©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Direct Interaction between G-Protein Subunits and the Raf-1 Protein Kinase (*)

Kevin M. Pumiglia (1) (3)(§), Harry LeVine (2), Taraneh Haske (2), Tania Habib (1), Richard Jove (3), Stuart J. Decker (1) (3)(¶)

From the (1)Parke-Davis Pharmaceutical Research Division, Departments of Signal Transduction and (2)Neurosciences, Ann Arbor Michigan, 48106 and the (3)Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan 48109

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Raf-1 is a serine/threonine protein kinase positioned downstream of Ras in the mitogen-activated protein kinase cascade. Using a yeast two-hybrid strategy to identify other proteins that interact with and potentially regulate Raf-1, we isolated a clone encoding the carboxyl-terminal half of the G subunit of heterotrimeric G-proteins. In vitro, purified G subunits specifically bound to a GST fusion protein encoding amino acids 1-330 of Raf-1 (Raf/330). Binding assays with truncation mutants of GST-Raf indicate that the region located between amino acids 136 and 239 is a primary determinant for interaction with G. In competition experiments, the carboxyl terminus of -adrenergic receptor kinase (ARK) blocked the binding of G to Raf/330; however, the Raf-1-binding proteins, Ras and 14-3-3, had no effect. Scatchard analysis of in vitro binding between Raf/330 and G revealed an affinity of interaction (K = 163 ± 36 nM), similar to that seen between G and ARK (K = 87 ± 24 nM). The formation of native heterotrimeric G complexes, as measured by pertussis toxin ADP-ribosylation of G, could be disrupted by increasing amounts of Raf/330, with an EC of approximately 200 nM, in close agreement with the estimated binding affinity. In vivo complexes of Raf-1 and G were isolated from human embryonic kidney 293-T cells transfected with epitope-tagged G The identification and characterization of this novel interaction raises several possibilities for signaling cross-talk between growth factor receptors and those receptors coupled to heterotrimeric G-proteins.


INTRODUCTION

Raf-1 is a serine-threonine protein kinase critically positioned in a kinase cascade linking activated growth factor receptors with the nucleus and ultimately regulating the mitogenic response. Structurally, Raf-1 can be subdivided into three regions, CR1, CR2, and CR3, which are conserved through evolution and among the various isoforms of Raf-1 (1). CR1 consists of a Ras binding domain (amino acids 53-132)(2, 3) and a zinc finger similar to those found in protein kinase C. A serine/threonine-rich region with multiple phosphorylation sites, including an autophosphorylation site (Thr)(4) , comprises CR2. The catalytic region of the protein, CR3, makes up most of the COOH-terminal half of the molecule. Previous studies have demonstrated that truncations or fusions which result in the loss of the amino-terminal portion of Raf-1 are oncogenically active(5, 6) . Thus, regions contained in the amino-terminal portion of Raf-1 are presumed to be critical for regulating the biological activity of this kinase.

Raf-1 has been shown to bind Ras, in a GTP-dependent fashion(2, 7, 8, 9) . This binding event appears to be a critical step in the activation of Raf-1(3, 10) . Current models of activation suggest that Raf-1 is bound in a native complex with 14-3-3 protein(s)(11, 12) and hsp50 and hsp90 proteins(13, 14) . Activated Ras is thought to recruit Raf-1 to the plasma membrane, where it interacts with other modulators of its activity and with relevant substrates(15, 16) . To identify other proteins that interact with and potentially regulate Raf-1, we utilized two-hybrid interaction screening with the amino-terminal regulatory domain of Raf-1 as the target protein. An interacting clone was identified which encodes the carboxyl-terminal half of the G subunit of heterotrimeric G-proteins.()In this report, we have characterized this interaction, raising the possibility that the binding of G to Raf-1 could play a role in regulation of the mitogen-activated protein kinase pathway by G-protein-coupled receptors and/or potentially provide a junction for integration of signals generated by tyrosine kinase growth factor receptors and G-protein-coupled receptors.


