©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of the 14.3.3 Domains Important for Self-association and Raf Binding (*)

(Received for publication, May 30, 1995; and in revised form, August 3, 1995)

Zhi-jun Luo (§) Xian-feng Zhang Ulf Rapp (1) Joseph Avruch (¶)

From the Diabetes Research Laboratory and Medical Services, Massachusetts General Hospital, Charlestown, Massachusetts 02129, the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02129, Institut für medizinische Strahlenkunde und Zellforschung (MSZ), Universität Wurzburg, Versbacher Strasse 5, 97078 Wurzburg, Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The 14.3.3 protein is a ubiquitous and abundant arachidonate-selective acyltransferase and putative phospholipase A(2), which self-assembles into dimers and binds to c-Raf-1 and other polypeptides in vitro and in intact cells. The 14.3.3 polypeptides endogenous to Sf9 cells associate in situ with both active and inactive recombinant Raf and copurify at a fairly reproducible molar ratio that is probably geq1. Purified baculoviral recombinant Raf, despite its preassociated 14.3.3 polypeptide, binds additional recombinant 14.3.3 polypeptide in vitro, in a saturable and specific reaction, forming a complex that is resistant to 1 M LiCl. A two-hybrid analysis indicates that 14.3.3 binds primarily to Raf noncatalytic sequences distinct from those that bind Ras-GTP, and in vitro 14.3.3 binds to Raf without inhibiting the Ras-Raf association or Raf-catalyzed MEK phosphorylation. Deletion analysis of 14.3.3 (1-245) indicates that the 14.3.3 domain responsible for binding to Raf extends over the carboxyl-terminal 100 amino acids, whereas 14.3.3 dimerization is mediated by amino-terminal sequences. As with Ras, the 14.3.3 polypeptide does not activate purified Raf directly in vitro. Moreover, expression of recombinant 14.3.3 in COS cells beyond the substantial level of endogenous 14.3.3 protein does not alter endogenous Raf kinase, as judged by the activity of a cotransfected Erk-1 reporter. Coexpression of recombinant 14.3.3 with recombinant Myc-tagged Raf in COS cells does increase substantially the Myc-Raf kinase activity achieved during transient expression, which is attributable primarily to an increased level of Myc-Raf polypeptide, without alteration of Myc-Raf specific activity or the activation that occurs in response to epidermal growth factor or 12-O-tetradecanoylphorbol-13-acetate. Nevertheless, evidence that 14.3.3 actively participates in Raf activation in situ is provided by the finding that although full-length 14.3.3 binds active Raf in situ, truncated versions of 14.3.3, some of which bind Raf polypeptide in situ nearly as well as full-length 14.3.3 , are recovered in association only with inactive Raf polypeptides. Thus, 14.3.3 polypeptides bind tightly to one or more sites on c-Raf. Overexpression of 14.3.3 enhances the expression of recombinant Raf, perhaps by stabilizing the Raf polypeptide. In addition, Raf polypeptides bound to truncated 14.3.3 polypeptides are unable to undergo activation in situ, indicating that 14.3.3 participates in the process of Raf activation by mechanisms that remain to be elucidated.


INTRODUCTION

An important insight into the initial step in Raf activation was the discovery that Raf binds directly to the GTP-bound form of Ras (1, 2, 3) . This Raf-Ras-GTP interaction does not result directly in Raf activation, inasmuch as addition of Ras GTP to inactive, baculoviral recombinant Raf in vitro does not alter Raf kinase activity. Presumably, Ras GTP functions in situ to translocate Raf to the surface membrane so as to enable its activation by other processes. Support for this model is provided by the demonstration that fusion of plasma membrane targeting (CAAX) sequences onto the Raf carboxyl terminus is transforming and bypasses the need for Ras in Raf activation; a large increase in the activity of membrane-associated Raf is observed in growth factor-deprived cells, and EGF stimulates Raf CAAX activity a further 10-fold in a Ras-independent reaction(4, 5) .

The inability of Ras to directly activate Raf, together with the finding that mitogen activation of Raf becomes Ras independent if Raf is targeted directly to the plasma membrane, implies that physiologic activation of (Ras bound) Raf requires Raf interaction with other plasma membrane components, e.g. lipids, polypeptides, or both. Ghosh et al.(6) reported that the Raf amino-terminal noncatalytic sequences bound to liposomes in a phosphatidylserine-dependent reaction that is independent of Ca and diacylglycerol. In this report, we describe the binding of c Raf-1 in vitro and in situ to the 14.3.3 polypeptide, an arachidonate-selective acyl transferase and putative phospholipase A2(7) . We define the Raf domain employed for the binding of 14.3.3 in situ and the 14.3.3 domains necessary for self-association and Raf binding; we find that while carboxyl-terminal fragments of 14.3.3 bind Raf in situ nearly as well as full-length 14.3.3, only the latter is found in association with catalytically active Raf polypeptides in situ.


MATERIALS AND METHODS

cDNAs encoding murine 14.3.3 were isolated from a murine T cell DNA library by two-hybrid expression cloning according to Durfee et al.(8) , using the c-Raf sequences 1-25/305-648 as bait (see ``Results''). cDNAs encoding rat Erk-1, human MEK-1, and human C-Ha-Ras were expressed in Escherichia coli as GST (^1)fusion proteins using the p-GEX kg vector (9) and purified by glutathione-agarose affinity chromatography. The free Erk-1 and 14.3.3 polypeptides were obtained after thrombin cleavage. Recombinant Raf polypeptide containing a hexahistidine tag at the carboxyl terminus was expressed in Sf9 cells using a recombinant baculovirus and purified by nickel chelate affinity chromatography. Active baculoviral Raf kinase was obtained by co-infection with baculoviruses encoding v-Ras and v-Src(10) .

