INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

The three ras proto-oncogenesencoding H-Ras, K-Ras4A, K-Ras4B, and N-Ras (2)have each been implicated in the regulation of differentiation, cell growth, and cell proliferation (33, 34). Ras proteins participate in various extracellular signaling cascades initiated from a number of receptor and nonreceptor tyrosine kinases and thus serve to communicate information from the cell surface to the nucleus (50). In this context, they operate as molecular switches activated by guanine nucleotide exchange factor-mediated enrichment of active, GTP-bound forms (45). A number of effector pathways are downstream of Ras, the best characterized of which is the Raf/MEK/MAPK kinase cascade. This mitogenic signaling pathway is perhaps the archetypal example of Ras-mediated communication from the extracellular milieu to the nucleus. Indeed, virtually all studies focused on Ras signaling pertain to the unidirectional flow of information from the cytoplasm to the nucleus.

Like Ras, the retinoblastoma protein pRb is also involved in regulation of cell proliferation during G₁ and differentiation processes (7, 17, 60). pRb controls cell cycle progression, at least in part, through regulation of the E2F family of transcription factors, which is in turn mediated by phosphorylation events (14, 60). During mid-G₁, the initial phosphorylation events on pRb are controlled by D-type cyclins (cyclins D1, D2, and D3) (55). Consistent with the notion of nuclear proteins being the ultimate target of Ras-mediated signaling, each member of the Ras/Raf/MEK/MAPK pathway has been implicated in the regulation of cyclin D1 and thereby the state of pRb phosphorylation and its cell cycle function (11). Indeed, Rb-deficient fibroblasts are resistant to the G₁ arrest induced by either Ras inactivation (29, 37, 44) or cyclin D1 neutralization (32). Thus, pRb appears a legitimate downstream target of Ras action. A connection between Ras and pRb has also recently been demonstrated genetically in Caenorhabditis elegans. However, the LIN-35 pRb/LET-60 Ras communication appears not to be involved in the regulation of proliferation in this system (31).

Both Ras and pRb also control differentiation (5, 7, 28, 34, 62, 63). Unlike cell cycle progression, a link between pRb and Ras during differentiation is not immediately apparent, although available evidence suggests that such a connection may exist. For example, either loss of pRb or ectopic expression of constitutively active Ras impairs the transcriptional functions of MyoD (20, 24, 38, 40, 47, 53). Thus, both the presence of pRb and the regulation of Ras activity appear to be important for proper MyoD function.

It has recently been demonstrated that Ras activation following restimulation of quiescent murine fibroblasts is biphasic, with peaks of activation soon after growth factor addition and then again in mid-G₁ (58). In the same body of work, it was shown that the mid-G₁ activation of Ras in a human cervical carcinoma line, HeLa, was independent of serum factors, but was dependent on de novo mRNA and protein synthesis (58). Since HeLa cells express the E7 oncoprotein, which binds to and inactivates pRb (15), one interpretation of these results is that pRb might influence Ras activation. We have directly addressed this possibility by using mouse embryo fibroblasts (MEFs) derived from Rb-deficient embryos and show that these cells display elevated levels of activated N- and K-Ras. Additionally, we provide evidence suggesting that the ability of pRb to regulate the activation of Ras may be linked to the influence of pRb on differentiation. These data suggest that signaling from Ras to the nucleus is bidirectional.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Cell culture. Rb^+/+ and Rb^/ MEFs and their immortalized 3T3 derivatives have been described previously (44). MEFs derived from p107-deficient mice were kindly provided by T. Jacks, N. Dyson, and E. Harlow. 3T3 derivatives of p107^/ MEFs were generated as described previously (59). All MEFs and their 3T3 derivatives were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). NIH 3T3 cells and their cyclin D1 derivatives (46) were cultured in DMEM containing 5% bovine calf serum. Rb^/ 3T3 cells expressing hemagglutinin (HA)-tagged versions of wild type and mutant pRb were created by cotransfecting pSG5L-HA-pRB, pSG5L-HA-pRBex22, pSG5L-HA-pRB;661W or pSG5L-HA-pRBex4 (53) together with pBABE-puro, with subsequent selection in puromycin (3 µg/ml). Both stable clones (see Fig. 2) and pooled populations (see Fig. 4) were used for analysis. pRb expression was reconstituted in Rb^/ MEFs by retroviral infection. The retroviral vector encoding human pRb, pBPJTR2-pRb, was constructed by subcloning the Rb cDNA from pSG5-Rb into pBPJTR2 (42). The packaging cell line, Bosc23 (43), was transfected with pBPJTR2-pRb, and the resulting retroviral supernatant was used for infections. After 3 days of puromycin selection, pooled populations were used for analysis. Rb^/ 3T3 cells expressing MyoD were similarly constructed by using a retroviral vector encoding MyoD (pBabe-MyoD [38]). Stable lines of Rb^+/+ 3T3 cells expressing T antigen and the K1 mutant were created by cotransfecting pSG5-WT T antigen (TAg) or pSG5-K1 TAg (64) together with pBABE-puro, followed by selection in puromycin. For serum starvation, cells were maintained in DMEM containing 0.2% FBS for 72 h.

