Rhob Mrna Is Stabilized by Hur After UV Light

RhoB mRNA is stabilized by HuR after UV light

Cara J Westmark1, Virginia B Bartleson1,2 and James S Malter*,1

1Department of Pathology and Laboratory Medicine, Waisman Center for Developmental Disabilities, University of Wisconsin, Madison, WI 53705, USA

RhoB is a small GTP-binding protein that is involved in et al., 2002) and lung carcinomas (Mazieres et al., 2004). apoptotic signal transduction. We have cloned the mouse Conversely, RhoB expression inhibits melanoma metas- RhoB mRNA including a 1377 nucleotide 30-untranslated tasis to the lung in a mouse model (Jiang et al., 2004b) region (UTR) that contains six AU-rich elements (AREs) and is an early marker for ischemic injury in degenerat- as well as several uridine-rich stretches. There is 94% ing hippocampal neurons (Trapp et al., 2003). homology overall between the mouse and rat RhoB genes RhoB codes for a 21 kDa GTPase involved in and 92% homology between the mouse and a putative multiple signaling events that influence transcriptional human clone. Ultraviolet light (UVL) induces RhoB activation, cell transformation, protein localization and production through regulated changes in gene transcrip- apoptosis. RhoB enhances transcription of the nitric tion and mRNA stabilization although the latter mechan- oxide synthase-2 (Delarue et al., 2001), preproendothe- ism is unknown. We observed that UVL increased the lin-1 (Hernandez-Perera et al., 2000) and COX-2 genes half-life of RhoB mRNA from 63 min to 3.3 h in NIH/3T3 (Shao et al., 2000), but antagonizes TGFb (Engel et al., cells and from 87 min to 2.7 h in normal human 1998)- and NFkb (Fritz and Kaina, 2001a)-mediated keratinocyte cells. In vitro mobility shift assays demon- transcriptional activation. This GTPase is required for strated that HuR bound the 30-UTR of RhoB at three transformation by oncogenic Ras (Prendergast et al., distinct locations (nucleotides 1342–1696, 1765–1920 and 1995), targets the serine/threonine kinase PRK1 to 1897–1977) suggesting a regulatory role for this RNA- endosomes (Mellor et al., 1998), and acting through binding protein. HuR immunoprecipitations were positive PRK1 regulates epidermal growth factor receptor for RhoB mRNA indicating an in vivo association, and (EGFR) traffic (Gampel et al., 1999). RhoB protein Western blot analysis and immunofluorescence demon- levels are upregulated in ischemia-damaged neurons strated that HuR rapidly partitions from the nucleus to (Trapp et al., 2001) and overexpression of RhoB causes the cytoplasm after UVL. Therefore, we propose a model alkylation-induced apoptosis in cells (Fritz and Kaina, in which UVL induces stress-activated signal transduction 2000). In knockout mice, the RhoB deletion does not leading to nuclear/cytoplasmic shuttling of HuR and affect mouse development, but mouse embryonic subsequent stabilization of RhoB mRNA. fibroblast (MEFÀ/À) cells exhibit defective motility, Oncogene (2005) 24, 502–511. doi:10.1038/sj.onc.1208224 decreased cell attachment and spreading, altered cytos- Published online 15 November 2004 keletal actin and proliferative responses to TGFb,as well as increased susceptibility to tumor formation in the Keywords: RhoB; HuR; ARE; mRNA stability; UVL presence of cancer-inducing agents (Liu et al., 2001). The kinetics of RhoB expression are typical for an early response gene (ERG) (Jahner and Hunter, 1991; Fritz et al., 1995; Jiang et al., 2004a). ERGs encode short-lived proto-oncogene, signaling molecule, cyto- Introduction kine and lymphokine mRNAs, which are transcribed in response to extracellular stimuli such as growth factors and DNA-damaging agents. Many ERG mRNAs The development of cell transformation involves the 0 overexpression of oncogenes or the downregulation of contain AU-rich elements (AREs) in their 3 -untrans- tumor suppressors (Jiang et al., 2004a). RhoB is a lated regions (UTRs) that target them for rapid member of the Ras family of small, GTP-binding degradation. Interaction of ARE mRNAs with stabiliz- proteins whose expression decreases substantially in ing (Hu family) or destabilizing (AUF, TTP) factors invasive and dedifferentiating head and neck (Adnane determines their mRNA half-life. Indeed, the combined effects of transcriptional and post-transcriptional regulation determine ARE mRNA expression. *Correspondence: JS Malter, University of Wisconsin, Waisman The embryonic lethal abnormal vision (ELAV)-like Center Rm. T509, 1500 Highland Ave, Madison, WI 53705, USA; family of RNA-binding proteins includes HuB (Hel-N1/ E-mail: [email protected] 2Current