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J Biol Chem, Vol. 274, Issue 28, 19992-20001, July 9, 1999

G_i Proteins Use a Novel - and Ras-independent Pathway to Activate Extracellular Signal-regulated Kinase and Mobilize AP-1 Transcription Factors in Jurkat T Lymphocytes^*

Karen E. Hedin^§¶, Michael P. Bell, Catherine J. Huntoon, Larry M. Karnitz, and David J. McKean

From the ^§ Department of Surgery,  Department of Immunology, and  Division of Radiation Oncology, The Mayo Clinic and Foundation, Rochester, Minnesota 55905

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Receptors coupled to pertussis toxin (PTX)-sensitive G_i proteins regulate T lymphocyte cytokine secretion, proliferation, and chemotaxis, yet little is known about the molecular mechanisms of G_i protein signaling in mammalian lymphocytes. Using the Jurkat T lymphocyte cell line, we found that a stably expressed G_i protein-coupled receptor (the -opioid receptor (DOR1)) stimulates MEK-1 and extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2) and transcriptional activity by an ERK target, Elk-1, via a mechanism requiring a PTX-sensitive G_i protein. Levels of -adrenergic receptor kinase-1 C-terminal fragment that inhibited signaling by G_i protein subunits in these cells had no effect on DOR1 stimulation of either MEK-1- or Elk-1-dependent transcription, indicating that this pathway is independent of . Analysis of this -independent pathway indicates a role for a herbimycin A-sensitive tyrosine kinase. Unlike -mediated pathways, the -independent pathway was insensitive to RasN17, inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase), and constitutive PI 3-kinase activity. The -independent pathway regulates downstream events, since blocking it abrogated both Elk-1-dependent transcription and mobilization of the mitogenic transcription factor, AP-1, in response to DOR1 signaling. These results characterize a novel, Ras- and PI 3kinase-independent pathway for ERK activation by G_i protein signaling that is distinct from ERK activation by and may therefore be mediated by the i subunit.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

G_i protein signaling in many cell types stimulates the activity of the extracellular signal-regulated kinases (ERKs),¹ ERK1 and ERK2 (Refs. 1-16; reviewed in Ref. 17). ERK and other mitogen-activated protein kinases phosphorylate transcription factors, thereby mediating transcriptional regulation in response to signaling by cell surface receptors (18-22). Like other G proteins, G_i proteins consist of an  subunit and a dimeric subunit and mediate activation of intracellular signaling pathways in response to the ligation of receptors with seven transmembrane-spanning domains. When stimulated by a ligand-bound receptor, the  subunit binds GTP and dissociates from . This event allows both -GTP and free to directly regulate the activities of downstream effector molecules. G_i proteins contain one of the closely related i subunits (i1, i2, i3) in addition to  and  subunits shared by other types of G proteins. Pertussis toxin (PTX) covalently modifies G_i protein i subunits, preventing G_i protein activation by ligated receptors. Besides G_o and G_t, only G_i proteins are inactivated by PTX.

In epithelial and fibroblast cells, certain G_i- or G_o-coupled receptors activate ERK by liberating G protein subunits that directly stimulate an isoform of phosphatidylinositol-3 kinase (PI 3-kinase) (p110-) (8, 12, 23). This event leads to activation of Ras and the Raf/MEK/ERK kinase cascade (1-3, 22) via a mechanism involving SHC tyrosine phosphorylation and the recruitment of GRB2-SOS (5, 6, 8, 12). Although tyrosine kinases related to PYK2 (6, 14), ZAP-70/SYK (16), and Src (6, 14-16) have been implicated in this process, a detailed mechanism explaining activation by Ras is presently unavailable.

The  subunits of PTX-sensitive G proteins also have the potential to activate ERK. The gip2 oncogenes encode constitutively active i2 subunits with either an R179C or a Q205L point mutation that inhibits GTP hydrolysis (24). gip2 expression constitutively stimulates ERK (25) and mediates the oncogenic transformation of Rat1a fibroblasts (24, 26). Unlike stimulation of ERK (1-3, 22), gip2 stimulates ERK via a mechanism that is independent of Ras (25), a result suggesting that i and activate ERK via distinct pathways. Moreover, the m1-muscarinic and platelet-activating factor receptors use the closely related PTX-sensitive o subunit to activate ERK via a Ras-independent pathway in the Chinese hamster ovary epithelial cell line (10). An interesting feature of this pathway is its cell type specificity; in COS-7 fibroblasts, these same receptors use and Ras to stimulate ERK (10). This resembles the cell-specific actions of gip2, which transforms Rat-1a cells but not NIH 3T3 or Swiss 3T3 fibroblasts (24). Recent evidence suggests that i and o activate ERK in Rat-1a and COS-7 cells via transactivation of the epidermal growth factor receptor tyrosine kinase (7, 13). Although Src family tyrosine kinases have been implicated in this process (13), additional mechanistic details of this pathway are obscure.

Lymphocytes express abundant heterotrimeric G_i proteins (27) that direct lymphocyte migration (28-31), proliferation (32, 33), and cytokine secretion (34, 35). Although recent work has uncovered novel mechanisms for G_i protein stimulation of ERK activity in fibroblast and epithelial cell types (above), little is known about the molecular mechanisms of ERK activation by G_i protein signaling in mammalian lymphocytes. We recently reported that signaling by a heterologously expressed G_i protein-coupled -opioid receptor (DOR1) uses a PTX-sensitive mechanism to stimulate the accumulation of AP-1 transcription factor complexes in the Jurkat human T lymphocyte cell line (35). Here, we demonstrate that DOR1 signaling activates ERK via a novel, Ras-independent, G_i protein-mediated pathway. We further present evidence that this pathway is independent of , characterize this pathway as distinct from previously characterized -mediated ERK activation pathways, and demonstrate that this pathway leads to PTX-sensitive AP-1 mobilization in these cells.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cells-- DOR1/Ju.1 cells were derived by fluorescence-activated cell sorting of the Ju.1 Jurkat T cell subline that was stably transfected with DOR1, as described previously (36). All cells were maintained in Medium B (RPMI 1640 supplemented with 5% fetal calf serum, 5% calf serum, 10 mM HEPES, pH 7.4, 2 mM L-glutamine, and 2 µM 2-mercaptoethanol). DOR1 expression by the DOR1/Ju.1 cell line was confirmed by assays every few months as described (35).

Assay for Active, Phosphorylated ERK1 and ERK2-- Cells were preincubated 16-24 h in Medium C (RPMI 1640 supplemented with 0.5% fetal calf serum, 10 mM HEPES, pH 7.4, 2 mM L-glutamine, and 2 µM 2-mercaptoethanol) and then washed and resuspended in Medium C at a density of 10⁶ cells/ml. Cells were stimulated as indicated for 5 min at 37 °C, pelleted by rapid centrifugation, and lysed in SDS-PAGE sample buffer. Lysates were fractionated by SDS-PAGE (using approximately 2 × 10⁶ cell equivalents/lane) and transferred to Immobilon-P membrane (Millipore, Corp., Bedford, MA). The presence of active ERK1 and ERK2 phosphorylated on threonine 202 and tyrosine 204 was detected by immunoblotting with phosphospecific p44/p42 ERK1 and ERK2 (Thr²⁰²/Tyr²⁰⁴) antiserum, while total ERK2 protein was detected using p44 ERK kinase antiserum (both antisera from New England Biolabs, Beverly, MA). Unless otherwise indicated, stimuli were 10⁷ M deltorphin (Research Biochemicals International (RBI), Natick, MA), 50 ng/ml PMA (Sigma), 5 µg/ml of biotinylated anti-CD3 mAb (OKT3; prepared as described (37) and cross-linked by incubation with 10 µg/ml avidin (final concentration) for 20 min at 25 °C just before use), or 0.1 mM pervanadate (applied from a freshly prepared 10 mM pervanadate stock (38, 39)). Before stimulation, some cultures were pretreated for 16 h with 50-100 ng/ml PTX or PTX-B control toxins (both from RBI), 16 h with 0.3 µM herbimycin A (RBI), 90 min with 50 µM PD098059 (RBI), or 30 min with 100-300 nM wortmannin (Sigma).