The transacylation activity of the recombinant 14.3.3 was measured according to Zupan et al.(7) . The Raf kinase assay was performed as previously described(11, 12) .

The binding in vitro of various polypeptides to GST or GST fusion proteins immobilized on glutathione-Sepharose was carried out at 30 °C for 30 min in buffer A containing 25 mM Tris-Cl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol; polypeptide concentrations are described in the figure legends. The beads were washed in excess binding buffer three times; the retained polypeptides were eluted directly into SDS-containing buffer, separated by SDS-PAGE, and analyzed by protein staining, immunoblot, or autoradiography as described.

The association of polypeptides in situ was assessed during transient expression in COS M7 cells and transfected by the DEAE-dextran method. The cDNA sequences encoding Raf were inserted into two mammalian expression vectors; Myc-Raf contains a 33-amino acid epitope from human c-Myc, known to be reactive with the monoclonal antibody 9B7.3(13) , appended to the Raf amino terminus, and inserted into pMT2. Raf was also expressed as a GST fusion protein using the vector pEBG, which encodes glutathione S-transferase driven by the EF1alpha promoter/enhancer. The cDNA encoding 14.3.3 was introduced unmodified into the vector CMV5, into the pEB vector (lacking the glutathione S-transferase sequences) with a 9-amino acid epitope from the influenza hemagglutinin (HA epitope, (14) ) added to its carboxyl terminus, and into pEBG for expression in situ as a GST fusion. Deletion mutation of the 14.3.3 was made by polymerase chain reaction from the 5`- and 3`-ends of the cDNA. The polymerase chain reaction products were subcloned into the pEBG vector, and the structures were verified by DNA sequence analysis.

All transfections utilized a total of 20 µg of DNA; 48 h after transfection, extracts were prepared by homogenization in a buffer containing 25 mM Tris-Cl, pH 7.5, 1 mM EDTA, 2 mM EGTA, 1 mM dithiothreitol, 25 mM beta-glycerophosphate, 1 mM sodium vanadate, 1% Triton X-100, and proteinase inhibitors. Immunoprecipitations were carried out for 1 h at 4 °C using the monoclonal antibody 12CA5 for the HA epitope or the anti-Myc monoclonal antibody 9B7; immune complexes were harvested with protein G-Sepharose. GST-Raf and GST-14.3.3 fusions were recovered using glutathione-Sepharose beads. Immunoblots of Raf were carried out using 9B7.3 for Myc-Raf or a polyclonal antipeptide antibody raised to the carboxyl-terminal 12 amino acids of human c-Raf-1. Immunoblot of 14.3.3 was carried out using a polyclonal antibody raised to cleaved, purified recombinant murine 14.3.3 .

L-alpha-1-palmitoyl-2-arachidonyl (arachidonyl-1-^14C) phosphatidylethanolamine, and [-P]ATP were obtained from DuPont NEN. Glutathione-Sepharose 4B was from Pharmacia Biotech Inc.


RESULTS

Seeking proteins other than Ras and related small GTPases that interact with Raf, we utilized the Raf sequences corresponding to 1-25/305-648 in a two-hybrid screening(8) . This truncated Raf polypeptide, also known as BXB-Raf(15) , is a transforming protein that encodes a dispensable region of the Raf amino terminus (residues 1-25) fused to the Raf carboxyl-terminal 344 amino acids, which includes the entire catalytic domain (residues 335-627) flanked by short segments of amino-terminal (residues 305-334) and carboxyl-terminal (residues 628-648) noncatalytic sequences. 20 His and LacZ cDNAs were recovered in a screen of 2.5 times 10^6 transformants. Five of these cDNAs gave much more rapid complementation of LacZ activity than the remaining cDNAs; the former all encoded MEK1 or MEK2. Among the remaining 15 cDNAs were 5 that encoded polypeptide sequences 100% identical to the rat isoform of the 14.3.3 polypeptide, and that differed from a human platelet PLA(2) polypeptide by a single conservative substitution(7) . Inasmuch as 14.3.3 proteins have been reported to copurify or associate with a relatively large number of proteins(16) , and 14.3.3 has been identified as a protein cofactor for a number of enzymes in vitro, including the ADP-ribosylation of Ras and other small GTPases by exoenzyme S(17) , we examined several proteins other than Raf for their ability to associate with 14.3.3 in the two-hybrid system. No interaction of 14.3.3 with p70 S6 kinase and amino-terminal regulatory domain of protein kinase C (residues 1-245) or c-Ha-Ras (1-185) was detected (data not shown). The relative selectivity of the 14.3.3-Raf association led us to undertake a further characterization of this interaction.

The two-hybrid method was employed to identify the region on Raf that interacts with 14.3.3 in comparison to MEK1 and Ras, proteins known to interact with Raf in a physiologic context. The Ras binding site has previously been localized to Raf residues 50-150(3, 18) , whereas neither MEK nor 14.3.3 interacts with Raf 1-257. MEK, a known Raf substrate, interacts strongly with the BXB-Raf and holo-Raf (1-648) but not at all with Raf 1-332 (Table 1). By contrast, 14.3.3 interacts weakly with BXB-Raf and holo-Raf (1-648) but associates strongly with the Raf 1-332 (Table 1). Thus, 14.3.3 binds in situ most avidly to a segment of Raf between amino acids 257 and 332, a noncatalytic region distinct from those that bind to Ras or MEK.