EXPERIMENTAL PROCEDURES

Constructs

GST-Raf fusion proteins were made by PCR amplification of sequences encoding amino acids 1-135 (Raf/135), 1-239 (Raf/239), 136-239 (Raf/136-239), and 1-330 (Raf/330) of human Raf-1. Amplified fragments were cloned into pCR-script (Stratagene) and subcloned into pGEX-KG(17) . Full-length Raf-1 was expressed as a MAL fusion protein using the pMALc2 vector (New England Biolabs). GST-14-3-3 was made by amplifying the complete coding sequence of human 14-3-3 . GST--adrenergic receptor kinase (ARK) contains amino acids 467-689 of rat ARK cloned into pGEX-KT (prepared from a cDNA clone kindly provided by Antonio DeBlasi, Instituto di Ricerche Farmalogiche Mario Negri). GST-Ras and GST-PEST tyrosine phosphatase (PTP-PEST) have been described previously(3, 18) . pc was generated by PCR using custom primers coding for a FLAG epitope tag (DYKDDDDK) in frame with the carboxyl terminus of G. The amplified fragment was cloned into the BamHI/EcoRI sites of the expression vector pcDNA3 (Invitrogen). The G subunit was also amplified by PCR and cloned into pcDNA3 at the EcoRI and XbaI sites. The expression plasmid pcRaf-KT3 codes for the full-length Raf-1 protein in pcDNA3 and was generously provided by Angus MacNicol (University of Chicago).

Yeast Two-hybrid Cloning

The plasmid pAY-Raf was constructed by amplifying the sequence coding for amino acids 1-320 by PCR and cloning into pAS1-CYH2 vector originally described by Durfee et al.(19) . This plasmid was co-transformed with a HeLa cell cDNA library, constructed in pACT(19) , into the Y190 lacZ/HIS3 reporter strain of yeast (20) using the lithium acetate procedure(21) . The transformation mix was plated onto dishes containing synthetic complete media lacking tryptophan, leucine, and histidine, in the presence of 3-aminotriazole, and incubated for 7-10 days at 30 °C. His colonies were analyzed for -galactosidase activity using a filter lift procedure(22) . Library-derived plasmids from His+/- gal+ clones were rescued and transformed into Escherichia coli for plasmid preparation and DNA sequencing.

The pAS1--integrin construct encodes the COOH-terminal 20 amino acids of the -integrin receptor as a fusion with the Gal-4 DNA binding domain in the pAS1-CYH2 vector. The pActPTP-PEST construct has been described previously(18) .

Purification of GSubunits and ADP-ribosylation Assay

The G subunits of heterotrimeric G-proteins were purified from bovine brain as described by Sternweis and Pang(22) . Proteins were stored in elution buffer at -70 °C at a protein concentration of 0.5-3.0 mg/ml. ADP-ribosylation reactions were carried out essentially as described previously(23) . Pertussis toxin was from List Biochemicals.

In Vitro Binding of Raf and G

The various fusion proteins were purified as described previously (17) on glutathione-Sepharose (Pharmacia Biotech Inc.) or amylose resin (New England Biolabs). Immobilized fusion protein (0.2-0.5 µg) was mixed with purified G subunits (300 nM final concentration) in phosphate-buffered saline containing 0.1% Lubrol. After a 60-min incubation, beads were washed three times with the same buffer followed by separation of bound proteins by SDS-polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose and immunoblotting was performed with anti-G antibodies (catalog number 261, Santa Cruz Biotechnology) and I-labeled protein A (Amersham Corp).

In experiments where binding affinities were estimated, various concentrations of G were used in the in vitro binding assay. The resulting immunoblots were quantified by exposure on a PhosphorImager (Molecular Dynamics) with known quantities of pure G subunits run as standards.

In experiments where proteins were added to compete with binding of G to Raf-1, G subunits were added at a concentration of 30 nM, and a 10-fold excess (mole/mole) of the competitor protein was used.

