©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The MEK Kinase Activity of the Catalytic Domain of RAF-1 Is Regulated Independently of Ras Binding in T Cells (*)

(Received for publication, September 27, 1994; and in revised form, December 1, 1994)

Charles E. Whitehurst (1) (2) Hajime Owaki (1) Joseph T. Bruder (3) Ulf R. Rapp (3) Thomas D. Geppert (1)(§)

From the  (1)Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75235-8884, the (2)Immunology Graduate Program, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9054, and the (3)Laboratory of Viral Carcinogenesis, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Maryland 21702-1201

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Deletion of the amino-terminal domain of Raf-1, which contains the Ras-binding region, results in the constitutive activation of the liberated Raf-1 catalytic domain in fibroblast cell lines. We demonstrate that the MEK kinase activity of the isolated Raf-1 catalytic domain, Raf-BXB, is not constitutively active, but is regulated in Jurkat T cells. Raf-BXB is activated by engaging the antigen receptor-CD3 complex, or treating cells with phorbol myristate acetate or okadaic acid. Increasing intracellular cAMP inhibits Raf-1 activation stimulated by phorbol myristate acetate, but not the activation of Raf-BXB. Serine 621, but not serine 499, is essential for Raf-BXB MEK kinase activity. Because Raf-BXB does not bind Ras, the data establishes a Ras-independent signal in directly regulating the activity of the Raf-1 catalytic domain.


INTRODUCTION

Raf-1, the product of the c-raf-1 proto-oncogene, is a serine/threonine protein kinase activated by numerous growth factors and cytokines(1, 2, 3, 4, 5, 6, 7, 8) . The stimulation of Raf-1 activity depends partly on the activation of the small G-protein Ras(6, 7, 8, 9, 10, 11, 12, 13) . When activated, Raf-1 phosphorylates and activates the microtubule-associated protein kinase kinases, MEK1 and MEK2(14, 15, 16, 17, 18, 19) . These enzymes in turn phosphorylate and activate the microtubule-associated protein kinases, ERK1 and ERK2(20, 21, 22, 23) . ERKs phosphorylate and regulate the activity of several cytosolic and nuclear proteins(24, 25, 26, 27, 28, 29, 30, 31) . Therefore, Raf-1 is pivotal in transmitting signals from Ras in the plasma membrane to cytosolic and nuclear compartments of the cell.

Raf-1 is composed of two functionally distinct domains, an NH(2)-terminal regulatory domain and a COOH-terminal kinase domain(32, 33, 34, 35, 36, 37) . Active GTP-Ras binds the NH(2)-terminal domain of Raf-1 in a region spanning amino acids 50 to 135(38, 39, 40, 41, 42, 43, 44) . This binding alone does not increase the kinase activity of Raf-1 in vitro(12) , but rather, may function to recruit Raf-1 to the plasma membrane, which is sufficient to cause its activation(12, 13) . However, membrane-anchored Raf-1 is further activated by a Ras-independent signal(13) , and in co-transfection experiments, additional signals besides v-ras are necessary to fully activate Raf-1(6, 45) . Therefore, these findings suggest that additional Ras-independent signals are required for the full activation of Raf-1.

Besides binding GTP-Ras, the NH(2)-terminal domain of Raf-1 has also been proposed to repress the phosphotransferase activity of the catalytic domain(33, 46) . For example, replacement of the NH(2)-terminal domain with a viral protein, as occurred in v-raf(47) , or its deletion, as represented by Raf-BXB(35) , is sufficient to cause constitutive activation of the Raf-1 catalytic domain in fibroblast cell lines. Expression of v-raf or Raf-BXB stimulates cell transformation(34, 36, 48) , the transactivation of AP1 regulatory elements(35, 49) , and the activation of MEKs and ERKs(16, 17, 48) . Therefore, based on these observations and the above Ras-binding studies, two separable signaling events may be necessary to activate Raf-1. Both events involve interactions with the NH(2)-terminal regulatory domain, one involving GTP-Ras binding, and the other involving modulation of the inhibitory effect exerted on the catalytic domain.