address: Mayo Clinic Medical School, Rochester, MN, USA N2), HuC and HuD, which are expressed in terminally Received 19 August 2004; revised 21 September 2004; accepted 25 differentiated neurons, and HuR, which is ubiquitously September 2004; published online 15 November 2004 expressed (Ma et al., 1996). The 36 kDa HuR protein RhoB is an ERG stabilized by UV light CJ Westmark et al 503 contains three RNA recognition motifs (RRMs) and a NM_022542 (Jahner and Hunter, 1991)) was selected novel shuttling sequence coined the HNS domain by ARE mRNA affinity chromatography with HuR (amino acids 190–244) between the second and third (Westmark et al., manuscript in preparation). Sequen- RRMs. The first two RRMs mediate ARE recognition cing revealed an extended 30-UTR (1377 bases) contain- (Chung et al., 1996), the third RRM has been implicated ing multiple AREs. RhoB is a known ERG induced by in poly(A) tail binding (Ma et al., 1997) and the HNS DNA damage, v-Fps, EGF and PDGF (Jahner and domain mediates nuclear/cytoplasmic shuttling (Fan Hunter, 1991; Fritz et al., 1995). Based on these data and Steitz, 1998a). HuR selectively binds to and and a recent publication (Malcolm et al., 2003), RhoB stabilizes ARE-containing mRNAs including c-fos appeared to be a promising candidate for a post- (Peng et al., 1998), cyclin A and cyclin B (Wang et al., transcriptionally regulated ERG. 2000a), vascular endothelial growth factor (VEGF) Our putative mouse clone was highly homologous and (Levy et al., 1998), cyclooxygenase-2 (COX-2) (Dixon partially overlapping with a mouse hippocampal cDNA et al., 2001), urokinase and urokinase receptor (Tran (GenBank accession number AK013784 (Carninci and et al., 2003) mRNAs. HuR has also been implicated in Hayashizaki, 1999)). In order to obtain the entire RhoB the decay of the G-protein-coupled b1-adrenergic cDNA, we designed primers against the beginning of the receptor (Kirigiti et al., 2001) and plasminogen activator mouse RhoB coding region (GenBank accession number inhibitor type 2 (PAI-2) mRNAs (Maurer et al., 1999). X99963(Nakamura et al., 1996)) and the terminus of the It has been proposed that HuR initially associates with 30-UTR of our cDNA and PCR amplified a 1.978 kb ARE-containing mRNAs in the nucleus and protects sequence that contained the entire RhoB coding and 30- them from RNase attack during and after export to the UTR. The 30-UTR sequence is 1377 nucleotides in cytoplasm (Fan and Steitz, 1998b). length, contains canonical AREs (Lagnado et al., 1994) We cloned the full-length cDNA for RhoB and noted at positions 1409 and 1844 and noncanonical AREs at the presence of 30-UTR AREs that are highly conserved positions 1084, 1706, 1926 and 1963as well as several between mouse, rat and human genes. These data uridine-rich regions (submitted to GenBank, accession suggested that RhoB mRNA would be regulated at a number AF481943) (Figure 1). Two recent GenBank post-transcriptional level by cell stress. Indeed, we now submissions (BC018275 and BC023334) are highly show that RhoB mRNA is stabilized by ultraviolet light homologous with our sequence. Our mouse RhoB gene (UVL) in both NIH/3T3 fibroblast cells and normal is 94% homologous with the rat gene and 92% human keratinocytes (NHEK). Based on mobility shift homologous with a human BAC clone (accession assays and immunoprecipitation studies, HuR interacts number AC023137 (1998)) (data not shown). The highly with RhoB mRNA in vitro and in vivo and this homologous 30-UTRs contain conserved AREs between interaction is increased by UVL. We also demonstrate species implicating this region in mRNA regulation. that HuR partitions from the nucleus to the cytoplasm In order to test this hypothesis, we first evaluated if in response to UV stimulation in both fibroblasts and UVL, a known inducer of RhoB gene expression, keratinocytes. These data suggest that RhoB mRNA is affected its steady-state mRNA levels. NIH/3T3 cells an authentic HuR ligand whose stability and localiza- were cultured on plastic in DMEM/10% FBS to 60– tion is determined by UVL-inducible interactions. 70% confluence and exposed to 60 J/m2 UVL. At times thereafter (0.5, 1, 2, 4, 6, 8 h), RNA was Northern blotted and hybridized with a radiolabeled, 30-UTR- specific RhoB cDNA probe. RhoB mRNA increased by Results 3.2-fold 1 h after UVL and returned to basal levels by 4 h (Figure 2). Alterations in mRNA steady-state levels A mouse mRNA highly homologous to the 30-UTR of could have resulted from transcriptional and/or post- the rat RhoB gene (GenBank accession number transcriptional regulation.