Ras activation assays. Cells were washed twice with ice-cold HBS (25 mM HEPES [pH 7.5], 150 mM NaCl) and lysed in Mg²⁺-containing lysis buffer (25 mM HEPES [pH 7.5], 150 mM NaCl, 1% NP-40, 0.25% Na deoxycholate, 10% glycerol, 25 mM NaF, 10 mM MgCl₂, 1 mM EDTA, 1 mM Na₃VO₄, 250 µM phenylmethylsulfonyl fluoride, 10 µg of leupeptin per ml, and 10 µg of aprotinin per ml). Lysates were clarified by centrifugation, and protein concentrations were determined (Bio-Rad protein assay). In all assays, 300 µg of the supernatants was incubated with the Ras binding domain (RBD) of cRaf-1 fused to glutathione S-transferase (GST-RBD) to isolate GTP-bound Ras (58). Glutathione-Sepharose beads (15 µl of packed beads [Pharmacia]) were preloaded with GST-RBD (10 µg). After incubation for 40 min at 4°C, beads were washed four times in lysis buffer. Bound proteins, separated on sodium dodecyl sulfate-polyacrylamide gels (12%), were transferred to polyvinylidene difluoride membranes, probed with pan Ras antibody (Ab-3; Oncogene Science), and visualized by enhanced chemiluminescence (ECL; Amersham).

Cell permeabilization and Ras guanine nucleotide binding. Rb^/ and Rb^+/+ 3T3 cells were serum starved and stimulated with DMEM containing 10% FBS for 5 min or 4 h before being washed with warm phosphate-buffered saline. To each dish, 0.36 ml of permeabilization buffer (Trans-port transient cell permeabilization kit; GIBCO/BRL) was added and diluted with Trans-port reagent, immediately followed by the addition of 10 µCi of [-³²P]GTP (3,000 Ci/mmol; NEN) (time zero). At various times thereafter, the supernatant was removed, and cells were lysed in 1% Triton X-100 buffer (50 mM HEPES [pH 7.4], 1% Triton X-100, 100 mM NaCl, 5 mM MgCl₂, 1 mg of bovine serum albumin per ml, 250 µM phenylmethylsulfonyl fluoride, 10 µg of leupeptin per ml, 10 µg of aprotinin per ml) containing 0.1 mM unlabeled GTP. Ras was immunoprecipitated from equal amounts of total cellular protein by using Y13-259 (American Type Culture Collection) as described previously (12). Total specific radioactivity was determined by Cerenkov counting for ³²P. Duplicate samples were analyzed, and values were averaged.