Transfections and Assays for Elk-1-dependent Transcriptional Activity-- Elk-1 dependent transcriptional activity was assayed using the PathDetect Elk trans-Reporting System (Stratagene, La Jolla, CA). Cells (10⁷) were mixed with 5 µg of pFR-Luc and 0.5 µg of pFA-Elk with or without transient expression constructs described below plus irrelevant plasmid DNA (pcDNAIII; Invitrogen, San Diego, CA), electroporated using a BTX Electro-square porator model T820 (BTX Inc., San Diego, CA), divided into 4-6 portions, and cultured in 2 ml of Medium B or Medium C in wells of a 24-well plate for 18-24 h at 37 °C. Results were similar following culture in Medium B or C, which contain 10% and 0.5% serum, respectively. Stimuli were added to some wells for an additional 5-6 h, and then the cells were harvested and assayed for luciferase using the Luciferase Assay System (Promega, Madison, WI) and a model LB 9501/16 lumat (Berthold Systems, Aliquippa, PA). DOR1-stimulated Elk1/GAL4 activity was calculated relative to that of unstimulated cells from the same transfection. To compare different transfections done the same day, the cells were additionally transfected with 20 ng of pRL-TK (Promega, Madison, WI), which encodes a Renilla luciferase gene downstream of a minimal HSV-TK promoter. Following stimulation of transfected cells, both Renilla luciferase and luciferase were measured using the Dual Luciferase Assay System (Promega), and the Renilla values were used for normalization.

Transient Expression Plasmids-- Where indicated, transfections included 5 µg each of plasmids encoding ₁ and/or ₂ in pcDNA-neo (a generous gift of E. J. Neer, Harvard Medical School, Boston, MA) (40), 5 or 10 µg of ARKct (generously provided by R. J. Lefkowitz, Duke University Medical Center, Durham, NC) (2), 5 µg of pcDNAIII-RasN17, 5 µg of pEF-BOS-FLAG-iSH2-CAAX, or 5 µg of pEF-BOS-AU1-AKT. pcDNAIII-RasN17 was created by subcloning the N17 mutant human Ha-Ras cDNA into pcDNAIII. pEF-BOS-FLAG-iSH2-CAAX was constructed by polymerase chain reaction to encode a fusion protein with an amino-terminal FLAG epitope (MDYKDDDDK), a flexible insert (GAGAGAP), the p110-binding iSH2 domain of p85 (amino acids 466-567) (4), a flexible linker (GGRGAGAGAA), and the isoprenylation and membrane-targeting CAAX sequence from human Ha-Ras (SGPGCMSCKCVLS) (41). The polymerase chain reaction product was subcloned into the pEF-BOS expression vector (42) and sequenced to yield pEF-BOS-FLAG-iSH2-CAAX. To make pEF-BOS-AU1-AKT, polymerase chain reaction was first used to isolate the human Akt cDNA and to substitute sequences encoding the AU1 epitope tag (MDTYRYI) and a flexible linker (GAGAGAP) for the initiating ATG. This polymerase chain reaction product was then subcloned into pEF-BOS and sequenced to yield pEF-BOS-AU1-AKT.

In Vitro Kinase Assays of Overexpressed, Myc-tagged MEK-1-- Cells (10⁷) were transfected with 5 µg of an expression vector encoding Myc-tagged MEK-1 (Myc-MEK-1) with or without other expression plasmids and plated in Medium B. 24 h later, Myc-MEK-1 was immunoprecipitated using an anti-Myc mAb (Babco, Berkley, CA), and an in vitro kinase assay was performed as described (43) in the presence of [-³²P]ATP, using as a substrate 1 µg/test of kinase-inactive ERK2 GST fusion protein (GST-ERK2-KD; prepared as described (44)). Results were visualized by SDS-PAGE, Western transfer to Immobilon P membrane, and autoradiography, and/or PhosphorImager analysis using a Storm model 840 (Molecular Dynamics, Inc., Sunnyvale, CA). Quantitation was performed by PhosphorImager analysis. Total immunoprecipitated Myc-MEK-1 was visualized by immunoblotting with anti-MEK-1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Infection with Recombinant Vaccinia Viruses-- To make the recombinant Vaccinia viruses, the coding sequence of human CD56 or human Ha-RasN17 was blunt end-cloned into the SmaI site of the Vaccinia virus expression vector, pSC11. The resulting vectors were introduced into the WR strain of Vaccinia via homologous recombination, and high titer viral stocks were prepared as described (45). Stocks of the WR (wild-type/parental) strain Vaccinia virus were a generous gift of Dr. Paul Leibson (Mayo Clinic, Rochester, MN). Mutant Ras expression by the RasN17 recombinant virus was confirmed by reverse transcriptase-polymerase chain reaction and DNA sequencing of the viral stock (data not shown) and by anti-Ras immunoblotting of infected cells (Fig. 5, B and C). Semipurified recombinant CD56, RasN17, or WR (control) viruses were used for infection. For each experiment, 4 × 10⁷ cells were infected in 8.0 ml of RPMI 1640 for 1 h at 37 °C at a multiplicity of infection of 20:1. One volume of RPMI 1640 supplemented with 1% fetal calf serum was then added, and the cells were incubated for an additional 1 h at 37 °C. Infected cells were washed twice with RPMI 1640 supplemented with 0.5% fetal calf serum and either immediately incubated with anti-CD56 mAb coupled to phycoerythrin (Becton-Dickenson, San Jose, CA) and analyzed by flow cytometry or divided into six equal portions and used for endogenous MEK-1 and/or ERK2 kinase assays as described below.

Kinase Assays of Endogenous ERK2 and MEK-1-- Cells (2 × 10⁶/test) were stimulated as indicated at 37 °C in medium consisting of RPMI 1640 supplemented with 1% bovine serum albumin and lysed as described by Kohn et al. (43). Endogenous MEK-1 and/or ERK2 was immunoprecipitated using specific antisera (Santa Cruz Biotechnology) and in vitro kinase assays performed as for the Myc-MEK-1 assay above, using as substrate either 1 µg/test of kinase-inactive ERK2-GST fusion protein (for MEK-1 assay) or 5 µg/test of myelin basic protein (MBP) (for ERK2 assay; Upstate Biotechnology, Inc., Lake Placid, NY). Immunoblotting was used to quantitate the amounts of ERK2 or MEK-1 immunoprecipitated for each test (anti-ERK2 and anti-MEK-1 antisera from Santa Cruz Biotechnology). Where indicated, of the total lysate was reserved and analyzed by immunoblotting for RasN17 expression using anti-Ha-Ras antisera (Santa Cruz Biotechnology).