Figure 1 Nucleotide sequence of the mouse RhoB 30-UTR. The RhoB gene was PCR ampliﬁed from mouse hippocampus and the sequence of the 30-UTR is shown. Numbering refers to the total cDNA but only nucleotides 30 to the stop codon are shown. Single AREs found at positions 1084, 1706, 1926 and 1963(magenta) and two canonical AREs at positions 1409 and 1844 (yellow) as well as several U-rich stretches (blue) are highlighted

Oncogene RhoB is an ERG stabilized by UV light CJ Westmark et al 504

Figure 2 UVL increases RhoB steady-state mRNA levels in NIH/ 3T3 cells. NIH/3T3 cells were treated with 60 J/m2 UVL and incubated for the indicated times. Total RNA was harvested for control cells at 0 min (lane 1, denoted ‘C’) and for UV-treated cells after 0.5 h (lane 2), 1 h (lane 3), 2 h (lane 4), 4 h (lane 5), 6 h (lane 6) and 8 h (lane 7). (a) Representative Northern blot of the RhoB and b-actin-specific hybridization signals. (b) Histogram depicting Figure 3 UVL stabilizes RhoB mRNA in NIH/3T3 cells. Cells RhoB mRNA levels normalized to b-actin and plotted as a 2 percentage of total RhoB mRNA versus time. The error bars were treated with 60 J/m UVL and cultured for 3h prior to the indicate the standard error of the mean for triplicate samples addition of 5 mg/ml Act D. Total RNA was harvested with TRI- Reagent at 0 min (lane 1), 20 min (lane 2), 40 min (lane 3), 80 min (lane 4), 160 min (lane 5) and 320 min (lane 6). (a) Representative Northern blots of the RhoB- and b-actin-specific hybridization signals. (b) Histogram depicting RhoB mRNA levels normalized to b-actin and plotted as a percentage of total RhoB mRNA versus In order to evaluate if RhoB mRNA decay was time. The open square symbols represent RhoB levels in untreated altered, NIH/3T3 cells were exposed to UVL and NIH/3T3 cells, while the open circle symbols designate RhoB cultured for an additional 1 or 3h prior to the addition mRNA levels for UV-treated cells. The error bars indicate the standard error of the mean for triplicate samples of actinomycin D (Act D). Time points were harvested up to 320 min and analyzed by Northern blotting. The decay rate of RhoB mRNA was rapid in untreated cells (t1/2 ¼ 35 min73.1), but substantially slowed after 1 h transcription was blocked with Act D and total RNA UVL (t1/2 ¼ 80 min77.2) (data not shown). Similarly, isolated and Northern blotted with radiolabeled RhoB there was a greater than threefold increase in the half- or b-actin probes. As shown in Figure 4, there is a very life of RhoB mRNA measured 3h after UV treatment, low level of RhoB mRNA expression in untreated which increased from 63 min to 3.3 h (Figure 3). The NHEK cells. We do not observe any RhoB signal (panel 63min half-life of RhoB in unstimulated NIH/3T3cells a) and a barely detectable signal (panels b and c) in is moderately higher than published values of approxi- untreated cells with Molecular Dynamics ImageQuant mately 20 min (Fritz et al., 1995, 1999) and we did software. RhoB mRNA levels were increased with 30 observe variability in the half-life of RhoB mRNA in and 60 J/m2 UVL relative to untreated controls at time control cells (35–63 min). Irrespective of control rates, zero (point of addition of Act D), but not with 10 J/m2 we consistently observed a substantial increase in UVL. RhoB mRNA decayed with a half-life of 87 min in mRNA half-life after UVL. The kinetics of UV-induced control cells, which was increased nearly twofold changes in RhoB mRNA steady-state levels (Figure 2) (162 min) in UVL-treated cells (60 J/m2 UVL). b-Actin and post-transcriptional stabilization (Figure 3) imply mRNA was unchanged over this time. This data is that UVL initially affects RhoB mRNA levels through a comparable to those obtained in NIH/3T3 cells where combination of transcriptional and post-transcriptional RhoB mRNA decayed with a half-life of 63min in regulation (1 h post-treatment). By 3h after UVL, post- resting cells and 200 min in UVL-treated cells and show transcriptional stabilization predominates. that RhoB mRNA is somewhat more stable in growing, In order to demonstrate similar regulation in un- control NHEK than in NIH/3T3 cells, but that transformed cells, we evaluated the effects of UVL on treatment with an equivalent dose of UVL induced RhoB gene expression in NHEK (Clonetics, Walkers- similar increases in RhoB mRNA stability. ville, MA, USA). These keratinocytes are a widely used, The underlying mechanism for UVL-induced mRNA nontransformed model system to study the effects of stabilization could be generic inactivation of cellular UVL. We exposed 4th passage NHEK at 80% RNases or a more specific blockade of RhoB mRNA confluence to 10, 30 or 60 J/m2 UVL. After 3h, decay by ARE-binding proteins such as HuR. Recent