Transcriptional transactivation assays. For MyoD transactivation assays, Rb^+/+ and Rb^/ 3T3 fibroblasts were plated onto 60-mm-diameter dishes at 1.5 × 10⁵ and 1 × 10⁵ cells per plate, respectively. The cells were transfected (44) as indicated with 1 µg of pCSA-MyoD (38), 2 µg of pMCK-Luc (gift from A. Lassar), 1 µg of pCMV-Gal, 2 to 4 µg of pSG5L-HA-RB (53), 0.25 to 0.75 µg of pMT-Ras^N17 (18), or empty vector plasmid. Forty-eight hours after transfection, the cell culture medium was changed to differentiation medium (DMEM containing 2% horse serum). Luciferase and -galactosidase activities were assayed 48 h later. For glucocorticoid receptor alpha (GR) transactivation assays, Rb^+/+ and Rb^/ 3T3 fibroblasts were plated onto 60-mm-diameter dishes at 2 × 10⁵ and 1.5 × 10⁵ cells per plate, respectively. The cells were transfected as indicated with 0.5 µg of pRS-hGR (19), 2 µg of pMMTV-GRE-Luc (gift from W. Chin), 1 µg of pCMV-Gal, 1 to 2 µg of pSG5L-HA-RB, 0.25 to 0.75 µg of pMT-Ras^N17, or empty vector plasmid. Twenty-four hours after transfection, dexamethasone (1 µM) was added. Luciferase and -galactosidase activities were determined 24 h later. For E2F transactivation assays, 2 × 10⁵ Rb^/ 3T3 cells were transfected as indicated with 150 to 450 ng of pRc/CMV-E2F-1 (gift from E. Flemington), 1 µg of 3X(E2F)DHFR-Luc (gift from E. Flemington), 1 µg of pCMV-Gal, 0.25 to 0.75 µg of pMT-Ras^N17, or empty vector plasmid. Luciferase and -galactosidase activities were determined 48 h later.

MHC induction. Rb^/ 3T3 cells stably expressing MyoD were transfected with plasmids encoding Ras^N17, pRb, or the vector control and subsequently placed in differentiation medium. Forty-eight hours later, cell lysates were prepared, and 10 µg was resolved on a denaturing gel. After transfer to a polyvinylidene difluoride membrane, the blot was probed with a monoclonal antibody to myosin heavy chain (MHC) (MF20 [1]; Developmental Studies Hybridoma Bank) followed by enhanced chemiluminescence detection (Amersham).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Rb-deficient fibroblasts display elevated levels of activated N- and K-Ras. To determine whether loss of pRb influences Ras activity, we analyzed the activation state of Ras during the G₁ interval in MEFs derived from genotyped Rb^/ and Rb^+/+ littermate embryos, and their derivatives were immortalized according to a defined 3T3 protocol. The activation state of Ras was determined with an assay that is based on the fact that active, GTP-bound forms of Ras bind to Raf-1, while inactive, GDP-bound Ras does not (58).

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View larger version (44K): [in this window] [in a new window]   FIG. 1.   Ras activation in Rb/ and Rb+/+ fibroblasts and other cell strains. (A to D) The indicated cell types were serum starved for 72 h and restimulated by the addition of serum. At the indicated times, lysates were prepared, and the presence of activated N- and K-Ras in equal amounts of total protein was assayed. Whole-cell lysates (WCL) at 15 µg (1X) or 30 µg (2X) were analyzed for total Ras protein; asterisks indicate that 30 and 60 µg were loaded. Each panel is representative of at least five independent experiments.

View larger version (34K): [in this window] [in a new window]   FIG. 2.   Reconstitution of pRb in Rb-deficient fibroblasts and the effect on Ras activation. (A) Individual clones of Rb/ 3T3 cells transfected with or without a pRb-encoding plasmid were analyzed for pRb expression by immunoprecipitation followed by Western blot analysis (pRb-clone 1 and -2 and vector). Rb/ MEFs were infected with a pRb retrovirus (JTR2-pRb) or control retrovirus (vector), and pooled populations were analyzed for pRb expression. (B) Rb/ 3T3 clones described for panel A were analyzed for the presence of activated N- and K-Ras as described in the legend to Fig. 1. Fifteen micrograms of whole-cell lysate (WCL) was analyzed for total Ras proteins; an asterisk indicates that 30 µg instead of 15 µg of WCL was loaded. (C) Same as panel B, except Rb/ MEFs were analyzed. (D) N- and K-Ras activation during reentry to the cell cycle from G0. Rb/ 3T3, Rb+/+ 3T3, and an Rb/ 3T3 clone in which pRb expression was reconstituted (clone 1) were used for the analysis. Ras activation was expressed as the ratio of activated N- or K-Ras to total N- or K-Ras expressed as a percentage following densitometric scanning of autoradiograms represented in Fig. 1 and 2B. Serum-starved and EGF-treated cells (10 ng/ml for 10 min) were analyzed in parallel. Bars represent the averages plus standard deviations for at least three independent experiments. (E) Same as panel D, except primary MEFs were used.

