©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Biochemical Analysis of MEK Activation in NIH3T3 Fibroblasts
IDENTIFICATION OF B-Raf AND OTHER ACTIVATORS (*)

(Received for publication, December 16, 1994; and in revised form, January 31, 1995)

Christoph W. M. Reuter (§) Andrew D. Catling Tomas Jelinek Michael J. Weber (¶)

From the Department of Microbiology and Cancer Center, University of Virginia Health Sciences Center, School of Medicine, Charlottesville, Virginia 22908

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Numerous potential activators of MEK have been identified, including c-Raf-1, B-Raf, c-Mos, and a family of MEK kinases. However, little information gives insight into the activators actually utilized in vivo. To address this, we have used column chromatography and a coupled MEK activation assay to identify in NIH3T3 cells, two major MEK activators, and a third insulin-specific activator. The first MEK activator has an apparent M(r) of 40,000-50,000, was immunologically distinct from A-Raf, B-Raf, c-Raf-1, c-MEKK, c-Mos, MEK1, and MEK2, and was rapidly activated by serum, platelet-derived growth factor (PDGF), insulin, thrombin, and phorbol ester. The second MEK activator was identified as B-Raf. Activation of 93-95 kDa B-Raf was observed in column fractions and B-Raf immunoprecipitates from cytosolic and particulate fractions after stimulation with serum or PDGF, but not insulin. c-Raf-1 from cytosol did not exhibit MEK activator activity; however, c-Raf-1 immunoprecipitates from the particulate fraction revealed MEK activator activity that was enhanced after stimulation with PDGF or phorbol ester, but not serum or insulin. Both c-Mos and c-MEKK were present in NIH3T3 fibroblasts but did not show MEK activator activity. These data provide direct evidence that 93-95-kDa B-Raf isozymes and an unidentified 40-50-kDa MEK activator are major agonist-specific MEK activators in NIH3T3 fibroblasts.


INTRODUCTION

The MAP kinase (^1)pathway is activated by diverse agonists that stimulate cell division, differentiation, and secretion(1) . MAP kinase activation requires phosphorylation on both tyrosine and threonine residues(2, 3, 4) , reactions that are catalyzed by dual-specificity kinases named MEKs (MAP or ERK kinases). MEKs have recently been cloned from mouse, rat, human, and Xenopus(5, 6, 7, 8, 9) and two different MEK cDNAs (MEK1 and MEK2) have been isolated(6, 9, 10) . MEK activity is in turn regulated by serine phosphorylation(9, 11, 12, 13, 14, 15, 16, 17) , a reaction catalyzed by MEK activators, which constitute a diverse group of protein kinases.

A number of candidate MEK-activating kinases have been reported, most notably c-Raf-1. A variety of biochemical(18, 19, 20, 21, 22, 23) , genetic(18, 19, 20, 24, 25, 26, 27, 28, 29, 30) , and regulatory (31, 32) evidence points to the importance of c-Raf-1 as a MEK activator. Recently, B-Raf was identified as a major MEK activator in neuronal tissue(33, 34, 35, 36) . In addition, c-Mos (37) and a MEK kinase related to the yeast MEK activators byr2 and ste11 (MEKK, (38) ) have been shown to be potential MEK activators in Xenopus oocytes and various mouse tissues including brain and fibroblasts, respectively. However, it is unclear which of these MEK activators functions under different physiological circumstances. Presumably, the selection of specific MEK activators provides specificity in a signaling pathway which activates the MAP kinases as a generic, nonspecific response.

c-Raf-1 is a 72-76-kDa cytoplasmic protein serine/threonine kinase which is ubiquitously expressed(39, 40, 41, 42) . Two related cellular raf genes have also been described, A-raf and B-raf(43, 44, 45, 46) . The Raf protein kinase family is characterized by three conserved domains (CR1, CR2, and CR3): (i) the N-terminal CR1 region is rich in cysteine residues and contains a putative zinc binding region, which is important for association with GTP-Ras; (ii) CR2 is also located in the N terminus of the molecule and is rich in serine and threonine residues; (iii) the C-terminal CR3 region contains the kinase domain(47, 48) . A-Raf and B-Raf are highly homologous to c-Raf-1 in the kinase domain (CR3) and CR1(42, 49) . However, a third region of sequence homology (94%) between A-Raf and c-Raf-1 is only 47% conserved in B-Raf(49) , and regions surrounding CR2 are largely isozyme-specific. A-Raf has been reported to be expressed primarily in urogenital tissues and kidney and lowest in brain, whereas B-Raf is reportedly expressed primarily in brain and testes(42) .

The regulation of c-Raf-1 activity is complex and incompletely understood(39, 50, 51, 52, 53) . The c-Raf-1 protein becomes phosphorylated during activation by kinases whose identity is unknown, but which may include protein kinase C isoforms and tyrosine protein kinases(51, 52, 54) . The major in vivo sites of c-Raf-1 phosphorylation are Ser-43, Ser-259, and Ser-621(52) . Coexpression of activated tyrosine kinases and c-Raf-1 in the Baculovirus Sf9 expression system revealed Tyr-340 and Tyr-341 as major tyrosine-phosphorylation sites accompanied by high activity(52) . Phosphorylation of c-Raf-1 on tyrosine residues has been detected in several systems, however, the physiological importance of this phosphorylation is unclear(52) . c-Raf-1 has been demonstrated to bind directly to GTP-Ras in vitro and in yeast two-hybrid systems(55, 56, 57, 58, 59, 60, 61, 62) . Recently, Arg-89 was identified as a critical residue in c-Raf-1 required for Ras/c-Raf-1 interaction(62) . However, the Ras-Raf interaction appears insufficient to activate c-Raf-1 kinase activity(63, 64) . Ras is suggested to function in the activation of c-Raf-1 by recruiting c-Raf-1 to the plasma membrane where a separate, Ras-independent, activation of c-Raf-1 occurs (63, 64, 65) . The activation of c-Raf-1 kinase after or during membrane attachment occurs by an unknown mechanism(63, 64, 65) .