Oncogene RhoB is an ERG stabilized by UV light CJ Westmark et al 505 fourth ARE; and nucleotides 1897–1977 (denoted D) containing the fifth and sixth AREs (see Figure 5a). HuR was expressed in bacteria as a maltose-binding protein (MBP) fusion and purified over amylose and DE52 resins. The final protein was greater than 95% pure based on Coomassie-stained acrylamide gels (data not shown). MBP/HuR bound to the RhoB 30-UTR in RNA gel mobility shift assays (Figure 5b). HuR interacts strongly with the 355 base fragment containing nucleotides 1342–1696 (fragment A) and with the 156 base fragment from nucleotides 1765–1920 (fragment C), but weakly with the 81 base fragment encompassing nucleotides 1897–1977 (fragment D) (Figure 5b). Spe- cific binding to these regions was confirmed by competition experiments with unlabeled RNAs (Figure 5c–e). Of note, full-length 30-UTR was fully competed by unlabeled fragment A or C (Figure 5c), suggesting that multiple HuR/RhoB 30-UTR interactions can occur. Fragment A was entirely competed with unlabeled fragment C (Figure 5d), whereas fragment C was only partially competed with unlabeled fragment A (Figure 5e). Both fragments A and C were partially competed with unlabeled fragment D (Figure 5d,e). While we have not unequivocally shown that HuR binds to AREs within these 30-UTR domains of RhoB mRNA, that is the most logical and likely conclusion given the known specificity of HuR for AREs and that AREs are the only common feature of fragments which cross compete for HuR binding. In addition, these results imply that the preferred (strongest) HuR-binding Figure 4 UVL stabilizes RhoB mRNA in NHEK cells. Cells were site in the RhoB 30-UTR is found within fragment C, treated with 10, 30 and 60 J/m2 UVL (panels a, b and c, respectively) and cultured for 3h prior to the addition of 5 mg/ml with other sites occurring in fragments A and D. Act D. Total RNA was harvested at 0 min (lane 1), 20 min (lane 2), Thus, HuR binds strongly to two elements in the 40 min (lane 3), 80 min (lane 4), 160 min (lane 5) and 320 min (lane RhoB 30-UTR as shown by RNA gel mobility shift 6). (a–c) Representative Northern blots of the RhoB- and b-actin- assays in Figure 5. Fragments A and C contain the two specific hybridization signals. (d) Histogram depicting RhoB canonical AREs (positions 1409 and 1844, respectively). mRNA levels normalized to b-actin and plotted as a percentage of total RhoB mRNA versus time. The open square symbols The absence of HuR binding to nucleotides 1–1423of 0 represent RhoB levels in untreated NHEK cells, while the open the RhoB RNA containing the coding and proximal 3 - circle symbols designate RhoB mRNA levels for UV-treated cells UTR (data not shown) suggests that the ARE at (60 J/m2). The error bars indicate the standard error of the mean for position 1409 is not involved in HuR binding or RhoB duplicate samples (different NHEK donors) mRNA regulation. Fragment A likely interacts with HuR through several uridine-rich regions downstream of nucleotide 1423. HuR binding to fragment C is presumably mediated by the ARE at position 1844. The data has shown that HuR can prevent the decay of 168 base fragment encompassing nucleotides 1653–1820 several ARE-containing mRNAs including VEGF (fragment B) does not bind to HuR, and fragment D, (Levy et al., 1998) and c-fos (Peng et al., 1998). Since which contains two isolated AREs immediately up- RhoB mRNA was originally selected from mouse brain stream of the poly(A) tail, binds very weakly. These data tissue by HuR affinity chromatography, we asked if are summarized in Figure 5a. HuR interacted with RhoB mRNA in vitro by electro- To determine if HuR binds to RhoB mRNA in vivo, phoretic mobility shift assays (EMSA). A truncated anti-HuR/19F12 monoclonal antibody was used to RhoB mRNA containing the coding region and the first immunoprecipitate HuR from cytoplasmic lysates of two AREs of the 30-UTR (positions 1084 and 1409) but control versus UV-irradiated NIH/3T3 cells. RNA that not downstream elements was produced. Additionally, coprecipitated with HuR was isolated followed by we PCR amplified and in vitro transcribed the entire reverse transcription (RT) and PCR amplification with RhoB 30-UTR (nucleotides 592–1978) and multiple primers specific for a 224 bp fragment of the RhoB 30- fragments of the 30-UTR-containing individual AREs UTR. The PCR reactions were analyzed by ethidium and uridine-rich stretches. These included nucleotides bromide–agarose gel electrophoresis (Figure 6), which 1342–1696 (denoted A) containing the second ARE; indicated that RhoB mRNA was specifically associated nucleotides 1653–1820 (denoted B) containing the third with HuR in resting as well as UV-treated cells. ARE; nucleotides 1765–1920 (denoted C) containing the The amount of RhoB mRNA coimmunoprecipitated

Oncogene RhoB is an ERG stabilized by UV light CJ Westmark et al 506

Figure 5 RhoB mRNA deletion constructs and HuR gel mobility shift binding data. RhoB 30-UTR (bases 592–1978) fragments A (bases 1342–1696), (bases 1653–1820), C (bases 1765–1920) and D (bases 1897–1977) were PCR amplified and used as templates for transcription reactions with T7 RNA polymerase. RNA probes (1 Â 105 c.p.m.) were incubated with recombinant MBP/HuR protein, crosslinked, digested with RNase T1 and analyzed by SDS–PAGE. (a) Summary of RhoB RNAs represented by starting and final nucleotides and their binding affinity. Fragments are denoted A–D and the asterisks mark the location of AU-rich domains. (b) EMSAs with radiolabeled 30-UTR (lane 1), fragment A (lane 2), fragment B (lane 3), fragment C (lane 4) and fragment D (lanes 5–6) are shown. Lane 6 is a darker exposure of lane 5. Each lane contained 100 ng MBP/HuR protein. (c) EMSAs with radiolabeled 30-UTR and 25 ng MBP/HuR in the presence of unlabeled fragment A (lanes 3–5), unlabeled fragment B (lanes 6–8), unlabeled fragment C (lanes 9–11) and unlabeled 30-UTR (lanes 12–14). In each set of three competitor lanes, 25-, 100- or 500-fold molar excess of cold RNA over probe was used. (d) EMSAs with radiolabeled fragment A probe and 20 ng MBP/HuR in the presence of unlabeled fragment A (lanes 3and 4), unlabeled fragment B (lanes 5 and 6), unlabeled fragment C (lanes 7 and 8), unlabeled fragment D (lanes 9 and 10) and unlabeled 30-UTR (lanes 11 and 12). Competitors were used at fivefold or 25-fold molar excess over probe. (e) EMSAs with radiolabeled fragment C probe and 20 ng MBP/HuR in the presence of cold competitors as described for panel d. All cold competitor RNAs were electrophoresed on ethidium-stained agarose gels to ensure they were full-length

