Ras-dependent Mitogen-activated Protein Kinase Activation by G Protein-coupled Receptors
CONVERGENCE OF Gi- AND Gq-MEDIATED PATHWAYS ON CALCIUM/CALMODULIN, Pyk2, AND Src KINASE*

(Received for publication, March 31, 1997, and in revised form, May 14, 1997)

Gregory J. Della Rocca Dagger , Tim van Biesen §, Yehia Daaka , Deirdre K. Luttrell , Louis M. Luttrell par and Robert J. Lefkowitz **

From the Howard Hughes Medical Institute and the Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710 and the  Department of Molecular Biochemistry, Glaxo Wellcome Research and Development, Research Triangle Park, North Carolina 27709

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Many receptors that couple to heterotrimeric guanine-nucleotide binding proteins (G proteins) have been shown to mediate rapid activation of the mitogen-activated protein kinases Erk1 and Erk2. In different cell types, the signaling pathways employed appear to be a function of the available repertoire of receptors, G proteins, and effectors. In HEK-293 cells, stimulation of either alpha 1B- or alpha 2A-adrenergic receptors (ARs) leads to rapid 5-10-fold increases in Erk1/2 phosphorylation. Phosphorylation of Erk1/2 in response to stimulation of the alpha 2A-AR is effectively attenuated by pretreatment with pertussis toxin or by coexpression of a Gbeta gamma subunit complex sequestrant peptide (beta ARK1ct) and dominant-negative mutants of Ras (N17-Ras), mSOS1 (SOS-Pro), and Raf (Delta N-Raf). Erk1/2 phosphorylation in response to alpha 1B-AR stimulation is also attenuated by coexpression of N17-Ras, SOS-Pro, or Delta N-Raf, but not by coexpression of beta ARK1ct or by pretreatment with pertussis toxin. The alpha 1B- and alpha 2A-AR signals are both blocked by phospholipase C inhibition, intracellular Ca2+ chelation, and inhibitors of protein-tyrosine kinases. Overexpression of a dominant-negative mutant of c-Src or of the negative regulator of c-Src function, Csk, results in attenuation of the alpha 1B-AR- and alpha 2A-AR-mediated Erk1/2 signals. Chemical inhibitors of calmodulin, but not of PKC, and overexpression of a dominant-negative mutant of the protein-tyrosine kinase Pyk2 also attenuate mitogen-activated protein kinase phosphorylation after both alpha 1B- and alpha 2A-AR stimulation. Erk1/2 activation, then, proceeds via a common Ras-, calcium-, and tyrosine kinase-dependent pathway for both Gi- and Gq/11-coupled receptors. These results indicate that in HEK-293 cells, the Gbeta gamma subunit-mediated alpha 2A-AR- and the Galpha q/11-mediated alpha 1B-AR-coupled Erk1/2 activation pathways converge at the level of phospholipase C. These data suggest that calcium-calmodulin plays a central role in the calcium-dependent regulation of tyrosine phosphorylation by G protein-coupled receptors in some systems.


INTRODUCTION

GTP-binding protein (G protein)1-coupled receptors (GPCRs) comprise a family of heptahelical membrane-bound receptors that mediate responses to a vast array of ligands (1). While the effects of these receptors on intermediary metabolism have been extensively studied, recent data have suggested that they play important roles in the regulation of cell growth and differentiation. Constitutively activating mutations of the thyrotropin and luteinizing hormone receptors are associated with hyperfunctioning thyroid adenomas and idiopathic male precocious puberty (1, 2). Expression of a constitutively active mutant of the alpha 1B-adrenergic receptor (AR) in myocardial cells induces myocardial hypertrophy in transgenic animals (3), and alpha 1-adrenergic agonists stimulate hypertrophy in cultured neonatal rat ventricular myocytes (4).

Mitogen-activated protein (MAP) kinases represent a point of convergence for cell surface signals regulating cell growth and division. The MAP kinases comprise a family of serine/threonine kinases, which include the extracellular signal-regulated kinases Erk1 and Erk2, the Jun N-terminal kinase/stress-activated protein kinase, and p38mapk (5). MAP kinases are regulated via protein phosphorylation cascades whose basic pattern has been highly conserved throughout evolution. In the mammalian Erk1/2 pathway, the proximal kinases Raf-1 and B-Raf phosphorylate and activate the dual function threonine/tyrosine kinases MAP/Erk kinases 1 and 2, which in turn phosphorylate Erk1/2. Once phosphorylated, activated Erk1/2 translocate to the cell nucleus, where they phosphorylate and activate nuclear transcription factors (6). Many signals received at the cell surface, including those mediated by growth factor receptor tyrosine kinases (7) and integrins, which mediate cell adhesion (8), initiate the MAP kinase cascade via activation of the low molecular weight GTP-binding protein, p21ras (9). Association with GTP-bound p21ras localizes Raf to the plasma membrane, which is sufficient to induce its activation (10).

Recently, receptors that couple to heterotrimeric G proteins, including the lysophosphatidic acid (LPA) (11, 12), bombesin (13), thromboxane A2/prostaglandin H2 (14), prostaglandin F2alpha (15), alpha -thrombin (16), angiotensin II (17, 18), alpha 1B-adrenergic (13), alpha 2A-adrenergic (13, 19), M1 muscarinic acetylcholine (13), D2 dopamine (13), and A1 adenosine (13) receptors, have been shown to activate MAP kinases (13, 20, 21). The signal transduction pathways employed by these receptors are heterogeneous. In Rat-1 and COS-7 cells, receptors coupled to pertussis toxin-sensitive G proteins mediate Erk1/2 activation via a Gbeta gamma subunit complex-mediated pathway that is dependent upon tyrosine protein phosphorylation and p21ras activation (13, 19, 22). These signals are independent of receptor-mediated effects on phosphatidylinositol hydrolysis, calcium influx, or inhibition of adenylyl cyclase (22, 23). In contrast, receptors coupled to pertussis toxin-insensitive G proteins mediate Erk1/2 activation via a Galpha subunit pathway that is p21ras-independent and may involve PKC (13). Direct activators of PKC, such as phorbol esters, have been reported to stimulate activation of MAP kinases through both p21ras-dependent and -independent pathways (4, 9).