In this report, we demonstrate that the MEK kinase activity of the isolated Raf-1 catalytic domain, Raf-BXB, is not constitutively active, but is regulated in Jurkat T cells. Because the defined Ras-binding region is deleted from Raf-BXB, these findings establish that the catalytic domain of Raf-1 is regulated independently of Ras binding in these cells.


EXPERIMENTAL PROCEDURES

Reagents and Monoclonal Antibodies

Okadaic acid and calyculin A (LC Service Corp., Woburn, MA) were dissolved in Me(2)SO and added to cells at the indicated concentrations. 4beta-Phorbol 12-myristate 13-acetate (PMA) (^1)and forskolin (Sigma) were dissolved in ethanol and added to cells as indicated. 8-Bromoadenosine 3`:5`-cyclic monophosphate (Sigma) was freshly dissolved in media before each experiment. The monoclonal antibodies (mAbs) used were 12CA5 (Berkeley Antibody Co., Richmond, CA), a murine IgG2b mAb that reacts with the hemagglutinin antigenic peptide YPYDVPDYA; and 3C10 (American Type Culture Collection, ATCC), a murine IgG2b mAb to human CD14. Rabbit polyclonal antiserum raised to ERK2 (A249), MEK1 (A2227), and MEK2 (A2228), and histidine-tagged recombinant murine MEK1 (rMEK1), and purified phosphatase 2A catalytic subunit were all generously provided by Dr. Melanie Cobb(50) . The rMEK1 bacterial expression vector was kindly provided by Dr. Gary Johnson. Purification of recombinant histidine-tagged human K71R mutated ERK1 (rERK1) was described previously(51) .

Generation of Raf-BXB Constructs and Cell Lines

The Raf-1 derived construct Raf-BXB has been previously described(35) . This construct was digested from a Rous sarcoma virus-driven expression vector and ligated into pBluescript KS(-) (Stratagene) using XhoI and XbaI restriction sites. This was then digested with HindIII and NotI enzymes and ligated into the metallothionine-driven expression vector pMEP (Invitrogen). pMEP is an episomal replicon vector containing the hygromycin resistance gene driven by the thymidine kinase promoter. Raf-HABXB is an epitope-tagged version of Raf-BXB with the hemagglutinin antigen (HA) at the amino terminus: MAYPYDVPDYAS. Raf-1 mutants containing serine 621 mutated to aspartate (S621D) and serine 499 mutated to alanine (S499A) were constructed by site-directed mutagenesis, and then subcloned into the Raf-HABXB construct and dideoxynucleotide sequenced to confirm mutation and sequence integrity. Jurkat cell lines expressing the control vector (pMEP), Raf-BXB, and Raf-HABXB were generated as described previously (52) . Before use, cells were cultured for 24-48 h in 7.5 µM ZnSO(4) and 0.75 µM CdSO(4), washed with 0.9% NaCl, resuspended in RPMI 1640 medium, and then equilibrated at 37 °C for 15 min before being stimulated. The ZnSO(4)/CdSO(4) concentrations utilized do not alter the activation of the microtubule-associated protein kinase pathway in Jurkat T cells or the observed regulation of Raf-BXB MEK kinase activity (data not shown).