Disruption of the GTP-Rasbulletc-Raf-1 complex correlates with inhibition of the MAP kinase pathway in fibroblasts (66, 67, 68) and smooth muscle cells(69) . Agents that elevate cyclic AMP can inhibit the GTP-Ras/c-Raf-1 interaction and decrease ligand-induced MAP kinase activation(54, 67, 68) . Recent studies suggest that in addition the cyclic AMP-dependent protein kinase A directly phosphorylates the c-Raf-1 kinase domain and inactivates the c-Raf-1 kinase activity(54) . In PC12 cells, however, elevation of cyclic AMP activates the MAP kinase cascade(70) . Furthermore, a single amino acid mutation in c-Raf-1 (Arg-89 to Leu), which disrupted the interaction with Ras in vitro and in the two hybrid system, prevented a dominant-inhibitory effect of kinase defective c-Raf-1 on Ras-mediated signal transduction in Xenopus laevis oocytes(62) .

Although it is widely believed that c-Raf-1 is the major MEK activator, several reports argue that c-Raf-1 may not be involved in MAP kinase activation in at least some circumstances(34, 36, 71, 72, 73, 74) . Additionally, most studies identifying c-Raf-1 as a major MEK activator suffer from one or more of the following weaknesses: (i) the use of partially purified c-Raf-1 and/or MEK utilized in high concentrations in MEK activation assays, where unspecific reactions might occur; (ii) the use of MEK phosphorylation as a read out of c-Raf-1 activation instead of MEK activation, which is problematic because of the variety of kinases which might phosphorylate MEK without necessarily enhancing its kinase activity; (iii) the use of dominant-negative c-Raf-1 to block MAP kinase activation which may be unspecific because it may result in sequestration of Ras, which prevents access of other Ras-dependent effectors; (iv) the use of genetic experiments which establish enzyme sequences, but not proximal partners.

Thus, the methodology used in these earlier studies has precluded the possibility of determining whether unknown MEK activators might exist. To address this issue we used a biochemical approach to identify MEK activators in fibroblasts before and after stimulation with serum or other agonists. We have detected active 93-95-kDa B-Raf in fibroblasts, contrary to prior reports, and have found that it phosphorylates MEK-1, is activated after serum-stimulation, binds Ras-GTP, and its binding is inhibited by pretreatment of cells with forskolin. We suggest that B-Raf fulfills all necessary criteria to be considered an agonist-specific alternative to c-Raf-1, as an effector of Ras in fibroblasts. In addition, two other MEK activators have been identified, which appear to be unrelated to c-Raf-1 and one of which is specifically induced by insulin.


MATERIALS AND METHODS

Cells

Mouse NIH3T3 fibroblasts were from the American Type Culture Collection (Rockville, MD). Recombinant kinase-defective (K52R) ERK2 was purified to homogeneity as described previously(76) . Histidine-tagged MEK1 expression was achieved under the control of a cytomegalovirus promoter in CCL39 fibroblasts (ATCC). Inactive histidine-tagged MEK1 from stable transfectants (CCL39hMEK1) was purified to homogeneity by sequential Ni-chelate (Ni-NTA, Qiagen, Chatsworth, CA) and strong anion exchange (Mono Q, Pharmacia, Uppsala, Sweden) chromatography as recently described(36) . MEK1 expression was verified by immunoblotting and activity measurements.

Partial Purification of MEK1 Activators from NIH3T3 Fibroblasts

10 confluent 150-mm plates of NIH3T3 cells were incubated for 12 h in Dulbecco's modified Eagle's medium containing 0.1% FCS. Five plates were stimulated for 2 min at 37 °C in Dulbecco's modified Eagle's medium containing 10% FCS. 2 ml of ice-cold hypotonic cell lysis buffer A (25 mM Tris/HCl, pH 7.5, 50 mM NaF, 1.5 mM dithiothreitol, 1 mM sodium orthovanadate, 3 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 2 mM EGTA) were added to each plate. After 10 min on ice cells were removed and homogenized in a 1-ml tissue culture homogenizer (Wheaton, Millville, NJ; 25 strokes). Cell extract was clarified by a 20-min centrifugation at 15,000 times g at 4 °C. The supernatants were collected and filtered using 0.2-µm syringe filters (Schleicher & Schuell). 10 ml of filtered cell extract (33 mg of protein) were loaded on a 5 times 50-mm Mono Q ion exchange column (volume 1.0 ml, Pharmacia) which was equilibrated with 20 ml of hypotonic lysis buffer A. The column was washed with 10 ml of buffer A and developed with a 40-ml linear gradient from 0 to 0.5 M NaCl in buffer A at 0.5 ml/min and 40 fractions (1 ml) were collected.

MEK activator was measured by a coupled MEK activation assay in which aliquots (10 µl) of each fraction were incubated either with or without 200 ng of purified inactive MEK1 (20 min at 30 °C) in 40 µl of reaction buffer containing 50 mM Hepes, pH 7.5, 10 mM magnesium acetate, 1 mM dithiothreitol, 100 µM ATP, 1 µM okadaic acid, 1 µg/ml leupeptin. After 20 min, 2 µg of purified recombinant kinase-defective K52R-ERK2 and 10 µCi of [-P]ATP (222 TBq/mmol (6000 Ci/mmol), DuPont) were added in 10 µl of 50 mM Hepes, pH 7.4 10 mM magnesium acetate. The reaction was terminated after an additional 20 min at 30 °C by the addition of 20 µl of 4 times electrophoresis buffer. Samples were incubated at 95 °C for 5 min and analyzed by SDS-PAGE.

