Rheb Is in a High Activation State and Inhibits B-Raf Kinase in Mammalian Cells

Rheb is in a high activation state and inhibits B-Raf kinase in mammalian cells

Edward Im1,4, Friederike C von Lintig1,4, Jeﬀrey Chen1, Shunhui Zhuang1, Wansong Qui1, Shoaib Chowdhury2,3, Paul F Worley2,3, Gerry R Boss1 and Renate B Pilz*,1

1Department of Medicine and Cancer Center, University of California, San Diego, La Jolla, California, CA 92093-0652, USA; 2Department of Neuroscience, Johns Hopkins University, Baltimore, Maryland, MD 21205, USA; 3Department of Neurology, Johns Hopkins University, Baltimore, Maryland, MD 21205, USA

Rheb (Ras homolog enriched in brain) is a member of the activity (Yamagata et al., 1994). Rheb was subsequently Ras family of proteins, and is in the immediate Ras/Rap/ found to be widely, if not ubiquitously, expressed in Ral subfamily. We found in three different mammalian human tissues, with highest levels found in skeletal and cell lines that Rheb was highly activated, to levels much cardiac muscle, testes, and ovary (Gromov et al., 1995; higher than for Ras or Rap 1, and that Rheb’s activation Clark et al., 1997; Mizuki et al., 1996). Within the state was unaffected by changes in growth conditions. extended group of Ras-related proteins, it is most Rheb’s high activation was not secondary to unique closely related to the Ras, Rap, Ral subfamily with glycine to arginine, or glycine to serine substitutions at 35.8% sequence identity to H-Ras and 525% identity positions 14 and 15, corresponding to Ras residues 12 with the Rho, Rab, and ARF subfamilies (Yamagata et and 13, since Rheb R14G and R14G, S15G mutants had al., 1994; Mach et al., 2000). Moreover, it shares with similarly high activation levels as wild type Rheb. These Ras and Rap six of the nine amino acids in the Ras core data are consistent with earlier work which showed that effector domain with two of the non-identical amino purified Rheb has similar GTPase activity as Ras, and acids being conservative substitutions (Yamagata et al., suggest a relative intracellular deficiency of Rheb 1994; Yee and Worley, 1997). However, almost all Ras GTPase activating proteins (GAPs) compared to Rheb family members have a conserved glycine at position 12 activators. Further evidence for relatively low intracel- in the G1 domain, but the corresponding residue in lular GAP activity was that increased Rheb expression mammalian Rheb (position 15) is arginine; any change led to a marked increase in Rheb activation. Rheb, like in this position in Ras results in activation which Ras and Rap1, bound B-Raf kinase, but in contrast to suggests that wild type Rheb could be constitutively Ras and Rap 1, Rheb inhibited B-Raf kinase activity and active (Barbacid, 1987; Yamagata et al., 1994). There, prevented B-Raf-dependent activation of the transcrip- however, have been no measurements in cells of Rheb tion factor Elk-1. Thus, Rheb appears to be a unique activation, i.e., the per cent of Rheb molecules in the member of the Ras/Rap/Ral subfamily, and in mamma- active GTP-bound state. lian systems may serve to regulate B-Raf kinase activity. Because of the high similarity of Ras and Rheb in Oncogene (2002) 21, 6356 – 6365. doi:10.1038/sj.onc. the effector domain, it was postulated that Rheb could 1205792 interact with Ras effectors and, indeed, two groups have shown that purified Rheb binds Raf-1 kinase in Keywords: Rheb; B-Raf kinase; MAP kinase; mamma- vitro (Yee and Worley, 1997; Clark et al., 1997). The lian cells Rheb-Raf-1 interaction appeared to be physiologically relevant because one of the groups showed that transfected Rheb cooperated synergistically with Raf- 1 to transform NIH3T3 cells, as potently as the Introduction combination of wild type Ras and Raf-1 (Yee and Worley, 1997). However, the other group demonstrated Rheb was first described in 1994 by differential cloning that Rheb antagonized transformation of NIH3T3 cells of neuronal activity-dependent genes in adult rat induced by an oncogenic constitutively-active Ras, and hippocampus; Rheb mRNA was found to be rapidly decreased transcription from a Ras-responsive promo- induced in hippocampal granule cells by seizures and by ter (Clark et al., 1997). One way, at least in part, to N-methyl-D-aspartate receptor-dependent synaptic reconcile these seemingly disparate results would be if Rheb inhibits B-Raf kinase. Compared with Raf-1, B- Raf has a higher basal kinase activity and interacts with their shared targets MEK-1 and MEK-2 with *Correspondence: RB Pilz; E-mail: [email protected] 4These two authors contributed equally to the work greater affinity; as a consequence, B-Raf appears to be Received 17 October 2001; revised 6 June 2002; accepted 18 June the major Ras effector and MEK activator in several 2002 cell types including some strains of NIH3T3 cells Rheb and B-Raf kinase EImet al 6357 (Moodie et al., 1994; Eychene et al., 1995; Papin et al., fibroblasts, and we found Rheb protein in baby 1995, 1996, 1998; Catling et al., 1994; Jaiswal et al., hamster kidney (BHK) cells (Figure 1, lane 6), but 1994; Pritchard et al., 1995; Reuter et al., 1995; Vossler we did not detect a definite signal for Rheb in CHOK- et al., 1997). MEK is the immediate upstream activator 1 cells (Figure 1, lane 1). of the mitogen-activated protein (MAP) kinases, ERK- 1 and ERK-2, which are required for many of the Activation levels of endogenous Rheb and of transfected growth-promoting effects of Ras (Campbell et al., Rheb 1998). Thus, in transfection experiments with over- expressed proteins, the combination of Rheb and Raf-1 We assessed the activation state of Rheb, both of the could be transforming if Rheb activates Raf-1, while endogenous protein in neuronal cells as well as of Rheb Rheb by itself could antagonize Ras functions by expressed from the pRK5-myc-Rheb vector in CHOK- inhibiting B-Raf kinase. 