Binding of Raf to 14.3.3 in Vitro

The ability of recombinant Raf to interact directly with recombinant 14.3.3 was investigated. The 14.3.3 polypeptide was expressed in E. coli as a glutathione S-transferase fusion protein(9) , purified by GSH affinity chromatography, and employed with and without thrombin cleavage (Fig. 1). The functional integrity of the recombinant 14.3.3 was evaluated by examining its ability to catalyze autoacylation from [^14C]arachidonate sn-2-phosphatidylethanalomine. The GST-14.3.3 fusion protein and thrombin-cleaved 14.3.3 polypeptides, although devoid of PLA(2) activity using a variety of substrates, each catalyze the cleavage of the sn-2 fatty acid of [^14C]arachidonyl sn-2-phosphatidylethanolamine and the formation of a covalent [^14C]arachidonyl-14.3.3 protein adduct, presumably an acyl-enzyme intermediate. Thus, the recombinant prokaryotic 14.3.3 exhibits an acyl transferase but not a PLA(2) function, similar to that described by Gross and colleagues(7) .


Figure 1: Recombinant 14.3.3 catalyzes autoacylation from [^14C]arachidonyl sn-2-phosphatidylethanolamine. GST, GST-14.3.3 , and cleaved 14.3.3 (5 µg each) were incubated with L-alpha-1-palmitoyl-2 ([^14C]arachidonyl phosphatidylethanolamine, 52.8 µM, 125,400 cpm/pmol) at 37 °C for 3 min, mixed rapidly with SDS sample buffer, and heated to 90 °C for 3 min. The samples were subjected to SDS-PAGE and subsequent fluorography. Lanes1-3, Coomassie Blue stain; lanes4-6, autoradiogram of [^14C]arachidonate. Lanes1 and 4, glutathione S-transferase; lanes2 and 5, GST-14.3.3 ; lanes3 and 6, thrombin cleaved and purified 14.3.3 .



Raf 1 containing a hexahistidine tag at its carboxyl terminus was expressed in Sf9 cells by baculoviral infection and purified by nickel chelate chromatography, either as a catalytically inactive polypeptide (Fig. 2, lane6) or in a catalytically active form, as a result of coinfection with baculoviral v-Ras plus v-Src (Fig. 2, lanes1 and 3-5)(10) . An antiserum against prokaryotic recombinant murine 14.3.3 , reactive primarily with epitopes in the amino-terminal half of 14.3.3 (Fig. 5C, upperpanel), readily immunoblots the 28- and 30-kDa 14.3.3 polypeptides endogenous to Sf9 (Fig. 2, lane2) and COS cell (Fig. 7A, upperpanel) extracts; immunoblot of several Raf-1 isolates (Fig. 2, lanes2-6) shows that both the active (lanes3-5) and inactive (lane6) Raf polypeptides purified from Sf9 cells are recovered in association with endogenous Sf9 14.3.3 polypeptides. Both the recombinant Raf and Sf9 14.3.3 polypeptides are readily visualized on Coomassie Blue-stained gels of the preparations of purified Raf (Fig. 2, lane1); although the relative Coomassie Blue binding per unit mass for c-Raf-1 and 14.3.3 is unknown, the comparable staining intensities observed for Raf and the co-purified 14.3.3, together with the general tendency of acidic polypeptides such as 14.3.3 to stain weakly with Coomassie Blue, suggests that the molar ratio of 14.3.3 to Raf in these isolates is at least 1. Moreover, a relatively constant ratio of the two polypeptides is recovered in different isolates of Raf, whether active (Fig. 2, lanes3-5) or inactive (Fig. 2, lane6).


Figure 2: Copurification of 14.3.3 and c-Raf-1 from insect Sf9 cells. Sf9 cells were infected with baculovirus encoding human (His) 6-tagged c-Raf-1 alone or plus baculoviral encoded v-Ras and/or v-Src. The recombinant Raf was purified by nickel chelate affinity purification and subjected to SDS-PAGE prior to immunoblotting. Lane1, Coomassie Blue stain of SDS-PAGE gel corresponding to the isolate shown in lane5. Lanes2-6, the upper part was immunoblotted with an antiserum against carboxyl-terminal 12 amino acids of human c-Raf-1, and the lower part was immunoblotted with an antiserum against mouse 14.3.3 . Lane2, Sf9 cell extract without infection; lane3, purified Raf coinfected with v-Src; lane4, Raf coinfected with v-Ras; lane5, Raf coinfected with v-Ras plus v-Src; lane6, inactive baculoviral Raf; mwm, molecular weight markers.




Figure 5: Deletion analysis of functional domains of 14.3.3 . Truncations of 14.3.3 were constructed by the polymerase chain reaction. The polymerase chain reaction products were subcloned to pEBG vector and expressed as GST fusions in COS cells. A, schematic diagram of GST-14.3.3 variants. B, dimerization domain of 14.3.3 . cDNAs encoding the GST-14.3.3 variants shown in A were cotransfected with a vector encoding a full-length 14.3.3 polypeptide tagged at its amino terminus with a Myc epitope. Extracts were prepared 48 h later; the GST fusion proteins were purified by GSH affinity chromatography and resolved on SDS-PAGE gel. A Coomassie Blue-stained gel is shown in the upperpanel, and an immunoblot using anti-Myc antibody 9E10.2 is shown in the lowerpanel. Lane1, GST; lane2, GST-14.3.3 (1-245); lane3, GST-14.3.3 (1-80); lane4, GST-14.3.3 (1-140); lane5, GST-14.3.3 (1-180); lane6, GST-14.3.3 (79-245); lane7, GST-14.3.3 (139-245); lane8, GST-14.3.3 (179-245). mwm, molecular weight markers (from the top) 200, 116, 97.4, 66.2, 45, 31, and 21.5 kDa. C, Raf binding domain of 14.3.3 . GST (lane1) or GST-14.3.3 fusions (lanes2-8 as in B) were coexpressed with Myc-Raf in COS cells and purified by GSH affinity chromatography. The toppanel is immunoblot of the GSH-Sepharose isolates using a polyclonal antiserum raised against recombinant 14.3.3 ; note that the major epitopes are in the amino-terminal half of the 14.3.3 polypeptide. The middlepanel is an immunoblot of the GSH affinity isolates using the anti-Myc antibody 9E 10.2. The bottompanel displays the Raf kinase activity of the Myc-Raf polypeptides associated with the GSH-Sepharose-bound, GST-14.3.3 polypeptides. After purification of GST-14.3.3-Raf complex, the Raf kinase activity was measured by the addition of magnesium, [-P]ATP (0.1 mM, 2000 cpm/pmol), GST-MEK-1 (20 µg/ml), and Erk-1 polypeptide. The basal activity of the substrate GST-MEK 1, incubated with Erk-1, magnesium, [-P]ATP, and GSH-Sepharose beads, is shown in lane0.