Active fractions (No. 15-20) were pooled (6 ml), diluted to 20 ml with buffer A, and applied to a 5 times 50-mm Mono S ion exchange column (volume 1.0 ml, Pharmacia) equilibrated with buffer A at 0.2 ml/min. The column was washed with 10 ml of buffer A and developed with a 10-ml linear gradient from 0 to 0.75 M NaCl in buffer A at 0.2 ml/min. 20 fractions (0.5 ml) were collected and assayed for MEK1 activators using purified histidine-tagged inactive MEK1 as substrate.

For gel filtration, 500 µl of active fractions (No. 5-9 or 15-20) (volume 2.5 and 3.0 ml, 0.5 and 0.4 mg/ml protein concentration, respectively) were concentrated to 250 µl by a 40-min centrifugation at 4,000 times g (4 °C) using a Centricon^R 30 microconcentrator (Amicon Div., WR Grace & Co. Beverly, MA). 200 µl of concentrated Mono Q pools were loaded on a 16 times 240-mm Superose 12 gel filtration column (volume 24 ml, Pharmacia) equilibrated in buffer A supplemented with 0.2 M NaCl, 0.2% Tween 20, and 1 µg/ml leupeptin. The flow rate was 0.2 ml/min and 0.25-ml fractions were collected. The column was calibrated using molecular weight standards from Pharmacia: M(r) 25,000, chymotrypsinogen A; M(r) 43,000, ovalbumin; M(r) 67,000, bovine serum albumin; M(r) 158,000, aldolase; M(r) 232,000, catalase; M(r) 440,000, ferritin; M(r) 669,000, thyroglobulin; M(r) 2,000,000, blue dextran 2000.

Immunoprecipitation of MEK Activators

For immunoprecipitation, cell lysates or column fraction pools were normalized for protein concentration before immunoprecipitation assays. 4 mg of protein A-Sepharose CL-4B (Pharmacia) were swollen in lysis buffer for 15 min at 4 °C. 1 mg of cellular protein was incubated with protein A-Sepharose beads and 10 µl of alpha-c-Raf-1 (C12), alpha-B-Raf (C19), alpha-A-Raf (C10, Santa Cruz Biotechnology, Santa Cruz, CA), alpha-MEKK rabbit peptide polyclonal antibodies (Transduction Laboratories, Lexington, KY), or alpha-c-Mos rabbit peptide polyclonal antibody (alpha-c-Mos[37-55], gift of R. B. Arlinghaus, MD Anderson Cancer Center, University of Texas) in a final volume of 200 µl. Samples were rotated for 2 h at 4 °C. The immunoprecipitates were washed twice with 1 ml of ice-cold lysis buffer and twice with 50 mM Hepes, pH 7.4, 10 mM magnesium acetate. For in vitro MEK activation assay, immobilized complexes were resuspended in 40 µl of reaction buffer containing 50 mM Hepes, pH 7.5, 10 mM magnesium acetate, 1 mM dithiothreitol, 100 µM ATP, 1 µM okadaic acid, 1 µg/ml leupeptin, 200 ng of purified histidine-tagged MEK1, and incubated at 30 °C. After 20 min, 2 µg of purified recombinant kinase-defective K52R-ERK2 and 10 µCi of [-P]ATP (222 TBq/mMol (6000 Ci/mmol), Du Pont, Wilmington, DE) were added in 10 µl of 50 mM Hepes, pH 7.4, 10 mM magnesium acetate. The reaction was terminated after an additional 20 min at 30 °C by the addition of 20 µl of 4 times electrophoresis buffer. Samples were incubated at 95 °C for 5 min and analyzed by SDS-PAGE. Under these conditions the MEK activation assay was linear with time and activator concentration(36) .

Biochemical Determinations

Protein concentration was determined using the Coomassie dye-binding assay according to Bradford (74) (Bio-Rad).

NIH3T3 fibroblasts were washed with ice-cold phosphate-buffered saline. Cells were lysed in 0.7 ml of 0.5 N HCl and protein concentrations were determined. cAMP was measured according to Brooker et al.(75) using an automated radioimmunoassay system.

MAP Kinase, MEK, and Ras-binding Assays

MAP kinase and MEK assays were done as described recently(76) . For binding of B-Raf to Ras, samples were processed essentially as described(56, 61) .


RESULTS

Identification of MEK Activators in NIH3T3 Fibroblasts

To test for MEK activators, we used as a substrate over-expressed MEK purified from quiescent mammalian cells, and assayed its enzymatic activation in a coupled reaction with purified recombinant, kinase-defective ERK2 as the ultimate substrate(36) . This assay is linear with time and activator concentration. Since physiological MEK activators would themselves be activated coincident with or prior to the in vivo activation of MEKs, we began our studies by performing time course experiments in which quiescent cells were stimulated with fetal calf serum for various lengths of time, cell lysates were prepared and separated by Mono Q FPLC, and the fractions were assayed for MEK activity and MEK activators. As expected, serum stimulation of NIH3T3 fibroblasts resulted in rapid activation of MEK (Fig. 1). Two cytosolic MEK activators were identified, one eluting early in the salt gradient, between fractions 5 and 12 (10-80 mM), the other in the middle of the gradient, between fractions 15 and 23 (50-180 mM NaCl) (Fig. 1). The first MEK activator peak appeared rapidly (0.5-2.0 min) after stimulation, whereas the second peak appeared to have a measurable basal activity. The most consistent activations were obtained at 2 min, and this time was used in subsequent analyses.