1 cells. As described in Materials and methods, we used In this study, we show that endogenous Rheb is highly a modification of a method for measuring Ras activated, even under basal conditions, and that the activation (Scheele et al., 1995; Dupuy et al., 2001; activation state of transfected Rheb increases progres- Prigent et al., 1996; Sharma et al., 1998; Suhasini et al., sively with the amount of Rheb expressed; the latter data 1998), and this assay measures the amount of GTP and would appear to have important implications concern- the sum of GTP plus GDP bound to Rheb. All ing the interpretation of data from experiments in which samples are split in half, with one-half of the sample Ras and other small G proteins are exogenously receiving an anti-Rheb or anti-myc antibody (the expressed in cells. In addition, we show that Rheb and experimental sample), and the other half of the sample B-Raf kinase are associated in vivo, and that Rheb receiving non-specific immunoglobulin (control inhibits B-Raf kinase activity and B-Raf-dependent sample). Values presented are the difference between activation of the transcription factor Elk-1. experimental and control samples with control samples generally being 510% of experimental samples. For logarithmically growing C6 cells, we found Results 0.5+0.04 fmol/mg DNA of GTP bound to Rheb, and 2.08+0.2 fmol/mg DNA of GTP+GDP bound to Rheb expression in NB2A, C6, PC-12, BHK, and Rheb (mean+s.d. of three independent experiments CHOK-1 cells performed in duplicate; corresponding values for the Because Rheb is widely expressed in brain tissue, we control samples were 0.03 and 0.15 fmol/mg DNA). examined Rheb expression in cultured cells of neuronal These values yield a Rheb activation level of 24.3+4% origin. Western blotting demonstrated that Rheb was (Figure 2a, open bar). Interestingly, Rheb’s activation expressed, albeit at relatively low levels, in NB2A state remained 420%, even after 24 – 48 h of serum mouse neuroblastoma cells, C6 rat glioma cells, and starvation, and serum stimulation of serum-starved PC-12 rat pheochromotycoma cells (Figure 1, lanes 3 – cells caused only a small 2 – 4% increase in Rheb 5, respectively; lanes 1 and 2 show Chinese hamster activation. We found similar results in NB2A and, as ovary (CHOK-1) cells transfected with empty vector or described below, in PC-12 cells; in NB2A cells, Rheb Rheb expression vector to positively identify Rheb on activation was similar under all growth conditions, and the blots). The blot in Figure 1 was generated using an was 22+4% in serum-starved, 27+5% in serum- anti-Rheb antibody directed against the C terminus, stimulated, and 26+3% in logarithmically growing but similar results were found with an anti-N terminus cells (mean+s.d. of at least three independent experi- Rheb antibody. Previous reports have found Rheb ments performed in duplicate). These Rheb activation mRNA induction by growth factors in Balb/c3T3 levels, particularly for serum-starved cells, are consid- fibroblasts (Yamagata et al., 1994), and Rheb protein erably higher than we and others have found for other has been detected by Western blotting in NIH3T3 cells wild type Ras-related proteins, including Ras and Rap (Clark et al., 1997). Thus, Rheb is expressed in 1: activation levels for serum-starved cells are generally in the range of 0.5 – 8%, depending on the cell type, and activation levels for logarithmically growing or serum-stimulated cells are generally between 10 – 18% (Scheele et al., 1995; Tian et al., 2000; von Lintig et al., 2000; Sharma et al., 1998; Prigent et al., 1996; Haugh et al., 1999; Satoh et al., 1990a,b). To perform experiments under identical conditions, we measured Figure 1 Western blot analysis for Rheb protein. Logarithmi- Rheb, Ras, and Rap 1 activation in serum-starved PC- cally growing cells were lysed and equal amounts of cell lysate were analysed for Rheb protein expression by Western immuno- 12 cells, and found activation levels of 23+4%, blotting using an anti-Rheb antibody directed against the C termi- 3+0.5%, and 5+0.7%, respectively. Thus, compared nus. Lane 1 untransfected CHOK-1 cells; lane 2, CHOK-1 cells to other Ras-related proteins, Rheb appears to be transfected with 50 ng of the pMT2-Rheb expression vector; lane constitutively in a highly activated state, and its 3, NB2A cells; lane 4, C6 cells; lane 5, PC-12 cells; and lane 6, BHK cells. Similar results were found in two other experiments activation state is unaffected by growth conditions and when blotting with an anti-Rheb antibody directed against (Scheele et al., 1995; Guha et al., 1996; Pilz et al., the N terminus 1997).