Significant heterogeneity may also exist between cell types. Activation of Erk1/2 by alpha 1-adrenergic receptors in neonatal rat ventricular myocytes (4) and by prostaglandin F2alpha receptors in NIH-3T3 cells (15) is Galpha q/11-mediated and p21ras-dependent, suggesting that Galpha q/11 subunits also activate p21ras in some cell types. This pathway differs markedly from the Gq/11-coupled receptor-mediated p21ras-independent MAP kinase activation that has been described in COS-7 cells (13). In this paper, we characterize the mechanisms of Erk1/2 activation employed by the Gi-coupled alpha 2A- and by the Gq/11-coupled alpha 1B-adrenergic receptors, heterologously expressed in HEK-293 cells. We find that both receptors mediate p21ras-dependent Erk1/2 activation via phospholipase C and calcium-dependent activation of Src family kinases. These data suggest that, in some cell types, the Gbeta gamma subunit complex-dependent alpha 2A-AR and Galpha q/11 subunit-dependent alpha 1B-AR signals converge at the level of PLC and proceed via a common, p21ras-dependent, signaling pathway.


EXPERIMENTAL PROCEDURES

Materials

Phorbol 12-myristate 13-acetate (PMA) and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) were from Sigma. UK-14304 was from Pfizer. Fluphenazine, calmidazolium, U73122, A23187, bisindolylmaleimide I (GFX), genistein, and herbimycin A were from Calbiochem. Ophiobolin A was from Biomol. Pertussis toxin was from List Biologicals. Rabbit polyclonal anti-Pyk2 IgG was a kind gift of J. Schlessinger.

DNA Constructs

The cDNAs for the alpha 1B- and the alpha 2A-adrenergic receptors were cloned in our laboratory (24, 25). The beta -adrenergic receptor kinase 1 carboxyl-terminal (beta ARK1ct) peptide-encoding minigene, containing cDNA encoding the carboxyl-terminal 195 amino acids of beta ARK1, and the dominant-negative SOS-Pro construct, encompassing the proline-rich carboxyl-terminal fragment of mSOS1, were prepared in our laboratory as described previously (19, 26). The cDNA encoding a constitutively active mutant of Galpha q (Galpha q-Q209L), as described previously (27), was prepared in our laboratory by R. Premont. The cDNA encoding a constitutively active mutant of Galpha i2 (Galpha i2-Q204L) was from H. Bourne. The cDNAs encoding Gbeta 1 and Ggamma 2 were from M. Simon. The p21N17ras dominant negative mutant was from D. Altschuler, the p74raf-1 dominant negative mutant (Delta NRaf) was from L. T. Williams, the p112pyk2 dominant negative mutant (PKM) was from J. Schlessinger, p60c-src was from D. Fujita, and p50csk was from H. Hanafusa. The constitutively activated Y530F p60c-src (TAC(Y) right-arrow TTC(F)), in which the regulatory carboxyl-terminal tyrosine residue has been mutated, and catalytically inactive K298M p60c-src (AAA(K) right-arrow ATG(M)) were prepared as described (28-31). All cDNAs were subcloned into pRK5, pcDNA, pCMV, or pRSalpha eukaryotic expression vectors for transient transfection.

Cell Culture and Transfection

HEK-293, Rat-1, and PC12 cells were from the American Type Culture Collection. HEK-293 cells were maintained in minimum essential medium with Earle's salts (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Life Technologies) and 100 µg/ml gentamicin (Life Technologies), at 37 °C in a humidified 5% CO2 atmosphere. Rat-1 cells were maintained in Dulbecco's modified Eagle's medium (Life Technologies) supplemented with 10% fetal bovine serum and 100 µg/ml gentamicin under similar conditions. PC12 cells were maintained in RPMI medium 1640 (Life Technologies) supplemented with 10% heat-inactivated horse serum (Life Technologies), 5% fetal bovine serum, 100 µg/ml gentamicin, and 20 µg/ml L-glutamic acid (Life Technologies) under similar conditions. Transfections of HEK-293 cells were performed on 80-90% confluent monolayers in six-well dishes. Cells were transfected using the calcium phosphate coprecipitation method as described previously (32). Empty pRK5 vector was added to transfections as needed to keep the total mass of DNA added per well constant within an experiment.

Prior to stimulation, transfected monolayers were serum-starved in minimum essential medium with Earle's salts (HEK-293 cells) or Dulbecco's modified Eagle's medium (Rat-1 cells) supplemented with 0.1% bovine serum albumin (fraction V, protease-free) (Boehringer Mannheim), 100 µg/ml gentamicin, and 10 mM HEPES, pH 7.4, for approximately 24 h.

Pyk2 Immunoblotting

Unstimulated PC12 and HEK-293 cell monolayers were lysed directly with 100 µl/well Laemmli sample buffer. Cell lysates were sonicated briefly, and approximately 30 µg of protein/lane were loaded for resolution via SDS-polyacrylamide gel electrophoresis. Pyk2 was detected by protein immunoblotting using a 1:1000 dilution of rabbit polyclonal anti-Pyk2 IgG with horseradish peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology) as secondary antibody. Chemiluminescent detection of Pyk2 was performed after development of membranes with ECL reagent (Amersham Corp.), according to the manufacturer's instructions and exposure to Biomax XR scientific imaging film (Eastman Kodak Co.).