Figure 1: Identification of MEK activating activity by Mono Q chromatography of extracts of serum-stimulated and untreated NIH3T3 cells. NIH3T3 fibroblasts (10^8) were treated with 10% serum for 0, 0.5, 2, and 10 min at 37 °C. Cell lysates (10 ml) were prepared and chromatographed on a Mono Q column with a 40-ml linear gradient from 0 to 500 mM NaCl, and 1-ml fractions were collected. MEK activator was measured by a coupled assay in which aliquots (10 µl) of alternate fractions were incubated at 30 °C for 20 min either with or without added inactive recombinant MEK (200 ng). Recombinant kinase-defective K52R-ERK2 was added in each sample for an additional 20 min in order to measure MEK activity (see ``Materials and Methods''). MEK activator activity is the difference in K52R-ERK2 phosphorylation, with or without added MEK1. Endogenous MEK (circle) and MEK activator (bullet) activity are given in units/ml (1 unit represents 1 pmol/min incorporation of [P]phosphate into kinase-defective, recombinant K52R-ERK2).



Western Analysis of Mono Q Column Fractions

In order to identify different members of the Raf/MAP kinase pathway, the Mono Q fractions were probed for immunoreactivity by Western blotting with antibodies against MEK1 and -2, B-Raf, c-Raf-1 (Fig. 2), as well as ERK1 and -2, A-Raf, c-Mos, and MEKK (data not shown). Endogenous MEK1 and MEK2 coeluted in a sharp, single peak at 50 mM NaCl corresponding with the MEK activity determined in the coupled kinase assay (Fig. 1). Endogenous ERK1 and ERK2 coeluted in a broad peak starting at 100 mM NaCl. c-Raf-1 was detected in a broad peak starting at fraction 16 (150 mM NaCl). B-Raf was detected in three portions: fractions 6-8, 12-20, and 22-26 (50, 100, and 250 mM NaCl, respectively). Fractions 6-8 contained a single B-Raf band at 95 kDa, fractions 12-20 two bands of 93 and 95 kDa, and fractions 22-26 a single 95-kDa band. In addition, a 65-kDa band was sometimes observed in fractions 22-32. These findings are in agreement with recent observations that B-Raf exists in at least two molecular mass forms, 65-70 and 95-100 kDa, in rat PC12 cells and crude mouse brain extracts(35, 49, 77, 78) . These two forms of B-Raf are thought to be alternatively spliced products of the B-Raf gene(35) . Similar alternatively spliced products of the B-raf gene, expressing a 93.5- and a 95-kDa protein, have previously been found at high levels in neural tissue (78) . Hypotonic, cytosolic lysates, and fractions did not show immunoreactivity with antibodies against c-Mos and MEKK.


Figure 2: Western analysis of Mono Q column fractions from NIH3T3 cell lysates. Proteins of Mono Q fractions obtained from lysates of serum-treated (2 min, upper panel) or untreated (lower panel) NIH3T3 cells were separated by SDS-PAGE on a 10% gel, the proteins were transferred to a nitrocellulose membrane and were probed with antibodies against B-Raf (A), c-Raf-1 (B), MEK-1 (C), or MEK-2 (D). The positions of the protein standards are indicated.



Characterization of the MEK Activator in Peak I

Fractions of maximal MEK1 activation (Mono Q peak I 5-9) were pooled and immune complex MEK activation assays for c-Raf-1, A-Raf, B-Raf, MEKK, and c-Mos were performed using activation of purified histidine-tagged MEK1 as a read-out. As shown in Fig. 3, immune complexes with antibodies against A-Raf, B-Raf, c-Raf-1, c-Mos, or MEKK peak I (fractions 5-9) did not show any MEK activating activity in the coupled immune complex MEK activation assays. The MEK activating activity remained in the supernatants of the immunoprecipitates. In some cases traces of MEK activator were found in B-Raf immunoprecipitates. This accounted for less than 5% of the total MEK activating activity in the supernatants, suggesting that the 95-kDa B-Raf protein, which is present in these fractions (see Fig. 2), does not account for the MEK activator.


Figure 3: MEK activator in Mono Q peak I is not immunoprecipitated with antibodies against A-Raf, B-Raf, c-Raf-1, MEKK, and c-Mos. Mono Q fractions 5-9 from serum-treated (+FCS, 2 min) and untreated (-FCS) NIH3T3 fibroblasts were immunoprecipitated with an anti-A-Raf, anti-B-Raf, anti-c-Raf-1, anti-MEKK, anti-c-Mos antiserum, or nonimmune serum. Immune complexes were assayed for MEK activator activity as described in the legend to Fig. 1. The phosphorylated proteins were analyzed by SDS-PAGE on a 10% gel, transferred to nitrocellulose and autoradiographs were prepared. Lanes: U and S, Mono Q pool I (fractions 5-9) of cell extracts from untreated (U) and serum-stimulated (S) NIH3T3 fibroblasts with (+) and without(-) added recombinant MEK1; 1-12, supernatants (SUP.) of immunoprecipitates (IP): Lanes 1, 2, and 13, A-Raf; 3, 4, and 14, B-Raf; 5, 6, and 15, c-Raf-1; 7, 8, and 16, MEKK; 9, 10, and 17, c-Mos; 11, 12, and 18, non-immune serum; 19, inactive MEK1 plus K52R-ERK2.



In order to further purify and characterize the MEK activating activity in peak I, Mono Q fractions 5-9 were pooled, diluted, and applied to a Mono S column. The column was developed with a linear NaCl gradient. The majority of the MEK activity and the MEK activator of peak I was found in the flow through (data not shown).