Oncogene Rheb and B-Raf kinase EImet al 6358 Table 1 Rheb-bound guanine nucleotides in Rheb-transfected CHOK-1 cells Rheb DNA transfected GTP GTP+GDP (ng) (fmol/mg protein)

15 1.0+0.2 4.1+0.5 30 3.8+0.4 7.0+0.9 60 8.0+0.9 12+1.5 150 17+2.1 21+2.6

CHOK-1 cells grown on 6-well cluster dishes and transfected with the indicated amounts of the pRK5-myc-Rheb expression vector were extracted and the samples were split in half, receiving either a mouse monoclonal anti-myc antibody (experimental sample) or mouse IgG (control sample). To both the control and experimental samples were added protein G agarose beads, and after a 1 h incubation, the beads were washed and heated to 1008C to elute bound nucleotides. GTP, and the sum of GTP plus GDP, were measured in coupled enzymatic assays as described in Materials and methods. The data are diﬀerent between the experimental samples and the control samples with the latter ranging between 0.02 and 0.1 fmol/mg protein for GTP, and 0.1 and 0.5 fmol/mg protein for the sum of GTP+GDP. Values are the mean+s.d. of three independent experiments performed in duplicate

GDP was not linear over the range of DNA transfected, it correlated well with the increase in the amount of Rheb protein expressed at each level of transfected DNA: Rheb protein, as assessed by Western blotting using either an anti-Rheb antibody (Figure 2b, lanes 2 – 5) or an anti-myc antibody (Figure 2c, lanes 2 – 5), showed a progressive but nonlinear increase over the range of DNA transfected. As can be noted in Table 1, the rate of increase in Rheb-bound GTP was greater than that for Rheb-bound GTP+GDP; thus, over the range Figure 2 Rheb activation levels. C6 cells grown to subconfluence of DNA transfected, there was a continual increase on 100 mm plates (a, open bar) and CHOK-1 cells grown on 6- well cluster dishes and transfected with the indicated amounts in Rheb activation from 26.2 to 85.3% (Figure 2a, of the pRK5-myc-Rheb expression vector (a, closed bars) were closed bars; again, the data are for CHOK-1 cells, lysed, and Rheb was isolated by immunoprecipitation using either but similar results were obtained in BHK cells). the anti-Rheb C antibody (C6 cells) or an anti-myc antibody These data provide further evidence that Rheb is (CHOK-1 cells). The activation state of Rheb, i.e., the per cent constitutively in a highly activated state, and suggest of Rheb molecules in the active GTP bound state, was measured by a coupled enzymatic assay as described in Materials and meth- that negative regulators of Rheb’s activation, e.g., ods, and the data are the means+s.d. of at least three indepen- Rheb GTPase activating proteins (GAPs), are at dent experiments performed in duplicate (a). Proteins recovered relatively limiting intracellular concentrations. from the immunoprecipitates were subjected to SDS – PAGE To examine further the cause of Rheb’s high and Western blotting using the anti-Rheb N antibody (b)or anti-myc antibody (c). In (b) and (c), lane 1 is from C6 cells, activation levels, we studied two mutants of Rheb in and lanes 2 – 5 are from CHOK-1 cells transfected with 15, 30, which arginine 15 and serine 16 were changed to 60 and 150 ng of the pRK5-myc-Rheb expression vector glycine. In Ras the corresponding residues at positions 12 and 13 are both glycines, and any mutation at position 12 is activating while some mutations at Since we found high Rheb activation in three position 13 are activating (Barbacid, 1987). Both the different neuronal cell lines, it seemed unlikely that single R15G and the double R15G, S16G Rheb the increased Rheb activation could be from genetic mutants transfected into logarithmically growing mutations in Rheb in these cells. To study Rheb CHOK-1 cells exhibited similar high activation levels activation further, we transfected variable amounts as wild type Rheb, i.e., 25+5% to 80+7% of myc-tagged wild type Rheb into CHOK-1 and (mean+s.d. of three experiments performed in BHK cells, and isolated Rheb using an anti-myc duplicate) over a range of 5 – 100 ng of transfected antibody. We found a progressive increase in total DNA. Thus, the arginine and serine at positions 15 nucleotides (GTP+GDP) bound to Rheb when we and 16 in Rheb do not appear to be responsible for transfected from 15 to 150 ng of the Rheb Rheb’s high activation state, and these data are expression vector (Table 1; data are for CHOK-1 consistent with the finding of Yamagata et al. (1994) cells, but similar results were found for BHK cells). that purified Rheb in vitro has a similar intrinsic Although the increase in Rheb-bound GTP and GTPase activity as Ras.