Akt Kinase Assay-- Cells (2 × 10⁷) were transfected with 5 µg of an expression vector encoding AU1-tagged Akt (pEF-BOS-AU1-AKT) with or without 10 µg of pEF-BOS-FLAG-iSH2-CAAX and plated in Medium B. 24 h later, Akt was immunoprecipitated using an anti-AU1 mAb (Babco, Berkley, CA), and an in vitro kinase assay was performed using myelin basic protein as a substrate (43). Results were visualized by SDS-PAGE, transfer to Immobilon P membrane, and autoradiography. Immunoprecipitated Akt was visualized by immunoblotting with sheep anti-Akt antiserum and horseradish peroxidase-conjugated anti-sheep IgG (both antisera from Upstate Biotechnology).

RNA Blot-- Cells were pretreated and stimulated as indicated in Medium B at a density of 5 × 10⁵ to 8 × 10⁵ cells/ml, and total RNA was prepared using Trizol reagent according to the manufacturer's instructions (Life Technologies, Inc.). RNA (10 µg/lane) was fractionated by electrophoresis in a 1% agarose-formaldehyde gel, transferred to Hybond-N membrane (Amersham Pharmacia Biotech), and hybridized with ³²P-labeled c-Fos and glyceraldehyde-3-phosphate dehydrogenase cDNA probes using standard procedures.

Detection of AP-1 DNA-binding Complexes-- Cells were stimulated for 4 h in Medium B at a density of 5 × 10⁵ to 8 × 10⁵ cells/ml. Nuclear extracts were prepared (35), and AP-1 DNA-binding complexes were assayed by electromobility shift assay as described (37). The double-stranded DNA probe was 5'-AAATCCAATGAGTCAGCGCGGAT-3', which contains a high affinity AP-1 binding site (underlined).

AP-1 Reporter Activity Assay-- Cells (10⁷) in Medium B were transfected with 10 µg of irrelevant plasmid DNA (pcDNAIII; Invitrogen) and 20 µg of the AP-1 reporter plasmid, pGL-AP1-Luc (generously provided by E. O'Neill (Merck)), which contains three tandem repeats of a sequence (5'-GTGACTCAGCGCG-3') with a high affinity AP-1 binding site (underlined) upstream of a minimal -fibrinogen promoter (46) and a luciferase gene. Following culture in Medium C for 18-24 h at 37 °C, cells were pretreated and stimulated for an additional 5-6 h before reporter activity was assayed by luciferase as above.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The G_i Protein-coupled Receptor, DOR1, Activates MEK-1, ERK1, and ERK2 in Jurkat T Cells via a PTX-sensitive Pathway-- To address the potential for mitogenic signaling by G_i proteins in lymphocytes, we first asked whether receptor-mediated activation of endogenous G_i proteins could stimulate ERK activity in a lymphoid cell line. For this purpose, we used DOR1/Ju.1 cells, a Jurkat T cell line stably transfected with an expression vector encoding the G_i protein-coupled receptor, DOR1 (35, 36). ERK activation was measured in response to deltorphin, a specific DOR1 agonist. Fig. 1A shows that treatment with 10⁷ M deltorphin stimulated the appearance of active, phosphorylated ERK1 and ERK2 in DOR1/Ju.1 cell lysates. This effect of deltorphin required G_i proteins since DOR1, but not PMA, activation of ERK was abrogated by pretreatment with PTX. DOR1 activation of ERK1 and ERK2 most likely requires upstream MEK activation, since ERK activation was sensitive to the specific MEK inhibitor, PD098059 (Fig. 1A) (47). In fact, DOR1 signaling activates MEK-1, shown by transiently transfecting a construct encoding Myc-tagged MEK-1 and then immunoprecipitating and assaying its kinase activity (Fig. 1B). DOR1 signaling also activates the kinase activity of endogenous MEK-1 (Fig. 5C). Like DOR1 activation of ERK1 and ERK2, DOR1 activation of either overexpressed or endogenous MEK-1 was completely sensitive to pretreatment with PTX (data not shown).

View larger version (31K): [in this window] [in a new window]   Fig. 1.   DOR1 uses a Gi protein to stimulate MEK-1, ERK1, and ERK2 activity in Jurkat T lymphocytes. A, DOR1 activates ERK1 and ERK2 via a PTX-sensitive pathway. DOR1/Ju.1 cells were pretreated with PTX or PD098059 (PD) and stimulated with 107 M deltorphin (Delt) or 50 ng/ml PMA for 5 min. Cells were then lysed and analyzed by SDS-PAGE and immunoblotting for active, Thr202/Tyr204 phosphorylated ERK1 and ERK2 (top) or total ERK2 protein (bottom), using specific antisera. The result shown is representative of six independent experiments. B, DOR1 activates MEK-1. DOR1/Ju.1 cells were transiently transfected with Myc-tagged MEK-1 (Myc-MEK-1), stimulated with 107 M deltorphin for the indicated times, and lysed. Myc-MEK-1 was immunoprecipitated and assayed for in vitro kinase activity toward a kinase-dead ERK2 fusion protein (GST-ERK2-KD) in the presence of [-32P]ATP. Results shown are representative of five independent experiments. Upper gel and graph, 32P incorporation into GST-ERK2-KD following deltorphin stimulation. Lower gel, immunoblot showing amounts of Myc-MEK-1 immunoprecipitated per lane. C and D, DOR1 activates Elk-1-dependent transcription via a PTX-sensitive pathway. DOR1/Ju.1 Jurkat T cells were transiently transfected with pFR-Luc and pFA-Elk plasmids, cultured for 24 h, and stimulated as indicated for 5 h. Elk-1-dependent transcriptional activity was assessed by measuring luciferase activity derived both from pFR-Luc and from a cotransfected control reporter plasmid constitutively expressing the Renilla luciferase (Con.). Where indicated, cells were pretreated with PD098059, PTX, or the PTX-B control toxin before being stimulated. Results are expressed in relative luciferase units (RLU). Stimuli were 107 M deltorphin or 50 ng/ml PMA. C, inset, results of stimulating with varying concentrations (M) of deltorphin, expressed as a percentage of the maximal response obtained with PMA. Each symbol denotes the mean of four determinations ± S.E. D, inset, summary of multiple experiments as in D, showing the -fold increase in Elk-1-dependent transcriptional activity with 107 M deltorphin compared with basal levels, following pretreatment with PTX or PTX-B toxins. Each bar represents the mean ± S.E. of three independent determinations.

Since the transcription factor, Elk-1, is activated by ERK phosphorylation (20, 21), we also tested the ability of DOR1-mediated G_i protein signaling to mediate Elk-1-dependent transcriptional activity. DOR1/Ju.1 cells were transiently transfected with a plasmid that constitutively expresses a chimeric Elk-1/Gal4 transcription factor (pFA-Elk), together with a plasmid containing the luciferase gene downstream of five Gal4 DNA binding sites (pFR-Luc). When activated by upstream signaling events, ERK phosphorylates the Elk-1 domain of the transfected Elk/Gal4 chimera, which then stimulates luciferase expression from the Gal4-responsive promoter of pFR-Luc.