mRNA in vivo and that binding is increased after UV- induced stress. The increased quantity of immunoprecipitated, cytoplasmic RhoB mRNA, after UVL, could be due to increased cytoplasmic localization and/or binding activity of HuR. To gather more data related to these events, NIH/3T3 cells were treated with UVL and fractionated Figure 6 HuR binds to RhoB mRNA in vivo. NIH/3T3 control followed by Western blot analysis of nuclear, cytoplas- lysates (lanes 1 and 3) versus UV-treated lysates (lanes 2 and 4) mic or whole-cell lysates. Equal cell equivalents were were immunoprecipitated with HuR/19F12 monoclonal antibody loaded on 12% SDS gels for the nuclear and whole-cell (lanes 1 and 2) and rabbit IgG (lanes 3and 4). RNA was harvested with Tri-Reagent 3h post-UV treatment, reverse transcribed and fractions, while 15 mg of protein was loaded per lane for PCR amplified with RhoB-specific primers. The ethidium bromide- the cytoplasmic lysates. The proteins were transferred to stained agarose gel depicting the 224 bp RhoB PCR product is nitrocellulose membrane and stained with ECL þ re- shown agents as previously described (Westmark and Malter, 2001b). Cytoplasmic HuR increased with UV treatment at all of the times tested (10 min, 1, 3, 6 h), while total and nuclear levels remained at near constant levels dramatically increased in UV-treated cells. Transfer of (Figure 7). Our data agree with the literature in which the PCR products from the agarose gel to nitrocellulose UV stimulation increases the nuclear to cytoplasmic membrane followed by Southern blot analysis confirmed shuttling of HuR cells (Wang et al., 2000b). We did that the PCR product was indeed RhoB (data not observe cyclic variations in basal HuR levels in the shown). These data indicate that HuR binds RhoB cytoplasm of control lysates. HuR levels increased after

Oncogene RhoB is an ERG stabilized by UV light CJ Westmark et al 507

Figure 7 UVL induces translocation of HuR from the nucleus to the cytoplasm. NIH/3T3 cells were left untreated (odd numbered Figure 8 Visualization of HuR translocation from the nucleus to 2 lanes) or treated with 60 J/m UVL (even numbered lanes) and the cytoplasm. NHEK cells were left untreated (controls) or treated harvested after 10 min (lanes 1 and 2), 1 h (lanes 3and 4), 3h (lanes with 60 and 100 J/m2 UVL, cultured for 3h, fixed with ethanol and 5 and 6) and 6 h (lanes 7 and 8). Cells were fractionated into analyzed by immunofluorescence. The primary antibody was anti- cytoplasmic versus nuclear lysates and Western blotted with anti- HuR/19F12 (1 : 1000) (Molecular Probes) and the secondary HuR/19F12 or anti-b-actin antibodies. (a) Cytoplasmic lysates. (b) antibody was fluorescein goat anti-mouse antibody (1 : 2000) Nuclear lysates. (c) Whole-cell lysates (Molecular Probes). Fluorescence microscopy with an FITC filter (left), a DAPI filter (center) and overlayed images (right) fresh medium was added to the cells, returned to basal and Ran) (Takai et al., 2001). The Ras proteins, the levels within 8 h and remained low for approximately 8 h most well-characterized members of the family, regulate before increasing again (data not shown). It appears that gene expression and show a high frequency of mutations HuR shuttles between the nucleus and cytoplasm on a in human cancers. The Rho family regulates cytoskele- cyclic basis in the absence of drugs that synchronize the ton reorganization and is involved in the regulation of cell cycle, which likely accounts for the difference in gene expression. The Rab and Arf/Sar families control cytoplasmic HuR levels in the control lysates in vesicle trafficking and budding, respectively, while the Figure 7a. Therefore, UVL augments cytoplasmic Ran family directs nuclear/cytoplasmic transport and localization of HuR, which is accompanied by enhanced microtubule organization. These guanine nucleotide binding of HuR to RhoB mRNA and increased stability exchange proteins transduce signals from the cell of the RhoB message. membrane through modulation of their GTP- and We also visualized HuR nuclear/cytoplasmic shuttling GDP-bound states. in NHEK cells in response to UVL (0, 10, 30, 60 and The Rho family consists of three subfamilies (Rho, 100 J/m2). After UVL, cells were stained with anti-HuR Rac and Cdc42) with at least 22 distinct members antibody and evaluated by immunofluorescence constituting the Rho subfamily (Wennerberg and Der, (Figure 6, only 60 and 100 J/m2 shown). With the higher 2004). The RhoA, RhoB and RhoC proteins are highly UVL doses (30–100 J/m2 UVL), there is considerable homologous at the amino-acid level (B85%) but RhoB movement of HuR protein into the cell cytoplasm and contains an unique 30-UTR consistent with post- with 60 and 100 J/m2 UVL, HuR containing granules, transcriptional regulation. Malcolm et al. (2003) have predominantly around the nucleus, are visible. Recent recently demonstrated that RhoB mRNA is stabilized in data also demonstrates that HuR dimerizes and forms serum-stimulated Rat-2 fibroblasts and that the rat granular structures in the cytoplasm that may serve as RhoB 30-UTR can confer serum- and EGF-inducible sites of post-transcriptional gene regulation (Kasashima expression to a luciferase reporter gene. We have sought et al., 2002). With the higher UV doses, granule to further define the mechanism of post-transcriptional formation is apparent in NHEK (Figure 8). regulation of RhoB mRNA. Sequence analysis reveals that there is high homology between the mouse, rat and human RhoB 30-UTR sequences and conservation of the Discussion AREs between species, suggesting common post-transcriptional regulation in all three species. The small GTP-binding proteins (G proteins) are In NIH/3T3 mouse fibroblast and NHEK keratino- categorized into five families (Ras, Rho, Rab, Arf/Sar cyte cells, the half-life of RhoB mRNA increases by 2–3-