Figure 7: Effect of 14.3.3 on the expression and activity of Myc-Raf in situ. Vectors encoding GST (lanes1, 5, 9), GST-14.3.3 (1-245) (lanes2, 6, 10), GST-14.3.3 (139-245) (lanes3, 7, 11), or Raf (1-257) (lanes4, 8, 12) were cotransfected with Myc-Raf (lanes1-12) into COS cells; 48 h later, the cells were placed in medium containing 0.5% serum, and after a further 18-h incubation period, they were treated with carrier (lanes1-4), EGF (10 ng/ml, lanes5-8), or TPA (500 nm) (lanes9-12) for 15 min, rinsed, and extracted. Aliquots of the cell extracts, normalized for protein content, were subjected to anti-Myc immunoprecipitation (A, 3rdpanel from top, and bottompanel) and GSH-Sepharose affinity chromatography (B, allthreepanels). A, the toppanel shows an anti-14.3.3 immunoblot of the cell extracts. The panel secondfromthetop shows an immunoblot of the cell extracts using the anti-Myc antibody 9 E10; the Myc-Raf band is indicated. The panelthirdfromthetop, and the bottompanels are analyses of the anti-Myc immunoprecipitates prepared from the extracts shown in the toptwopanels; the panelthirdfromthetop is a Myc immunoblot of the Myc immunoprecipitate; the Myc-Raf polypeptide is indicated. The bottompanel displays an assay of the Raf kinase activity found in the Myc-Raf immunoprecipitates, estimated by the phosphorylation of recombinant GST-MEK 1, coupled to the phosphorylation of recombinant Erk-1 as described in Fig. 5C, bottompanel. B, the proteins retained by GSH-Sepharose affinity chromatography of extracts prepared from serum-deprived (lanes1-3), EGF-treated (lanes4, 5), and TPA-treated (lanes6, 7) COS cells, transfected with a vector encoding Myc-Raf (lanes1-7) together with GST (lane1), GST-14.3.3 (1-245) (lanes2, 4, 6), or GST-14.3.3 (139-245) (lanes3, 5, 7)), are analyzed for 14.3.3 content (by immunoblot with anti-14.3.3, upperpanel), Myc-Raf content (by anti-Myc immunoblot, middlepanel), and Myc-Raf kinase activity (by coupled phosphorylation assay with [-P]ATP, GST MEK, and Erk-1 substrates, lowerpanel). Lane8 in the lowerpanel contains GST-MEK, [-P]ATP substrates with empty GSH-Sepharose beads.



Despite the presence of considerable Sf9-derived 14.3.3 already bound to the baculoviral recombinant Raf-1 polypeptide, such Raf preparations bind in vitro to a GST-14.3.3 fusion protein but not to GST alone (Fig. 3, upperpanel, lanes1 and 2); preincubation of Raf with cleaved, purified E. coli recombinant 14.3.3 polypeptide prevents the subsequent binding of Raf by immobilized GST-14.3.3 , whereas preincubation of Raf with bovine serum albumin has no effect (Fig. 3, upperpanel, lanes1, 3, 4). The ability of 14.3.3 to bind active baculoviral Raf was verified by the demonstration that GST-14.3.3 can specifically immobilize essentially all of the MEK phosphorylating activity in a preparation of active Raf (Fig. 3, lowerpanel, lanes1-4), and the complex of active Raf and GST-14.3.3 is resistant to washing with 1 M LiCl. Extensive Raf autophosphorylation in vitro did not interfere with Raf binding to GST-14.3.3 (Fig. 3, upperpanel, lanes6 and 7); inactive Raf polypeptide (identifiable by its slightly faster mobility in SDS-PAGE) also binds specifically to GST-14.3.3 (Fig. 3, upperpanel, lane5).


Figure 3: Binding of Raf to GST-14.3.3 in vitro. Upperpanel, baculoviral Raf activated by coinfection with v-Ras and v-Src (0.2 µM, lanes1-4, 6, and 7) or unactivated (0.3 µM, lane5) and purified by nickel chelate chromatography was incubated with immobilized GST-14.3.3 (20 µg/ml settled beads, lanes1 and 3-6) or GST (20 µg/ml beads, lanes2 and 7) directly (lanes1, 2, and 5-7) or after preincubation with a 100-fold molar excess of purified 14.3.3 (lane3) or bovine serum albumin (lane4). In lanes6 and 7, the activated Raf was subjected to autophosphorylation in vitro in the presence of magnesium (10 mM), [-P]ATP (100 µM, 30 °C for 30 min) prior to addition of GST-14.3.3 (lane6) or GST (lane7). In lanes1-5, Raf was detected by anti-Raf immunoblot; in lanes6 and 7, P-Raf was detected by autoradiography. Lower panel, baculoviral Raf, activated in situ by coinfection with v-Ras and v-Src, was purified and mixed with GST-14.3.3 (lanes1 and 3) or GST (lanes2 and 4) immobilized on GSH-Sepharose. Aliquots of the supernatants (lanes1 and 2) and washed GSH-Sepharose beads (lanes3 and 4) were assayed for Raf kinase activity by phosphorylation of GST-MEK (20 µg/ml) [-P]ATP (0.1 mM, 2000 cpm/pmol).