As shown in Fig. 1C, treatment of transiently transfected DOR1/Ju.1 cells with 10⁷ M deltorphin increased Elk-1-dependent luciferase activity. Elk-1 may potentially be regulated by several mechanisms; however, the deltorphin-mediated response is sensitive to the specific MEK inhibitor, PD098059. In contrast, PD098059 had little effect on luciferase activity derived from a cotransfected control Renilla reporter gene (Con.). Elk-1-dependent transcription in response to deltorphin was dose-dependent (Fig. 1C, inset) in a manner consistent with its reported K_d of 3.3 nM for DOR1 (21). Subsequent experiments used 10⁷ M deltorphin for stimulation. Fig. 1D shows that the Elk-1-dependent transcription stimulated by deltorphin was completely inhibited by pretreatment with PTX but not by pretreatment with the PTX B-oligomer, which lacks the ADP-ribosyltransferase subunit that targets G protein i subunits. Fig. 1D, inset, shows that these effects of PTX and PTX-B were observed in multiple experiments. In contrast, PTX had no effect on Elk-1-dependent transcription stimulated by PMA. Moreover, deltorphin had no effect on luciferase expression from the control Renilla reporter gene or on luciferase expression in DOR1/Ju.1 cells cotransfected with pFR-Luc and a plasmid encoding only the Gal4 DNA binding domain (data not shown). Together, these results demonstrate that DOR1 uses a PTX-sensitive G_i protein to activate MEK-1, ERK1, and ERK2 and to mediate transcriptional activation by an ERK target, Elk-1, in Jurkat T cells.

Overexpression of G_i Protein Subunits Stimulates MEK-1 Activity and Elk-1-dependent Transcription in Jurkat T Cells-- Overexpression of the G protein ₁ and ₂ subunits mimics signaling that follows the ligation of some G_i protein-coupled receptors (1-6, 8, 10, 12). We therefore examined the ability of to activate Elk-1-dependent transcription in Jurkat T cells. Cells were transiently transfected with pFR-Luc and pFA-Elk, together with expression vectors encoding ₁ and ₂. Fig. 2A shows typical results. Transient expression of both ₁ and ₂ subunits significantly elevated Elk-1-dependent luciferase activity (solid bars) without affecting expression from the control Renilla reporter gene (open bars, Con.). In contrast, transient expression of either ₁ or ₂ alone had little effect on Elk-1-dependent or control luciferase activity. In multiple experiments, overexpressing both ₁ and ₂ elevated Elk-1-dependent transcriptional activity by 5.3 ± 0.6-fold compared with basal levels (n = 7; p < 0.01) (Fig. 2B). Elk-1-dependent transcriptional activity that was stimulated by overexpressed was sensitive to the MEK inhibitor, PD098059, but PD098059 had little effect on the transcription of luciferase from the control Renilla reporter gene (Fig. 2, A and B, and data not shown).

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Transient expression of G protein subunits stimulates Elk-1-dependent transcriptional activity in Jurkat T lymphocytes. A, DOR1/Ju.1 Jurkat T cells were transiently transfected with pFR-Luc and pFA-Elk with or without plasmids encoding G protein 1 and/or 2 subunits, plated in 0.5% serum containing medium with or without PD098059 for 30 h, and Elk-1-dependent transcriptional activity was measured by assaying for luciferase activity derived both from pFR-Luc (solid bars) and from a cotransfected control reporter plasmid constitutively expressing the Renilla luciferase (Con., open bars). B, results of multiple experiments performed as in A. The results of individual experiments were derived from transfections performed the same day, normalized using the control Renilla luciferase values. Each bar represents the mean ± S.E. of 3-7 independent experiments.

To directly examine the regulation of MEK activity by overexpressed , we transiently transfected together with a construct encoding Myc-tagged MEK-1 and then immunoprecipitated and assayed the kinase activity of the Myc-tagged MEK-1. Results shown in Fig. 3C show that overexpressing in Jurkat T cells resulted in an approximately 2-fold activation of Myc-MEK-1 kinase activity. These results show that overexpressing G protein subunits in Jurkat T cells increases both MEK-1 activity and Elk-1-dependent transcription, suggesting that is capable of activating ERK in these cells.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   DOR1 stimulates MEK-1 via a pathway that is insensitive to ARKct. A, DOR1/Ju.1 Jurkat T cells were transiently transfected with Myc-tagged MEK-1 with or without a plasmid encoding ARKct, stimulated as indicated for 5 min with 107 M deltorphin, and the in vitro kinase activity of Myc-MEK-1 was determined as in Fig. 1B. Upper gel, 32P incorporation into the MEK-1 substrate (GST-ERK2-KD) following deltorphin stimulation. Lower gel, immunoblot showing the amounts of Myc-MEK-1 immunoprecipitated per test. Graph, 32P incorporation into the MEK-1 substrate normalized to the total amount of Myc-MEK-1 per test. B, DOR1/Ju.1 cells were transiently transfected with Myc-MEK-1 with or without vector alone (circles) or 5 µg of ARKct (triangles) and stimulated for the indicated times with 107 M deltorphin. Myc-MEK-1 activity was determined as in Fig. 1B. C, DOR1/Ju.1 cells were transiently transfected with Myc-MEK-1 and with or without 10 µg of ARKct as indicated, and the Myc-MEK-1 activity of unstimulated cells was determined as in Fig. 1B. The results shown are representative of 3-6 (A and C) or two (B) individual experiments.

DOR1 Stimulates MEK-1 Activity and Elk-1-dependent Transcription via a -Independent Pathway-- We next asked if mediates MEK-1 activation by DOR1 in Jurkat T cells. ARKct binds free , and ARKct expression can block stimulation of ERK that is initiated by either receptor ligation or overexpression (2, 3, 10). We therefore tested the effects of ARKct cotransfection on the ability of DOR1 to activate MEK-1. Fig. 3A shows that cotransfection of ARKct had no detectable effect on DOR1 signaling that increased the kinase activity of Myc-MEK-1. In multiple experiments, DOR1 activation of Myc-MEK-1 in the presence of ARKct was 105 ± 25% (n = 3) of that in the absence of ARKct (100%), which represents no significant inhibition (p = 0.86). In the same experiments, ARKct cotransfection significantly reduced activation of Myc-MEK-1 to only 33 ± 6.8% (n = 3; p = 0.01; Fig. 3C) of that stimulated by alone. Fig. 3B additionally shows that DOR1 activation of Myc-MEK-1 kinase activity was unaffected by ARKct throughout its time course.

We next tested the ability of cotransfected ARKct to inhibit DOR1 stimulation of the Elk-1-dependent transcriptional activity that is downstream of MEK-1 and ERK. While cotransfecting ARKct clearly inhibited the ability of overexpressed subunits to stimulate Elk-1-dependent transcription, ARKct had little effect on the transcriptional activity that resulted from DOR1 ligation by deltorphin (Fig. 4A). Fig. 4B summarizes the results of multiple experiments. ARKct exerted no significant effect on the ability of deltorphin to stimulate Elk-1-dependent transcriptional activity (n = 3; p = 0.96), while in the same experiments ARKct significantly inhibited activity in response to overexpressed (n = 3; p < 0.02). Together, the results in this section indicate that DOR1 uses a -independent mechanism to stimulate MEK-1 kinase activity and transcriptional activation by Elk-1. Since this pathway is also sensitive to PTX (Fig. 1), these results suggest that DOR1 uses i rather than to stimulate MEK-1 and downstream events in Jurkat T cells.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   ARKct has no effect on DOR1 stimulation of Elk-1-dependent transcription. DOR1/Ju.1 Jurkat T cells were transfected with pFR-Luc and pFA-Elk with or without plasmids encoding 1, 2, or 5 µg of ARKct. Deltorphin or stimulation of Elk-1-dependent transcriptional activity was measured as in Figs. 1 and 2. A, results of a representative experiment, with results expressed as a percentage of the PMA response in the same transfection. B, summary of multiple experiments as in A, with deltorphin or stimulation in the absence of ARKct normalized to 100%. Each bar denotes the mean ± S.E. of three independent determinations. *, the mean is significantly different from 100% (p < 0.02).