Oncogene Rheb and B-Raf kinase EImet al 6359 Rheb associates with B-Raf kinase in vivo Previous work has shown that Rheb binds Raf-1 in vitro, and B-Raf kinase was found together with Raf-1 in a complex bound to Rheb (Yee and Worley, 1997; Clark et al., 1997). These data suggest that Rheb may interact with B-Raf kinase in vivo, although B-Raf- kinase could have been associated with other proteins found in the complex including MEK and 14-3-3 proteins (Yee and Worley, 1997). To study a possible direct Rheb-B-Raf kinase interaction, we performed co-immunoprecipitation studies. We transfected CHOK-1 cells with EE-tagged B-Raf kinase, myc- tagged Rheb, or the combination of the two, and after immunoprecipitating with an anti-EE antibody or an anti-myc antibody, we analysed the precipitates by Western blotting with an anti-B-Raf kinase or anti- Rheb antibody (Figure 3). We found B-Raf kinase in the anti-myc immunoprecipitates only when we co- transfected myc-tagged Rheb with B-Raf kinase (Figure 3a, upper panel, compare lane 3 with lanes 1 and 2). Similar amounts of myc-tagged Rheb were in the immunoprecipitates in the absence or presence of co-transfected B-Raf kinase (Figure 3a, lower panel, compare lanes 2 and 3). Based on Western blots of the cell extract, approximately 2 – 3% of cellular B-Raf kinase was associated with Rheb (Figure 3a top panel, compare lanes 3 and 4). Similarly, when B-Raf kinase was immunoprecipitated using the anti-EE antibody, Rheb was detected in the reciprocal experiment (Figure 3b, upper panel, compare lane 3 with lanes 1 and 2; transfected Rheb yielded two bands, possibly repre- senting fully processed protein in the lower band and unmodified protein in the upper band). Similar amounts of EE-tagged B-Raf kinase were in the immunoprecipitates in the absence or presence of co- transfected Rheb (Figure 3b, lower panel, lanes 2 and 3), and Western blots of the cell lysate showed that about 1 – 2% of cellular Rheb was associated with B- Raf kinase (Figure 3b, upper panel, compare lanes 3 and 4). These experiments were performed transfecting 100 ng of pRK5-myc-Rheb DNA, but similar results were obtained when 50 ng of Rheb DNA was used. Figure 3 In vivo association of Rheb and B-Raf kinase. In (a) The association of endogenous Rheb and B-Raf kinase CHOK-1 cells were transfected with pcDNA-B-Raf (lane 1), pRK5-myc-Rheb (lane 2), or the combination of the two (lanes was below the level of detection due to the fact that the 3 and 4), and 40 h later cells were lysed and Rheb was iso- efficiency of immunoprecipitation was very low with lated by immunoprecipitation using an anti-myc antibody the available antibodies. (lanes 1 – 3). The immuno-precipitates were subjected to SDS – PAGE, and Western blots were generated using an anti-B-Raf kinase antibody (upper blot) or an anti-Rheb anti- Rheb inhibits B-Raf kinase activity and MAP kinase body (lower blot). A small aliquot (5%) of cell lysate from activation cells transfected with both the Rheb and B-Raf kinase vectors was analysed in parallel (top panel, lane 4). In (b) the recipro- Since Rheb associates with B-Raf kinase in vivo,we cal experiment to (a) was performed. CHOK-1 cells were examined the effect of Rheb on B-Raf kinase activity. transfected with pMT2-Rheb (lane 1), pcDNA3-EE-B-Raf (lane 2), or the combination of the two (lanes 3 and 4), and 40 h We transfected logarithmically-growing CHOK-1 cells later cells were lysed, and B-Raf kinase was isolated by immu- with expression vectors encoding B-Raf kinase, with or noprecipitation using an anti-EE antibody (lanes 1 – 3). Immu- without 100 ng of Ras G12V or Rheb expression noprecipitates were analysed by SDS – PAGE/Western blotting vectors, isolated B-Raf kinase by immunoprecipitation, using an anti-Rheb antibody (upper blot) or an anti-B-Raf ki- and measured B-Raf kinase activity using MEK as a nase antibody (lower blot). Cell lysate (5% of total) from cells transfected with both the B-Raf kinase and Rheb vectors was substrate (Figure 4). As described by other investiga- analysed in parallel (upper blot, lane 4). For both (a) and (b) tors in different cell types (Kuroda et al., 1996; Marais similar results were obtained in two other independent experi- et al., 1997; Jelinek et al., 1996), we found that co- ments

Oncogene Rheb and B-Raf kinase EImet al 6360

Figure 4 Effect of Ras and Rheb on B-Raf kinase activity. Lanes 1 – 3. CHOK-1 cells were co-transfected with 50 ng of pcDNA3- EE-B-Raf either with empty vector (lane 1) or in combination with 100 ng pDCR-H-Ras(Val12) (lane 2) or 100 ng (lane 3) of pMT2-Rheb, and were cultured in medium containing 10% FBS. Lanes 4 – 7. CHOK-1 cells were co-transfected with 10 ng of pcDNA3-EE-B-Raf with empty vector (lane 4) or with 12.5 ng (lane 5), 25 ng (lane 6) or 50 ng (lane 7) of pMT2-Rheb; cells were transferred to medium containing 0.1% FBS and 0.1% bovine serum albumin. Cells were harvested 36 h later, and B-Raf kinase was isolated from cell lysates by immunoprecipitation. B-Raf kinase activity (a) was measured in the immunoprecipitates using cat- 32 alytically-inactive MEK-1 and [g- PO4]ATP as substrates; reaction products were analysed by SDS – PAGE/autoradiography with a longer exposure time for samples in lanes 4 – 7. The amount of B-Raf kinase present in the immunoprecipitates was determined by Western blotting using a B-Raf-speciﬁc antibody (b). Similar results were obtained in two other independent experiments

