the P53 Tumor Suppressor Targets A Novel Regulator of G Protein Signaling
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Length: 5 pages
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- Buckbinder, L.
- Chen, Y.
- Gao, J.
- Gelbert, L.
- Gutkind, J.S.
- Kley, N.
- Seizinger, B.R.
- Talbott, R.
- Velascomiguel, S.
- Xu, N.
- PSCI, PUBLICATION SCIENTIFIC
- BIBL, BIBLIOGRAPHY
- EXTR, EXTRA
- MARG, MARGINALIA
- Named Organization
- Princeton Univ
- Author (Organization)
- Proc Natl Acad
- Bristol Meyers Squibb Pharmaceutical Res
- Genome Therapeutics
- Lab of Cellular Development + Oncology
- Molecular Signaling Unit
- Nas, Natl Academy of Sciences
- Natl Inst of Dental Research
- NIH, Natl Inst of Health
- Named Person
- Kley, N.
- Shenk, T.E.
- Master ID
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Proa Natl. Acad. Sd. USA Vol. 94, pp. 7868-'/872, 3-1y 1997 Biochermstry The p53 tumor suppressor targets a novel regulator of G protein signaling LEONARD BUCKBINDER*, SUSANA VELASCO=IV[IGUEL*, YAN CHEN*, N~N~Z~ XU$, RANDY TALBOTr*, LARRY GELBERT~, JIZONO OAO*, BERND R. SEIZINOER*§, J. SILVIO GIYlXIND~', AND NIKOLM KLEY*§¶ • Department of Molecular Genefic~ and *Biomolecular Drug Discovery, Oncology, Bri~toi-Myer~ Squibb Pharmaceutical Research Institute, D.O. Box 4000, Princeton, NJ 08543; and ~'Molecuiar Signaling Unit, Laboratory of Cellular Development and Oncology, National Institute of Dental Research, National rmstitute~ of Health. Bethesda, MD 20892 . . Communicated by Thomas E. Shenk, Princeton University, Princeton, NJ, May & 1997 (received for review December 20, 1996) ABSTRACT Heterotrimeric G proteins transduce multi- ple growth.factor.receptor-initiated and intracellular signals that may lead to activation of the mitogen-activated or stress- activated protein kinases. Herein we report on the identifica- tion of a novel p53 target gene (A28-RGS14) that is induced in response to genotoxic stress and encodes a novel member of a family of regulators of G protein signaling (RGS) proteins with proposed GTPase-activating protein activity. Overex- • pression of A28.RGS14p protein inhibits both G~- and Gq- coupled growth.factor-receptor-mediated activation of the mitogen-activated protein kinase signaling pathway in mam- malian cells. Thus, through the induction ofA28-RGS14, p53 may regulate cellular sensitivity to growth and/or survival factors acting through G protein-coupled receptor pathways. Inactivation of the p53 tumor suppressor protein is the most common aberration known to occur in human cancers (1). As a consequence of loss of wild-type p53 functions, cells are defective in critical, cell cycle checkpoints as well as intracel- lular and extracellular pathways regulating cellular growth and programmed cell death (2-5). Several pS3-induced target genes that encode a complex spectrum of regulators of such pathways have been identified. For instance, p21wA~t (6) mediates pS3-induced cell cycle arrest and may exert protective effects against apoptosis (7), whereas bax (8) encodes a positive effector of cell death. Induction of IGF-BP3, an inhibitor of insulin-like growth factors, provides a mechanism whereby pS3 may interfere with the mitogenic and survival functions of insulin-like growth factors, thereby further sen- sitiT./ng ceils to apoptotic stimuli (S). Cell-specific integration of the activity, of such and yet to be identified p53-regulated pathways is intimately associated with cell fate of normal and tumorigenic cells. To gain further insight into p53 signaling pathways, we undertook a screen to clone nov.el pS3 target genes. Herein we report the identification of a novel factor induced by p53 that can inhibit G protein-coupled mitogenic signal