In Vitro Protein Kinase Assays

To measure Raf-1, Raf-BXB, and Raf-HABXB activity, cells were lysed in RIPA-H buffer (20 mM Tris (pH 8.0), 1.0% Nonidet P-40, 0.5% deoxycholic acid, 0.1% SDS, 150 mM NaCl, 3 mM MgCl(2), 6 mM EGTA, 1 mM dithiothreitol, 20 mMp-nitrophenyl phosphate, 50 mM NaF, 50 µM Na(3)VO(4), 10 µg/ml aprotinin, 1 mM leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 5 mM benzamidine), clarified by centrifugation (15,000 times g; 10 min), and equivalent aliquots were then incubated with 4 µl of antiserum or 10 µg of purified monoclonal antibody. Immune complexes formed using Staphylococcal protein A-agarose were washed twice with RIPA-H buffer, twice with 0.5 M LiCl, 100 mM Tris (pH 7.6), and twice with 10 mM PIPES (pH 7), 100 mM NaCl, 10 µg/ml aprotinin, and then incubated for 30 min at 30 °C in reaction buffer (20 mM PIPES (pH 7), 10 mM MgCl(2), 10 mM MnCl(2), 50 µM cold ATP, or [-P]ATP (8 cpm/fmol), 20 µg/ml aprotinin, 40 µg/ml rMEK1). To measure the activation of the rMEK1 substrate by immunoprecipitated Raf-HABXB, 7 µl of the supernatant was transferred to a 25-µl reaction (10 mM HEPES (pH 7.5), 10 mM MgCl(2), 50 µM [-P]ATP (8-16 cpm/fmol), 1 mM dithiothreitol, 1 mM EGTA, 1 mM benzamidine, 25 µg/ml rERK1) for 30 min at 30 °C. To measure MEK1 and MEK2 activity, immunoprecipitates were incubated for 30 min at 30 °C in a reaction buffer containing 20 mM HEPES (pH 7.5), 10 mM MgCl(2), 1 mM dithiothreitol, 1 mM benzamidine, 1 mM EGTA, 25 µg/ml rERK1, and 50 µM [-P]ATP (5 cpm/fmol). ERK2 activity was assessed as described(50) . All of the above reactions were terminated by boiling in gel loading buffer (30 mM Tris (pH 6.8), 1% SDS, 2.5% beta-mercaptoethanol, 0.5 mg/ml bromphenol blue, 5% glycerol) and samples were analyzed by 12% SDS-PAGE, Coomassie Blue staining, autoradiography, and liquid scintillation counting of excised substrates. To measure cAMP-dependent protein kinase, 5 µl of cell extract was mixed in a 20-µl reaction (20 mM Tris (pH 7.5), 50 µM Kemptide, 100 µM ATP (2 cpm/fmol), 250 µM EGTA, 10 mM MgCl(2)) and incubated for 20 min at 30 °C. Samples were spotted onto p81 Whatman filters, washed in 75 mM H(3)PO(4), and the filters subjected to liquid scintillation counting.

Immunoblotting

Proteins were immunoprecipitated, eluted from the immune complexes, electrophoresed as described above, and subjected to immunoblotting using antiserum SP63 raised to the COOH terminus of Raf-1(52, 53) . Reactive proteins were detected as described(50) .


RESULTS

The MEK Kinase Activity of Raf-BXB Is Regulated in Jurkat T Cells

The isolated Raf-1 catalytic domain, Raf-BXB, was generated by an in-frame deletion of amino acids 26-302 of Raf-1(35) . Raf-BXB was expressed under the control of the metallothionine promoter in a permanently transfected Jurkat T cell line(52) . The MEK1 kinase activities of Raf-1 and Raf-BXB from this cell line were measured by an immune complex kinase assay. Endogenous p72 Raf-1 and Raf-BXB were both effectively immunoprecipitated (Fig. 1, lower panel). The basal activity of Raf-1 and Raf-BXB combined was not elevated over that of Raf-1 from control cells, suggesting Raf-BXB was inactive in these cells (Fig. 1, upper panel). Previous studies in T cells have demonstrated that phorbol esters stimulate Raf-1 autokinase activity and the phosphorylation of an exogenous substrate(4, 54) . PMA also stimulated the MEK kinase activity of Raf-1. Interestingly, markedly greater activity was detected from Raf-BXB-expressing cells stimulated with PMA, indicating a substantial contribution of activity from Raf-BXB. As detected by immunoblotting, a fraction of Raf-BXB underwent an SDS-PAGE mobility shift, typical of post-translation modification (Fig. 1, lower panel). Okadaic acid, a cell-permeable inhibitor of protein phosphatase 1 and phosphatase 2A(55) , did not stimulate Raf-1 activity in control cells; however, activity was detected from the immune complexes containing Raf-BXB, suggesting that the activity of Raf-BXB was increased (Fig. 1). Okadaic acid also stimulated an SDS-PAGE mobility shift of Raf-BXB detectable by immunoblotting (Fig. 1, lower panel).