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Reconstitution of pRb in Rb-deficient fibroblasts decreases levels of activated N- and K-Ras. To rule out the possibility that the effect of pRb on the levels of GTP-bound N- and K-Ras might be due to a genetic event other than loss of Rb, we reintroduced Rb into Rb-deficient fibroblasts and analyzed Ras activity. The expression of pRb in two such representative clones is shown in Fig. 2A. These clones showed a pattern of Ras activation during G₁ that was similar to that found with Rb-positive fibroblasts (Fig. 2B). Similar results were obtained when a pooled population of Rb^/ primary MEFs, in which pRb expression had been reconstituted by retroviral infection, was analyzed (Fig. 2A and C). In both the primary and immortalized Rb^/ cells, reintroduction of pRb decreased the level of N- and K-Ras activation in mid-G₁ by approximately 13- and 10-fold, respectively (Fig. 2D and E). Thus, at this level of analysis, the effect of pRb loss on N- and K-Ras activation appears to be reversible and not attributable to an adaptive mutation.

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Viral oncoproteins induce activation of Ras. To further exclude the possibility that a pRb-independent mechanism might be responsible for the observed aberrant levels of active Ras in Rb^/ cells, we inactivated pRb function in Rb^+/+ cells and determined the effect on Ras. To this end, Rb^+/+ 3T3 cells expressing simian virus 40 large TAg which can bind to and inactivate pRb, were generated (Fig. 3A). The profile of Ras activation during the G₁ interval in Rb-positive cells expressing T antigen was similar to that observed in Rb^/ 3T3 cells (Fig. 3B). Analogous results have been reported for murine C3H10T1/2 cells expressing TAg (48). In contrast, Rb^+/+ 3T3 cells expressing a mutant of TAg (K1) that fails to bind to pRb (10) did not exhibit elevated levels of activated N- and K-Ras (Fig. 3). Similar observations were made with Rb^+/+ and NIH 3T3 cells expressing human papillomavirus E7 (data not shown). Thus, functional inactivation of pRb through the expression of viral oncoproteins results in increased levels of Ras activity.

View larger version (33K): [in this window] [in a new window]   FIG. 3.   Ras activation in Rb+/+ 3T3 cells expressing simian virus 40 TAg. (A) Rb+/+ 3T3 lines expressing wild-type (wt) TAg mutant TAg (K1), or the vector control were analyzed for expression of TAg. (B) Rb+/+ 3T3 lines described for panel A were analyzed for the presence of activated N- and K-Ras as described in the legend to Fig. 1. WCL, whole-cell lysate. Results are representative of at least five independent experiments.