transduction and activation of the mitogen-activated protein kinase (MAPK) signaling cascade implicated in cel- lular proliferation, transformation, and oncogenesis. MATERIALS AND METHODS Cell Culture. EBI colon carcinoma cells (9) were cultured as described (S). RKO and RKO E6 colon carcinoma cells were cultured at 37~C and 5% CO2/95% air in modified Eagle's medium supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin (GIBCO/BRL). NIH 3T3 MI and M2 cells were cultured as described (10). T98G glioblastoma, The publication costs of this article wer, defrayed in part by page charge payment. This article must therefore be hereby marked "advertiaement'" in accordance with 18 U,S.C. §1734 solely to indicate this fact. ~) 1997 by The National Academy of Sciences 0027.8424/97/947868-552.00/0 PNAS is available, online at http://www.pnas.org. U-87 astrocytoma, HL-60 promyelocytic leukemia, and MCF7 breast carcinoma cells were obtained from American Type Culture Collection and maintained at 37°C and 5% CO2/95% air in RPMI 1640 medium supplemented with 10% FBS and penicillin-streptomycin (100 units/ml) (GIBCO/BRL). MCF7 Adr (11) and MCF 7 clone 6 (clonal population derived from the parental cells) were cultured as the parental MCF7 cells were cultured. RNA and Northern Blot Analysis. RNA preparation and Northera blot analysis were as described (12). Quantitation of "Northern blots was performed with laser densitometry (Mo- lecular Dynamics) of the autoradiograms or by exposing the blots to phosphorimaging plates followed by analysis on a phosphorimager (Fuji). eDNA Isolation and Cloning. A PCR-based h'brary subtrac- tion procedure was used to enrich for eDNA fragments • representing RNAs induced by p53 (12). One fragment, A28, detected an ,-2.5-kb p53-regulated transcript and was used as a probe to screen a human brain eDNA library in X ZAPII (Stratagene). Several independent clones were identified and isolated as pBluescript plasmids by phagemid rescue (Strat- agene). A28-15B, the longest clone, was sequenced in both directions by automated DNA sequencing (Applied Biosys- terns) using vector- and gene-specific primers. A28-15B was 1969 nt and all other clones were found to be 5' truncated versions of this sequence. Thus, none of the identified clones appeared to be full-length. Additional upstream sequence was obtained by using 5' rapid amplification of eDNA ends (CLONTECH) and RNA obtained from cadmium chloride- stimulated (10 h) EBI cells (12). This additional 416 nt of eDNA sequence was confirmed by sequencing the correspond- ing genomic region from a cosmid clone (L.B., R.T., N.K., and L.G., unpublished results). Plasmid Construction. The 5' fragment obtained from rapid amplification of eDNA ends was subcloned into a unique BglII restriction site near the 5' end of A28-15B, yielding a recom- binant full-length clone (2383 nt). The entire A28-RGS14 sequence (where RGS is regulators of G protein signaling) was excised by EcoRI digestion and subeloned into the/n vivo/in v/tro expression vector (pCDNA3), yielding pIGIL4 (sense) or pAS3 (antisense). In V'ffro Interaction of A28.RGS14 with Ga Protein. A28- RGS14 was expressed in baculovirus as a polyhistidine fusion protein (pBlueBacHis, Invitrogen) and purified by chroma- tography using nickel-agarose (Qiagen, Chatsworth, CA). Ga Abbreviations: MAPK. rnitogen-activated protein kinase; RGS, reg- ulators of G protein signaling; HA, hemagglutinin. Data deposition: The sequences reported in this paper have been deposited in the C~nBank database (accession nos. U70426 and U70427). §Current address: Oenome Therapeutics Corporation, Waltham, MA 02154. ~tTo whom reprint requests should be addressed, e-maiL Kley@ genomecorp.com. 7868