MAP Kinase Assay and Immunoblotting

Stimulations were carried out at 37 °C in serum-starving medium as described in the figure legends. After stimulation, monolayers were lysed directly with 100 µl/well Laemmli sample buffer. Cell lysates were sonicated briefly to disrupt DNA, and proteins (30 µg/lane) were resolved by SDS-polyacrylamide gel electrophoresis. Phosphorylation of Erk1/2 was detected by protein immunoblotting using a 1:1000 dilution of rabbit polyclonal phospho-specific MAP kinase IgG (New England Biolabs) with alkaline phosphatase-conjugated goat anti-rabbit IgG (Amersham) as secondary antibody. Quantitation of Erk1/2 phosphorylation was performed after development of membranes with Vistra ECF reagent (Amersham) by scanning on a Storm PhosphorImager (Molecular Dynamics). After scanning, membranes were treated for 30 min with 40% methanol to remove the Vistra ECF reagent, stripped by treatment with stripping buffer (62.5 mM Tris-Cl, pH 6.8, 2% SDS, 100 mM beta -mercaptoethanol) for 30 min at 50 °C, and reprobed with rabbit polyclonal anti-Erk2 IgG (Santa Cruz Biotechnology) to quantitate total p42mapk.


RESULTS

HEK-293 and Rat-1 Cells Exhibit Distinct Patterns of Erk1/2 Activation by Endogenous GPCRs

LPA receptor-mediated Erk1/2 activation in Rat-1 fibroblasts is mediated by Gbeta gamma subunits derived from PTX-sensitive G proteins (33) and is independent of changes in intracellular cAMP, calcium, or PKC (23). As shown in Fig. 1A, stimulation of endogenous LPA or thrombin receptors in these cells resulted in a 3-6-fold increase in Erk1/2 phosphorylation, which was completely inhibited by treatment with PTX. Acute activation of PKC by treatment with phorbol ester resulted in a less than 2-fold increase in Erk1/2 phosphorylation. Exposure to the calcium ionophore A23187 resulted in a less than 2-fold increase in Erk1/2 phosphorylation.


Fig. 1. Differential PTX sensitivity of GPCR-mediated Erk1/2 phosphorylation in Rat-1 and HEK-293 cells. Left panels, serum-starved Rat-1 (A) and HEK-293 (B) cells were preincubated overnight with PTX (100 ng/ml) or vehicle (control). Cells were stimulated for 5 min with LPA (10 µM), thrombin agonist peptide (SFLLRN, 100 µM), or EGF (10 ng/ml) prior to determination of Erk1/2 phosphorylation. Right panels, serum-starved Rat-1 (A) and HEK-293 (B) cells were stimulated for 5 min with PMA (1.0 µM) or calcium ionophore (A23187; 10 µM) prior to determination of Erk1/2 phosphorylation. Data are expressed as -fold Erk1/2 phosphorylation, in which the Erk1/2 phosphorylation produced in unstimulated, PTX-untreated cells was defined as 1.0. Values shown represent means ± S.E. from three separate experiments each performed in duplicate. Immunoblots represent phospho-Erk1/2 (top) and total Erk2 (bottom) from single representative experiments. NS, not stimulated.
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HEK-293 cells exhibit a distinct pattern of Erk1/2 activation. As shown in Fig. 1B, Erk1/2 phosphorylation via endogenous LPA and thrombin receptors is mediated via both PTX-sensitive and -insensitive G proteins in these cells. LPA and thrombin induce a similar 8-10-fold increase in Erk1/2 phosphorylation. Like Rat-1 cells, the LPA signal is PTX-sensitive. In contrast, the thrombin receptor mediates PTX-insensitive Erk1/2 phosphorylation, indicating that a distinct Erk1/2 activation pathway, mediated by PTX-insensitive G proteins, exists in these cells. Also, acute stimulation of HEK-293 cells with phorbol ester or with A23187 resulted in 24- and 15-fold stimulations of Erk1/2 phosphorylation, respectively, in stark contrast to the above results obtained in Rat-1 cells.

Erk1/2 Phosphorylation in HEK-293 Cells Is Mediated by Galpha Subunits for Gq/11-coupled Receptors and by the Gbeta gamma Subunit Complex for Gi-coupled Receptors

To characterize the mechanisms of Erk1/2 activation via PTX-sensitive and insensitive G proteins in HEK-293 cells, we employed a transiently transfected model system in which Gi-coupled alpha 2A-AR and Gq/11-coupled alpha 1B-AR were heterologously expressed. As shown in Fig. 2A, stimulation of the alpha 2A-AR resulted in PTX-sensitive Erk1/2 phosphorylation, while alpha 1B-AR-mediated Erk1/2 phosphorylation in response to stimulation was insensitive to pretreatment with PTX. Since Gbeta gamma subunits mediate Erk1/2 activation by several GPCRs, we determined whether cellular expression of a Gbeta gamma -sequestrant polypeptide derived from beta ARK1ct would inhibit alpha 1B-AR- and alpha 2A-AR-mediated MAP kinase activation. As shown in Fig. 2B, expression of the beta ARK1ct peptide attenuated Erk1/2 phosphorylation in response to alpha 2A-AR, but not alpha 1B-AR, stimulation. EGF-stimulated Erk1/2 phosphorylation was not sensitive to pretreatment of cells with PTX or to overexpression of cDNA coding for the beta ARK1ct peptide. This suggests that the alpha 2A-AR signals primarily via the Gbeta gamma subunit complex from PTX-sensitive G proteins, whereas the alpha 1B-AR signal is mediated by the Galpha subunit from PTX-insensitive G proteins. As shown in Fig. 2C, overexpression of a constitutively active mutant of Galpha q (Galpha q-Q209L), but not of Galpha i2 (Galpha i2-Q204L), was sufficient to induce Erk1/2 phosphorylation. Overexpression of Gbeta 1gamma 2 resulted in a consistent 2-fold stimulation of Erk1/2 phosphorylation, unlike the 6-8-fold stimulations of MAP kinase activity observed previously in COS-7 cells (13, 34).