These results demonstrate that baculoviral recombinant Raf, although purified as a complex with Sf9 14.3.3, binds additional 14.3.3 in vitro, and this binding is saturable. The ability of GST-14.3.3 to specifically adsorb essentially all the Raf polypeptides, both active (Fig. 3) and inactive, from such preparations indicates that GST-14.3.3 must bind Raf polypeptides that already contain bound Sf9 14.3.3. Although displacement of or dimerization with the preassociated Sf9 14.3.3 is possible, the most plausible model envisions more than one binding site on Raf for 14.3.3. Thus, the ability of 14.3.3 to interact with Raf in the two-hybrid yeast expression system certainly reflects the direct binding of the two polypeptides; however, the number of 14.3.3 binding sites on Raf and their precise localization remain to be more fully defined.

Binding of Raf to 14.3.3 in Situ

The interaction of recombinant 14.3.3 with Raf in situ was examined further by cotransfection (Fig. 4). An epitope-tagged recombinant 14.3.3 polypeptide was created by fusion of a 9-amino acid epitope from the influenza hemagglutinin to either the amino terminus or carboxyl terminus, enabling the selective immunoprecipitation of the tagged 14.3.3 polypeptide with anti-HA monoclonal antibody 12CA5(14) . After transfection of either tagged version of 14.3.3 , the anti-HA immunoprecipitates contain, in addition to a 30-kDa band of HA-tagged recombinant 14.3.3 , a 28-kDa polypeptide that is not reactive with the HA antibody but is reactive with the anti-14.3.3 antiserum (data not shown). Thus, the recombinant HA-tagged 14.3.3 forms complexes (presumably dimers) with endogenous 14.3.3 polypeptides, and free amino or carboxyl termini are not crucial for dimerization.


Figure 4: Association of recombinant Raf and 14.3.3 in intact COS cells. Two cDNA encoding tagged Raf polypeptides were constructed, one in the vector pEB, encoding full-length Raf fused at its amino terminus to the carboxyl terminus of glutathione S-transferase, and a second in the vector pMT2 encoding a Myc epitope fused to the Raf amino terminus. In lanes1-4, pEB encoding GST-Raf (lanes1, 3, 4) or GST (lane2) was cotransfected into COS cells with pMT2 encoding an HA-tagged 14.3.3 (lanes1-3) or the pMT2 HA vector (lane4). Similarly in lanes5-8, pMT2 encoding Myc-Raf (lanes5, 7, 8) or pMT2 Myc vector (lane6) was cotransfected with vector encoding HA-tagged 14.3.3 (lanes5-7) or empty pMT2 HA vector (lane8). After 48 h, cells were extracted, and recombinant polypeptides were purified using GSH-Sepharose (lanes1 and 2), anti-Myc monoclonal antibody 9B7.3 (lanes5 and 6) for direct isolation of recombinant Raf, or anti-HA epitope monoclonal antibody 12CA5 for isolation of recombinant 14.3.3 (lanes3, 4, 7, and 8). Raf polypeptide in each isolate was detected by immunoblot with an anti-COOH-terminal Raf peptide antibody. Note that recovery of recombinant Raf using GSH-Sepharose (lane1) or anti-Myc immunoprecipitation (lane5) is comparable to that achieved by immunoprecipitation of HA 14.3.3 (lanes3, 7).



Two tagged versions of recombinant Raf-1 were constructed by introduction at the Raf amino terminus of a Myc epitope (13) or by fusion to GST. After cotransfection of HA-tagged 14.3.3 with either version of Raf into COS cells, anti-HA 14.3.3 immunoprecipitates (Fig. 4, lanes3, 4, 7, 8) contain substantial immunoreactive Raf-1, ranging from 30 to 100% of the amount of Raf polypeptide recovered from an identical aliquot of the COS cell extract by anti-Myc immunoprecipitation (Fig. 4, lanes5 and 6) or by binding to GSH-Sepharose ( Fig. 4lanes1 and 2). The anti-Myc immunoprecipitates of Raf-1 contained in addition to the recombinant 30-kDa HA-tagged 14.3.3 polypeptide some endogenous 28-kDa 14.3.3 polypeptide, as visualized by immunoblot with anti-14.3.3 antiserum (not shown). Thus, a substantial portion of recombinant Raf is recovered in association with the coexpressed 14.3.3 , indicating clearly that the 14.3.3 associates in situ with most or all of the recombinant Raf-1 in COS cells.

