anti-phosphotyrosine antibody (05-321). B, association
of Grb-2 with Shc in response to IL-3. The same membrane used in A was blotted with anti-Grb-2 antibody
(MS-20-3).
Figure 6:
Effects of herbimycin A on BaF3 cells. A, inhibition of IL-3-stimulated tyrosine phosphorylation of
Shc and Shc/Grb-2 association. BaF3 cells were treated with herbimycin
A (1 µg/ml) for 24 h prior to IL-3 stimulation. Tyrosine
phosphorylation of p46 and p52
and association of Grb-2 with Shc in response to IL-3 were
detected as described in Fig. 5. B, inhibition of cell
number increase. Cell number increase of BaF3 cells cultivated within
the culture medium in the presence (
) or absence (
) of
herbimycin A (1 µg/ml) is shown.
Although Ras is not essential for IL-3-dependent growth stimulation, it is possible that Ras may be involved in the growth signaling pathway. To clarify this point, we next studied the effects of dominant-active Ras(G12V) using V2 cells. In V2 cells, Ras(G12V) was induced after a 4-h treatment with IPTG as shown in Fig. 1. We measured MEK kinase activity of immunoprecipitated endogenous c-Raf-1 protein and the mobility retardation of ERK2 following the induction of Ras(G12V) in IL-3-deprived V2 cells. As shown in Fig. 7, both MEK kinase activity of c-Raf-1 and the hyperphosphorylation of ERK2 were markedly enhanced by Ras(G12V) even in the absence of IL-3 stimulation. In addition, we detected increased transcription from c-fos promoter in IPTG-induced V2 cells in the absence of IL-3 using the luciferase assay (data not shown). Under these conditions, we tested whether the cells are able to survive and proliferate.
Figure 7:
Ras(G12V)-induced activation of c-Raf-1
and ERK2 in V2 cells. A, Ras(G12V) induction of c-Raf-1
activation. V2 cells were treated with IPTG (5 mM) for the
indicated periods. During the last 3 h, the cells were starved in RPMI
1640 containing bovine serum albumin (1 mg/ml). Then, c-Raf-1 protein
was immunoprecipitated with a specific antibody (sc-227) from the cell
lysates (2 10
cells per lane), and the precipitates
were subjected to MEK kinase assay. Phosphorylated GST-MAPK(K57D) was
detected by SDS-PAGE (9% acrylamide) and the following autoradiography. B, Ras(G12V) induction of ERK2 hyperphosphorylation. V2 cells
were treated as described in A. The cellular proteins were
separated by SDS-PAGE (10% acrylamide, 5
10
cells
per lane), and ERK2 was detected by immunoblotting with a specific
antibody (05-157).
Fig. 8illustrates the fragmentation of chromosomal DNA characteristic of apoptotic cell death. IL-3 starvation for 48 h caused apoptosis in both N6 and V2 cells. Whereas Ras(S17N) showed no effect on the induction of DNA fragmentation, Ras(G12V) effectively prevented the cells from the apoptotic cell death. The findings imply the participation of Ras protein in antiapoptosis signaling although Ras is not essential for IL-3-dependent survival of the cells. Fig. 9shows growth properties of V2 cells in the presence or absence of IL-3. Like parental BaF3 and N6 cell lines, V2 cells were incapable of proliferating in the absence of IL-3. When Ras(G12V) was induced, V2 cells were able to survive and continue to proliferate in the absence of IL-3 although the growth rate was significantly decreased in comparison with the normal growth; doubling times were 48 h in the presence of Ras(G12V) and 15 h in the presence of IL-3, respectively. The results suggest that Ras is implicated not only in antiapoptotic signal transduction, but also in growth-promoting signaling to some extent.
Figure 8:
Effects of Ras mutants on DNA
fragmentation induced by IL-3 deprivation. V2 or N6 cells were
cultivated for 48 h, in the culture medium in the presence or absence
of mouse IL-3 and IPTG (5 mM). The cells were harvested, and
DNA fragmentation of the cells (3 10
cells per
lane) was examined as described under ``Experimental
Procedures.'' kbp, kilobase
pair(s).
Figure 9:
Effects of Ras(G12V) on proliferation of
V2 cells. Numbers of V2 cells were measured in the culture medium with
neither mouse IL-3 nor IPTG (5 mM) (), in the presence
of mouse IL-3 (
), or in the presence of IPTG (5 mM)
(
).
Although Ras is activated upon IL-3 stimulation, it has not been clear whether the function of Ras is required for IL3-induced proliferation of hematopoietic cell lines. A dominant-negative mutant Ras(S17N), which binds to Ras-GEFs tightly to prevent its action toward endogenous Ras, has been utilized widely to evaluate whether Ras is essential for a particular signal transduction pathway (Feig and Cooper, 1988). In fibroblast cell lines, for example NIH3T3 cells, the dominant-negative Ras(S17N) efficiently blocks growth factor- or serum-induced DNA synthesis and cell cycle progression (Feig and Cooper, 1988; Cai et al., 1990). The mutant also inhibits nerve growth factor-induced neuronal differentiation of PC12 cells without affecting the cell growth (Szeberényi et al., 1990). The results indicate that the function of Ras signaling pathway may differ depending on cell types.
IL-3 triggers the activation of multiple signal transducing molecules. In the present study, we took advantage of an inducible expression system for dominant-negative and dominant-active mutant Ras proteins to assess the function of Ras in these pathways. Only Ras-independent pathways are activated when dominant-negative Ras(S17N) is induced in the presence of IL-3, whereas only Ras-dependent pathways are activated when dominant-active Ras(G12V) is induced in the absence of IL-3. The system allowed us to evaluate the function of Ras-dependent and Ras-independent signaling pathways in IL-3-dependent pro-B cell line BaF3.