Fig. 2.

PTX and beta ARKct sensitivity of alpha 1B-AR- and alpha 2A-AR-mediated Erk1/2 phosphorylation in HEK-293 cells and effect of overexpression of constitutively active mutant forms of Galpha q and Galpha i2 and of wild type Gbeta and Ggamma on Erk1/2 phosphorylation. A, HEK-293 cells were transiently transfected with plasmid DNA encoding alpha 2A-AR (0.2 µg/well), alpha 1B-AR (0.2 µg/well), or empty vector. Where indicated, cells were preincubated overnight with PTX. Serum-starved cells were stimulated with 10 µM UK-14304 (alpha 2A-AR), 20 µM phenylephrine (alpha 1B-AR), or EGF for 5 min prior to determination of Erk1/2 phosphorylation. B, HEK-293 cells were transiently transfected as in A with the addition of either 1 µg/well plasmid DNA coding for beta ARK1ct or 1 µg/well empty vector, and stimulated as described. C, HEK-293 cells were transiently transfected with plasmid DNA encoding Galpha q-Q209L (1 µg/well), Galpha i2-Q204L (1 µg/well), empty vector (1 µg/well), or both Gbeta 1 (0.5 µg/well) and Ggamma 2 (0.5 µg/well) as described. Erk1/2 phosphorylation in these cells was determined after 24 h of serum starvation. Data are expressed as -fold Erk1/2 phosphorylation, in which the Erk1/2 phosphorylation produced in unstimulated, empty vector-transfected cells was defined as 1.0. In the absence of transfected alpha 1B-AR and alpha 2A-AR, phenylephrine and UK-14304 resulted in 2.3- and 0.9-fold stimulation of Erk1/2 phosphorylation, respectively. Values shown represent means ± S.E. from three separate experiments each performed in duplicate. *, greater than not stimulated (NS) (p < 0.05, two-tailed T test).


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Both alpha 1B-AR- and alpha 2A-AR-mediated Erk1/2 Phosphorylation in HEK-293 Cells Is Dependent upon p21ras Activity

In COS-7 cells, alpha 2A-AR stimulation results in Erk1/2 activation by a p21ras-dependent mechanism, whereas alpha 1B-AR-mediated Erk1/2 activation is insensitive to overexpression of a p21ras dominant-negative mutant and is inhibited by down-regulation of PKC (34). To determine the role of p21ras in adrenergic receptor-mediated Erk1/2 phosphorylation in HEK-293 cells, cDNA coding for either the alpha 1B-AR or alpha 2A-AR was coexpressed with cDNA coding for dominant-negative mutant forms of p21ras (N17-Ras), mSOS1 (SOS-Pro), or p74raf-1 (Delta N-Raf). As shown in Fig. 3, phosphorylation of Erk1/2 in response to stimulation of both the alpha 1B-AR and the alpha 2A-AR was attenuated in cells coexpressing N17-Ras, SOS-Pro, or Delta N-Raf. Acute stimulation with phorbol esters was attenuated by overexpression of Delta N-Raf, but not by overexpression of N17-Ras or SOS-Pro, indicating that PKC-mediated Erk1/2 activation is Ras-independent. As expected, EGF-stimulated Erk1/2 phosphorylation was sensitive to the effects of overexpressed N17-Ras, SOS-Pro, and Delta N-Raf. Phosphorylation of Erk1/2 as a result of Galpha q-Q209L expression was similarly attenuated in cells coexpressing N17-Ras (data not shown). These data suggest that in HEK-293 cells, stimulation of both Gq/11- and Gi-coupled receptors leads to Erk1/2 phosphorylation in a manner that is dependent upon mSOS, p21ras, and p74raf-1 activation.


Fig. 3. N17-Ras, SOS-Pro, and Delta N-Raf sensitivity of alpha 1B-AR- and alpha 2A-AR-mediated Erk1/2 phosphorylation in HEK-293 cells. HEK-293 cells were transiently cotransfected with plasmid DNA encoding alpha 1B-AR (0.2 µg/well) or alpha 2A-AR (0.2 µg/well) plus plasmid DNA encoding N17-Ras (1.0 µg/well), SOS-Pro (1.0 µg/well), or Delta N-Raf (1.0 µg/well). Serum-starved cells were stimulated for 5 min with phenylephrine (alpha 1B-AR), UK-14304 (alpha 2A-AR), EGF, or PMA prior to determination of Erk1/2 phosphorylation. Data are expressed as -fold Erk1/2 phosphorylation, in which the Erk1/2 phosphorylation produced in unstimulated, empty vector-transfected cells was defined as 1.0. Values shown represent means ± S.E. from four separate experiments, each performed in duplicate.
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Erk1/2 Phosphorylation Mediated by alpha 1B-AR and alpha 2A-AR in HEK-293 Cells Is Phospholipase C- and Calcium-dependent

Pertussis toxin-sensitive Gbeta gamma subunit-mediated activation of p21ras in COS-7 cells is sensitive to inhibitors of tyrosine kinases and requires recruitment of the Ras guanine-nucleotide exchange factor, mSOS (19). The Gbeta gamma subunit effectors responsible for these signals are unknown. Since PLCbeta isoforms are regulated by both Galpha q/11 and Gbeta gamma subunits and PLCbeta overexpression results in Erk1/2 activation in COS-7 cells (34), we tested whether PLC activation was required for alpha 1B-AR- and alpha 2A-AR-mediated Erk1/2 phosphorylation in HEK-293 cells. As shown in Fig. 4, pretreatment of HEK-293 cells with the PLC inhibitor, U73122, markedly attenuated both alpha 1B-AR- and alpha 2A-AR-mediated Erk1/2 phosphorylation. Phorbol ester-mediated Erk1/2 phosphorylation was insensitive to the effects of U73122. The results suggest that one or more isoforms of PLC are required for both Gq/11- and Gi-coupled receptor-mediated MAP kinase activation in HEK-293 cells.