expression of Ras with B-Raf kinase caused a marked 75 ng of pMT2-Rheb DNA were co-transfected with increase in B-Raf kinase activity (Figure 4a, compare B-Raf kinase and HA-tagged ERK-1 expression lanes 1 and 2). These findings were in sharp contrast to vectors into CHOK-1 cells, there was a 44, 64, and results obtained with Rheb: when we co-expressed 93%, respectively, decrease in ERK-1 activation (mean Rheb with B-Raf kinase, we found a marked of duplicates from a representative experiment). Under diminution in B-Raf kinase activity (Figure 4a, left identical conditions, co-transfecting pDCR-H- panel, compare lanes 1 and 3). Inhibition of B-Raf Ras(Val12) with the B-Raf kinase and HA-tagged kinase activity by Rheb was observed even when lesser ERK-1 expression vectors caused a more than 10-fold amounts of Rheb DNA were transfected (Figure 4a, increase in ERK-1 activity. right panel, which is a longer exposure than shown in the left panel; compare cells transfected with B-Raf Rheb inhibits B-Raf kinase-dependent transcriptional kinase plus empty vector (lane 4) to cells transfected activation of Elk-1 with B-Raf kinase plus 12.5, 25, and 50 ng of Rheb DNA (lanes 5, 6 and 7)). The differences in B-Raf Since Rheb inhibited B-Raf kinase activity, we decided kinase activity were not secondary to differences in the to study the effect of Rheb on B-Raf kinase-dependent amount of B-Raf kinase present in the assay, as similar transcription. Because B-Raf kinase is an important amounts of B-Raf kinase were in the immunoprecipi- activator of Erk-1 and Erk-2 (Vossler et al., 1997; tates under all conditions (Figure 4b). In addition, Jaiswal et al., 1994) and the transcription factor Elk-1 Rheb inhibition of B-Raf kinase activity was observed is a physiological target of these two MAP kinases under different culture conditions because Figure 4a, (Johnson et al., 1996), we used a chimeric Elk-Gal4 left panel, shows results for cells maintained in medium construct to measure B-Raf kinase activation of a Gal- containing 10% fetal bovine serum (FBS), whereas the 4-dependent reporter gene (Qiu et al., 2000). We right panel shows results for cells cultured in 0.1% performed these studies in two of the neuronal cell FBS. Thus, Rheb is a potent inhibitor of B-Raf kinase lines which have endogenous Rheb, i.e., NB2A and activity. PC-12 cells, and in two fibroblastic cell lines, one with Since B-Raf kinase activates the MAP kinases ERK- and one without endogenous Rheb, i.e., BHK and 1 and -2 through MEK-1/2, we studied the effects of CHOK-1 cells. To minimize the effects of growth Rheb on B-Raf kinase-induced activation of ERK-1. factors on B-Raf kinase activation, these studies were Consistent with its inhibitory effect on B-Raf kinase performed in serum-starved cells. Because of changes in activity, we found that Rheb markedly diminished B- Rheb activation at different levels of Rheb expression Raf kinase activation of ERK-1: when 12.5, 25, or (Figure 2a), we performed these studies transfecting

Oncogene Rheb and B-Raf kinase EImet al 6361 both high and low amounts of Rheb DNA, i.e., 100 ng reporter gene activity with about a 30 – 60-fold increase (Figure 5a) and 5 – 25 ng of pMT2-Rheb (Figure 5c); in NB2A, BHK, and CHOK-1 cells, and more than a at the low levels of DNA, the activation state of the 1000-fold increase in PC-12 cells (Figure 5a,c, bars with transfected Rheb is similar to that of endogenous Rheb wide cross-hatching). Co-expression of wild type Rap (Figure 2a). 1A caused a modest decrease in reporter gene activity Expression of B-Raf kinase increased reporter gene in NB2A and PC-12 cells, and had no effect in BHK activity 2 – 3-fold in NB2A, PC-12, and BHK cells, and cells (Figure 5a, bars with narrow cross-hatching). In approximately ninefold in CHOK-1 cells (Figure 5a,c, all four cell lines, co-expression of wild type Rheb bars with narrow diagonal stripes). There was no significantly inhibited B-Raf-kinase-dependent tran- significant difference in transfection efficiency or in B- scription with Elk-Gal4 activity suppressed below Raf kinase expression between BHK and CHOK-1 basal levels in NB2A cells (Figure 5a,c, filled bars). cells (data not shown). As expected, co-expression of The different effects of Ras, Rap 1A, and Rheb an activated Ras protein caused a marked increase in observed in these studies were not secondary to

Figure 5 Effect of Rheb, Rap-1, and Ras on B-Raf Kinase- and Raf-1 kinase-dependent activation of Elk-Gal4. Cells were grown in 12-well culture dishes, and transfected pGAL4-Luc, pRSV-bGal, and pELK-Gal4, with the total amount of transfected DNA kept constant by adding pRC/CMV (empty vector). (a) NB2A, PC-12, and BHK cells were co-transfected with empty vector (open bars), or experimental vectors encoding B-Raf (bars with narrow diagonal stripes), B-Raf plus Rheb (filled bars), B-Raf plus Rap 1A (bars with narrow cross-hatching), or B-Raf plus H-Ras(Val12) (bars with wide cross-hatching); the amount of DNA used was 50 ng of pcDNA-B-Raf and 100 ng of pMT2-Rheb, pCMV-Rap 1A, and pDCR-H-Ras(Val12). (b) NB2A cells were transfected as described in (a); equal amounts of cellular protein were subjected to SDS – PAGE, and a Western blot was generated using an anti- B-Raf kinase antibody. Cells were transfected with empty vector (lane 1), or expression vectors encoding B-Raf (lane 2), B-Raf plus Rheb (lane 3), B-Raf plus Rap 1A (lane 4), or B-Raf plus H-Ras(Val12) (lane 5). (c) CHOK-1 cells were transfected with empty vector (open bars), 10 ng of pcDNA-B-Raf in the absence (bars with narrow diagonal stripes) or presence of pMT2-Rheb (filled bars, 5, 10, and 25 ng) or 10 ng of pDCR-H-Ras (Val12) (bars with wide cross-hatching). (d) BHK cells were transfected with empty vector (open bars) or 25 ng pCMV5-Raf-1 in the absence (bars with wide diagonal stripes) or presence of 100 ng of pMT2-Rheb (filled bars) or 100 ng of pDCR-H-Ras(Val12) (bars with wide cross-hatching). Post transfection, all cells were cultured in 0.1% FBS and 0.1% bovine serum albumin for 40 h, and then lysed. In (a), (c) and (d), luciferase and b-galactosidase activities were measured as described in Materials and methods; luciferase activity was normalized to b-galactosidase activity, with cells transfected with empty vector alone assigned a value of one. The data are the means+s.d. of at least three independent experiments performed in duplicate (in some cases error bars are too small to be seen). In (a) and (c), the values for cells transfected with B-Raf kinase and Ras are shown above the bars