Figure 1: MEK kinase activity of Raf-1 and Raf-BXB measured from Jurkat T cells. The MEK kinase activity of Raf-1/Raf-BXB (upper panel) from control (pMEP) and Raf-BXB cells stimulated with 10 ng/ml PMA, 20 µM okadaic acid (OA), or 0.1% Me(2)SO carrier (CONT) for 15 min was measured by an in vitro kinase assay as described under ``Experimental Procedures.'' NIH 3T3 lysate was a positive control. Autophosphorylation by rMEK1 was measured by incubating rMEK1 alone in reaction buffer (rMEK1 CONT). Samples were immunoblotted with anti-Raf-1 antiserum SP63 (lower panel).



To measure Raf-BXB activity exclusively, an expression vector encoding an epitope-tagged version of Raf-BXB (hemagglutinin antigen; Raf-HABXB) was constructed and stably transfected into Jurkat T cells. Raf-HABXB activity was examined using a monoclonal antibody specific for the epitope tag (12CA5; alphaHA). MEK1 kinase activity was only immunoprecipitated from Raf-HABXB cells; no activity was found in immunoprecipitates from control cells. The activity of Raf-BXB was stimulated by PMA and okadaic acid (Fig. 2A). The recombinant MEK1 substrate phosphorylated by Raf-HABXB became activated, in turn phosphorylating exogenously added recombinant ERK1 (Fig. 2A). Although MEK1 has been shown to form a stable complex with the kinase domain of Raf-1 (56) , the ERK kinase activity detected in these assays was not from co-immunoprecipitated endogenous MEKs, since the addition of exogenous recombinant MEK1 to the immune complexes was essential to detect any activity in our assay system (Fig. 2B).


Figure 2: MEK kinase activity of epitope-tagged Raf-BXB. A, Raf-HABXB MEK kinase activity was measured from stable transfectants stimulated with PMA, okadaic acid (OA), or carrier as described in the legend to Fig. 1by an in vitro kinase assay as described under ``Experimental Procedures.'' Supernatant from the initial reaction was transferred to a new reaction to measure recombinant MEK1 (rMEK1)-catalyzed recombinant ERK1 (rERK1) phosphorylation. Autophosphorylation (AUTO) was measured by incubating rMEK1 alone, and then adding rERK1 substrate. B, assay demonstrating that exogenously added rMEK1 is essential for measuring the coupled ERK kinase activity. After cells were stimulated with 10 ng/ml PMA and 10 µM okadaic acid for 15 min, Raf-HABXB activity was measured as described in A, except with and without the addition of rMEK1.



We next examined the consequence of okadaic acid and PMA together on the activity of Raf-1 and Raf-BXB. Interestingly, the combination of okadaic acid and PMA stimulated less Raf-1 activity than did PMA alone (Fig. 3, left). In contrast, okadaic acid and PMA together synergistically stimulated Raf-HABXB activity (Fig. 3, right). Therefore, the activation of intact Raf-1, but not Raf-BXB, was inhibited by okadaic acid.


Figure 3: Inhibition of Raf-1 activation and augmentation of Raf-BXB activation by okadaic acid. Raf-1 activity measured from Jurkat T cells stimulated with 0.1% Me(2)SO carrier (CONT), 10 ng/ml PMA, 20 µM okadaic acid, or the combination for 10 min (left). In a separate experiment, Raf-HABXB activity was measured from stable transfected Jurkat T cells stimulated for 15 min as indicated (right). Raf-1 and Raf-HABXB activities were measured by the activation of rMEK1 catalytic activity toward rERK1 substrate as described under ``Experimental Procedures.''



Engagement of the T cell antigen receptor-CD3 complex with a monoclonal antibody mimics the normal physiological stimulus, and has been demonstrated to rapidly and transiently stimulate Raf-1 activity in peripheral blood T cells(54) . Therefore, we examined the effect of this stimulus on Raf-HABXB activity. Raf-HABXB was activated within 30 s of engaging the CD3 complex, and activation was maximal at 2 min (Fig. 4A, upper panel). After 2 min the activity subsided, approaching unstimulated activity by 15 min (Fig. 4B, upper panel). The addition of PMA with anti-CD3 for 2 min even further activated Raf-HABXB (Fig. 4C, left upper panel). As with Raf-1(4) , treating cells with a Ca ionophore alone did not stimulate Raf-HABXB activity, or significantly influence Raf-HABXB activity stimulated by PMA (Fig. 4C, right upper panel). These observed changes in Raf-HABXB activity were not due to differences in the levels of Raf-HABXB immunoprecipitated as uniformity of the immunoprecipitations was confirmed by immunoblotting (Fig. 4, lower panels).