Protein products encoded by partially penetrant alleles of Rb retain the ability to regulate Ras activity. Classical familial retinoblastoma is attributable to germ line mutations in the Rb gene. These mutations result in bilateral tumors in 90% of carriers. However, Rb mutations have been identified in which the carriers are either absent of disease, develop unilateral retinoblastoma, or suffer benign retinomas (13, 25, 30). The protein products encoded by such "partially penetrant Rb alleles" have been shown to be defective for a subset of known pRb functions (25, 53, 61), including the ability to bind E2F. In an effort to gain further insight into which functions of pRb might be involved in the regulation of Ras activity, we determined whether the protein products encoded by two partially penetrant Rb alleles, 661W (amino acid substitution) and ex4 (deletion of exon 4), retain the ability to affect Ras activation. Expression of either of these pRb mutants in Rb^/ 3T3 cells resulted in a marked downmodulation of Ras activation, similar to that observed upon reintroduction of wild-type pRb (Fig. 2 and 4). In contrast, ectopic expression of a pRb mutant defective in all known functions of pRb, ex22 (deletion exon 22), had no effect on Ras activation (Fig. 4). At this level of analysis, these results indicate that the protein products of two partially penetrant alleles of Rb retain the ability to regulate the activation state of Ras and suggest that this function of pRb is separable from its role in E2F regulation.

View larger version (74K): [in this window] [in a new window]   FIG. 4.   Effect of protein products encoded by partially penetrant mutants of Rb on Ras activation. Individual lines of Rb/ 3T3 cells expressing pRb;661W, pRbex4, or pRbex22 and control (empty vector) lines were analyzed for the presence of activated N- and K-Ras as described in the legend to Fig. 1. Whole-cell lysates (WCL) at 15 µg (1X) or 30 µg (2X) were analyzed for total Ras protein; an asterisk indicates that 30 and 60 µg were loaded. wt, wild type. Results are representative of five independent experiments.

Inhibition of Ras activity restores pRb-dependent transcription and the expression of a late marker of skeletal muscle differentiation. In an effort to demonstrate a biological consequence of elevated Ras activation resulting from loss of pRb, we considered the known properties of Rb-deficient fibroblasts. If aberrant Ras activation was indeed responsible for the characteristic cell cycle or differentiation defects associated with pRb loss, we would predict that inhibition of Ras activity in a pRb-negative background should restore these functions. Although not ruling out the possibility that the effect of pRb loss on Ras activation does, in some way, impinge upon cell cycle and/or growth control, it has already been established that Ras inactivation in Rb-deficient fibroblasts is without significant impact on G₁ cell cycle progression (29, 37, 44). The mechanisms by which pRb controls differentiation are less well characterized than those of pRb-mediated regulation of E2F, although pRb appears to regulate a number of transcription factors involved in promoting these processes (5, 6, 20, 57). Importantly, the pRb mutants 661W and ex4, which have lost the capacity to regulate E2F while retaining the ability to potentiate certain differentiation processes (53), behave essentially like the wild-type protein in relation to Ras activation (Fig. 4). We considered the possibility that loss of pRb leading to aberrant levels of N- and K-Ras activation might be linked to the inability of Rb-deficient cells to support certain aspects of differentiation.

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View larger version (25K): [in this window] [in a new window]   FIG. 5.   Effect of inhibition of Ras activity on transcriptional activation in Rb/ and Rb+/+ 3T3 cells. (A) Rb/ 3T3 cells were transfected with plasmids encoding MyoD, RasN17, and pRb. A MyoD-responsive reporter, pMCK-Luc, and a -galactosidase-encoding plasmid were included in each case. After transfection, cells were placed in differentiation medium, and luciferase and -galactosidase activities were determined 48 h later. Relative luciferase activities, normalized for -galactosidase activity, were calculated. Bars represent the averages plus standard deviations for four independent experiments. (B) Same as panel A, except Rb+/+ 3T3 cells were used. (C) Rb/ 3T3 cells were transfected with plasmids encoding GR, RasN17, and pRb. A GR-responsive reporter, MMTV-GRE-Luc, and a -galactosidase-encoding plasmid were included. After transfection, cells were treated with dexamethasone for 24 h, at which time, relative luciferase activities, normalized for -galactosidase activity, were determined. Bars represent the averages plus standard deviations for five independent experiments. (D) Same as panel C, except Rb+/+ 3T3 cells were used. (E) Rb/ 3T3 cells were transfected with plasmids encoding E2F-1 and RasN17. An E2F-responsive reporter, 3X(E2F)DHFR-Luc, and a -galactosidase-encoding plasmid were included. Twenty-four hours later, relative luciferase activities, normalized for -galactosidase activity, were determined. Bars represent the averages plus standard deviations for three independent experiments. (F) Rb/ 3T3 cells infected with a retrovirus encoding MyoD were transfected with plasmids encoding RasN17 (0.5, 1, 2.5, or 5 µg), pRb (0.5 or 1 µg), or a vector control (V). After transfection, cells were placed in differentiation medium for 48 h. At this time, the expression of MHC and CDK4 (as a loading control) was monitored by Western blot analysis. The results are representative of at least four independent experiments.