To assess the molecular weight of the MEK activator in peak I, the pooled Mono Q fractions 5-9 (Mono Q peak I) from serum-stimulated and unstimulated NIH3T3 fibroblasts were concentrated and chromatographed on a Superose 12 gel filtration column. MEK activating activity from Mono Q peak I eluted in a single peak with an apparent M(r) of 40,000-50,000 together with MEK activity (Fig. 4, left). The peak of MEK activating activity from serum-stimulated NIH3T3 cells was 7-fold higher as compared to the MEK activator peak from unstimulated cells. Superose 12 fractions 50-54 containing MEK activator were pooled and immunoprecipitations were performed using antibodies against A-Raf, B-Raf, c-Raf-1, MEKK, and c-Mos. Immobilized immunocomplexes were assayed for MEK activating activity in a coupled MEK activation assay. MEK activating activity was only found in the supernatants but not in the immunoprecipitates, suggesting again that this MEK activator is distinct from A-Raf, B-Raf, c-Raf-1, MEKK, or c-Mos (data not shown).


Figure 4: Gel filtration chromatography of MEK activator of Mono Q peak I. Mono Q fractions 5-9 (MEK activator peak I) from serum-treated (right) and untreated (left) NIH3T3 cells were pooled, concentrated to a final volume of 300 µl, and then applied (200 µl) to a Superose 12 column, and fractions (0.25 ml) were collected. MEK (circle) and MEK activator (bullet) was measured in a coupled MEK activation assay as described in the legend to Fig. 1. The void volume (V(0)) of the column and the elution positions of protein standards are indicated by arrows.



As this MEK activator migrates with an M(r) close to that of MEK itself, we asked whether MEK could auto-activate. However, immunoprecipitates of activated MEK1 or MEK2 do not activate exogenously added MEK1, and antibodies against MEKs did not clear these fractions of MEK activator activity (data not shown). Thus, we believe that the MEK activator in peak I, which is the major regulated MEK activator in cell cytosols, is distinct from known members of the Ras MAP kinase cascade.

Identification of B-Raf in MEK Activator Peak II

In order to identify the MEK activator in Mono Q peak II (see Fig. 1), fractions 15-20 were pooled and immune complex kinase assays for A-Raf, B-Raf, c-Raf-1, MEKK, and c-Mos were performed. In contrast to Mono Q peak I, immunoprecipitates of B-Raf from Mono Q peak II showed highest MEK activator activity. Surprisingly, no activity was found in A-Raf, c-Raf-1, MEKK, and c-Mos immunoprecipitates (Fig. 5). The efficiency of the immunoprecipitation was demonstrated by sequential immunoprecipitation of B-Raf and c-Raf-1 from Mono Q peak I (data not shown). Immunoprecipitates and supernatants were monitored by Western analysis and coupled kinase assays for MEK activator. B-Raf and c-Raf-1 were quantitatively immunoprecipitated from these samples. Only B-Raf immunoprecipitates contained MEK activating activity, suggesting that c-Raf-1 is not enzymatically active in the cytosolic fraction of these cells. Furthermore, B-Raf immunoprecipitates did not contain c-Raf-1 and vice versa (data not shown).


Figure 5: Identification of MEK activator in Mono Q peak II as B-Raf. Mono Q fractions 15-20 from serum-treated (left, +FCS for 2 min) and untreated (right, -FCS) NIH3T3 fibroblasts were immunoprecipitated with an anti-A-Raf, anti-B-Raf, anti-c-Raf-1, anti-MEKK antibody, anti-c-Mos antiserum, and nonimmune serum. Immune complexes were assayed for MEK activator activity in a coupled assay as described in the legends to Fig. 1and Fig. 3. Lanes: 1 and 2, 10 µl of Mono Q pool II (fractions 15-20) of cell extracts from untreated (1) and serum-treated NIH3T3 fibroblasts (2); immunoprecipitates (IP) with: 3 and 9, anti-A-Raf antibody; 4 and 10, anti-B-Raf antibody; 5 and 11, anti-c-Raf-1 antibody; 6 and 12, anti-MEKK antibody; 7 and 13, anti-c-Mos antiserum; 8 and 14, nonimmune serum.



To further characterize the MEK activator in Mono Q peak II, Mono Q fractions 15-20 were pooled, diluted, and resolved on a Mono S column. The MEK activator was found in fractions 8-16 (peak, fraction 12) (300 mM NaCl) (Fig. 6, upper panel), and co-migrated with B-Raf, as determined by Western blots (data not shown). c-Raf-1 was present in this fraction, but peaked slightly ahead (fraction 10) of the activity peak. Fractions containing maximal MEK activating activity were pooled(8, 9, 10, 11, 12, 13, 14) and immunocomplex kinase assays for A-Raf, B-Raf, c-Raf-1, MEKK, and c-Mos were performed. MEK activating activity above background was only found in immobilized B-Raf immunoprecipitates (Fig. 6, lower panel), and showed a 2.5-fold increase after stimulation with serum.


Figure 6: Mono S chromatography of MEK-1 activator from Mono Q peak II. Mono Q column fractions 15-20 from NIH3T3 fibroblasts were 1:1 diluted and chromatographed (10 ml) on a Mono S column with a 10-ml linear gradient from 0-750 mM NaCl, and 0.5-ml fractions were collected. MEK activator activity was measured by a coupled MEK activation assay ( Fig. 1and Fig. 3). A, Mono S chromatography of MEK-1 activating activity from serum-treated (bullet) and untreated (circle) NIH3T3 fibroblasts. Counts/min were determined by Cerenkov counting of the excised K52R-ERK2 bands. B, autoradiogram of the coupled MEK activation assay of immunoprecipitates with anti-A-Raf, anti-B-Raf, anti-c-Raf-1, anti-MEKK, and anti-c-Mos antibodies. Lanes: 1, 10 µl of Mono S pooled fractions 9-15 of cell extracts from untreated (-FCS) and serum-treated (+FCS) NIH3T3 fibroblasts; 2-7, supernatants (SUP); or 8-13, immunoprecipitates (IP) with antibodies against: 2 and 8, A-Raf; 3 and 9, B-Raf; 4 and 10, c-Raf-1; 5 and 11, MEKK; 6 and 12, c-Mos; 7 and 13, nonimmune serum; 14, inactive MEK1 plus K52-R-ERK2.