Oncogene RhoB is an ERG stabilized by UV light CJ Westmark et al 508 fold within several hours of UV treatment. Fritz et al. 1 (CRM1)-dependent cytoplasmic accumulation of these (1995) measured a 3–4-fold increase in the amount of complexes (Gallouzi et al., 2001). We have confirmed RhoB mRNA 30 min after UV treatment that could be that UV light increases the cytoplasmic localization of blocked by Act D. This transcriptional activation was HuR in NIH/3T3 and NHEK cells. The increase in not dependent on p53or c-fos, but did require an intact cytoplasmic HuR levels is accompanied by enhanced CCAAT element in the RhoB promoter (Fritz and binding of HuR to RhoB mRNA as determined by Kaina, 2001b). Therefore, UV irradiation appears to immunoprecipitation and Southern blot analyses. These stimulate transcriptional upregulation and post-tran- data are in agreement with a previous reports in the scriptional stabilization of the RhoB gene. Similarly, literature that UVC increases the binding of HuR to a UV irradiation controls c-fos mRNA levels transcrip- cis-element in the 30-UTR of the tumor suppressor tionally and post-transcriptionally in NIH/3T3 cells. p21WAF1 mRNA (Giles et al., 2003), resulting in Within 15 min of cell stimulation, transcription is increased mRNA expression (Lal et al., 2004). transiently upregulated followed by a return to basal AREs are classified into three groups: class I AREs mRNA levels and a second wave of c-fos accumulation contain one to three copies of dispersed AUUUA due to mRNA stabilization, which begins by 4–5 h pentamers located in uridine-rich regions (for example, postirradiation and persists for several hours thereafter c-fos mRNA), class II AREs possess multiple copies of (Blattner et al., 2000). clustered AUUUA motifs (for instance, GM-CSF UV radiation likely triggers these events by first mRNA) and class III AREs lack AUUUA pentamers, activating intracellular signaling pathways. In particular instead containing uridine-rich regions. HuR distin- is rapid activation of the extracellular-regulated kinase guishes between class I, II and III AREs by blocking (ERK) (Chen et al., 2001), c-jun N-terminal kinase decay mediated by the c-fos ARE (class I), but not (JNK) (Assefa et al., 1997) and p38 mitogen-activated affecting mRNA decay directed by either the c-myc, protein kinase (Pillaire et al., 2000) (MAPK) cascades, GM-CSF (class II) or c-jun AREs (class III) (Peng et al., which constitute the major secondary cellular signaling 1998; Chen et al., 2002). The RhoB 30-UTR contains six pathway in cells. The MAPK are involved in cell isolated AREs at positions 1084, 1409, 1706, 1844, 1926 differentiation, movement, division and death and are and 1963as well as several uridine-rich stretches (class activated by phosphorylation in response to environ- III AREs). The AREs at positions 1409 and 1844 fit the mental stress, proinflammatory cytokines and mitogenic definition of a canonical ARE (UUAUUUA(U/A)(U/ stimuli (Schaeffer and Weber, 1999). JNK and p38 A)) (Lagnado et al., 1994), whereas the others are mediate mRNA stabilization through the AREs located noncanonical (AUnA). Only the ARE at position 1844 is in the 30-UTRs of numerous ERG mRNAs (Montero embedded in a uridine-rich region identifying it as a and Nagamine, 1999; Winzen et al., 1999; Brook et al., class I ARE. In gel mobility shift assays, HuR-binding 2000; Lasa et al., 2000). The MAPKs also regulate the activity was the greatest for a 30-UTR fragment nuclear/cytoplasmic transport of many RNA-binding harboring the class I ARE, but weaker interactions proteins including heterogeneous nuclear ribonucleo- were observed with fragments containing noncanonical protein A1 (hnRNP A1), which shuttles continuously AREs and uridine-rich stretches. HuD, HuB and HuR between the nucleus and cytoplasm with p38-mediated preferentially bind to poly(U) regions (Park-Lee et al., phosphorylation regulating its cytoplasmic accumula- 2003; Lopez de Silanes et al., 2004) suggesting that HuR tion (van der Houven van Oordt et al., 2000). Therefore, interacts with the RhoB 30-UTR through uridine-rich UVL stimulates cellular signaling pathways that alter regions. The presence of multiple HuR-binding sites in the cytoplasmic accumulation of RNA-binding proteins RhoB mRNA could indicate that (1) multiple 30-UTR and the amounts and decay of their mRNA targets. elements may interact to regulate mRNA decay or (2) Ultimately, these interactions are instrumental in HuR regulates mRNA transport and decay via distinct influencing ERG mRNA accumulation and regulation elements. in the cytoplasm. Our data lends itself to a model in which environ- HuR localization varies during the cell cycle. In mental stress, such as UV irradiation, mediates stabili- synchronously growing cells, HuR is almost exclusively zation of RhoB mRNA through the ARE-binding localized in the nucleus during G1 and moves into the protein HuR. There is accumulating evidence that UV cytoplasm during late G1, S and G2 (Wang et al., radiation induces nuclear/cytoplasmic transport of 2000a). UV irradiation induces an immediate arrest of RNA-stabilizing proteins. For example, A18 hnRNP the cell cycle at the G1 phase due to the disappearance of translocates from the nucleus to the cytoplasm (Yang cyclin D1 mRNA and clearance of the protein and Carrier, 2001), whereas YB1 moves into the nucleus (Miyakawa and Matsushime, 2001). It has previously upon UV radiation (Koike et al., 1997). Others and we been shown that UV irradiation has no significant effect have observed that HuR accumulates in the cytoplasm on total cellular HuR levels, but it does substantially in response to UVL. It is likely that UV-induced post- enhance the quantity of cytoplasmic HuR (Wang et al., translational modifications (phosphorylation and/or 2000b). Heat shock causes localization of 12–15% of methylation) of HuR regulate its nuclear/cytoplasmic HuR to discrete foci in the cytoplasm (Gallouzi et al., shuttling and hence the accumulation and stabilization 2000) by increasing the association of HuR with the of ERG mRNAs in the cytoplasm. Our data with RhoB nuclear/cytoplasmic shuttling proteins pp32 and APRIL mRNA fits a model developing in the literature in which leading to the chromosomal region maintenance protein HuR binds to newly transcribed ERG mRNAs in the