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Increased Ras activation in Rb-deficient fibroblasts occurs at the level of guanine nucleotide exchange. To explore the mechanism by which loss of pRb leads to increased levels of Ras activation, we considered processes known to be involved in the regulation of Ras. There are two complementary ways in which wild-type Ras activation can be upregulated: (i) increased guanine nucleotide exchange, mediated by guanine nucleotide exchange factors (45); and (ii) decreased GTPase activity through downmodulation of GTPase activating proteins (3). We attempted to discriminate between these two alternative mechanisms by measuring the rate of guanine nucleotide binding to Ras, an event thought to be regulated by exchange factors.

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View larger version (14K): [in this window] [in a new window]   FIG. 6.   Ras guanine nucleotide binding in Rb/ and Rb+/+ 3T3 cells. Rb/ and Rb+/+ 3T3 cells were serum starved for 72 h before restimulation. Five minutes (top panel) or 4 h (bottom panel) later, cells were permeabilized and incubated with [-32P]GTP. At the indicated times thereafter, cell lysates were prepared and subjected to immunoprecipitation with anti-Ras antibody. The recovered radioactivity was quantified and plotted as described in Materials and Methods. Points represent the averages plus standard deviations for three independent experiments.

Ras activation in Rb-deficient fibroblasts is cycloheximide sensitive. The mid-G₁ activation of Ras in HeLa cells, unlike growth factor-induced activation of Ras, has been reported to be sensitive to cycloheximide-induced inhibition of protein synthesis (58). This information was used to further characterize the pathway leading to aberrant levels of Ras activity induced by loss of pRb. Specifically, we sought to determine the effect of cycloheximide treatment on Ras activation in our experimental system induced by either loss of pRb or growth factor stimulation. However, even a moderate inhibition of protein synthesis can have a profound impact upon fibroblast cell cycle progression (4, 41). So, in an attempt to minimize possible effects secondary to cell cycle perturbation and to map the periods of cycloheximide sensitivity with high resolution, cells were treated for 30 min at various times after release from quiescence to determine the effect of cycloheximide on Ras activation during G₁.

View larger version (55K): [in this window] [in a new window]   FIG. 7.   Effect of cycloheximide treatment of Ras activation. (A) Quiescent Rb/ and Rb+/+ 3T3 cells were restimulated with serum. At the indicated times, the activation state of Ras was measured. Cycloheximide (CHX) was added (25 µg/ml) to cultures 30 min before the indicated time point when levels of GTP-bound Ras were determined. Also shown are the levels of GTP-bound Ras determined 10 min after the addition of EGF alone (lane 17) or EGF with cycloheximide (lane 18) at time zero. In lane 11, cycloheximide was added at 3.5 h after serum stimulation and EGF was added 20 min later. Analysis of the activation state of Ras was performed at 4 h. The results shown are representative of at least 10 independent experiments. (B) Same as panel A, except primary MEFs were used, and the cycloheximide treatments at 30 min and 1 h were not included.