Figure 4: Raf-BXB activity stimulated by engagement of the antigen receptor-CD3 complex. A, early kinetics of Raf-HABXB activation. Stable Raf-HABXB expressing Jurkat T cells were incubated with 1 µg/ml anti-CD3 monoclonal antibody for the time periods indicated and Raf-HABXB activity was measured as in Fig. 2(upper panel). Samples were immunoblotted with anti-Raf-1 serum SP63 (lower panel). B, late kinetics of Raf-HABXB activation. Cells were stimulated and activity measured as in A. C, additive Raf-HABXB stimulation by anti-CD3 and PMA, but not ionomycin and PMA. Stable Raf-HABXB expressing Jurkat cells were stimulated with 1 µg/ml anti-CD3 mAb and/or 10 ng/ml PMA for 2 min (left panel), and with 1 µM ionomycin and/or 10 ng/ml PMA for 15 min (right panel). Activity was measured as in A.



Effect of Raf-BXB Expression on the Microtubule-associated Protein Kinase Pathway

The above data demonstrated that Raf-BXB was only active in stimulated Jurkat T cells. Therefore, we examined the activity of endogenous MEK1, MEK2, and ERK2 in cells expressing Raf-BXB. As shown in Fig. 5, basal MEK1 (left panel) and MEK2 (right panel) activities were essentially the same in control and Raf-HABXB-expressing cells. In cells stimulated with an anti-CD3 monoclonal antibody or PMA, greater MEK1 and MEK2 activities were evident in the Raf-HABXB cells. Okadaic acid and calyculin A treatment also markedly enhanced MEK1 and MEK2 activities in Raf-HABXB cells. Basal and PMA-stimulated ERK2 activities were approximately the same in control and Raf-BXB cells, but okadaic acid caused a greater increase of ERK2 activity in Raf-BXB cells (Fig. 6A). Anti-CD3 stimulated ERK2 activity was also greater in Raf-BXB cells, but only at the time of maximal activation (Fig. 6B). These findings are consistent with the changes noted in Raf-BXB MEK kinase activity in Jurkat T cells and demonstrated that active Raf-BXB does couple to MEK1, MEK2, and ERK2 in these cells.


Figure 5: Augmentation of MEK1 and MEK2 activation by Raf-BXB expression. Effect of Raf-HABXB expression on MEK1 and MEK2 activation. The kinase activity of MEK1 and MEK2 was measured as described under ``Experimental Procedures'' from control (pMEP) and Raf-HABXB-expressing Jurkat T cells stimulated with 1 µg/ml anti-CD3 monoclonal antibody for 5 min, or 10 ng/ml PMA, 20 µM okadaic acid (OA), or 1 µM calyculin A (CLA) for 15 min.




Figure 6: Effect of Raf-BXB expression on ERK2 activation. A, the kinase activity of ERK2 was measured as described under ``Experimental Procedures'' from control (pMEP) and Raf-BXB expressing Jurkat T cells stimulated with 10 ng/ml PMA or 20 µM okadaic acid for 15 min. B, effect of Raf-BXB expression on anti-CD3 stimulated ERK2 activation. ERK2 activity was compared in control (pMEP) and Raf-BXB expressing Jurkat T cells stimulated with 1 µg/ml of a control or anti-CD3 monoclonal antibody for various time periods.