Fig. 4. Sensitivity of alpha 1B-AR- and alpha 2A-AR-mediated Erk1/2 phosphorylation to the PLC inhibitor U73122 in HEK-293 cells. HEK-293 cells were transiently transfected with plasmid DNA encoding alpha 1B-AR (0.2 µg/well), alpha 2A-AR (0.2 µg/well), or empty vector (0.2 µg/well). Prior to stimulation, serum-starved cells were treated for 15 min with Me2SO (1%, control) or U73122 (10 µM). Cells were stimulated with the appropriate agonist for 5 min, and Erk1/2 phosphorylation was determined. Data are expressed as -fold Erk1/2 phosphorylation, in which the Erk1/2 phosphorylation produced in unstimulated, empty vector-transfected cells was defined as 1.0. Values shown represent means ± S.E. from three separate experiments each performed in duplicate. NS, not stimulated.
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Recent reports have suggested that Ras-dependent Erk1/2 activation in vascular smooth muscle cells (35) and in neuronal cells (36) may be calcium-dependent. As shown in Fig. 5, treatment of HEK-293 cells with the calcium ionophore A23187 resulted in 5-10-fold increases in Erk1/2 phosphorylation, similar to that observed after 5-min stimulation of cells expressing transfected alpha 1B- and alpha 2A-AR. Pretreatment of HEK-293 cells with the cell membrane-permeable Ca2+ chelating agent BAPTA abrogated alpha 1B-AR- and alpha 2A-AR-mediated, as well as A23187-induced, Erk1/2 phosphorylation. Erk1/2 phosphorylation after stimulation of BAPTA-pretreated cells with EGF was unaffected. These data suggest that increased intracellular Ca2+ concentration, resulting from Gbeta gamma - or Galpha -mediated PLC activation, is required for Erk1/2 activation in HEK-293 cells.


Fig. 5. Sensitivity of alpha 1B-AR- and alpha 2A-AR-mediated Erk1/2 phosphorylation to the intracellular calcium chelator BAPTA in HEK-293 cells. HEK-293 cells were transiently transfected with plasmid DNA encoding alpha 1B-AR, alpha 2A-AR, or empty vector. Prior to stimulation, serum-starved cells were treated for 15 min with phosphate-buffered saline (control) or BAPTA (50 µM). Cells were stimulated as indicated for 5 min, and Erk1/2 phosphorylation was determined. Data are expressed as -fold Erk1/2 phosphorylation, in which the Erk1/2 phosphorylation produced in unstimulated, empty vector-transfected cells was defined as 1.0. Values shown represent means ± S.E. from three separate experiments each performed in duplicate. NS, not stimulated.
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alpha 1B-AR-, alpha 2A-AR-, and Calcium Ionophore-stimulated Erk1/2 Phosphorylation Requires Tyrosine Kinase Activity

Tyrosine phosphorylation of the Shc adaptor protein, which supports the SH2 domain-mediated recruitment of Grb2-Sos to the plasma membrane, has been implicated in both receptor-tyrosine kinase- and GPCR-mediated Erk1/2 activation in some cell types (37). To test whether the calcium-dependent alpha 1B- and alpha 2A-AR signals in HEK-293 cells are also dependent upon tyrosine protein phosphorylation, we determined the effects of two tyrosine kinase inhibitors, genistein and herbimycin A, on alpha 1B- and alpha 2A-mediated Erk1/2 phosphorylation in HEK-293 cells. As shown in Fig. 6A, pretreatment of HEK-293 cells with the tyrosine kinase inhibitors markedly attenuated alpha 1B-AR-, alpha 2A-AR-, and EGF-R-mediated Erk1/2 phosphorylation. Erk1/2 phosphorylation induced by the calcium ionophore A23187 was also tyrosine kinase inhibitor-sensitive, suggesting that elevation of intracellular Ca2+ levels is sufficient to induce tyrosine phosphorylation in these cells.


Fig. 6.

Involvement of the c-Src tyrosine kinase in alpha 1B-AR- and alpha 2A-AR-mediated Erk1/2 phosphorylation in HEK-293 cells. A, HEK-293 cells were transiently transfected with plasmid DNA encoding alpha 1B-AR, alpha 2A-AR, or empty vector. Prior to stimulation, serum-starved cells were treated with Me2SO (0.1%; control) or herbimycin A (1 µM) for 24 h or with Me2SO (0.1%; control) or genistein (50 µM) for 15 min. Cells were stimulated with the appropriate agonist for 5 min, and Erk1/2 phosphorylation was determined. B, HEK-293 cells were transiently transfected with plasmid DNA encoding either wild-type c-Src (0.5 µg/well) or Src-Y530F (0.5 µg/well) as described. Erk1/2 phosphorylation in these cells was determined after 24 h of serum starvation. C, HEK-293 cells were transiently cotransfected with plasmid DNA encoding alpha 1B-AR, alpha 2A-AR, or wild-type c-Src (0.5 µg/well) plus plasmid DNA encoding either Src-K298M (1.0 µg/well) or Csk (1.0 µg/well). Cells were serum-starved for 24 h and stimulated as indicated for 5 min, and Erk1/2 phosphorylation was determined. Data are expressed as -fold Erk1/2 phosphorylation, in which the Erk1/2 phosphorylation produced in unstimulated, empty vector-transfected cells was defined as 1.0. Values shown represent means ± S.E. from three separate experiments each performed in duplicate. NS, not stimulated.