The domains of 14.3.3 responsible for dimerization and Raf association were determined by coexpression in COS cells of Myc-Raf or Myc-14.3.3 with GST-14.3.3 (1-245) and a series of 14.3.3 fragments constructed as GST fusions (Fig. 5A). The transiently expressed GST-14.3.3 fusions were purified from COS cell extracts using GSH-Sepharose, and the isolates were evaluated for the presence of the cotransfected full-length Myc-tagged 14.3.3 polypeptide (Fig. 5B) or Myc-Raf (Fig. 5C). The Coomassie Blue stain of the purified, COS recombinant GST-14.3.3 fusions and their associated polypeptides is shown in Fig. 5B, upperpanel. The variation in Coomassie Blue staining of the GST-14.3.3 fusions reflects the variability in their expression in situ rather than in their recovery, inasmuch as recovery of the irrelevant endogenous COS polypeptides that bind to GSH-Sepharose (designated as A and B, Fig. 5B, upperpanel) is identical in all lanes. The binding of cotransfected Myc-tagged 14.3.3 polypeptides to the GST-14.3.3 fusions is evident as the 30-kDa Coomassie band seen just above bandA in lane2 (absent in lane1) and more faintly in lanes3-5. The identity of this band as Myc-14.3.3 is verified by anti-Myc immunoblot (Fig. 5B, lowerpanel). These results indicate that although a free NH(2) terminus is not critical for 14.3.3 self-association, the amino-terminal 80 amino acids of 14.3.3 are sufficient to confer some 14.3.3 self-association, and nearly full self-association is seen with GST-14.3.3 (1-140). Reciprocally, deletion of the amino-terminal amino acids from 14.3.3 markedly reduces its ability to self-associate; little (Fig. 5B, lowerpanel, lane7) or no (Fig. 7B) self-association is detectable with deletion of the amino-terminal 138 amino acids, and the carboxyl-terminal 14.3.3 fragment 179-245, although very well expressed, is unable to associate at all with full-length Myc-14.3.3 (Fig. 5B, lowerpanel, lane8).

As to the effects of truncation on the ability of GST-14.3.3 to bind cotransfected Myc-Raf, the 14.3.3 amino-terminal 139 residues are largely dispensable; optimal Myc-Raf recovery is observed with GST-14.3.3 (139-245) (Fig. 5C, middlepanel, lanes2, 7), and considerable Myc-Raf is recovered with GST-14.3.3 (179-245) (Fig. 5C, middlepanel, lane8), whereas no binding of Myc-Raf occurs to GST-14.3.3 (1-140) (Fig. 5C, middlepanel, lane4). Each of these GST-14.3.3 isolates was also assayed for the presence of active Raf kinase, estimated by its ability to phosphorylate and activate GST-MEK (Fig. 5C, bottompanel). Notably, only the GST-14.3.3 (1-245), i.e. full-length 14.3.3, is associated with catalytically active Raf polypeptide. Thus, although GST-14.3.3 (139-245) retains as much Myc-Raf polypeptide as GST-14.3.3 (1-245), the Raf associated in situ with the truncated 14.3.3 is devoid of catalytic activity. The failure of active Raf to bind to truncated 14.3.3 polypeptides in situ probably cannot be attributed to a lack of 14.3.3 dimerization, inasmuch as the Myc-Raf bound to GST-14.3.3 (1-180) (Fig. 5C, middlepanel, lane5), which dimerizes quite well with Myc-14.3.3 (Fig. 5B, bottompanel, lane5), also lacks detectable kinase activity (Fig. 5C, bottompanel, lane5). Taken together, these results establish that although the carboxyl-terminal 65 amino acids of 14.3.3 are sufficient to mediate association with Raf, only the full-length 14.3.3 sequences are found in association with catalytically active Raf polypeptides in situ.

Effect of 14.3.3 on Raf Kinase Activity

We sought to determine whether the binding of 14.3.3 to Raf directly alters Raf regulation or catalytic function. Baculoviral recombinant Raf-1, activated in situ by coinfection with v-Ras plus v-Src, catalyzes a brisk autophosphorylation in vitro, as well as the phosphorylation of recombinant MEK1 in vitro. The recombinant 14.3.3 polypeptide at concentrations up to 3 µM is not phosphorylated at all by Raf, whereas GST MEK1 at 0.57 µM is phosphorylated by this amount of Raf kinase to a stoichiometry of 0.4 mol of PO(4)/mol of MEK (not shown) and activated to 25% of maximal. The 14.3.3 polypeptide does not inhibit Raf autophosphorylation when added at >100 molar excess to Raf, nor does 14.3.3 inhibit Raf-catalyzed MEK phosphorylation when present at 10-50-fold excess over MEK (not shown). Thus, 14.3.3 is neither a substrate nor an inhibitor of the Raf kinase in vitro.

We next examined the ability of 14.3.3 to modulate the activation of Raf kinase in vitro and in situ. An initial step in Raf activation in situ involves its binding to GTP-Ras. The effects of 14.3.3 polypeptide on the Ras-Raf interaction was assessed in vitro. The full-length Raf protein binds specifically to immobilized GST-Ras-GTP. Addition of 14.3.3 at >50-fold molar excess to Raf does not inhibit the binding of Raf to GST-Ras-GTP (Fig. 6, upperpanel, lanes1 and 2). In addition, after washing the immobilized GST-Ras-Raf complex, 14.3.3 is seen to have been retained by the GST-Ras, but only if Raf is present (Fig. 6, lowerpanel, compare lanes1 and 2). These data demonstrate that 14.3.3 does not bind to Ras directly nor displace Raf from Ras, but rather it is capable of binding Raf in vitro so as to allow the formation of a ternary complex with Ras and Raf.


Figure 6: Raf binds 14.3.3 and Ras simultaneously. Upperpanel, GST-Ras, charged with GTP (71 nM, lanes1, 2) or GST (lanes3, 4), were incubated with baculoviral recombinant Raf alone (45 nM, lanes1, 3) or Raf (45 nM) plus 14.3.3 (3.6 µM) (lanes2, 4). After washing, the polypeptides bound to GSH-Sepharose were resolved by SDS-PAGE, and the retained Raf polypeptides were detected by immunoblot with an anti-Raf COOH-terminal peptide antibody. Lowerpanel, the recombinant 14.3.3 polypeptide was incubated with GST-Ras-GTP (lanes1, 2) or GST (lane3) in the presence of Raf (lane1) or without Raf (lanes2, 3). After washing the bead, bound polypeptides were resolved by SDS-PAGE, and the retained 14.3.3 polypeptides were detected by immunoblot with an antibody to GST-14.3.3. Lane4 contains an aliquot of 14.3.3 as a positive control. Residual reactivity of this antiserum toward GST polypeptide sequences is evident in the blot.