Oncogene RhoB is an ERG stabilized by UV light CJ Westmark et al 509 nucleus, mediates transport of the mRNA/protein with 60 J/m2 UVC (254 nm) in an UV Stratalinker 2400 complexes across the nuclear membrane and protects (Stratagene, La Jolla, CA, USA), the growth medium was the messages from RNase attack in the cytoplasm. replaced, and the cells were cultured for 3h prior to the addition of 5 mg/ml Act D. Total RNA was harvested with TRI-Reagent at the indicated times, separated on formalde- hyde–agarose gels, transferred to nitrocellulose membrane, Materials and methods and hybridized with radiolabeled cDNA probes as described (Westmark and Malter, 2001a). The membranes were exposed Materials to a phosphorimager screen and the signals were quantitated The HuR/19F12 monoclonal antibody was a kind gift from Dr with ImageQuant software from Molecular Dynamics, Inc. (Sunnyvale, CA, USA). The radiolabeled cDNA probe was Henry Furneaux (Memorial Sloan-Kettering Cancer Center, 0 New York, USA) and also purchased from Molecular Probes specific for the 3 -UTR of the RhoB gene. The samples were (Eugene, OR, USA). NIH/3T3 cells were bought from ATCC analyzed in triplicate (NIH/3T3) and duplicate (NHEK), and (Manassas, VA, USA), NHEK) from Clonetics (Walkersville, the RhoB-specific signals were normalized to the signal for b- MA, USA), pGEM-T vector from Promega (Madison, WI, actin mRNA and plotted as a percentage of total RhoB USA) and TRI-reagent from Molecular Research Center, Inc. mRNA. Error bars depict the standard error of the mean. (Cincinnati, OH, USA). The b-actin monoclonal AC-15 antibody, protease inhibitor cocktail (catalog #P2714), protein RNA gel mobility shift assays A sepharose (catalog #9424) and Act D-mannitol were from The protocol for the binding reactions has been previously Sigma (St Louis, MO, USA). Oligos were synthesized by described (Westmark and Malter, 2001b). Details regarding Gibco BRL Life Technologies (Gaithersburg, MD, USA). the RNA probes and recombinant HuR concentrations are HotStarTaq DNA Polymerase and Omniscript Reverse given in the figure legend. Transcriptase were acquired from Qiagen (Valencia, CA, USA). The fluorescein goat anti-mouse secondary antibody Immunoprecipitations and RT–PCR (catalog number F-2761) was purchased from Molecular Probes (Eugene, OR, USA) and the Vectashield from Vector NIH/3T3 were grown in six-well dishes, treated 7UV, Laboratories (Burlingame, CA, USA). cultured for the indicated time, washed with ice-cold DPBS, scraped from tissue culture wells, spun at 2000 g for 30 s in a Amplification of the full-length mouse RhoB gene Stratagene picofuge microcentrifuge, resuspended in ice-cold buffer containing 50 mM Tris (pH 8), 100 mM NaCl, 10% Poly(A)-mRNA (27.5 ng) isolated from mouse hippocampal glycerol, 1 Â protease inhibitor cocktail and 5 mM DTT and tissue 1 h after seizure induction with PTZ was reverse frozen at À801C. The cells were lysed by the addition of NP-40 transcribed with Omniscript RT in a 20 ml reaction as per the to 1% final concentration on ice for 30 min with occasional manufacturer’s protocol. The RT reaction was diluted fivefold mixing. The lysates were spun at 10 000 g for 10 min at 41C. and 1 ml was amplified by PCR (1 min at 941C, 1 min at 501C The supernatants were transferred to fresh tubes and frozen at and 2 min at 721C for 35 cycles) in a 50 ml reaction with the À801C. The protein concentration of the lysates was quantita- forward 50-ATGGCGGCCATCCGCAAGAA-30 and reverse tively determined with Bio-Rad protein assay dye reagent as 0 0 5 -T10ATAAATGGCATGATCATAGTC-3 primers and Qia- per the manufacturer’s recommendations. Lysates (50 mg) were gen HotStarTaq DNA Polymerase. The PCR product was gel- incubated in buffer (50 mM Tris, pH 8, 100 mM NaCl, 10% purified on a low-melt agarose gel, ligated into the pGEM-T glycerol, 1 Â protease inhibitors, 5 mM DTT and 2 units/ml vector and transformed into competent XL-1 Blue Escherichia RNasin) with 7.5 mg HuR/19F12 antibody and mixed at 41C coli cells via electroporation. for 4 h. For the control immunoprecipitation reactions, rabbit IgG (10 mg) replaced the HuR/19F12 antibody. An equal Cell culture volume of protein A sepharose (50% slurry in DPBS) was added per immunoprecipitation and mixed overnight at 41C. NIH/3T3 Swiss mouse embryo fibroblast adherent cells The protein A sepharose was pelleted at 5000 g for 2 min at (ATCC item number CRL-1658) were grown in Dulbecco’s 41C, washed with 1 ml ice-cold DPBS and resuspended in 1 ml modified Eagle’s medium containing L-glutamine and 4.5 gm/l of TRI-Reagent. Total RNA was harvested, resuspended in glucose supplemented with 10% fetal bovine serum and 10 ml depc-water, denatured for 5 min at 651C and chilled on penicillin/streptomycin. Fresh medium was added every 2–3 ice before RT with Omniscript RT and a random 9-mer primer days and the cells were subcultured with trypsin at 75% in a 20 ml reaction as per the manufacturer’s recommendations confluency. Cells were grown to 60–75% confluency prior to Qiagen (Valencia, CA, USA). The RT reaction (5 ml) was used removal of the culture medium and treatment with UVL as as template for a 50 ml PCR reaction (1 min at 941C, 1 min at described below. 601C and 30 s at 721C for 25 cycles) with RhoB-specific primers NHEK cells (Clonetics catalog number CC-2501) were (forward: 50-CCTCCGGCAGAGGATCCAGG-30 and re- grown in KGM medium (containing 0.1 ng/ml hEGF, 5.0 mg/ verse: 50-GTGTGGTCAGAATGCTGTCT-30). The PCR re- ml insulin, 0.5 mg/ml hydrocortisone, 50 mg/ml gentamicin, actions (10 ml each) were run on 1% agarose gels and visualized 50 ng/ml amphotericin-B and 0.15 mM calcium) supplemented by EtBr staining. with 7.5 mg/ml bovine pituitary extract. The keratinocytes were propagated and subcultured to passage 4 as per the Protein lysate fractionation and Western blot analysis manufacturer’s detailed growth instructions. At 80% confluency, cells were treated with UVL as described below. NIH/3T3 cells were treated with UV as described above, removed from the polystyrene tissue culture plates (100 mm Â 20 mm) by scraping, spun at 1300 r.p.m. for 5 min UV stimulation, RNA purification and Northern blotting at 41C, washed with ice-cold DBPS, and lysed for 15 min on ice NIH/3T3 and NHEK cells were grown as described above, the in hypotonic lysis buffer (10 mM HEPES (pH 8), 10 mM KCl, growth medium was removed and saved, the cells were treated 1.5 mM MgCl2,1Â protease inhibitors, 1% NP-40) with gentle