Ras Is Required for Stimulation but Not DOR1 Stimulation of ERK-- To characterize and compare the mechanisms used by and DOR1 to stimulate ERK, we examined the effects of the dominant-negative mutant of Ras, RasN17 (48), on DOR1 and signaling leading to ERK activity and downstream events in Jurkat T cells. To facilitate direct assay of the kinase activities of endogenous MEK-1 and ERK2, we employed a recombinant Vaccinia virus that expresses RasN17. Unlike transient transfection, recombinant Vaccinia virus infection of Jurkat T cells results in >95% of the cells expressing high levels of the recombinant protein. This is demonstrated by a flow cytometric assay of cell surface CD56 following infection of DOR1/Ju.1 Jurkat T cells with a CD56-expressing recombinant Vaccinia virus (Fig. 5A). Using identical infection conditions, we infected Jurkat T cells with recombinant Vaccinia viruses that express either RasN17 or nothing (WR, or wild-type, strain). Following stimulation with deltorphin, the presence of active, phosphorylated ERK1 and ERK2 was assayed by immunoblotting as in Fig. 1A. As shown in Fig. 5B, RasN17 had no detectable effect on the time course of ERK1 or ERK2 phosphorylation in response to deltorphin. RasN17 also had no effect on the ability of deltorphin to activate endogenous MEK-1 or ERK2 activity, as measured by immunoprecipitating these kinases and measuring their kinase activities in vitro (Fig. 5C). Fig. 5D shows that in multiple experiments, we observed no significant inhibition of DOR1-mediated activation of endogenous ERK2 or MEK-1 kinase activity in cells expressing RasN17.

View larger version (40K): [in this window] [in a new window]   Fig. 5.   DOR1 activation of ERK is not sensitive to RasN17. A, >95% of DOR1/Ju.1 Jurkat T cells express recombinant protein following infection with a recombinant Vaccinia virus. Cells were infected at a multiplicity of infection of 20:1 with either WR (wild-type) or CD56-expressing recombinant Vaccinia. 2 h later, cell surface CD56 was assayed by incubation with anti-CD56 mAb and flow cytometry. The gray histogram shows CD56 cell surface expression by CD56-infected cells. The two open histograms are controls: unstained cells and cells infected with wild-type Vaccinia virus and stained for cell-surface CD56 expression. B, DOR1 stimulation of active, phosphorylated ERK1 and ERK2 is not sensitive to RasN17. DOR1/Ju.1 Jurkat T cells were infected either with WR (wild-type) or RasN17-encoding recombinant Vaccinia viruses as in A. 2 h later, cells were stimulated with 107 M deltorphin for the indicated times. Cells were then lysed, and whole-cell lysates were analyzed by SDS-PAGE and immunoblotting for active, Thr202/Tyr204-phosphorylated ERK1 and ERK2 (upper gel). Total ERK2 (middle gel) or RasN17 (bottom gel) in the lysates was assayed by stripping the same blot and immunoblotting with antisera specific for ERK2 or Ras. A 5-fold longer exposure of the Ras immunoblot shows endogenous Ras in the wild-type virus-infected lanes. Results are representative of three independent experiments. C and D, DOR1 stimulates the activity of MEK-1 and ERK2 via a RasN17-insensitive pathway. Cells were infected with either WR (wild-type) or recombinant Vaccinia viruses encoding RasN17 as in B. 2 h postinfection, cells were stimulated with 107 M deltorphin for the indicated times and lysed, and endogenous MEK-1 or ERK2 was immunoprecipitated and assayed for kinase activity in the presence of [-32P]ATP. Substrates were kinase-deficient ERK2 fusion protein (GST-ERK2-KD; for MEK-1 assay) or MBP (for ERK2 assay). C, results of a representative experiment. Western blotting shows the amounts of MEK-1 or ERK2 immunoprecipitated in each test. The bottom gel shows the amount of RasN17 detected by immunoblotting of the cell lysates. D, summary of multiple experiments as in C, with the deltorphin response of RasN17-infected cells expressed as a percentage of the response of cells that were infected with wild-type Vaccinia virus in the same experiment. Each bar denotes the mean ± S.E. of three or four independent determinations.

We also tested the effects of RasN17 on DOR1 signaling that leads to Elk-1-dependent transcriptional activity. An expression plasmid encoding RasN17 was transfected into DOR1/Ju.1 cells together with pFR-Luc and pFA-Elk, and the cells were stimulated and assayed for Elk-1-dependent transcriptional activity as in Fig. 1, C and D. Typical results are shown in Fig. 6A. RasN17 inhibited Elk-1-dependent transcription in response to either overexpression or stimulation of the T lymphocyte antigen receptor-CD3 complex (TCR-CD3) with anti-CD3 mAb. In addition, cotransfection of this RasN17-encoding plasmid inhibited (by approximately 80%) transcription from an IL-2 promoter construct in response to TCR-CD3 and CD28 stimulation (n = 3; p = 0.02; data not shown). These results are consistent with previous reports that Ras participates in signaling by (2, 3, 10) and TCR-CD3 (49, 50). In contrast to its effects on TCR-CD3 and signaling, RasN17 had little or no effect on Elk-1-dependent transcriptional activity stimulated by DOR1 or PMA (Fig. 6A). Fig. 6B summarizes the results of multiple experiments. RasN17 consistently inhibited , but not deltorphin, stimulation of Elk-1-dependent transcription.

View larger version (32K): [in this window] [in a new window]   Fig. 6.   , but not DOR1, stimulation of Elk-1-dependent transcription is sensitive to RasN17. A and B, DOR1/Ju.1 Jurkat T cells were transfected with pFR-Luc, pFA-Elk, and a control plasmid expressing Renilla luciferase with or without plasmids encoding 1, 2, or RasN17, plated in 0.5% serum-containing medium for 24 h, stimulated as indicated for 5 h, and assayed for Elk-1-dependent luciferase activity as in Figs. 1 and 2. A, results of a representative experiment. B, summary of multiple experiments as in A. Each bar denotes the mean ± S.E. of 3-9 independent experiments.

To summarize, the results in this section demonstrate that DOR1 uses a RasN17-insensitive mechanism to activate MEK-1-, ERK1- and ERK2-, and Elk-1-dependent transcriptional activity in Jurkat T cells. In contrast, stimulation of MEK-1 and Elk-1-dependent transcription in these cells is sensitive to RasN17. Since DOR1, but not , signaling is also insensitive to ARKct, these results suggest that DOR1 and subunits stimulate ERK activation via independent signaling pathways that differ in their requirements for Ras.