Oncogene Rheb and B-Raf kinase EImet al 6362 differences in B-Raf kinase expression, since similar (1994) that purified Rheb hydrolyzed [g-32P]GTP at a amounts of B-Raf kinase were found under all rate comparable to that of Ras; this is in marked conditions (Figure 5b; data are shown only for contrast to Ras with a glycine to valine mutation at NB2A cells, but similar results were found for the position 12 which hydrolyzes GTP at a rate approxi- other cell lines). Thus, Rheb inhibits B-Raf kinase mately 1/50th that of wild type Ras (Cale´ s et al., 1988). function in several different cell types, both when Thus, unlike one would have originally predicted, the expressed at high and low levels. This inhibitory effect data suggest that Rheb’s high activation state is not of Rheb appeared to be specific for B-Raf kinase intrinsic to the protein, but rather may be secondary to because Rheb had no effect on Raf-1 kinase-dependent a relatively high intracellular ratio of activating activation of the Elk-Gal 4 construct in BHK cells, proteins, i.e., guanine nucleotide exchange factors while activated Ras stimulated Raf-1-dependent tran- (GEFs), to inactivating proteins, i.e., GAPs. scription (Figure 5d). The inhibition of B-Raf kinase- Further evidence that there may be relatively more dependent transcription by Rheb may explain why intracellular GEF than GAP activity for Rheb is that similar levels of B-Raf kinase expression in four increased levels of Rheb expression caused a progres- different cell types resulted in greater transcriptional sive increase in Rheb activation, with the increased activation of Elk-Gal4 in Rheb-deficient CHOK-1 cells Rheb activation occurring at Rheb protein concentra- compared to the other three cell types which contain tions that were only several-fold higher than endogenous Rheb. endogenous Rheb. Since we observed the same progressive increase in Rheb activation in both CHOK-1 cells, which do not express endogenous Discussion Rheb, and BHK cells, which do express Rheb, the data suggest that Rheb GEF(s) and GAP(s) may be In addition to being widely expressed in rat and human shared with other Ras family members. However, the tissues, Rheb homologs have been identified in the GAP(s) is/are unlikely to be RasGAP or neurofibromin budding yeast Saccharomyces cerevisiae, the fission since neither of these proteins increased the intrinsic yeast Schizosaccharomyces pombe, the sea squirt Ciona GTPase activity of Rheb in vitro (Yamagata et al., intestinalis, the zebrafish Danio rerio, and the fruit fly 1994). These data additionally provide a cautionary Drosophila melanogaster (Mach et al., 2000; Urano et note to experiments in which Ras-related proteins are al., 2000). The rat and human protein have 98.9% over-expressed in cells: at levels only a few-fold higher amino acid identity, while across all species Rheb than that of the endogenous protein, excessively high exhibits 26% amino acid sequence identity and 57% degrees of activation could occur due to saturation of similarity (Urano et al., 2000; Mizuki et al., 1996). normal regulatory mechanisms, which could result in Data from the yeast protein and from studies in non-physiological effects. NIH3T3 cells have provided indirect evidence that the Functional analyses of Rheb in S. cerevisiae and S. activation state of wild type Rheb is at a relatively high pombe have revealed that Rheb plays rather diverse level, and that it apparently cannot be increased further roles. In S. cerevisiae, Rheb regulates cellular uptake of by a genetic mutation. Thus, in S. pombe mutation of arginine and lysine, and this appeared to be a specific the glutamine at position 64 in Rheb, which corre- Rheb function since no other phenotypic abnormalities sponds to position 61 in Ras and leads to constitutive were found in Rheb-deficient cells (Urano et al., 2000). Ras activation (Barbacid, 1987), did not enhance Rheb This appears to be the first example of a Ras-related function, and the phenotype of NIH3T3 cells trans- protein regulating amino acid transport. In S. pombe, fected with wild type Rheb or Rheb Q64L was virtually Rheb inhibits the entry of cells into stationary phase indistinguishable (Clark et al., 1997). We now provide when extracellular nitrogen levels are adequate for cell direct evidence from three different types of neuronal growth (Mach et al., 2000); this is in contrast to Ras cells, i.e., C6, NB2A, and PC-12 cells, that wild type which regulates mating in response to limiting Rheb is constitutively in a highly activated state, even nutrients. in serum-starved cells. Thus, compared to its close Compared to the yeast proteins, the function of relatives Ras and Rap 1, Rheb appears to be unique. mammalian Rheb is less clear. Rheb expression was The degree of Rheb activation in the neuronal cells was initially found to be rapidly and transiently induced at similar to what we have found for mutated constitu- the transcriptional level by seizures in hippocampal tively-activated Ras proteins, either H-Ras G12V granule cells, and Rheb was found to be an immediate expressed in NIH3T3 cells or N-Ras Q61L, the early gene in PC12 cells and Balb/c 3T3 fibroblasts transforming protein in HL-60 promyelocytic leukemic (Yamagata et al., 1994). Rheb was subsequently found cells (Scheele et al., 1995; Guha et al., 1996; Pilz et al., to bind Raf-1 kinase, and the combination of Rheb 1997). The basis for the high activation state of Rheb and Raf-1 synergistically transformed NIH3T3 fibro- was not, however, from the arginine in position 15, nor blasts as potently as the combination of Ras and Raf-1 the serine in position 16 (corresponding to Ras residues (Yee and Worley, 1997). Another group found, 12 and 13), since mutation of these two residues to however, that Rheb, like Rap 1A, antagonized Ras- glycine had no effect on Rheb’s activation level when induced transformation of NIH3T3 cells, and proposed transfected into CHOK-1 cells. These data are that this was most likely through competition for Raf-1 supported by the original finding of Yamagata et al. (Clark et al., 1997). We now report that Rheb binds to