Oncogene RhoB is an ERG stabilized by UV light CJ Westmark et al 510 mixing. An aliquot (whole-cell lysate) was quick frozen at stained with fluorescein goat anti-mouse secondary antibody À801C, the nuclei were pelleted at 3500 r.p.m. for 4 min at 41C, (1 mg/ml) for 1 h at room temperature in the dark. The and the supernatants (cytoplasmic lysates) were transferred to coverslips were washed with DBPS, fixed to glass slides with fresh tubes and flash frozen at À801C. Cytoplasmic protein Vectashield and examined by fluorescent microscopy. concentrations were determined by the Bradford assay. Equal cell equivalents were loaded on 12% SDS gels for the nuclear and whole-cell fractions, while 15 mg of protein was loaded per lane for the cytoplasmic lysates. The proteins were transferred Abbreviations to nitrocellulose membrane and stained with ECL þ reagents as Act D, actinomycin D; ARE, AU-rich element; ERG, early previously described (Westmark and Malter, 2001b). The response gene; EMSA, electromobility shift assay; ELAV, primary antibodies were anti-HuR/19F12 (1 : 2000) and anti-b- embryonic lethal abnormal vision; NHEK, normal human actin (1 : 5000). keratinocyte; PTZ, pentylenetetrazole; RNP, ribonucleopro- tein; RRM, RNA recognition motif; UVL, ultraviolet light; Immunofluorescence UTR, untranslated region. NHEK cells (passage 4) grown on glass coverslips were treated with increasing doses of UVL (0, 10, 30, 60 and 100 J/m2), Acknowledgements cultured for 3h, fixed with absolute ethanol ( À201C) and We thank Dr Henri Furneaux for pGEX2T/HuR plasmid and stored at 41C. The fixed cells were rehydrated by washing the anti-HuR antibody (Memorial Sloan-Kettering Cancer Cen- coverslips 7 Â with DPBS, blocked in DPBS containing 5% ter, New York), Drs Syrus Soltani-nassab and Meggan BSA and 0.05% Triton X-100 for 90 min at room temperature Czapiga for help with the fluorescence microscopy and and stained with anti-HuR/19F12 antibody (0.2 mg/ml) over- members of the laboratory, particularly Amit Dande, for their night at 41C. The cells were washed 3 Â with DPBS, 3 Â with thoughtful comments. This work was supported by National DBPS containing 5% BSA and 0.05% Triton X-100 and Institutes of Health Grant RO1 MH61666 (to JSM).

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Oncogene