Addition of 14.3.3 to inactive Raf purified from Sf9 cells does not activate Raf-catalyzed MEK phosphorylation (not shown). This negative result was obtained despite preincubation of Raf and 14.3.3 polypeptide in the presence of various combinations of GTP-Ras (both bacterial and baculoviral, fully processed Ras), phospholipid micelles prepared from bovine brain lipids, Mg (10 mM), Ca (0.1 mM), and ATP (100 µM), for 30 min prior to and after the addition of recombinant GST-MEK1. Inasmuch as a reliable in vitro assay for the activation of Raf kinase is not yet available and 14.3.3 and Raf associate in situ during transient expression, the influence of 14.3.3 on Raf activation was examined in situ by cotransfection. To examine the effects of 14.3.3 overexpression on the regulation of endogenous Raf (and other mitogen-responsive MEK activators), 14.3.3 was cotransfected in COS cells with an HA-tagged Erk-1 reporter. Overexpression of 14.3.3 severalfold above the already substantial level of endogenous 14.3.3 polypeptides did not alter recombinant Erk-1 activity in serum-deprived cells or in response to TPA or EGF (data not shown). We next examined the effects of 14.3.3 on the activity of recombinant Myc-Raf. COS cells transfected with Myc-Raf and various truncated forms of 14.3.3 were either serum deprived or stimulated with mitogens prior to harvest, and the Myc-tagged Raf-1 recombinant was immunoprecipitated and assayed for MEK kinase activity. Coexpression of Myc-Raf with GST-14.3.3 (1-245) increased the MEK kinase activity recovered in a Myc immunoprecipitate by about 2-3-fold, both in serum-deprived cells (Fig. 7A, bottompanel, compare lane1 to lane 2) and in response to EGF (Fig. 7A, bottompanel, compare lane5 to lane 6); EGF itself gave 2-3-fold activation of Myc-Raf (Fig. 7A, bottompanel, compare lane1 to lane 5). The ability of GST-14.3.3 to increase Myc-Raf activity is not due to Raf activation, as occurs with EGF or TPA, but appears to be attributable to an increased Myc-Raf polypeptide abundance, as seen by immunoblot of the whole cell extract (Fig. 7A, 2ndpanel from top, compare lanes1 to 2, 5 to 6, 9 to 10) and in the Myc immunoprecipitates (Fig. 7A, 3rdpanel from top). Such an increase in Myc-Raf expression and recovery of activity was observed repeatedly on cotransfection with GST-14.3.3 (1-245) and was present but less pronounced with the truncated 14.3.3 GST fusion proteins that were capable of binding Myc-Raf in proportion to their somewhat lesser expression than GST-14.3.3 (1-245) (Fig. 7A, toppanel and 2ndpanel from top, compare lanes2 to 3, 6 to 7, 10 to 11).

Based on the finding that GST-14.3.3 (139-245) bound Myc-Raf strongly but did not associate in situ with active Raf (Fig. 5C, lowerpanel), we examined whether GST-14.3.3 (139-245) could interfere with Myc-Raf activation. Fig. 7B demonstrates that although nearly equal amounts of Myc-Raf are recovered with GST-14.3.3 (1-245) and GST-14.3.3 (139-245) (Fig. 7B, middlepanel), the latter is completely devoid of kinase activity (Fig. 7B, lowerpanel, lanes3, 5, and 7). Nevertheless, whereas coexpression with GST-14.3.3 (1-245) increased Myc-Raf abundance (Fig. 7A, 2nd and 3rdpanels from top) and activity (Fig. 7A, bottompanel) in parallel, the expression of GST-14.3.3 (139-245) had a lesser effect on Myc-Raf expression (Fig. 7A, 2nd and 3rdpanels from top), and neither inhibited nor activated overall Myc-Raf activity (Fig. 7A, bottompanel). It is likely that the high levels of 14.3.3 endogenous to COS (and other cells) prevent the recombinant GST-14.3.3 (139-245) fragment from interfering with the activation of most Raf polypeptides, whereas those that become associated with GST-14.3.3 (139-245) are clearly excluded from the activation process. The inactive Raf polypeptides bound to GST-14.3.3 (139-245) were not activated by the addition of recombinant 14.3.3 (1-245) in vitro (not shown).