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Both pertussis toxin-sensitive (38) and -insensitive (39) activation of Src family nonreceptor tyrosine kinases have been described by several laboratories, and Src activity appears to be required for Gbeta gamma subunit-mediated Erk1/2 activation in COS-7 cells (40). To determine whether Src family tyrosine kinases are involved in GPCR-mediated Erk1/2 activation in HEK-293 cells, we measured alpha 1B-AR- and alpha 2A-AR-mediated Erk1/2 phosphorylation in cells coexpressing either a catalytically inactive mutant of p60c-src, Src-K298M, or a negative regulatory protein of p60c-src, p50csk, which phosphorylates and inactivates p60c-src (41). As shown in Fig. 6B, overexpression of either a constitutively activated mutant form of p60c-src (Src-Y530F) or of wild-type p60c-src in HEK-293 cells was sufficient to induce Erk1/2 phosphorylation. As shown in Fig. 6C, overexpression of either Src-K298M or p50csk significantly attenuated Erk1/2 phosphorylation induced by either alpha 1B-AR and alpha 2A-AR stimulation or treatment with calcium ionophore. Erk1/2 phosphorylation induced by acute stimulation with phorbol ester or by overexpression of wild-type c-Src was unaffected. The inability of overexpressed p50csk to significantly inhibit Erk1/2 phosphorylation mediated by overexpressed wild-type c-Src probably reflects ineffective competition between the two overexpressed proteins. These data suggest that both alpha 1B- and alpha 2A-AR signals in HEK-293 cells are calcium-dependent and mediated by Src family tyrosine kinase activity.

Calcium-dependent Erk1/2 Phosphorylation in HEK-293 Cells Is Sensitive to Inhibitors of both Calcium-calmodulin and the Calcium-regulated Tyrosine Kinase, Pyk2, but Not Inhibitors of PKC

Acute stimulation of PKC with phorbol ester is sufficient to induce Erk1/2 phosphorylation in HEK-293 cells. Unlike the alpha 1B-AR- and alpha 2A-AR-mediated signals, the acute PMA signal is insensitive to the effects of N17-Ras, SOS-Pro, overexpressed p50csk, Src-K298M, and tyrosine kinase inhibitors. As shown in Fig. 7, the PKC inhibitor GFX, which abolished acute PMA-stimulated Erk1/2 phosphorylation, had no effect on Erk1/2 phosphorylation after stimulation of cells with adrenergic agonists, EGF, or calcium ionophore. Similar results were obtained through down-regulation of endogenous PKC expression after chronic treatment of cells with phorbol ester. These data suggest that PKC activation mediates Erk1/2 phosphorylation via a pathway that is distinct from the calcium- and tyrosine kinase-dependent pathway employed by alpha 1B- and alpha 2A-ARs.


Fig. 7. Insensitivity of alpha 1B-AR- and alpha 2A-AR-mediated Erk1/2 phosphorylation to GFX, an inhibitor of PKC, and to phorbol ester-induced intracellular PKC depletion in HEK-293 cells. HEK-293 cells were transiently transfected with plasmid DNA encoding alpha 1B-AR, alpha 2A-AR, or empty vector. Prior to stimulation, serum-starved cells were treated with Me2SO (0.1%, control) or PMA (1 µM) for 24 h or with Me2SO (0.1%, control) or GFX (2 µM) for 15 min. Cells were stimulated as indicated (PMA; 10 µM) for 5 min, and Erk1/2 phosphorylation was determined. Data are expressed as -fold Erk1/2 phosphorylation, in which the Erk1/2 phosphorylation produced in unstimulated, empty vector-transfected cells was defined as 1.0. Values shown represent means ± S.E. from three separate experiments each performed in duplicate. NS, not stimulated.
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Calcium-dependent activation of a novel focal adhesion kinase family protein-tyrosine kinase, Pyk2, has been shown to mediate calcium ionophore-, phorbol ester-, and Gq/11-coupled receptor-stimulated Erk1/2 activation in neuronal cells (36) via a direct interaction with c-Src (42). Although Pyk2 is expressed at high levels only in cells of neuronal origin (36), it is possible that this or a related kinase might link calcium flux to tyrosine kinase signaling pathways in other cell types. However, as shown in Fig. 8A, protein immunoblots of HEK-293 cell lysates using anti-Pyk2 antisera detect only low levels of Pyk2 expression. To determine whether Pyk2 is involved in the calcium-dependent activation of Erk1/2 in these cells, alpha 1B-AR- and alpha 2A-AR-mediated Erk1/2 phosphorylation was assayed in cells expressing a dominant negative mutant of Pyk2 (PKM; Ref. 42). As shown in Fig. 8B, Erk1/2 phosphorylation in response to adrenergic receptor stimulation or treatment with calcium ionophore was significantly attenuated, with no effect on EGF- or phorbol ester-induced signals.