Oncogene Rheb and B-Raf kinase EImet al 6363 B-Raf kinase and inhibits its activity, and that Rheb vector. Clones were analysed by restriction analysis and inhibits downstream functions of B-Raf, i.e., activation DNA sequencing. of the transcription factor Elk-1 (other workers apparently have found similar results, because in a Western blotting recent chapter in Methods of Enzymology, the authors Western blots were generated as described previously (Scheele cite unpublished data that Rheb inhibits B-Raf kinase et al., 1994) using the following antibodies: two different (Urano et al., 2001)). Rheb did not affect Raf-1 polyclonal goat anti-Rheb antibodies (anti-Rheb C and anti- activation of the Elk-Gal 4 reporter construct, Rheb N, Santa Cruz Biotechnology), a rabbit polyclonal anti- indicating that Rheb’s effect on B-Raf kinase was B-Raf kinase antibody (Santa Cruz Biotechnology), and specific, and not secondary to global inhibition of the mouse monoclonal anti-myc and anti-EE epitope antibodies reporter system. These latter studies were performed in (original clones were from the American Type Culture BHK cells, and the lack of stimulation of Raf-1- Collection, and G Walter, UCSD, respectively). Briefly, cells dependent transcriptional activation by Rheb may be were extracted in a sodium dodecyl sulphate (SDS)-based cell-type specific, similar to Rap 1’s stimulation of B- buffer, and proteins were separated by SDS-polyacrylamide Raf kinase (Vossler et al., 1997). gel electrophoresis (PAGE). The proteins were transferred to polyvinylidene fluoride membranes which were blocked with The inhibition of B-Raf kinase activity and function 1% milk protein and incubated with the indicated antibodies. by Rheb is in stark contrast to Ras and Rap 1, both of After washing, the membranes were incubated with appro- which activate B-Raf kinase and increase B-Raf kinase priate horseradish peroxidase-complexed secondary signaling. Thus, although Rheb is a close relative of antibodies, and proteins were detected by enhanced chemi- Ras and Rap 1, and is in the immediate Ras/Rap/Ral luminescence. family (Reuther and Der, 2000), Rheb appears to function differently from these two other proteins. Measurement of Rheb activation Future studies will be aimed at determining the molecular basis for the differences between Rheb and The activation state of Rheb, i.e., the per cent of Rheb Ras/Rap 1, which could be secondary to amino acid molecules in the active GTP-bound state, was determined both for the endogenous protein, and for myc-tagged Rheb differences in the extended effector domain. expressed from the pRK5-myc-Rheb vector. Both of these assays are based on well-established methods for measuring Ras activation (Scheele et al., 1995; Dupuy et al., 2001; Prigent et al., 1996; Sharma et al., 1998; Suhasini et al., Materials and methods 1998). For endogenous Rheb, cells were rapidly extracted in an Cell culture and transfection experiments ice-cold HEPES-based buffer containing 10 mM MgCl2, BHK, CHOK-1, NB2A, and PC-12 cells were cultured as protease inhibitors, and 1% Nonidet P-40 (Scheele et al., described previously (Suhasini et al., 1998; Qiu et al., 2000). 1995). The extracts were clarified by a brief centrifugation, For transfection experiments, cells were plated in either 6- or and to the supernatants were added protein G agarose and 12-well cluster dishes and 24 h later were transfected with a either the anti-Rheb C antibody or goat IgG; all samples total of either 1.2 or 0.5 mg of DNA using LipofectAMINE received 500 mM NaCl, 0.5% deoxycholate, and 0.05% SDS. PlusTM or LipofectAMINE 2000TM (Life Technologies, Inc.) After shaking for 1 h at 48C, the agarose beads were washed as described previously (Suhasini et al., 1998; Qiu et al., four times in lysis buffer containing NaCl and detergents, and 2000). The following plasmids were used: pMT2-Rheb (Yee two times in 20 mM TrisPO4,5mM Mg2SO4. The beads were and Worley, 1997), pRK5-myc-Rheb (Yee and Worley, resuspended in 20 mM TrisPO4,1mM DTT, 1 mM EDTA, 1997), pDCR-H-Ras(Val12) (from M Wigler (White et al., and heated at 1008C for 3 min to elute GTP and GDP bound 1995)), pCMV-Rap 1A (from L Quilliam (Quilliam et al., to the immunoprecipitated Rheb. GTP, and the sum of GTP 1990)), pcDNA3-B-Raf (from P Stork (Vossler et al., 1997)), plus GDP, were measured in coupled enzymatic assays. GTP pcDNA3-EE-B-Raf (Qiu et al., 2000), pCMV5-Raf-1 (from was converted to ATP by nucleoside diphosphate kinase in H Mischak (Mischak et al., 1996)), pELK-Gal4 (from G the presence of excess ADP with the resulting ATP measured Johnson (Johnson et al., 1996)), and pCMV-HA-ERK-1 and by the luciferase method; this assay is sensitive to 1 fmol, and pGAL4-Luc (both from M Karin (Johnson et al., 1996)). because the second reaction is irreversible due to light generation, both reactions go to completion making the entire system quantitative (Sharma et al., 1998). The sum of Site-directed mutagenesis and DNA sequencing GTP plus GDP was measured by converting GDP to GTP Rheb R15G or R15G/S16G expression constructs were made using pyruvate kinase and phosphoenolpyruvate; GTP, which with a two step polymerase chain reaction (PCR) amplifica- now represents the sum of GDP plus GTP, was measured as tion process using wild type pRK5-myc-Rheb as template. In just described. We define our lower limits of detection as the first PCR, the SP6 primer was used as the sense primer experimental sample values being twice that of control together with respective mutant primers 5’-GAGGAC- sample values, and in these studies, experimental samples TTTCCCACAGACCCATAGCCCAGGATGGCGATCT-3’ were generally 10 times higher than the corresponding control and 5’-ATGAGGACTTTCCCACACCACCATAGCCCAG- samples. GATGGCGATCT-3’ that were used as antisense. The first For measuring the activation state of myc-tagged Rheb, PCR products were then used as sense primers and 5’- CHOK-1 cells were transfected with 15 – 150 ng of pRK5- G GCGCGTCACGCGGCCGCTCACGAAGACTTCCCTT- myc-Rheb. Thirty-six hours later cells were harvested and GTGAAGC-3’ (NotI) were used as antisense to amplify the processed as described for measuring endogenous Rheb full-length mutant. The amplified products were digested with except the anti-Rheb antibody was replaced by an anti-myc SalI and NotI, and subcloned into the pRK5-myc expression antibody.