Raf-BXB Is Not Sensitive to the Inhibitory Effect of Increased Intracellular Adenosine 3`,5`-Monophosphate

Increasing cAMP levels in certain cell types inhibits Raf-1 activation stimulated by phorbol esters and growth factors(57, 58, 59, 60) . This blockade may result from a PKA-mediated phosphorylation of serine 43, located in the NH(2)-terminal regulatory domain of Raf-1(39, 57) . Because Raf-BXB lacks the entire NH(2)-terminal regulatory domain, we compared the effects of increased intracellular cAMP on the ability of PMA to increase Raf-1 and Raf-BXB activities. Pretreatment of Jurkat T cells with 8-bromoadenosine 3`:5`-cyclic monophosphate (8-Br-cAMP) or forskolin inhibited PMA-induced Raf-1 activation by approximately 50% (Fig. 7A, left panel). MEK1 activity measured from the same PMA-stimulated cells was similarly reduced (Fig. 7A, right panel). In contrast, these cAMP elevating agents did not significantly reduce Raf-HABXB activity stimulated by PMA (Fig. 7B). Therefore, unlike Raf-1, Raf-BXB was relatively insensitive to the inhibitory effect of increased cAMP.


Figure 7: Inhibition of Raf-1, but not Raf-BXB activation by increased cAMP. A, effect on Raf-1 and MEK1 activation by PMA. Jurkat T cells were preincubated for 15 min in medium alone, 2 mM (8-Br-cAMP), or 100 µM forskolin and an aliquot of cells was removed to measure cAMP-dependent protein kinase activity. The cells were then stimulated with 10 ng/ml PMA for an additional 15 min. Raf-1, MEK1, and cAMP-dependent protein kinase activities were measured from the same cells as described under ``Experimental Procedures.'' cAMP-dependent protein kinase activities (pmol/min) were: CONT, 17.6; 8-Br-cAMP, 55.7; forskolin (FORSK), 45.1. B, effect on Raf-HABXB. Stable Raf-HABXB expressing Jurkat T cells were preincubated with 2 mM 8-Br-cAMP or 100 µM forskolin and then stimulated with PMA as described above. cAMP-dependent protein kinase activities (pmol/min) were: Experiment 1, CONT, 0.1; 8-Br-cAMP, 3.9. Experiment 2, cont, 2.6; forskolin (FORSK), 10.4.



Serine 621 Is Essential for Raf-BXB Activity

In cells stimulated with PMA an increase in the serine phosphorylation of Raf-BXB was observed (data not shown). However, when immunoprecipitated Raf-BXB was extensively treated with high concentrations of phosphatase 1 (20 units/ml) or phosphatase 2A (30 µg/ml), only a portion of the serine phosphorylation was removed and Raf-BXB activity was uneffected (data not shown). Therefore, the roles of 2 serine residues that have been implicated as potential regulatory sites on the Raf-1 catalytic domain were specifically examined by site-directed mutagenesis. Constitutively active Raf-BXB expressed in NIH 3T3 cells is constitutively phosphorylated on serine 621, and mutation of serine 621 to alanine leads to the complete inactivation of Raf-1(64) . Serine 499 was mapped as a regulatory site on Raf-1 phosphorylated by protein kinase Calpha(65) . The mutation of serine 621 to aspartate (S621D) abolished Raf-HABXB MEK kinase activity (Fig. 8), whereas mutation of serine 499 to alanine did not effect its activation by PMA and okadaic acid (Fig. 8).


Figure 8: Effect of mutating serine 621 and serine 499 on Raf-HABXB activity. Cells transiently transfected with vectors encoding wild type (WT), S499A, and S621D variants of Raf-HABXB were cultured for 20 h and then stimulated and assayed for MEK kinase activity as described in the legend to Fig. 2A.




DISCUSSION

Previous studies have demonstrated that Raf-BXB is constitutively active when expressed in fibroblast cell lines(17, 35, 48, 61) . In this report, we have demonstrated that, in contrast to its behavior in fibroblasts, Raf-BXB is not constitutively active when expressed in Jurkat T cells. Our data clearly establishes that in T cells, removal of the NH(2)-terminal regulatory domain of Raf-1 is insufficient to activate the catalytic domain. One or more additional signals, provided by PMA, okadaic acid, or anti-CD3, directed to the Raf-1 catalytic domain are required for its activation. Interestingly, engagement of the antigen receptor-CD3 complex stimulated an extremely rapid and transient activation of Raf-BXB that resembled the time course of activation of full-length Raf-1(54) . This suggests a close coupling of this signal to the antigen receptor-CD3 complex. It is unlikely that Ras provides this signal because the defined Ras-binding region is deleted from Raf-BXB, the kinase domain of Raf-1 itself does not associate with Ras(38, 39, 40, 42, 48) , and the activating signal is resistant to the effect of increased cAMP, which inhibits the binding of Raf-1 to Ras-GTP(39, 57, 58, 59, 60) . Therefore, based on these observations, we conclude that the activity of the Raf-1 catalytic domain is regulated independently of Ras binding, by one or more signals acting directly on the catalytic domain.