Fig. 8. Involvement of Pyk2 in alpha 1B-AR- and alpha 2A-AR-mediated Erk1/2 phosphorylation in HEK-293 cells. A, Pyk2 immunoblot of whole-cell lysates from untransfected PC12 cells, untransfected HEK-293 cells, and HEK-293 cells transiently transfected with cDNA coding for PKM. B, HEK-293 cells were cotransfected with plasmid DNA encoding alpha 1B-AR or alpha 2A-AR, plus plasmid DNA encoding either a dominant-negative mutant of Pyk2 (PKM; 1.0 µg/well) or with empty pRK5 vector (1.0 µg/well). Cells were serum-starved for 24 h and stimulated for 5 min as indicated, and Erk1/2 phosphorylation was determined. Data are expressed as -fold Erk1/2 phosphorylation, in which the Erk1/2 phosphorylation produced in unstimulated, empty vector-transfected cells was defined as 1.0. Values shown represent means ± S.E. from two separate experiments each performed in duplicate. NS, not stimulated.
[View Larger Version of this Image (22K GIF file)]

The mechanism whereby calcium influx regulates the activity of the focal adhesion kinase family member Pyk2 is unknown. Neither Ca2+ nor PKC directly activate Pyk2 in vitro (36). Recently, calmodulin inhibitors have been shown to inhibit p21ras-dependent Erk1/2 activation in cultured rat vascular smooth muscle cells (35). To determine whether calmodulin might play a role in AR-mediated Erk1/2 activation in HEK-293 cells, we determined the effect of three different calmodulin inhibitors on alpha 1B- and alpha 2A-AR-stimulated Erk1/2 phosphorylation. As shown in Fig. 9, pretreatment of HEK-293 cells with fluphenazine, calmidazolium, or ophiobolin resulted in marked attenuation of the phospho-MAP kinase signal, compared with Me2SO-pretreated controls. Erk1/2 phosphorylation resulting from stimulation of endogenous EGF receptors was unaffected. These data suggest that calcium-calmodulin may directly or indirectly contribute to the regulation of Pyk2 kinases and the Src-dependent activation of Erk1/2 by GPCRs.


Fig. 9. Effect of calcium/calmodulin inhibitors on alpha 1B-AR- and alpha 2A-AR-mediated Erk1/2 phosphorylation in HEK-293 cells. HEK-293 cells were transfected with plasmid DNA encoding alpha 1B-AR, alpha 2A-AR, or empty vector. Prior to stimulation, serum-starved cells were treated with Me2SO (0.1%, control), fluphenazine (10 µM), calmidazolium (10 µM), or ophiobolin A (10 µM) for 15 min. Cells were stimulated as indicated for 5 min, and Erk1/2 phosphorylation was determined. Data are expressed as -fold Erk1/2 phosphorylation, in which the Erk1/2 phosphorylation produced in unstimulated, empty vector-transfected cells was defined as 1.0. Values shown represent means ± S.E. from three separate experiments each performed in duplicate. NS, not stimulated.
[View Larger Version of this Image (28K GIF file)]


DISCUSSION

These data suggest a model for alpha 1B-AR- and alpha 2A-AR-mediated Erk1/2 activation that is mediated by calcium-dependent regulation of protein-tyrosine kinases. In HEK-293 cells, as in COS-7 cells (34), the alpha 1B-AR-mediated signal is dependent upon the alpha  subunit of a pertussis toxin-insensitive G protein, while the alpha 2A-AR-mediated signal is sensitive to PTX treatment and is dependent upon the release of free Gbeta gamma subunit complexes. Fig. 10 depicts a model of GPCR-mediated Erk1/2 activation in HEK-293 cells that is consistent with our data. Galpha q/11- and Gbeta gamma -dependent activation of PLC increases cytoplasmic levels of inositol 1,4,5-trisphosphate, resulting in an increase in cytoplasmic calcium concentration. High intracellular concentrations of calcium, perhaps through calmodulin, lead to activation of Pyk2 or a closely related tyrosine kinase, which regulates the activity of p60c-src. Src-dependent tyrosine phosphorylation of adaptor proteins, such as Shc, results in recruitment of the Grb2-SOS complex to the plasma membrane, where it catalyzes p21ras guanine nucleotide exchange. Ras-dependent recruitment of p74raf-1 kinase to the membrane initiates the phosphorylation cascade leading to activation of Erk1/2. In this system the Gbeta gamma subunit- and Galpha q/11 subunit-mediated pathways each require the PLCbeta -dependent stimulation of calcium influx. This early convergence is distinct from findings in COS-7 and CHO cells (34) and more closely resembles the calcium- and Ras-dependent activation of Erk1/2, which has been reported in primary cultures of vascular smooth muscle cells and ventricular myocytes (35, 43, 44). The observation that dominant interfering mutants of p21ras, p74raf-1, and SOS do not fully attenuate alpha 1B-AR- and alpha 2A-AR-mediated Erk1/2 phosphorylation may reflect incomplete inhibition of receptor-mediated p74raf-1 activation. Alternatively, these data may be indicative of another, p21ras-independent, mechanism of Erk1/2 phosphorylation, as has been described for Gq- and Go-coupled MAP kinase activation in Chinese hamster ovary cells (34, 45).