In Vivo Association

Human embryonic kidney 293-T cells (donated by Akilesh Pandey, University of Michigan) were transfected with expression plasmids encoding Raf-1 (pcRaf-KT3), epitope-tagged G (pc), and G (pc) proteins, either alone or in combination using a calcium phosphate procedure (Stratagene). At 24 h after transfection, cells were washed twice with ice-cold phosphate-buffered saline prior to lysis in buffer A (50 mM HEPES [pH 7.5], 1% Nonidet P-40, 0.5% sodium deoxycholate, 150 mM NaCl, 50 mM NaF, 1 mMpara-nitrophenyl phosphate, 1 mM orthovanadate, 20 nM calyculin-A, 1 mM phenylmethylsulfonyl fluoride, 1 µM leupeptin, 1 µM antipain, and 0.1 µM aprotinin). Lysates were clarified by centrifugation and incubated for 1.5 h with 25 µl of FLAG-specific monoclonal antibody M2 immobilized on beads (Kodak). After washing three times with lysis buffer, precipitates were analyzed by immunoblotting with anti-Raf (catalog number 227; Santa Cruz) or anti- (catalog number 378; Santa Cruz) antibodies as outlined above, except enhanced chemiluminescence (Amersham Corp.) was used for detection(4) . Aliquots of the clarified extracts were also routinely analyzed to monitor expression levels.


RESULTS

Two-hybrid screening in yeast was used to identify cDNAs coding for proteins which bind the regulatory region of Raf-1 protein kinase. One such cDNA encoded amino acids 194-340 of the G subunit of heterotrimeric G-proteins. Specificity of this interaction was tested by measuring the ability of the two-hybrid constructs, pAY-Raf and pAct- to support His prototropy and lacZ expression when expressed either with the corresponding Gal-4 domain lacking a fusion, or with irrelevant proteins. summarizes these data and demonstrates a specific interaction between the amino terminus of Raf-1 and the carboxyl terminus of the G subunit.

To further characterize the interaction between Raf-1 and G, we tested the ability of purified G subunits to bind to Raf/330, to GST alone, or other GST-fusion proteins, in an in vitro binding assay (Fig. 1A). Raf/330 and GST-ARK (which has previously been shown to bind G(24, 25) ) bound G in this assay. Little or no binding, however, was detected between G subunits and GST alone, GST-Ras or GST-PTP-PEST, suggesting that the Raf/G interaction is specific.


Figure 1: In vitro binding of GST-Raf fusion proteins to purified G subunits. A, purified G subunits were incubated with GST alone or various GST-fusion proteins immobilized on glutathione-Sepharose. After washing, bound G subunits were detected by immunoblotting. A small aliquot of purified G protein was run (Std) to confirm the migration of the G subunit. B, glutathione beads containing either GST alone or various GST-Raf fusion proteins were incubated with purified G subunits in an in vitro binding assay. Binding of the bound G was assessed as described above. Quantitation of the immunoblot by PhosphorImager analysis provided values of: GST, 100 counts; GST-Raf 1-(1-330), 14,145 counts; GST-Raf-(1-135), 2,682 counts; GST-Raf-(1-239), 13,927 counts. C, amylose resin preincubated with either maltose-binding protein alone (Mal) or maltose-binding protein as a fusion with full-length Raf-1 (pMAL-Raf-1) was incubated with purified G subunits in the absence (-) or presence of potential competitors as described under ``Experimental Procedures.'' Bound proteins were detected by immunoblotting with anti-G antibodies. Figures are typical results from two or three independent experiments.



To better define the region of the Raf-1 amino terminus responsible for the binding of G subunits, we constructed additional GST-Raf proteins: one encodes amino acids 1-135, which includes the Ras binding domain, but excludes the cysteine rich region and CR2; the other codes for amino acids 1-239, which includes all of CR1 but stops just prior to the beginning of CR2. These constructs were compared with the full amino terminus (Raf/330) for their ability to bind Gin vitro. As shown in Fig. 1B, deletion of the CR2 region has no effect on the binding of G subunits to GST-Raf/259. In contrast, the additional removal of amino acids 136-239 results in over 80% loss of G binding. A fusion protein coding for amino acids 136 through 239 (Raf/136-239) bound G to the same extent as Raf/330 or Raf/239 (not shown), further demonstrating the importance of this region in binding G. We cannot exclude, however, the possibility that other sequences in Raf-1 (e.g. 1-135) may participate in and contribute to the stable interaction of Raf-1 and G.

c-H-Ras (2, 7, 9) and 14-3-3 proteins (11, 12) have also been reported to bind to the amino terminus of Raf-1. The carboxyl terminus of ARK, which contains a PH (pleckstrin homology) domain, is known to bind to G. We tested whether these proteins could effect the interaction between Raf/330 and G in the in vitro binding assay (Fig. 1C). GST-ARK, but not GST-Ras or GST-14-3-3, strongly inhibited binding of G to Raf/330.