DISCUSSION

The 14.3.3 class of 29-33-kDa polypeptides has been found to copurify with a broad array of proteins and has been repeatedly rediscovered as activators (e.g. of tyrosine hydroxylase), inhibitors (e.g. of protein kinase C), or cofactors (exoS) for a number of enzymes in vitro(16) . Several recent reports (19, 20) have described an interaction of Raf with 14.3.3 polypeptides in vitro and in intact yeast similar to that described here. All studies of the Raf 14.3.3 interaction including the present report have employed one or both as recombinant polypeptides, and it is possible that the interaction observed in vitro, or even in situ examining the overexpressed recombinant polypeptides, may not accurately reflect certain aspects of the interaction between the endogenous 14.3.3 and Raf polypeptides in situ. An apparent activation of recombinant human c-Raf-1 kinase in intact yeast occurs concomitant with overexpression of recombinant mammalian 14.3.3. Furthermore, disruption of the gene encoding the Saccharomyces cerevisae 14.3.3 homolog, Bmh1, abrogates the ability of recombinant c-Raf-1 to rescue a Ste11-deficient strain containing a Raf-responsive mutant Ste7. Fantl et al.(21) found that microinjection or overexpression of 14.3.3 in Xenopus oocytes led to an increase in the activity of endogenous or recombinant Raf-1. Li et al.(22) reported that transient expression of 14.3.3 in NIH 3T3 cells had little effect on the activity of cotransfected reporters known to be responsive to active Raf-1; however, the ability of transiently expressed Raf-1 or BXB Raf-1 to activate these reporters was substantially augmented by cotransfection with 14.3.3. We find that although overexpression of 14.3.3 in cultured mammalian cells (COS or 293) has no effect on endogenous c-Raf-1 abundance or activity, measured directly or by the activity of a cotransfected Erk-1 reporter (not shown), coexpression of 14.3.3 with recombinant Myc-Raf results in greater Raf kinase activity (Fig. 7), much as found by Li et al.(22) Significantly however, we find that this increase in Raf kinase activity is largely or entirely attributable to a 14.3.3-induced increase in the expression and abundance of recombinant Raf rather than an increase in Raf-1 specific activity. It seems probable that the ability of recombinant mammalian or endogenous yeast 14.3.3 to enhance the activity of mammalian Raf-1 when the latter is expressed in the heterologous milieu of S. cerevisae or Xenopus oocytes may be attributable, in part or in whole, to an ability of 14.3.3 to enhance Raf-1 polypeptide abundance, perhaps e.g. by stabilizing the recombinant Raf-1 polypeptide.

Several observations nevertheless suggest that 14.3.3 may participate more directly in Raf-1 activation. Irie et al.(20) observed that addition of a maltose binding protein-14.3.3 fusion protein directly to mammalian c-Raf-1, immunoprecipitated from yeast extracts, gave a 3-4-fold increase in MEK kinase activity. Takai and colleagues (23) have purified a MEK activator (REKS) from Xenopus oocyte cytosol that is activated in vitro approximately 2-fold by direct addition of GTP Ras and about 1.3-fold by 14.3.3 protein; addition of 14.3.3 in the presence of maximal GTP Ras gives a further 2-fold activation in REKS activity. Li et al.(22) observed that although recombinant 14.3.3 and Ras added directly to inactive baculoviral Raf do not alter Raf kinase activity, the further addition of a crude cellular extract results in a Ras-14.3.3-dependent, 2-3-fold activation of Raf-1 kinase. The biochemical mechanisms that underlie in vitro ``activation'' of Raf by 14.3.3 are not known, and the extent to which they reflect the ability of 14.3.3 to ``stabilize'' the Raf polypeptide in vitro, comparable to the effects that underlie the enhanced expression seen on cotransfection, is also not known. The present results, however, provide one persuasive piece of evidence that 14.3.3 participates in Raf activation beyond its ability to bind to and ``stabilize'' the Raf polypeptide. Carboxyl-terminal fragments of 14.3.3, which bind Raf nearly as well in situ as full-length 14.3.3 and which also provide some enhanced Raf expression in situ, are nevertheless recovered from cells in association only with catalytically inactive Raf polypeptides, whereas full-length 14.3.3 is recovered with catalytically active Raf kinase. This result suggests that the Raf-binding 14.3.3 fragments have lost a function critical to the activation of Raf. This function does not appear to be their ability to dimerize, inasmuch as the Raf polypeptides associated with GST-14.3.3 (1-180), which dimerizes normally (Fig. 5B, lane5), are not catalytically active (Fig. 5C, lane5). We suggest that full-length 14.3.3 contributes to Raf activation either by recruiting an as yet unidentified polypeptide, by providing an intrinsic catalytic function, or both. The present data show that in addition to its ability to bind Raf concomitantly with Ras, the recombinant 14.3.3 polypeptide binds phospholipid and cleaves the sn-2 acyl bond. Whether the acyl transferase function of 14.3.3 is contributory to its role in the regulation of Raf function is not known. Thus, 14.3.3 polypeptides participate in the regulation of Raf activity; however, their specific biochemical function in Raf activation remains to be elucidated.

Subsequent to the completion of these studies, the structures of 14.3.3 (24) and (25) crystals were reported. Both 14.3.3 isoforms exhibited dimeric structures; each monomer was composed of nine helical segments arranged in antiparallel arrays. The dimer interface is created by highly conserved, primarily hydrophobic residues from the four amino-terminal helices, suggesting that 14.3.3 heterodimers will form readily. The carboxyl-terminal five helices of each monomer (which provide the Raf binding domain as demonstrated in the present report) are folded so as to provide within the dimer a cavity whose internal face is composed of (primarily hydrophilic) residues that are highly conserved in all 14.3.3 isoforms and whose external surface is provided by nonconserved residues. Future studies will determine the contributions of specific residues on each of these surfaces to the interactions of 14.3.3 with its polypeptide partners and phospholipids.


FOOTNOTES

*
This work was supported by an award from the American Cancer Society. 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.

§
Supported by a National Research Service Award from NCI, National Institutes of Health.

To whom all correspondence should be addressed: Diabetes Research Laboratory, Massachusetts General Hospital, MGH-East, 149 13th St., Charlestown, MA 02129. Tel.: 617-726-6909; Fax: 617-726-5649.

(^1)
The abbreviations used are: GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin; TPA, 12-O-tetradecanoylphorbol-13-acetate; EGF, epidermal growth factor.


ACKNOWLEDGEMENTS

We thank K. L. Guan for the gift of human MEK-1 in pGEX-kg. We thank J. Bonventre and D. K. Kim for reagents and helpful discussion. We thank K. Andrabi and M. Kozlowski for gifts of monoclonal antibodies 9B7.3 and 12 CA5 and J. Prendable for preparation of the manuscript.


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