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Biochemistry: Buckbindcr et al. Proc.-Natl. Acad. Sci. USA 94 (1997) 7869 proteins (13) were expressed and labeled with [35S]mcthionine by using a coupled in vitro transcription-translation system (TNT, Promcga). Conditions for in vitro interaction and eo- immunoprecipitation were as described (rcf. 16 and Y.C., I.,B., and N.tC, unpublished results). ~luscarinic Receptor Signaling and MAPK Activity. Plas- :~..5 encoding the M1 or M2 muscarinic receptors (1 tzg per plate) were eotransfected with a pcDNA3-A28-RGS14 eDNA expression vector and a plasmid encoding hemagglutinin (HA)-taggcd MAPK (ERK2). Total transfccted DNA was maintained constant with an empty expression vector (pCDNA.3). After treatment with Carbachol (100 /~M) or phorbol 12-myristate 13-acetate (100 ng/ml), ceils were lysed and ERK activity was assayed on anti-HA immunoprecipitates as described (13), Radioactivity incorporated into myelin basic protein was quantitated with a Molecular Dynamics Phospho- rlmagcr. Total input MAPK was monitored by Western blot ~ .~.Iysis using anti-HA (Boehringer Mannhcim) monoclonal ~ ,~ibody as described (13). A28-RGS14 expression was mon- itored with a monoelonal antibody (Y.C., L.B., and N.K., unpublished results). RESULTS AND DISCUSSION As described (12), a 'differential eDNA cloning approach was used to identify novel p53 target genes potentially implicated in cellular growth control. This led to the isolation of a eDNA fragment derived from a novel gene induced by both exoge- nous and endogenous wild-type p53. Fig. 1 describes studies ~. ated to the regulation of the expression of this gene, called A28-RGS14, by wild-type p53. A28-RGS14 transcript expres- sion is rapidly induced upon activation of an inducible p53 transgene in human EB1 colon carcinoma cells (Fig. 1A). The magnitude and time course of induction is comparable to that observed for other known p53 response genes, such as p21w~a~l, and similar to that observed in other human cells types carrying inducible p53 transgenes (12). Subsequent studies addressed the inducibility of the A28-RGS14 gene by endogenous p53. Consistent with the A28-RGS14 gene being induced upon activation of endogenous wild-type p53, and thus "o in response to genotoxie stress, treatment of RKO colon ~.,rcinoma cells with the anticancer and DNA damaging agent doxorubiein leads to induction of A28-RGS14 transcripts (Fig. 1B). No such induction was observed in RKO-E6 cells in which p53 signaling is defective as a result of the human papilloma- virus E6 viral protein promoting degradation of p53 (Fig. 1B). Importantly, experiments using an anti-A28-RGS14p mono- clonal antibody have shown that A28-RGS14p protein is only detected in RKO and EB1 cells after p53 induction (Y.C., L.B., and N.K., unpublished results). As observed for other known p53 target genes, such as the bax and p21wAFt genes, the basal ~wels of A28-RGS14 mRNA were found to vary somewhat th cell type, tissue type, and cell culture conditions (Figs. 1C, 2C, and 3B), which is likely to reflect the complex regulation of this gene by p53-dependent and p53-independent mecha- nisms. However, eousistent with results obtained with RKO cells (Fig. 1B), A28-RGS14 transcripts were found to be induced by doxorubicin only in ceils expressing wild-type p53 and not in p53 mutant or null cells (Fig. 1C). Thus, these findings suggest a role for endogenous p53 in the induction of the A28-RGS14 gene in response to genotoxie stress in human cells. Curiously, no induction of A28-RGS14 mRNA was ~bserved in mouse embryonic fibroblasts in response to p53 duction. Whether this reflects a cell-specific or species- pccific phenomenon remains to bc determined. A A28-RGS14 