We next compared the relative affinities of Raf/330 and the carboxyl terminus of ARK (a protein dependent upon binding to G(24) for carrying out its physiological function) for binding to G. Scatchard analysis revealed a K of approximately 163 (±36; n = 3) nM for binding of G to Raf/330 compared to a K of approximately 87 (±24; n = 3) nM for G binding to ARK (Fig. 2).


Figure 2: Scatchard analysis of G binding to Raf/330 and ARK-CT. Increasing amounts of G were incubated with beads containing immobilized GST-Raf-(1-330) or GST-ARK-CT in an in vitro binding assay. Bound G was detected by immunoblotting with anti-G antibodies and quantified by PhosphorImager analysis using comparison with known amounts of purified G as described under ``Experimental Procedures.'' Constants were derived from three independent experiments. A representative experiment is shown.



ADP-ribosylation of G by pertussis toxin requires the intact heterocomplex between G and G subunits(26) . To determine if Raf/330 could inhibit association of G and G, we investigated the effects of increasing amounts of Raf/330 on the pertussis toxin-catalyzed ADP-ribosylation of G in the presence of G (Fig. 3). We found that the Raf/330 inhibited the ADP-ribosylation of G with an EC of approximately 200 nM. GST alone had no effect on ADP-ribosylation (not shown).


Figure 3: Inhibition of pertussis toxin-mediated ADP-ribosylation of G. Increasing amounts of GST-Raf-(1-330) were added to an ADP-ribosylation reaction containing G and G subunits purified from bovine brain, P-NAD, and the active subunit of pertussis toxin. Reactions were carried out as described under ``Experimental Procedures.'' Following separation by SDS-polyacrylamide gel electrophoresis and autoradiography, the band corresponding to G and [P]ADP-ribose was quantified by densitometry on a Bioimager (Millipore). Results represent duplicate determinations (±range).



The ability of G subunits to form complexes with Raf-1 in vivo was examined in human embryonic kidney 293-T cells. In order to facilitate the efficient and specific precipitation of G subunits, we engineered a FLAG epitope tag onto the carboxyl terminus of G 293-T cells were transfected with expression plasmids coding for Raf-1, G and G subunits, or combinations of these proteins. When equal amounts of Raf-1 protein are overexpressed (Fig. 4A), Raf-1 was specifically co-precipitated with anti-FLAG antibody only with concomitant expression of G. Significantly, the co-precipitation of endogenous Raf-1 protein with anti-FLAG antibody is detected in 293-T cells transfected with G and G alone (Fig. 4B). Thus, as the levels of G expression are quite modest (2-3-fold over endogenous), the formation of in vivo complexes between Raf-1 and G does not require significant overexpression of the proteins. Under the conditions employed we estimate that between 2 and 4% of the Raf-1 protein is co-precipitated with G, similar to the values previously reported for in vivo complexes between Ras and Raf-1(27) .


Figure 4: Co-immunoprecipitation of Raf-1 with G. 293-T cells were transfected, either alone or in combination, with expression plasmids coding for Raf-1, G, or G or with vector alone as a control. A, top: anti-FLAG immunoprecipitates from 293-T cells transfected as shown were analyzed for the presence of G and Raf-1 proteins by simultaneous immunoblotting with anti-G and anti-Raf antibodies. A, bottom: aliquots of the clarified whole cell extracts (WCL) were monitored for the expression levels of Raf-1 and G. B, top: anti-FLAG immunoprecipitates from cells transfected either with vector or a combination of G and G were analyzed for the co-precipitation of G and Raf-1 proteins by simultaneous immunoblotting with anti-G and anti-Raf antibodies. B, bottom: portions of the clarified whole cell lysates (WCL) were immunoblotted to determine the expression levels of Raf-1 and G Similar results were obtained in three other experiments.