Elk-1-dependent Transcription in Response to , but Not DOR1, Synergizes with Constitutive PI 3-Kinase Activity-- In fibroblast and epithelial cells, -dependent stimulation of ERK requires activity of the p110- isoform of PI 3-kinase (8, 12, 23). We therefore asked whether - or DOR1-mediated Elk-1-dependent transcription synergizes with constitutive PI 3-kinase activity. The binding of p85 to activated receptor tyrosine kinases normally stimulates the PI 3-kinase activity of associated p110/ PI 3-kinases by mediating their membrane localization (51, 52). We therefore constructed an expression plasmid encoding iSH2-CAAX, which consists of the p110/-binding iSH2 domain of p85 fused to the membrane targeting and isoprenylation domain of Ha-Ras. Transient expression of iSH2-CAAX enhanced the kinase activity of Akt, a downstream target of PI 3-kinase (53, 54) (Fig. 7A), indicating that like other fusion proteins that mediate membrane localization of p110/ (51, 52, 55), iSH2-CAAX stimulates PI 3-kinase activity. Consistent with this idea, iSH2-CAAX activation of Akt was abrogated by pretreatment with the PI 3-kinase inhibitor, wortmannin. iSH2-CAAX also mimicks PI 3-kinase signaling when transiently expressed in other cell types (56).

View larger version (35K): [in this window] [in a new window]   Fig. 7.   No role for PI 3-kinase activity in DOR1 activation of ERK- or Elk-1-dependent transcription. A, DOR1/Ju.1 Jurkat T cells were transfected with a plasmid encoding epitope-tagged Akt with or without a plasmid encoding iSH2-CAAX as indicated. 24 h later, the indicated cells were pretreated for 30 min with 100 nM wortmannin, and then all cells were lysed and Akt was immunoprecipitated and assayed in the presence of [-32P]ATP for kinase activity toward MBP. Phosphorylated MBP (pMBP) was visualized by SDS-PAGE and PhosphorImager analysis (upper gel image and graph), and the amount of immunoprecipitated Akt in each lane was assessed by immunoblotting (lower gel). B and C, DOR1/Ju.1 Jurkat T cells were transfected with pFR-Luc and pFA-Elk with or without plasmids encoding 1, 2, RasN17, or iSH2-CAAX, plated in 0.5% serum-containing medium for 24 h, stimulated as indicated for 5 h, and assayed for Elk-1-dependent luciferase activity as in Figs. 1 and 2. B, results of a representative experiment. Stimuli were 107 M deltorphin (Delt) or 5 µg/ml cross-linked biotinylated anti-CD3 mAb (CD3). C, summary of multiple experiments as in B, showing the mean -fold increases in Elk-1-dependent transcription compared with basal levels. Where indicated, transfected cells were cultured in the presence of 50 µM PD098059. Each bar denotes the mean ± S.E. of 3-10 independent determinations. D, DOR1/Ju.1 cells were pretreated for 30 min with wortmannin as indicated, stimulated for 5 min with 107 M deltorphin or 50 ng/ml PMA, and assayed for Thr202/Tyr204-phosphorylated ERK1 and ERK2 (top) or total ERK2 (bottom) as in Fig. 1A.

While transient expression of iSH2-CAAX increased the level of Elk-1-dependent transcriptional activity in unstimulated cells, iSH2-CAAX did not synergistically enhance Elk-1-dependent transcription in response to either deltorphin or anti-CD3 mAb (Fig. 7B). In contrast, cotransfection with iSH2-CAAX dramatically enhanced Elk-1-dependent transcriptional activity in response to overexpression. In multiple experiments, cotransfection of and iSH2-CAAX consistently elevated luciferase activity approximately 45-fold (n = 10; Fig. 7C). This effect was synergistic, since it exceeded by 3-fold (n = 10; p = 0.02) the calculated additive effects of expressing either or iSH2-CAAX alone. PD098059 inhibited Elk-1-dependent transcriptional activity stimulated by either iSH2-CAAX alone or iSH2-CAAX plus , suggesting that these pathways require MEK-1. Moreover, like the activity that arises from overexpressing alone (Fig. 6B), Elk-1-dependent transcriptional activity resulting from coexpressing and iSH2-CAAX was sensitive to RasN17 (Fig. 7, B and C).

We also tested the effects of the PI 3-kinase inhibitor, wortmannin, on ERK activation by DOR1. As shown in Fig. 7D, wortmannin applied at doses that completely inhibit p110/ or p110- PI 3-kinase activity (12, 57), as well as iSH2-CAAX-stimulated Akt activity (Fig. 7A), had little effect on ERK activation by deltorphin. Similarly, Elk-1-dependent transcriptional activity in response to deltorphin was unaffected by pretreatment with wortmannin (data not shown). Since the iSH2-CAAX fusion protein enhanced Elk-1-dependent transcription in response to signaling by , but not DOR1, these results provide additional support for the idea that DOR1 and stimulate ERK activity via distinct pathways. In contrast, the results in this section indicate that the DOR1-mediated pathway of ERK activation is independent of PI 3-kinase activity.

MEK-1 Activation Is Essential for DOR1 Signaling That Leads to Increased Levels of c-Fos mRNA and Transcriptionally Active AP-1 Transcription Factors-- When Elk-1 is phosphorylated by ERK, it can act together with serum response factor to mediate increased expression of c-Fos (18-21). Fig. 8A shows that deltorphin stimulated the accumulation of c-Fos mRNA but not control glyceraldehyde-3-phosphate dehydrogenase mRNA. Furthermore, this increase was abrogated by pretreatment with the MEK inhibitor, PD098059. Since PD098059 also blocks ERK activation and Elk-1-dependent transcription in response to deltorphin (Fig. 1), these results suggest that DOR1 requires MEK-1 and ERK activation to increase c-Fos mRNA. Consistent with this idea, DOR1 signaling leading to c-Fos mRNA induction was insensitive to wortmannin (Fig. 8A).

View larger version (30K): [in this window] [in a new window]   Fig. 8.   DOR1 signaling in Jurkat T cells leads to increased c-Fos mRNA and AP-1 transcription factor complexes. A, DOR1/Ju.1 cells were pretreated with PD098059 or 100 or 300 nM wortmannin. Cells were then stimulated for 20 min with 107 M deltorphin (Delt), and total RNA was isolated and analyzed for c-Fos mRNA (top) or control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA (bottom) by Northern (RNA) blotting. B, cells were stimulated with 107 M deltorphin for 4 h, and nuclear extracts were prepared and assayed for AP-1 by electromobility shift assay using a consensus AP-1 binding site probe. Control, assay performed in the presence of 100-fold excess unlabeled probe, No Ex., assay performed in the absence of nuclear extract. C, cells were transfected with a plasmid containing an AP-1-responsive promoter upstream of a luciferase gene, plated in 0.5% serum-containing medium for 24 h, pretreated 30 min with 100 nM wortmannin where indicated, stimulated as indicated for 5 h, and assayed for luciferase activity as in Fig. 1. Results shown are from a representative experiment performed in duplicate; similar results were obtained in three independent experiments. D, results of multiple experiments assaying AP-1 promoter activity performed as in C except that pretreatments were with PTX-B, PTX, or PD098059 (PD). Stimuli were 107 M deltorphin or 50 ng/ml PMA for 5 h. Each bar denotes the mean ± S.E. of 3-11 independent determinations. *, significantly different from deltorphin or PMA alone (p < 0.001).