To assess the molecular size of this activator activity, Mono Q fractions containing MEK activator were pooled, concentrated, and chromatographed on a Superose 12 gel filtration column. Using immune complex kinase assays with anti-B-Raf antibody, MEK activator was detected with an apparent molecular mass of >200 kDa (Fig. 7). Similar large sizes have been reported for a MEK activator from Xenopus(57, 79) , c-Raf-1(80) , and B-Raf (34, 36) .


Figure 7: Gel filtration chromatography of MEK activator in Mono Q peak II. Mono Q fractions 15-20 from serum-treated (bullet) and untreated (circle) NIH3T3 cells were pooled, concentrated to a final volume of 300 µl, and then applied (200 µl) to a Superose 12 column, and fractions (0.25 ml) were collected. The void volume (V(0)) of the column and the elution positions of protein standards are indicated by arrows. Superose 12 fractions were immunoprecipitated with an anti-B-Raf antibody. Immune complexes were assayed for MEK activating activity of the immune complexes as described.



Regulation of B-Raf and c-Raf-1 Activity in Particulate Fractions

Several recent observations suggest that agonist stimulation results in translocation of c-Raf-1 to the plasma membrane where enzymatic activation may take place. To look specifically at MEK activators in membrane-containing cell fractions, NIH3T3 cells were hypotonically lysed, the membrane-containing particulate fraction was prepared and resuspended in HLB containing 0.5% Triton X-100. MEK activating activity was assayed in A-Raf, B-Raf, c-Raf-1, MEKK, and c-Mos immunoprecipitates (Fig. 8). Only c-Raf-1, A-Raf, and B-Raf immunoprecipitates revealed MEK activating activity in the coupled MEK activation assay, with B-Raf immunoprecipitates displaying by far the most activity (20-40-fold higher than c-Raf-1). Serum stimulation increased B-Raf activity 2-3-fold in the particulate fraction of NIH3T3 cells, but had little if any effect on c-Raf-1 activity. In contrast, serum activated c-Raf-1 2.5-5-fold in particulate lysates of Rat-1 fibroblasts (data not shown). c-Raf-1 and B-Raf immunoprecipitates revealed a low MEK activity, suggesting a coimmunoprecipitation of endogenous MEK. Similar amounts of B-Raf or c-Raf-1 were detected by Western blotting in lysates of the particulate fraction before or after stimulation of NIH3T3 fibroblasts (data not shown), suggesting that the serum-induced increases in B-Raf and c-Raf-1 activity were due to an activation of these enzymes in the particulate fraction rather than a seruminduced translocation.


Figure 8: MEK activators in whole cell lysates and in the membrane-containing particulate fraction of serum-treated and untreated NIH3T3 cells. Untreated (-FCS) and serum-stimulated (+FCS) NIH3T3 fibroblasts (2 min at 37 °C) were lysed using Triton-lysis buffer (0.5%) or hypotonic lysis buffer and insoluble material was pelleted by centrifugation. Hypotonic lysis buffer pellets were frozen, thawed, and lysed again in hypotonic lysis buffer containing 0.5% Triton X-100. Insoluble material was removed by centrifugation. The supernatants were adjusted for protein concentration and immunoprecipitations (IP) with antibodies against A-Raf, B-Raf, c-Raf-1, MEKK, c-Mos, or non-immune serum were performed. Immobilized immunocomplexes were assayed for MEK activator activity. Upper panel, MEK activation assays from immunoprecipitates of whole cell detergent lysates. Lower panel, MEK activation assays from immunoprecipitates of the particulate fraction.



Activation of MEK Activators by PDGF, Insulin, Thrombin, and Phorbol Ester Stimulation of NIH3T3 Fibroblasts

In order to determine the effect of different agonists on MEK activators, serum-deprived NIH3T3 fibroblasts were stimulated for 2 min with 1 unit/ml thrombin, 100 nM PDBu, 10 µg/ml insulin, or 50 ng/ml PDGF-BB. All agonists induced 2-12-fold activation of MAP kinases p44 and p42 (data not shown). Hypotonic cell lysates were fractionated by anion exchange chromatography on a Mono Q column. As shown in Fig. 9A, MEK and MEK activator increased upon stimulation by PDGF, thrombin, insulin, and phorbol ester. A major increase in the second MEK activating activity (peak II) was only observed in column fractions from PDGF-treated NIH3T3 fibroblasts. To assay B-Raf and c-Raf-1 translocated to the membrane, detergent lysates of the membrane-containing pellets were prepared and assayed for MEK activating activity in immunoprecipitates with B-Raf and c-Raf-1 antibodies (Fig. 9B). The MEK activating activity of B-Raf immunoprecipitates was significantly increased after FCS, PDGF-BB, and phorbol ester stimulation. c-Raf-1 immunoprecipitates showed enhanced MEK activating activity after PDGF-BB, thrombin, and PDBu stimulation. No MEK or MEK activating activity was found in immunoprecipitates with A-Raf antibody (data not shown).