DISCUSSION

These experiments provide evidence for a direct and specific interaction between the Raf-1 protein kinase and the G subunits of heterotrimeric G-proteins. The affinity of this interaction is similar to that of G for ARK in the in vitro binding studies. There are several conceivable physiological ramifications of such an interaction. One possibility is that G is directly involved in the activation of Raf-1. G-protein receptor-coupled agonists, such as platelet-activating factor (28) and lipopolysaccharide(29) , have been shown to stimulate Raf-1 and the mitogen-activated protein kinase pathway in a Ras-independent manner. Robbins et al.(30) have shown that AlF-induced mitogen-activated protein kinase activation was only minimally inhibited by dominant negative Ras. In these cases, free G could act analogously to Ras, functioning to recruit Raf-1 to the plasma membrane where it could interact with other regulators and with substrates. A parallel interaction between certain protein kinase C isoforms and a G-related protein termed RACK1 has been reported by Ron et al.(31) .

A similar G-dependent mechanism has been proposed for the recruitment of ARK to the membrane. Once translocated by G, ARK phosphorylates agonist-bound G-protein receptor, resulting in receptor desensitization (24). Here we show that Raf/330 and ARK can compete for binding to G. This result implies that Raf-1 could potentiate receptor signaling by interfering with the capacity of ARK to desensitize activated receptor. In this way activation of a tyrosine kinase growth factor receptor pathway could impinge upon G-protein receptor signaling. For example, Raf-1 could be involved in regulation of cAMP levels by binding to G subunits and inhibiting ARK-induced down-regulation of G-linked receptors.

A third avenue through which Raf-1 could influence G-protein receptor signaling is provided by our finding that Raf/330 can inhibit association of G and G subunits. By influencing the ratio of free G and G subunits, Raf-1 might exert either a positive or a negative effect on signaling. G and G can act in concert to potentiate effector function as in the case of adenyl cyclase type II and IV(32) . In other systems, G can be either directly stimulatory or inhibitory by interaction with effectors such as adenyl cyclase, phospholipase C-, or potassium channels(33) . In addition, G retains the potential to indirectly dampen signals by sequestering G(33) .

Another consequence of Raf/G interaction might be negative regulation of Raf-1. The recruitment of Raf-1 to the membrane by free G subunits might prevent it from interacting with GTP-Ras and subsequently activated. Although our data do not support a direct competition between Ras and G, they do support a high affinity interaction between Raf-1 and G. As the intracellular concentrations of G can be as high as 500 µM in some tissues(33) , and the percentage of Ras that is GTP bound is typically low (10-25%), it is possible that liberated G subunits could effectively recruit and sequester Raf-1. This mechanism would allow G-protein agonists which stimulate cAMP production to dampen growth factor-mediated signals through parallel and potentially synergistic pathways(34, 35) .

In conclusion, we have identified and characterized a novel interaction between the serine-threonine protein kinase Raf-1 and the G subunits of heterotrimeric G-proteins. This interaction represents a potentially important point of cross-talk and regulation between the growth factor receptor pathway and signals emerging from G-protein receptors. Given the diversity of cellular effects in which G subunits have been implicated, the functional relationship between these proteins may be quite complex. The identification and characterization of this interaction allows the functional significance of this interaction to be tested in the various model systems.

  
Table: Yeast two-hybrid interactions between Raf, G, and control constructs

Constructs encoding either the NH terminus of Raf (pAY-Raf), the fragment of G isolated in a library screen (pACT-G), irrelevant proteins (PTP-PEST; pAS1--integrin), or fusion vector alone (pACT) were co-transformed into yeast, and co-transformants were selected by plating on media lacking leucine and tryptophan. Two independent co-transformants were evaluated for the ability to support growth on media lacking histidine (His3) and to induce the expression of -galactosidase (-gal). SNF1 and SNF4 plasmids were included as a positive control.



FOOTNOTES

*
This work was funded in part by Grant CA55652 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a Parke-Davis/University of Michigan Biotechnology Fellowship.

To whom correspondence should be addressed: Parke-Davis Pharmaceuticals, 2800 Plymouth Rd., Ann Arbor, MI 48106. Tel.: 313-998-5945; Fax: 313-996-5668; E-mail, deckers@aa.wl.com.

The abbreviations used are: G-protein, GTP-binding regulatory protein; PCR, polymerase chain reaction; GST, glutathione S-transferase; ARK, -adrenergic receptor kinase.


ACKNOWLEDGEMENTS

Roman Herrera for the generous gift of the pAS1--integrin construct, Stanley Fields for the yeast two-hybrid reagents, and Alan Saltiel for critical reading of the manuscript.


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