In light of the above observations, we propose that Raf-1 may require two or more separable signaling events for activation. At least one signal directly regulates the MEK kinase activity of the catalytic domain of Raf-1. First, by binding Ras-GTP (event 1), Raf-1 is likely translocated to the inner surface of the plasma membrane(12, 13, 38, 62) . In this environment, it may receive a Ras-independent signal that modulates the repression exerted by the NH(2)-terminal regulatory domain (event 2). Lastly, the importance of an additional signal or signals that directly activate the Raf-1 catalytic domain is implicated by our findings in Jurkat T cells (event 3). The last two events may be coupled and initiated by one signal acting on the catalytic domain. Therefore, in NIH 3T3 fibroblasts, this model predicts that the last signal is constitutively activated, explaining why Raf-BXB is active and transforming in these cells(17, 35, 48) .

The nature and source of the signal regulating the MEK kinase activity of Raf-BXB is unknown. A definitive role for serine phosphorylation in regulating the activity Raf-BXB is currently not evident. Our data excludes the importance of serine 499 as a regulatory site, but suggests that serine 621 is essential for Raf-BXB kinase activity. Whether the phosphorylation of serine 621 regulates the MEK kinase activity of Raf-BXB is unclear.

Interestingly, okadaic acid inhibits Raf-1 activation while activating Raf-BXB. Increased cAMP also inhibits the activation of Raf-1, but not Raf-BXB. The resistance of Raf-BXB to okadaic acid and cAMP antagonism suggests that the inhibition of Raf-1 is mediated entirely through the NH(2)-terminal regulatory domain. A precedent for such a mechanism exists, wherein cAMP-dependent protein kinase phosphorylates Raf-1 on serine 43 and inhibits its association with Ras-GTP(39, 57, 58, 59, 60) . Okadaic acid may inhibit Raf-1 activation by a similar mechanism in that okadaic acid does stimulate abundant serine and threonine phosphorylation of Raf-1 and Raf-BXB. (^2)

Finally, our data reveals a correlation between the activation of Raf-BXB in Jurkat T cells and it effects on interleukin-2 production. Raf-BXB expression enhances interleukin-2 production only in cells stimulated by engaging the antigen receptor-CD3 complex or treated with phorbol esters(52) . However, considering the modest effect Raf-BXB expression has on stimulated MEK1, MEK2, and ERK2 activities in these cells, it seems plausible that Raf-1 activity may trigger alternative biochemical events that promote interleukin-2 production, such as the activation of NFkB(63) , or other parallel kinase pathways that can regulate the interleukin-2 promoter(64) .


FOOTNOTES

*
This work was supported by the American Cancer Society, the American Heart Association, a National Institutes of Health Cancer Immunology Training Grant, and the Texas Department Ladies Auxiliary Veterans of Foreign Wars. 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.

§
To whom correspondence should be addressed. Tel.: 214-648-8351; Fax: 214-648-7995.

(^1)
The abbreviations used are: PMA, phorbol myristate acetate; mAb, monoclonal antibody; HA, hemagglutinin antigen; PAGE, polyacrylamide gel electrophoresis; PIPES, 1,4-piperazinediethanesulfonic acid.

(^2)
C. E. Whitehurst, H. Owaki, J. T. Bruder, U. R. Rapp, and T. D. Geppert, unpublished observation.


ACKNOWLEDGEMENTS

We thank Dr. Melanie Cobb for comments on the manuscript and for generously providing antisera, recombinant MEK1, and various other reagents from her laboratory.


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