Figure 9: Differential activation of B-Raf and c-Raf-1 in agonist-stimulated NIH3T3 fibroblasts. A, identification of MEK activators by Mono Q chromatography of extracts of serum-deprived, PDGF-BB, insulin, thrombin, or phorbol ester-stimulated NIH3T3 fibroblasts. Serum-deprived NIH3T3 fibroblasts were stimulated for 2 min with 1 units/ml thrombin, 100 nM PDBu, 10 µg/ml insulin, or 50 ng/ml PDGF-BB. Hypotonic lysates were prepared as described under ``Materials and Methods,'' chromatographed on a Mono Q column and assayed for MEK (circle) and MEK (bullet) activator activity as described in the legend to Fig. 1. B, MEK activator activity in immunoprecipitates with B-Raf and c-Raf-1 antibodies of the membrane-containing particulate fraction from NIH3T3 cells. Pellets were resuspended in hypotonic lysis buffer containing 0.5% Triton X-100 and immunoprecipitated and assayed for MEK activator as described in Fig. 8. B-Raf, unstimulated controls, n = 6; serum-stimulated (10% FCS), 2.8 ± 1.9-fold, n = 6, p = 0.051; platelet-derived growth factor (50 ng/ml PDGF-BB), 1.7 ± 0.9-fold, n = 5, p = 0.067; insulin (10 µM), 0.95 ± 0.50, n = 5, p = 0.797; thrombin (1 unit/ml), 1.4 ± 0.7, n = 5, p = 0.225; PDBu (100 nM PDBu), 2.8 ± 1.8, n = 5, p = 0.035. c-Raf-1, unstimulated controls, n = 6; serum-stimulated, 1.2 ± 0.26-fold, n = 6, p = 0.147; PDGF, 4.7 ± 2.1-fold, n = 5, p = 0.002; insulin, 1.3 ± 0.4-fold, n = 5, p = 0.118; thrombin, 2.3 ± 0.5-fold, n = 5, p < 0.001; PDBu, 6.5 ± 3.7-fold, n = 5, p = 0.009.



Regulation by cAMP of Raf Activation and the Association of B-Raf with Ras

Previous investigations have shown that c-Raf-1 and active Ras physically interact(56, 57, 58, 59, 61) , and that this interaction and the regulation of c-Raf-1 activity are inhibited when cellular cAMP levels are elevated(67) . In order to gain further understanding of the role played by various MEK activators in the regulation of the MAP kinase pathway, we analyzed the effects of forskolin on the Ras/B-Raf interaction and the activation of c-Raf-1 and B-Raf in NIH3T3 fibroblasts. Ras binding experiments were carried out using isotonic, detergent-free homogenates of NIH3T3 fibroblasts which had either been serum-deprived or stimulated with fetal bovine serum. B-Raf association was observed with GMP-PNP-Ras, with low detectable B-Raf associating with GDP-Ras, in agreement with previous results (34) (Fig. 10). Association of B-Raf with immobilized Ras occurred independently of prior stimulation of cells with serum, suggesting that primarily the production of GTP-Ras by mitogen stimulation is critical for the formation of B-RafbulletGTP-Ras complexes. Treatment of fibroblasts with forskolin, which activates adenylate cyclase(81) , decreased binding of B-Raf to immobilized Ras by approximately 60% (Fig. 10).


Figure 10: Binding of B-Raf from cultured NIH3T3 cells to immobilized Ras in a GMP-PNP-dependent manner, independently of serum stimulation and effect of forskolin. NIH3T3 fibroblasts were serum-deprived for 16 h and stimulated with 20% fetal calf serum for 5 min (A, lanes 3 and 4) or not stimulated (A, lanes 1 and 2). Cytosolic fractions (100 µg) were incubated with 50 µg of immobilized Ras loaded with GDP or GMP-PNP, and precipitated. Precipitated samples were analyzed by SDS-PAGE and assayed by Western blot analysis with anti-B-Raf antibodies. In order to study the effect of forskolin on B-Raf binding to immobilized Ras, NIH3T3 fibroblasts were serum-starved for 16 h, then left untreated (B, lanes 1 and 2) or treated with 50 µM forskolin for 15 min (B, lanes 3 and 4). Cytosolic fractions were assayed for Ras binding.



In order to determine the effect of forskolin on c-Raf-1, B-Raf, MEK1, and MEK2, as well as ERK1 and ERK2 activity, NIH3T3 cells were treated with or without forskolin, and PDBu-stimulated or untreated. Hypotonic lysates were assayed for ERK1, ERK2, MEK1, and MEK2 activity in immunocomplex kinase assays. Forskolin treatment reduced the basal activities of MEK1 and -2 as well as ERK2 approximately 25-50% as compared to enzyme activities from untreated controls (Table 1). After PDBu stimulation, MEK1 and MEK2 activity increased 2-3-fold in untreated cells, and this activation was only partly blunted in forskolin-treated fibroblasts. C-Raf-1 and B-Raf activity were determined in immunocomplex kinase assays of detergent lysates of the particulate fraction of NIH3T3 cells. c-Raf-1 activity increased 2-2.5-fold after PDBu stimulation and was reduced to undetectable levels in the presence of forskolin. In contrast, B-Raf activity was reduced approximately 70-80% in membranes from forskolin-treated NIH3T3 cells, comparable to the extent of inhibition of binding to Ras. Thus, the extent of inhibition of MEKs and MAP kinases by cAMP in these cells correlates more closely with changes in B-Raf activity than activity of c-Raf-1. Similar degrees of forskolin-induced inhibitions of B-Raf and c-Raf-1 MEK activator activity from PC12 cells have recently been described(33) .