The c-Fos protein can participate in forming the AP-1 transcription factor complex (21, 58); therefore, we examined the regulation of AP-1 in response to DOR1 signaling. Fig. 8B shows that DOR1 signaling elevated levels of DNA-binding AP-1 complexes. Like DOR1-mediated activation of ERK and c-Fos mRNA induction, DOR1 stimulation of DNA-binding AP-1 complexes was sensitive to PD098059 but not wortmannin. In addition, we previously showed that like DOR1 activation of ERK, the DOR1-mediated increase in DNA-binding AP-1 is abrogated by PTX (35). The AP-1 complexes mobilized by DOR1 include those that are transcriptionally active, since DOR1 ligation also elevated transcription from an AP-1 reporter plasmid in a manner sensitive to PTX or PD098059 but not to wortmannin (Fig. 8, C and D). These results indicate that the novel -, Ras-, and PI 3-kinase-independent pathway of ERK activation described above is essential for DOR1 signaling that leads to increased levels of c-Fos mRNA and transcriptionally active AP-1 transcription factors.

A Role for a Tyrosine Kinase in ERK Activation by DOR1/i Signaling-- Several reports indicate that tyrosine kinases can participate in G_i protein-mediated stimulation of ERK (6, 7, 13-16); therefore, we examined the effects on DOR1 signaling of a tyrosine kinase inhibitor, herbimycin A. Pretreatment with herbimycin A partially inhibited the activation of ERK in response to DOR1 signaling (Fig. 9A). Herbimycin A pretreatment also partially inhibited the increase in DNA-binding AP-1 transcription factors in response to deltorphin (Fig. 9B). In contrast to its partial inhibition of DOR1 effects, herbimycin A almost completely blocked ERK activation and AP-1 mobilization in response to pervanadate, an inhibitor of tyrosine phosphatases that activates Src family tyrosine kinases in Jurkat T cells (39). Densitometric analysis shows that the DOR1-mediated increases in ERK2 phosphorylation and AP-1 DNA binding activity were inhibited 50 and 70%, respectively, by herbimycin A. These results suggest that the -independent, DOR1-initiated pathway that stimulates ERK and increases AP-1 transactivation is regulated by a herbimycin A-sensitive tyrosine kinase.

View larger version (14K): [in this window] [in a new window]   Fig. 9.   DOR1 signaling stimulates ERK and mobilizes AP-1 via a pathway sensitive to herbimycin A. DOR1/Ju.1 Jurkat T cells were pretreated for 16 h with 3 µM herbimycin A as indicated. A, cells were then stimulated for 5 min with 107 M deltorphin (Delt) or 0.1 mM pervanadate (Perv.) and assayed for active, Thr202/Tyr204-phosphorylated ERK1 and ERK2 as in Fig. 1A. B, cells were stimulated with 107 M deltorphin or 0.1 mM pervanadate for 4 h, and nuclear extracts were prepared and assayed for AP-1 by EMSA as in Fig. 8B.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Signaling by PTX-sensitive G proteins leads to activation of ERK1 and ERK2 (1-16; reviewed in Ref. 17), events that are important for mobilizing AP-1 and for stimulating cell cycle entry in response to growth factors (17, 20-22, 58). Most studies on the molecular mechanisms that underlie this response have focused on ERK activation by G_i or G_o protein subunits (reviewed in Ref. 17), yet recent evidence for -independent pathways (10, 13) and the oncogenic potential of mutant G_i protein i subunits (24-26) indicate that alternative mechanisms exist. Here, we present results that establish the existence in Jurkat T cells of distinct -mediated and -independent pathways for G_i protein-mediated stimulation of MEK-1 and ERK activity. In addition, we present evidence that the -independent pathway is independent of Ras and PI 3-kinase activity and demonstrate that it is required for Elk-1-dependent transcription and the mobilization of AP-1 transcription factors in response to signaling by a G_i protein-coupled receptor.

We analyzed the molecular mechanisms of G_i protein-mediated ERK stimulation using the DOR1/Ju.1 subline of the human T lymphocyte cell line, Jurkat. DOR1/Ju.1 cells are stably transfected with the neuronal G_i protein-coupled receptor, DOR1, which we previously showed signals via a PTX-sensitive G_i protein in these cells (35, 36). Here, we show that agonist stimulation of DOR1 increases ERK activity and Elk-1-dependent transcriptional activity via a PTX-sensitive pathway, establishing that this pathway requires a G_i protein. We further show that these effects of DOR1 are sensitive to the MEK inhibitor, PD098059, and that DOR1 activates MEK-1, indicating that this PTX-sensitive pathway activates ERK and downstream transcriptional events via MEK-1.

Since both MEK-1 activity and Elk-1-dependent transcription were elevated by transiently transfecting G_i protein ₁ and ₂ subunits into Jurkat T cells, we asked if mediates DOR1 coupling to ERK. Interestingly, ARKct inhibited Elk-1-dependent transcriptional activity in response to overexpressed but had no effect on Elk-1-dependent transcription that followed ligation of DOR1. Similarly, ARKct inhibited constitutive MEK-1 activation by overexpressed but had no effect on MEK-1 activation in response to DOR1 signaling. In other cell types, ARKct inhibits ERK activation in response to ligation of certain G_i protein-coupled receptors, apparently because ARKct sequesters free (2, 3, 10). Our results therefore indicate that although signaling by subunits can stimulate MEK-1 and downstream events in Jurkat T cells, the G_i protein-coupled receptor, DOR1, stimulates MEK-1-, ERK-, and Elk-1-dependent transcription via a -independent pathway.

We next asked if signaling intermediates that generally participate in ERK regulation also participate in the -independent pathway. G protein subunits (1-3, 22), as well as receptors that signal using tyrosine kinases (22, 49), stimulate ERK via activation of Ras, a GTP/GDP-binding oncoprotein that stimulates the Raf/MEK/ERK kinase cascade. Although the DOR1- and -mediated pathways were each sensitive to the MEK inhibitor, PD098059, both ERK activity and Elk-1-dependent transcription in response to DOR1 ligation were insensitive to transient expression of the dominant-negative Ras, RasN17. In contrast, RasN17 inhibited Elk-1-dependent transcription in response to either overexpression or TCR-CD3 ligation, consistent with previous reports that these stimuli use Ras to activate ERK (1-3, 11, 49, 50). We also explored the role of PI 3-kinase activity in the -independent pathway. Interestingly, DOR1 stimulation of ERK or Elk-1-dependent transcription was insensitive to the PI 3-kinase inhibitor, wortmannin, and did not synergize with constitutively elevated PI 3-kinase activity that enhanced stimulation of Elk-1-dependent transcription.

Together, these results indicate that DOR1 activates MEK-1-, ERK1- and ERK2-, and Elk-1-dependent transcriptional activity in Jurkat T cells via a mechanism that requires neither Ras nor a PI 3-kinase. In contrast, -mediated stimulation of MEK-1- and Elk-1-dependent transcription in these cells is sensitive to RasN17 and synergizes with PI 3-kinase activation. Since DOR1, but not , signaling is also insensitive to ARKct, these results suggest that DOR1 and subunits stimulate ERK activity via independent signaling pathways that differ in their requirements for Ras. Since DOR1 stimulation of ERK was partially sensitive to the tyrosine kinase inhibitor, herbimycin A, the -independent pathway may involve a tyrosine kinase. Experiments aimed at identifying the tyrosine kinase(s) associated with DOR1 signaling are in progress. However, it seems unlikely that tyrosine kinases associated with the TCR-CD3 receptor complex are involved via transactivation in DOR1 activation of ERK, since RasN17 inhibited Elk-1-dependent transcription in response to signaling by TCR-CD3 but not DOR1.