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	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Both Ras and pRb regulate proliferation and differentiation. It would seem appropriate then, that in order to bring about orderly changes in cell behavior, each regulated these cellular processes in a coordinate manner. One means of achieving this is through communication. Indeed, in the context of cell cycle progression, the G₁ arrest induced by Ras inactivation is pRb dependent, placing pRb downstream of Ras (29, 37, 44). Here we provide evidence that this communication can also operate in the other direction because pRb has been shown to regulate N- and K-Ras activation.

pRb, Ras activation, and cell cycle control. A current model of pRb function known as the pRb pathway indicates that the upstream regulators of pRb function (G₁ cyclins, cyclin-dependent kinases [CDKs], and CDK inhibitors) influence the phosphorylation status of pRb and thereby its ability to regulate its best characterized downstream target, the E2F family of transcription factors. This model explains how pRb regulates proliferation and describes how deregulation or inactivation of each component of this pathway can confer a selective proliferative advantage and thus predispose to cancer (54). The results presented here suggest that the effect of pRb on N- and K-Ras activation may not be a function of the status of pRb phosphorylation, but rather the absence of pRb or its functional inactivation by viral oncoproteins (Fig. 1 and 3). Ectopic expression of cyclin D1 in fibroblasts has been shown to lead to premature activation of CDK4 and phosphorylation of pRb (26, 36, 49). However, enforced expression of cyclin D1 does not appear to significantly alter the activation state of Ras (Fig. 1D). Furthermore, in these cells as well as in parental NIH 3T3 cells, Ras activation peaks at approximately 2 h after growth factor stimulation (Fig. 1), several hours before the activation of cyclin D1-CDK4 in these cells (26).

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pRb, Ras activation, and the control of differentiation-specific transcription. The role of pRb in differentiation has been studied mostly in the context of skeletal muscle. Levels of pRb increase during myoblast differentiation (9, 16, 35), and although Rb^/ embryos die at approximately day 13 of gestation with what appears to be normal skeletal muscle (8, 22, 27), partial restoration of pRb function does reveal critical defects in muscle differentiation later in development (63). MyoD is comparatively inactive in a pRb-negative background, and in such cells, wild-type pRb and a partially penetrant mutant of pRb have been shown to restore MyoD-dependent transactivation and the expression of late markers of differentiation (20, 38, 53). Given these observations, we considered the possibility that the ability of pRb to regulate the activation state of Ras may be part of the mechanism by which pRb operates as a regulator of differentiation. To this end, we tested whether the effect of pRb loss on the activation state of Ras was causally related to the inability of MyoD to promote differentiation of Rb-deficient cells. Our data suggest that reintroduction of pRb and inhibition of Ras activity in Rb-deficient fibroblasts are, to a certain degree, functionally equivalent with respect to MyoD function (Fig. 5). Additionally, we show that the expression of MyoD together with dominant-negative Ras in Rb^/ fibroblasts can induce the expression of MHC, a late marker of muscle differentiation (Fig. 5).

Clues to the mechanism of Ras activation following pRb loss. Our data indicate that the rate of guanine nucleotide binding to Ras is higher in Rb^/ fibroblasts than in Rb^+/+ fibroblasts (Fig. 6). This suggests that the observed elevation of activated N- and K-Ras resulting from loss of Rb is due to an enhanced rate of GTP binding to these proteins. However, we have not ruled out the possibility that the intrinsic GTPase activity of N- and K-Ras is modulated as a function of Rb status. In preliminary studies, we have compared the levels of mSos1, mSos2, EGF receptor, Shc, GRB2, and p120RasGAP in Rb^/ and Rb^+/+ 3T3 cells and found no significant difference in the abundance of these proteins (data not shown). Thus, at this level of analysis, the abundance of the well-characterized upstream regulators of Ras activation does not appear to explain why Ras activation is elevated in Rb-deficient fibroblasts.

Concluding remarks. We have demonstrated that loss of pRb leads to a significant elevation in the levels of GTP-bound, active N- and K-Ras in murine fibroblasts, suggesting communication from the nucleus to the inner plasma membrane. The mid-G₁ activation of Ras in Rb-deficient fibroblasts requires de novo protein synthesis, indicating that immediate-early activation and mid-G₁ activation of Ras occur via distinct mechanisms. These observations likely have direct bearing on the roles of pRb and Ras in differentiation and possibly restriction point control. We have provided evidence suggesting that the aberrant levels of activated Ras in Rb-deficient cells are causally linked to the failure of these cells to execute a differentiation program. These findings reveal an additional component of pRb function in tumor suppression.