In this paper, it was demonstrated that the cells displayed normal phenotypes, that is IL-3-dependent survival and proliferation even when the dominant-negative form of Ras(S17N) was expressed sufficiently to inhibit the Ras-dependent signal transduction. On the other hand, when the dominant-active form of Ras(G12V) was present, the cells were capable of escaping apoptotic cell death and proliferating without IL-3 stimulation although the growth rate was significantly lowered. Furthermore, it was shown that tyrosine kinase-specific inhibitor, herbimycin A, blocked the cell growth as well as Shc/Grb-2 interaction. Thus, in conclusion, it is likely that tyrosine kinase-mediated, but Ras-independent signaling pathways are essential for transmitting sufficient signals for the induction of cell growth, whereas the Ras-dependent pathway is dispensable.
Several signaling
molecules other than Raf family proteins, for instance,
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), and MEKK (Lange-Carter and Johnson, 1994), have been proposed to
be direct targets of Ras although it is not clear whether these
molecules function within BaF3 cells. Furthermore, Rac-mediated
pathways, which are also downstream from Ras, were recently reported to
be independent of the Raf kinase cascade and essential for malignant
transformation of fibroblast cells (Qiu et al., 1995). We
consider that signaling pathways targeted by RasGTP other than
the Raf/MAPK pathway must be blocked as well if the Raf/MAPK pathway is
completely abolished because all effectors of Ras are thought to
interact with only GTP-bound active conformation of Ras, and Ras(S17N)
inhibits extracellular signal-dependent accumulation of this
conformation. We actually observed that the formation of Ras
GTP
within N6 cells upon IL-3 stimulation was limited to a lower level when
Ras(S17N) was induced. However, if the affinities between Ras and
different target molecules are different, we cannot exclude completely
the possibility that the residual Ras activity may be sufficient for
stimulating a downstream signaling pathway other than the Raf/MAPK
pathway, which is essential for growth stimulation.
We described that the activation of the endogenous c-Raf-1 and ERK2 elicited by IL-3 treatment was reduced considerably when Ras(S17N) was induced. The results also indicate that, in BaF3 cells, the Raf/MAPK pathway is dependent on Ras, which is similar to the signal transduction of platelet-derived growth factor, insulin, and nerve growth factor (de Vries-Smits et al., 1992; Thomas et al., 1992; Wood et al., 1992), but distinct from the case of epidermal growth factor, in which multiple pathways are responsible for the activation of MAPK (Burgering et al., 1993). Likewise, the transcriptional activation of the c-fos gene is dependent on Ras function in BaF3 cells.
Okuda et al.(1994) recently reported that 32D myeloid cells failed to proliferate in response to IL-3 when the dominant-negative Ras(S17N) was induced, whereas they were able to survive for more than 2 weeks in the presence of Ras(S17N) and IL-3. In addition, they found that granulocyte colony-stimulating factor (G-CSF)-induced differentiation to neutrophiles was not affected by Ras(S17N) (Okuda et al., 1994). The discrepancy between their results and ours may be due to the difference in the signal transduction networks between the two types of hematopoietic cells.
IL-3 and GM-CSF receptors share the common subunit, which
plays a pivotal role in signal transduction. From analyses using a set
of deletion mutants of the common
subunit, a cytoplasmic region
responsible for Ras activation was identified (Sato et al.,
1993). Kinoshita et al.(1995) have recently reported that
mutant GM-CSF receptors lacking the ability to stimulate the Ras/Raf
pathway failed to suppress apoptosis, but they were still able to exert
DNA synthesis. In addition, they showed that the activated Ras(G12V)
could overcome the mutants' inability to prevent the cell death.
In our experiments, the expression of Ras(S17N) did not result in
apoptosis (Fig. 8), and the cells could proliferate continuously (Fig. 4). Hence, it is possible that a Ras-independent pathway
that is sufficient for the survival of the cells may be activated
through the C-terminal domain of the common
subunit.
In IL-2
signal transduction, it has been proposed that three distinct signaling
pathways mediated by Bcl-2, Myc, and a tyrosine kinase Lck,
respectively, are regulated by IL-2 receptor (Miyazaki et al.,
1995). Analyses using deletion mutants of the IL-2 receptor
subunit have revealed that the activation of a tyrosine kinase is
required for the induction of Ras
GTP formation, as well as the
fact that a mutant that is incapable of stimulating the Ras pathway can
stimulate DNA synthesis (Satoh et al., 1992a). The results
imply that Ras-dependent signals may not crucial for proliferation also
in IL-2 signaling system.
Several kinds of Ras-independent pathways,
for example the JAK/STAT pathway and myc-related pathway, have
been reported. A member of the JAK family tyrosine kinases, JAK2, is
known to bind to the membrane-proximal region of the common
subunit of IL-3 receptor. Following IL-3 stimulation, JAK2 is activated
and subsequently phosphorylates STAT5, which induces the transcription
of specific genes (Silvennoinen et al., 1993; Quelle et
al., 1994; Azam et al., 1995; Mui et al., 1995)
although the role of this pathway has not been fully manifested.
Furthermore, it is possible that adaptors, such as Shc and Grb-2, which
function downstream from tyrosine kinases, may also regulate a
Ras-independent signaling cascade for cell growth. Clarification of the
role of Ras in the network of multiple signaling pathways, including
proliferation and differentiation, stimulated through lymphokine
receptors must await further investigation.