EB1 B Dox. (uM) c A28-RGS14 A28-RGS14 p21 GAPDH MCF-7 T98G U.87 HL-60 ~ #6 AdrR C D C D C 0 C D C O C D hdm-2 FIO. 1. Regulation of A28-RGS14 gene expression by p53 and genotoxie stress. (A) Regulation by exogenous p53. The metallothio- nein-promoter driven p53 transgene in EB1 colon carcinoma cells (9) was induced by the addition of CdClz (6 t~M) to the cell culture medium. Total RNA or protein was prepared from cells at the times (in hours) indicated. Northern blots (A) were prepared and analyzed by hybridization with 32P-labeled eDNA probes corresponding to A28-RGS14, p21wAl:l, p53, or GAPDH. (B) Induction of A28-RGS14 expression by endogenous p53. Northern blot analysis of RKO (colon carcinoma ceils, wild-type p53) and clonal RKO-E6 cells (express human papillomavirus E6 viral protein and consequently do not express significant levels of p53) treated with increasing concentra- tions of doxorubicin for 16 h. (C) Induction of A28-RGS14 transcripts in human cells that are wild type (WT) (MCF-7, breast carcinoma cells; MCF-7 subclone 6; U87, astrocytoma cells) but not mutant (T98G; glioblastoma cells, MCF-7Adr.: doxorubicin-resistant MCF7 subelone) or null (HL-60, promyeloeytie leukemia cells) for p53. Cell lines were treated with (lanes D) or without (lanes C) doxorubicin (1 /~M) for 16 h. RNA was prepared and analyzed by Northern blot as described in A. p53 in a human osteosareoma cell line, even in the absence of ongoing protein synthesis, as we have shown (12); and (iii) the induction of A28-RGS14 transcripts by p53 is not a general consequence of cells arresting in a particular stage of the cell The following observations indicate that the A28-RGS14 cycle, as demonstrated by the lack of induction in ceils blocked gene might be a direct p53 response gene: (i) A28-R~S14 in G~ (serum deprived), S (thymidine treated) or G~/M transcripts are rapidlv induced in response to p53 expression (nocodozole treated) phases of the cell cycle (data not shown). (Fig. 1A); (ii) A28-~GS14 transcripts are induced by the Thus, induction of A28-RGS14 is an early p53 response. To conditional activation of a temperature-sensitive mutant of determine whether this might be mediated via direct activation
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7870 Biochemistry: Buckbinder et al. Prec. Natl. Acad. Sci. USA 94 (1997) A mu28-RG814 1 M~RTLAAFPTTCLERAKEFKTRLGIFLHKSELGCDTG~TGKSEWGSKHSKENRNF~EDVLGWRESFDLLLSSKNGVAA~HA~LKTEFSEENLEFWLAcEE F I01 1 MCRTLATFPNTCLERAKEFKTRLGIFLHKSELSSDTGGISKFEWASKHNKENRSFSEDVLGWRESFDLLLNSKNGVAAFHAFLKTESSEENLEFWLACEE F 101 h~ B A28-RGSI4 RGS2 GAIP RGS 1 A28-RGSI4 RGS2 GAIP RGSI EGL 10 SS~2 A28-RGSI4 RGS2 GAIP RGSI EGLI0 SsU2 102 KK7RSATKLASRAHQIFEEFICSEAPKEVNIDHETRELTRMNLQTATATCFDAAQGKTRTLMEKDSYPRFLKSPAYRDLAAQASAASATLSSCSLDEPSH T 202 102 KKIRSATKLASRAHQIFEEYIRSEAPKEVNIDHETRELTKTNLQAATTSCFDVAQGKTRTLMEKDSYPRFLKSPAYRDLAAQASATSTSVPSGSPAEPSH T 202 1 ,M~-~L...~PT~ ........ ~ ................ ~-~H ,E~ELGCD~GS T~/G~ ................ ..... HSKEAXlRNF S~--~GWR~ SFDLLL SSKNGV~ZiKTEFSEENLEFW'gdkC EEF K~I RSATI~.~.~kSRAHQ:I:F E EP QQAF "r KP S P~E~LWS,~dLFDEL/.~SKYGZdMkFRAF/.,KSF.~IF_~WIdkCEDFKKTKS PQKL S S~K~YTDF C EW'CAT P S P~ SWAQ SFDKLI~-IS PAGRSVFRA~LRTEYS EF, NM~F~CEELIrOkF.NkNQHVVDEIrOkRLIYEDY ........ 2~q]~IQWSQSLEKL~NM~IQTC-~NV~GS~LKSESSE~NI EFWI.dkCEDYKKTES . DLLPCKiLEEIYKA~ ......... 417. LWEDS~'EELZd~DSLGRET~QK~LDKER'SGENLR~INgP~QI~RKCSS o RblVP~EI~IF-~ ......... 416 . SNI.a'ZK~DYVLTDP~IRYLPRRtL~EKELCVENLDVF IEIKRFLK. 45 8 ~ ............ ~ .... L E EN r CSEAP. KEVNZ.DrrSTR~-,TRtm'~QTATATC~'D~Q~TRTY.a.flZKD~P~KSPA~QAS~TLS S 193 "r EK~P. KEINZ~FQTKTL ~QNIQF-,A~SGC~'I~tQt~tVY~PR~LESEL~'YQDLCKK PQ ~ ~ P~T 211 V S :I:L S P. ICdZVS LD S RVREG:~Clr,.I~QEPS~h'I~'DD~Qr.Q~SY~RF~SSP TYRA~ L L QG P S (~S S F.dk 217 Wr~SDAA. KQ~IDF~TREST~m~:~:Ir~PTP~DFdtQ~v.