To our knowledge, this is the first evidence for a G_i protein-mediated but -independent pathway of ERK activation that is independent of both Ras and PI 3-kinase activity. Since DOR1 uses a PTX-sensitive mechanism to stimulate this pathway and since lymphoid cells do not express other  subunits that are PTX targets (28), it is most likely that DOR1 uses uses i2 and/or i3, rather than , to activate MEK-1, ERK, and downstream transcriptional events. Consistent with this idea, previous biochemical studies have shown that DOR1 can bind and promote the activation of G proteins containing both i2 and i3 subunits (Ref. 59 and references therein). The DOR1-mediated pathway resembles the pathway that is stimulated by the i2 oncogenic gip2 mutants, which activates ERK (25) and transforms Rat1a fibroblasts via RasN17-resistant pathways (24). However, unlike G_i protein signaling that activates ERK via the epidermal growth factor receptor in COS-7 cells (13), DOR1 activates ERK in Jurkat cells via a pathway resistant to the PI 3-kinase inhibitor drug, wortmannin. A Ras- and -independent pathway was recently described in Chinese hamster ovary epithelial cells, but this pathway appears to be mediated by o subunits and is inhibited by down-regulation of PMA-sensitive protein kinase C isoforms (10). DOR1 signaling most likely stimulates ERK via a distinct pathway, since DOR1 signaling was not sensitive to protein kinase C inhibitor drugs that inhibit PMA activation of ERK,² and since lymphocytes express the G_i protein subunits i2 and i3 but are deficient in other PTX-sensitive G proteins such as o (28).

Interestingly, DOR1 signaling showed no evidence of initiating the -mediated pathway of ERK activation, although receptor activation of the heterotrimeric G protein theoretically produces equal numbers of i and signaling moieties. Other reports of putative i/o-mediated ERK activation also do not address the question of why -mediated signaling is not detected in these cases (10). It is possible, however, that the molecules required for -mediated ERK activation, including the p110- PI 3-kinase and Ras, are inaccessible to that is produced in the vicinity of the activated DOR1 receptor. It is also possible that free is itself efficiently sequestered when it is produced in membrane subdomains associated with specific receptors. Further research will be necessary to adequately address the question of what happens to the free subunits during -independent signaling by DOR1 in Jurkat cells.

Although constitutively active i2 subunits (i2Q205L) have been shown to activate ERK in Rat1a fibroblasts (24, 25), we observed only a small elevation of Elk-1-dependent transcription and MEK-1 activity following transient transfection of such mutant i2 or i3 subunits into Jurkat T cells.² This may indicate that the i-mediated ERK activation pathway in these cells is rapidly down-regulated in response to chronic stimulation. An alternative explanation for our results is that i stimulates the -independent pathway by acting together with a second DOR1-activated  subunit. Biochemical studies show that besides PTX-sensitive i and o subunits, DOR1 is capable of physically interacting with PTX-insensitive G proteins that contain _q, _z/x, and ₁₆ (59). However, it is unlikely that DOR1 signaling in Jurkat T cells activates these  subunits in addition to i. Signaling by q-type subunits (including ₁₆) can directly activate PLC- (60). In contrast, DOR1 signaling increases calcium in Jurkat T cells via a mechanism that is fully sensitive to PTX (36). Similarly, the predominantly brain-expressed _z/x subunit permits adenylyl cyclase regulation in a PTX-insensitive manner, while DOR1 uses a PTX-sensitive mechanism to decrease cAMP levels in Jurkat T cells (36). While we are directly addressing this question by identifying the G proteins that co-purify with DOR1 in Jurkat T cells, we consider it most likely that the -independent ERK activation pathway we describe here is mediated by i2 and/or i3.

We also report here that -mediated but not DOR1-mediated activation of ERK is synergistically enhanced by iSH2-CAAX, a fusion protein designed to constitutively activate p110/ PI 3-kinases. Since stimulates ERK via a mechanism that requires Ras (1-3, 22) and the p110- PI 3-kinase (8, 12, 23), we hypothesize that the D3-phosphorylated lipids produced by constitutive PI 3-kinase activity synergize with signaling. RasN17 blocked ERK activity in response to either alone or plus iSH2-CAAX, consistent with iSH2-CAAX acting on the pathway upstream of Ras. The lipid products of p110/ PI 3-kinases may therefore participate in cross-talk that enhances -mediated activation of Ras and ERK.

Finally, we show that the -independent pathway for ERK activation is required for DOR1 to mobilize AP-1 transcription factors in the Jurkat T cell line, a result indicating that this novel -independent pathway contributes to the regulation of a downstream event relevant to mitogenic signaling. DOR1 signaling increased mRNA for the AP-1 subunit, c-Fos, and levels of transcriptionally active AP-1, via mechanisms sensitive to a MEK inhibitor drug that also blocked DOR1 stimulation of ERK. Moreover, neither c-Fos transcription nor AP-1 mobilization in response to DOR1 signaling was sensitive to the PI 3-kinase inhibitor, wortmannin. We previously showed that DOR1 increases AP-1 in these cells via a PTX-sensitive pathway (35). Together with signals from TCR-CD3 and CD28, the DOR1-mobilized AP-1 complexes enhance the transcriptional activity of the NF-AT/AP-1-binding element of the interleukin-2 (IL-2) promoter and increase IL-2 synthesis and secretion (35). Others have also shown that G_i protein-coupled receptors enhance the secretion of IL-2 (33, 34). IL-2 secretion occurs coincident with T lymphocyte immune activation and cell cycle entry and acts on T cells to promote their growth and continued immune activation. The -independent pathway of ERK activation may, therefore, form part of a mechanism that permits G_i protein-coupled receptors to regulate IL-2 and other AP-1-responsive genes in T lymphoid cell types.

	ACKNOWLEDGEMENTS

We are extremely grateful to Dr. Paul Leibson and his laboratory for generosity and advice on using recombinant Vaccinia viruses.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Supported by a grant from the Admadjaja Thymoma Research Foundation. To whom correspondence should be addressed: Dept. of Immunology, The Mayo Clinic, GUGG-3, Rochester, MN 55905. Tel.: 507-284-8178; Fax: 507-284-1637; E-mail: hedin@mayo.edu.

² K. E. Hedin, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: ARKct, -adrenergic receptor kinase-1 C-terminal fragment; DOR1, -opioid receptor; deltorphin, D-Ala²-deltorphin II; ERK, extracellular signal-regulated kinase; IL-2, interleukin-2; PI, phosphatidylinositol; PTX, pertussis toxin; PTX-B, control pertussis toxin containing only the B oligomer; PMA, phorbol 12-myristate 13-acetate; TCR-CD3, the T lymphocyte antigen receptor-CD3 complex; PAGE, polyacrylamide gel electrophoresis; mAb, monoclonal antibody; GST, glutathione S-transferase; MBP, myelin basic protein.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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