Oncogene Rheb and B-Raf kinase EImet al 6364 or 75 ng of pMT2-Rheb; in some experiments, 25 ng of Assessment of B-Raf kinase association with Rheb pDCR-H-Ras(Val12) was substituted for the pMT2-Rheb CHOK-1 and BHK cells were transfected with either myc- DNA. The cells were incubated for 40 h post transfection in tagged Rheb and non-tagged B-Raf kinase, or with EE- 0.1% FBS plus 0.1% bovine serum albumin, harvested, and tagged B-Raf kinase and non-tagged Rheb, and 36 h later ERK-1 was isolated by immunoprecipitation using an anti- cells were extracted in a HEPES-based buffer containing 1% HA antibody (BabCO). After washing the beads, ERK-1 Nonidet P-40 and protease and phosphatase inhibitors. The activity was measured by following the phosphorylation of 32 extracts were incubated with either an anti-myc antibody or myelin basic protein using [g- PO4]ATP. The assay was anti-EE antibody, and the immunoprecipitates were collected linear with time and extract protein, and the data are on agarose beads. After four washes in lysis buffer, the expressed as a per cent change in B-Raf kinase-dependent proteins were eluted in SDS sample buffer and separated by ERK-1 activity. SDS – PAGE. Proteins were transferred to membranes, and Western blots were generated as described above using either Reporter gene assays an anti-B-Raf kinase antibody or an anti-Rheb antibody. NB2A, PC-12, BHK, and CHOK-1 cells grown in 12-well cluster dishes were co-transfected with 100 ng of the reporter Measurement of B-Raf kinase activity plasmid pGAL4-Luc, 50 ng of pRSV-bGal (which served as B-Raf kinase activity was measured as described previously an internal control for transfection efficiency), and 1 – 2 ng of (Suhasini et al., 1998; Qiu et al., 2000). Briefly, CHOK-1 cells pELK-Gal4. As indicated, cells additionally received 10 – were co-transfected with EE-tagged B-Raf kinase expression 50 ng of pcDNA3-B-Raf with or without 5 – 100 ng of vector, and empty vector, Rheb, or Ras expression vectors. pMT2-Rheb, 10 – 100 ng of pDCR-H-Ras(Val12), or 100 ng The cells were harvested 36 h later and extracted as described of pCMV-Rap 1A; a variable amount of pRC/CMV was for assessing B-Raf kinase association with Rheb. B-Raf included for a total of 500 ng of transfected DNA. In some kinase was isolated by immunoprecipitation on agarose experiments, pcDNA-B-Raf was replaced by 25 ng of beads, and the beads were washed three times in lysis buffer, pCMV5-Raf-1. Cells were incubated in low serum-containing and two times in lysis buffer lacking detergent. The beads medium (0.1% FBS plus 0.1% bovine serum albumin) and were then incubated for 5 – 15 min at 378Cin20mlof harvested 40 h post transfection; the experiments could not 32 reaction buffer containing 50 mM [g- PO4]ATP (New be performed in the complete absence of serum because of a England Nuclear), and 300 ng of recombinant catalytically- high rate of cell death. Luciferase and b-galactosidase inactive MEK-1 (Santa Cruz Biotechnology). The reaction activities were measured in cell lysates as described previously was terminated by adding SDS sample buffer and heating to (Qiu et al., 2000) in the presence of luciferin and the 1008C for 3 min. The samples were centrifuged and the chemiluminescent substrate Galacton (Tropix, Inc.), respec- supernatants were subjected to SDS – PAGE/autoradiogra- tively. phy. The assay was linear with time and with the amount of extract protein, and in each assay two timepoints were measured. Acknowledgments This work was supported in part by USPHS Grants Measurement of ERK-1 activation GM55586 (to RB Pilz), CA76968 and CA81115 (to GR ERK-1 activity was measured in serum-starved CHOK-1 cells Boss) and MH53608 (to PF Worley); E Im was supported as described previously (Suhasini et al., 1998). Briefly, cells by a UCSD Institute for Research on Aging Scholarship. were transfected with 100 ng of HA-tagged ERK-1 in the We thank G Johnson, M Karin, L Quilliam, P Stork and absence or presence of 10 ng pcDNA3-B-Raf, and 12.5, 25, M Wigler for generously providing the indicated plasmids.

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Oncogene