~k'T~YP~LKS D ~L~LQ~K 196 IDIOTS ~C~ED~P~W~D~YC~SYQ~LRS E~L 536 ........................ . .... ..~ ...... 66~ FEIV~SF~TQS~AS~IEIQE 688 F ~ S F S Y D + + ~ + + + + + . 97.4Kd .... ' 66Kd --- _~ 46Kd -- 30Kd -- 8012 (antt-A28) His-A28 35 Gu(IVT-S ) Abl(anti-p53) His-p53 hdm2 (IVT-S35) FIG. 2. A28-RGS14 gcne encodes a novel conserved RGS protein. (A) Predicted protein sequences of human and mouse A28-RGS14p prote~ (B) Alignment of A28-RGS14p amino acid sequence and that of related mammalian, yeast, and C. elegans RGS protein family members. Shaded-~:~ areas indicate a 130-amino acid cor~ domain with highest sequence conservation and a predicted a-helical structure. (C) Tissue expression pattem:~ of the A28-RGS14 gent. A human multitissuc Northern blot (CLONTECI-I) was hybridized with a 32p-labeled A28-RGS14 eDNA probe a~_~ described in Fig. IA. (D) In vitro interaction of A28-RGS14p with (~oti2, but not Gc~ subunits of heterotrimerie G proteins. The in vitro-translated=,~ 3~S-labeled Ga proteins were coimmunoprecipitated with purified His-tagged A28-RGS14p (His-A28) and anti-A28-RGS14p antibody 801~ (Ab-8D12; Y.C., LB., and N.K., unpublished results). Lanes: 1, in vitro-translated Ga; 2, coirnmunopreeipitation of Ga with His-A28-RGS14p tm~ Ab-SD 12; 3, coimmunoprccipitation of Ga without His-A28-RGSI4p; 4, coimmunopreeipitation of/n v/tro-translated Ga with purified His-tagl~ p53 (His-p53) and its specific antibody (Ab-1, Oncogenc Science); 5, eoimmunoprceipitation of in vitro-translated hdm2 protein wi~ His-A28-RGS14p and Ab-8DI2. m.w.m., M.olecular mass markers. ~ by p53, genomie analysis of the A28-RGS14 gene was per- other putative p53 binding sites in sequences extending to ~ I_""~ kb upstream of the putative transcription start site N.K., unpublished data). putative p53-binding sites are encoded by sequences further upstream of the transcription start site, the p21wA~t gene (6), and whether any putative binding may cooperate in p53-mediated induction of A28-RGS14 expression. Screening of eDNA libraries, 5' rapid amplification eDNA ends (RACE) analyses, and genomic analyses formed. Purified p53 was found to bind to DNA elements resembling the consensus p53-binding sequence (14) and located in intron 3 (not shown) and the 3' untranslated region (5'- TGGGCTAGCCCAGAGTCCCTtAGCTTGTaC-Y, where underlined type represents p53 half sites and lowercase type represents divergent nuctcotides) of the 7.5-kb A28-RGS14 gene, although it is as yet unclear whether these mediate p53 responsiveness in vivo. Computer analysis did not reveal any
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A Biochemistry: Buckbinder et al. ~ MBP Fold Induction FtG. 3. (4) A28-RGS14 coexpression blocks agonist-indueed ac- tivation of ERK2 in M1- or M2-transfected COS-7 cells. Plasmids encoding M1 or M2 muscarinie receptors were cotransfceted with a peDNA3-A28-RGS14 eDNA vector or empty, expression vector, as indicate& and a plasmid encoding HA-tagged MAPK (ERK2). Ceils were exposed to Carbaehol (100 txM) or phorbol 12-myristate 13- acetate (100 ng/ml) for 5 min and lysed, and ERK activity was assayed on anti-HA immunoprecipitates. After autoradiography, radioactivity incorporated into myelin basic protein (MBP) was quantitated with a ' lecular D.vnamies Phosphorlmagcr. Data represent the mean - ":M of six to eight experiments, expressed as fold increase with respect to veetor-transfectcd ceils (control). Fifty. micrograms of total. lysate proteins was subjected to Western blot analysis using anti-HA or anti-A28-RGS14 mouse monoclonal antibodies (Ab-ICS; Y.C., L.B., and N.K., unpublished results). (B) Induction of A28-RGS14 