Fig. 10. Model of Gi- and Gq/11-coupled receptor-mediated Ras-dependent MAP kinase activation in HEK-293 cells. Stimulation of the Gi-coupled alpha 2A-AR and the Gq/11-coupled alpha 1B-AR leads to phospholipase C activation via liberation of Gbeta gamma and Galpha q/11-GTP subunits, respectively. Increases in intracellular calcium as a result of phosphoinositide hydrolysis by PLC cause activation of calmodulin (CaM) and the focal adhesion kinase family protein-tyrosine kinase Pyk2. Pyk2 activates c-Src, which results in phosphorylation of the Shc adaptor protein, recruitment of the Grb2-Sos complex to the membrane, and activation of Ras through guanine nucleotide exchange. Subsequent activation of Raf initiates the cascade of phosphorylation events leading to MAP kinase (Erk1/2) activation.
[View Larger Version of this Image (20K GIF file)]

Activation of p60c-src is required for Gi-coupled receptor-mediated, Gbeta gamma subunit-dependent activation of Erk1/2 in COS-7 cells (37), and Gi- and Gq/11-coupled receptor-stimulated Erk1/2 activation in PC12 cells (42). Src family kinase recruitment into Shc-containing protein complexes has been demonstrated following stimulation of formyl-methionyl peptide receptors in human neutrophils (46) and following stimulation of LPA and alpha 2-adrenergic receptors in COS-7 cells (37). Our data indicate that Src kinases function as key intermediates in calcium-dependent regulation of Erk1/2, mediated by both Gbeta gamma and Galpha q/11 subunits in some cell types. Collectively, these findings indicate that regulation of Src family protein-tyrosine kinase activity, potentially via multiple mechanisms, is a common requirement for GPCR-mediated Erk1/2 activation.

In neuronal cells, association of p60c-src with the calcium-regulated focal adhesion kinase family member Pyk2 mediates both Shc phosphorylation and Erk1/2 activation (42). Pyk2 was previously thought to be active only in neuronal cells. The detection of Pyk2 in HEK-293 cell lysates as well as the sensitivity of alpha 1B-AR- and alpha 2A-AR-mediated Erk1/2 activation in HEK-293 cells to both the dominant negative mutant of Pyk2 and specific inhibitors of p60c-src suggest that a Pyk2-mediated Src-dependent mechanism of p21ras activation may represent a paradigm for mitogenic signaling in a variety of non-neuronal cell types. The mechanism of Ca2+-mediated Pyk2 activation remains unclear, however, since calcium does not directly modulate Pyk2 activity (36). Perhaps significantly, both adrenergic receptor- and calcium ionophore-mediated Erk1/2 phosphorylation in HEK-293 cells is sensitive to chemical inhibitors of calmodulin. Eguchi et al. (35) have suggested that calmodulin regulates Erk1/2 activation in cultured rat vascular smooth muscle cells. In NG108 cells, depolarization induces calcium-dependent Erk1/2 activation, which is mediated by calmodulin-dependent kinase IV (47). Our data suggest that the calcium-mediated regulation of Src family tyrosine kinases proceeds through a calmodulin-dependent mechanism. These data also suggest that, if Pyk2 directly activates p60c-src in HEK-293 cells, then perhaps calcium/calmodulin is involved in activation of Pyk2, either directly or through a calcium/calmodulin effector protein.

The elucidation of GPCR-mediated mitogenic signaling pathways has revealed significant degrees of heterogeneity between cell types. In Rat-1 fibroblasts, Gi-coupled, but not Gq/11-coupled, receptors mediate tyrosine kinase-dependent Erk1/2 activation via a calcium- and PLC-independent mechanism (23). In Chinese hamster ovary cells, Gq/11-coupled receptor stimulation leads to Galpha q/11-mediated activation of PKC, p74raf-1, and Erk1/2 in a tyrosine kinase- and p21ras-independent manner (34). In PC12 neuroblastoma cells, both Gi- and Gq/11-coupled receptors have been shown to activate Erk1/2 via calcium-dependent regulation of p112pyk2, p60c-src, and p21ras (42). Our data suggest that calcium-dependent regulation of Ras by both Gi- and Gq/11-coupled receptors may represent a common mechanism of GPCR-mediated Erk1/2 activation in many non-neuronal cell types. Indeed, Gq/11-coupled receptors mediate calmodulin inhibitor-sensitive, Ras-dependent Erk1/2 activation in cultured vascular smooth muscle cells (35), a calcium-sensitive tyrosine kinase has been cloned from calf uterus (48), and Gq/11-coupled receptor-mediated hypertrophy of cultured rat ventricular myocytes is reportedly Ras-dependent (43). Characterization of receptor- and kinase-specific differences in the mechanisms of GPCR-mediated mitogenic signal transduction may permit the development of strategies for selective antagonism of distinct G protein-coupled receptor-mediated mitogenic signaling pathways, which ultimately may permit selective modulation of cell proliferation in a variety of pathophysiologic states.


FOOTNOTES

*   This work was supported in part by National Institutes of Health (NIH) Grant HL16037 (to R. J. L.).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.
Dagger    Supported by NIH Medical Scientist Training Program Grant T32GM-07171.
§   Present address: Neuroscience Research, Abbott Laboratories, D-4PM, AP10, 100 Abbott Park Rd., Abbott Park, IL 60064.
par    Recipient of an NIH Clinical Investigator Development Award.
**   To whom correspondence should be addressed: Howard Hughes Medical Institute, Box 3821, Duke University Medical Center, Durham, NC 27710. Tel.: 919-684-2974; Fax: 919-684-8875.
1   The abbreviations used are: G protein, GTP-binding protein; MAP, mitogen-activated protein; GPCR, G protein-coupled receptor; PLC, phospholipase C; beta ARK1ct, the beta -adrenergic receptor kinase 1 COOH-terminal peptide; Galpha and Gbeta gamma , the alpha  and beta gamma subunits, respectively, of G proteins; PKC, Ca2+-dependent protein kinase; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; PMA, phorbol 12-myristoyl 13-acetate; GFX, bisindolylmaleimide I; AR, adrenergic receptor; HEK, human embryonic kidney; PTX, pertussis toxin; LPA, lysophosphatidic acid; EGF, epidermal growth factor; SFLLRN, thrombin agonist peptide.

ACKNOWLEDGEMENTS

We thank Donna Addison and Mary Holben for expert secretarial assistance, Sameena Rahman for technical assistance, and Dr. John Raymond for insightful discussion.


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