expression in response to mitogenic signals--a potential role as negative feedback regulator. NIH 3T3 cells expressing the musearinie M1 receptor (10) were grown to confluence, transferred to serum-free medium (DMEM containing 0.1% BSA) for 16 h. and then stimulated with or without Carbachol (50 tzM) or fetal bovine serum (10%) for • in Fig. L4). Autoradiograms were analyzed by laser densitometric ~anning (Applied Biosystems) and the signal was normalized to GAPDH control. that this novel gene encodes a new member of an evolutionarily highly conserved gene family that encodes proteins implicated in the regulation of G protein signaling (regulators of G protein signaling or RGS proteins: refs. 15-17). It is in keeping Prec. Natl. Acad. Sci. USA 94 (1997) 7871 with the numbering system for. predicted RGS proteins that this gene was named A28-RGS14 and the protein product be named A28-RGS14p. On the basis of translation of cloned eDNA sequences, both human and mouse A28-RGS14 genes encode predicted proteins of 202 amino acids with 86% amino add identity and 90% similarity (Fig. 2A). These share high homology over a core region of 130 amino acids to other members of this family found in species as distant asAspergillus nidulans (18), yeast (19), and Caenorhabditis elegans (17) (Fig. 2B). The predicted A28-RGS14p protein is closely related to the human RGSl (BL34) and RGS2. (GOSS) proteins, en- coded by genes i~iduced in activated B cells and peripheral blood mononudca.r cells, respectively (20, 21). However, RNA expression stutlfes indicate that the A28-RGS14 gene is widely expressed in human tissues (Fig. 2C), in contrast to some other members of this family, including RGS1 and RGS2, which show more tissue-specific expression patterns. As reported for RGS1 (20), expression of A28-RGS14 was also detected in activated B cells (data not shown), but of several RGS genes analyzed to date only A28-RGS14 was found to be induced by p53 (LB. and N.K, unpublished data). First indications as to a potential role of this new class of proteins in cellular signaling came from studies in yeast (19). In Saccharorayces cerevisiae, pheromone signaling is mediated through a seven-transmcmbrane-domain receptor and is cou- pled via a G protein to downstream events leading to activation of the MAPK pathway. It is the/3T moiety of the G protein that activates downstream signaling leading to growth arrest in the (St phase of the cell cycle. However, pheromone signaling also promotes expression of the RGS protein Sst2p, which in turn mediates subsequent desensitization to pheromone and recov- ery from growth arrest. Genetic and biochemical studies ~dicate that Sst2p controls pheromone signaling through direct interaction with the coupling G protein Gpalp (19, 22). Importantly, recently reported studies showed that the mam- malian RGS1 and RGS4 proteins can complement the func- tion of the yeast homologue Sst2p in a yeast pheromone desensitization assay. Alternatively, they regulate signaling by G protein-coupled receptors in human B cells (15), suggesting that these proteins encode structurally and functionally con- served regulators of G protein-linked signaling pathways. These may, however, not necessarily involve plasma- membrane receptor-coupled signaling pathways. Thus, the subunit of G~, a G protein involved in intracellular trafficking through the Golgi, directly interacts with GAIP in yeast and in vitro (16). GAIP does not appear to interact with indicating specificity for interaction with G~xi3 (16). As dem- onstrated for the yeast Sst2p protein, these findings also indicate that mammalian RGS proteins may operate by directly interacting with a subunits of specific G proteins, disrupting receptor-G protein interaction or acting at the level of the G protein itself. Recent biochemical studies with GAIP and RGS4 have shown that RGS proteins can act as GTPase activating proteins, accelerating the rate of hydrolysis of all tested members of the Gi subfamily of G protein a subunits (23). Preliminary studies indicate that A28-RGS14p can also' interact with members of the Gi/Go subfamily of G proteins, including Gaiz, but not Gas in vitro (Fig. 2D and Y.C., L.B., and N.K., unpublished results). Thus, A28-RGS14p may possibly act as a more general inhibitor of Gi/Go transduced signaling. Accordingly, we tested whether A28-RGS14p could regu- late plasma-membrane receptor-initiated and G protein- transduced signaling in intact cells. We chose to study a well characterized system in which activation of Gi-coupled M2 musearinie receptor results in G protein/33,-subunit-mediated Ras-dependent activation of the MAPK pathway (13) (the mammalian pathway related to the yeast pheromone response pathway). In addition, we tested the effects on Gq-coupled M1 muscarinic receptor signaling, which is also associated with induction of growth signals and activation of the MAPK
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7872 Biochemistry: Buckbinder et al. pathway. COS-7 cells were transiently cotransfected with mammalian expression constructs encoding M1 or M2 recep- tors, an HA-tagged-MAPK fusion protein, and a pcDNA3 plasmid expressing A28-RGS14p or pcDNA3 control vector. Cells were stimulated with the muscarinic agonist Carbachol and MAPK activity was assayed in anti-HA immunoprecipi- tates from cell lysates (Fig. 3A). Pronounced activation of MAPK was observed upon activation of either M1 or M2 receptors. This activation was markedly reduced (-70%) in ceils coexpressing exogenous A28-RGS14p (Fig. 3,4). Immu- noblot data indicate that the inhibition of MAPK activity by A28-RGS14p did not result from a change in the level of expressed HA-MAPK. Furthermore, the inability of A28- RGS14p to inhibit phorbol 12-myristate 13-acetate-induced activation of MAPK (Fig. 3A) indicates that inhibition is specific and occurs upstream of Raf Idnase, consistent with a proposed role for RGS proteins as direct regulators of G proteins. The findings that A28-RGS14p interacts with (Fig. 2/) and data not shown) and regulates signal transduction implicating both Grtike and Gq proteins, suggest that it may encode an important negative regulator of extra and intracel- lular mitogenic signals associated with the activation of diverse G protein-coupled signaling cascades (among which the MAPK pathway would only be one example). These may include/3~- and o,-subtmit-activated pathways. In this context, it is ~ of interest to note that (3i2a, which also interacts with A28-RGS14p in vitro (Fig. 2D), is encoded by the gip2 protooncogene found mutated in certain human tumors (24). In its capacity as an RGS protein, A28-RGS14p may not only act as a mechanism for p53 to exert cellular growth control by acting upstream of the ras-raf-MAPK pathway but also as a negativ¢ feedback regulator in response to mitogenic signals. Thus, similar to regulation of the Sst2p RGS protein upon pheromone stimulation in yeast, A28-RGS14 gene expression i~ induced by serum growth factors and activation of G protein-coupled receptors (Fig. 3B). This activation is con- served between human and monse and appears to be p53- independent (S.V., L.B., and N.K., unpublished data). Thus, these data implicate A28-RGS14p in a mammalian desensiti- zation response and as a new signaling molecule whereby p53 may regulate cellular sensitivity to mitogenie and possibly apoptotic signals in human cellff. 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