DISCUSSION

In conclusion, we have shown for the first time that 65-70-and 93-95-kDa B-Raf is expressed in NIH3T3 and Rat-1 fibroblasts, and that B-Raf exerts MEK activating activity which is serum- and agonist-dependent. In addition, we have evidence for two other MEK activators, with unusual properties. The first MEK activator has a molecular mass of 40-50 kDa and is distinct from the other known enzymes which are candidate MEK activators, A-Raf, B-Raf, c-Raf-1, MEKK, and c-Mos, as determined by Western analysis and immunoprecipitation. The second novel MEK activator has not been characterized biochemically, but was only detectable after insulin stimulation of NIH3T3 fibroblasts, a finding which is in agreement with observations suggesting that c-Raf-1 does not act upstream of MAP kinase and RSK in insulin-signaling pathways in adipocytes(71, 82) . Our ability to detect these novel activators was dependent on the establishment of a reliable, quantitative assay for MEK activation, in which purified recombinant MEK was used as a substrate and phosphorylation of purified recombinant MAP kinase was the read-out (36) . Recently, similar MEK activation assays have been used successfully to screen cellular lysates and column fractions for unusual MEK activators in adipocytes and PC12 cells(34, 71) . Experiments are currently under way to identify the MEK activators found in NIH3T3 cells.

Although it is widely believed that c-Raf-1 is the major MEK activator in proliferating fibroblasts, its role in the MAP kinase signal transduction pathway is still uncertain. We were unable to detect MEK activator activity of c-Raf-1 in immune precipitates with anti-c-Raf-1 polyclonal antibody from whole cell lysates and hypotonic lysates of NIH3T3 and Rat1 fibroblasts, although c-Raf-1 was clearly detectable in the immune precipitates by Western analysis. However, c-Raf-1 immune precipitates from the particulate fraction of cells activated MEK1 in the coupled MEK activation assay. This result indicates that the cytosolic fraction of c-Raf-1 does not exhibit MEK activator activity. In contrast, B-Raf shows MEK activator activity in immune precipitates and column fractions from both the cytosolic and the particulate fraction of NIH3T3 fibroblasts, suggesting differences in the cellular regulation and function of c-Raf-1 and B-Raf kinase activity. This hypothesis is supported by the agonist-specific differential regulation of c-Raf-1 and B-Raf activity in immunoprecipitates from lysates of the particulate fraction of NIH3T3 fibroblasts.

Our findings are of particular interest as substantial evidence suggests that MEK activators distinct from c-Raf-1 exist and that c-Raf-1 might not be involved in the MAP kinase pathway under many circumstances. In PC12 cells, expression of an activated c-Raf-1 mutant resulted in the induction of neurite formation and gene expression similar to those induced by nerve growth factors but failed to activate MAP kinases(83, 84) . In addition, c-Raf-1 did not exhibit a MEK activator activity in neuronal tissue(34, 36) . Similarly, in Rat1a cells, c-Raf-1 was found not to be coupled to the MAP kinase cascade (85, 86) . Furthermore, c-Raf-1 was found not to be a major upstream regulator of MAP kinases in NRK rat fibroblasts expressing c-Raf-1 antisense RNA(72) . c-Raf-1 depleted cell lysates from nerve growth factor-treated PC12 cells and epidermal growth factor-treated Swiss 3T3 fibroblasts were shown to phosphorylate GST-MEK1, suggesting that different growth factor-sensitive c-Raf-1 independent MEK activators might exist(73) . This finding is supported by an observation of Chao et al.(87) who reported that epidermal growth factor and phorbol ester stimulation of stably transfected dominant-negative c-Raf-1 Balb/c3T3 fibroblasts is followed by activation of the MAP kinase pathway, suggesting an alternative c-Raf-1-independent pathway for MAP kinase activation. In 3T3L1 cells expression of transfected Raf oncogenes did not induce MAP kinase or RSK activation and transfected dominant-negative Raf mutants did not block the insulin-induced activation of these kinases, indicating that c-Raf-1 does not act upstream of MAP kinase and RSK in insulin-signaling pathways leading to 3T3L1 adipocyte differentiation(82) . Correspondingly, Haystead et al.(71) found a 56-kDa insulin-stimulated MEK kinase (I-MEKK) in rat adipocytes which is distinct from c-Raf-1 and MEK kinase.

In addition to c-Raf-1, MEK kinase (MEKK), which is ubiquitously expressed in mouse tissue and in Rat1a, NIH3T3, and PC12 cells, was recently identified (38) as a novel MEK activator. However, the role of MEKK in the MAP kinase cascade is even less clear than is the role of c-Raf-1, and may be involved in regulation of stress-induced ERK-related kinases(17, 96) . We were unable to detect 50- or 78-kDa MEKK in hypotonic lysates and column fractions of NIH3T3 fibroblasts, even in those fractions which contained MEK activating activity. Conversely, even though we found a 75-78-kDa form of MEKK in membrane lysates by immunoblotting, suggesting that MEKK associates with the plasma membrane, immunoprecipitates of these membrane fractions with rabbit polyclonal antibody against MEKK did not exhibit MEK activating activity. Thus, we obtained no evidence for a role of MEKK in the serum-dependent activation of MEK in these cells.

In addition to MEKK and c-Raf-1, c-Mos, which is preferentially expressed in germ cells, has recently been identified as a MEK activator in Xenopus oocyte extracts(37, 88) . Interestingly, p43 was also found in somatic cells of mouse kidney, liver, spleen, and brain as well as in NIH3T3 fibroblasts, NRK, and Chinese hamster ovary cells(89) . Using Western blotting, we were unable to identify p43 in column fractions of hypotonic lysates of NIH3T3 fibroblasts which contained MEK activating activity. Moreover, immune complex kinase reactions with c-Mos antibodies as described by Nebreda et al.(88) did not reveal any MEK activating activity. However, in detergent cell extracts of NIH3T3 cells a 43-kDa protein was detected by immunoblotting with this anti-c-Mos antibody (data not shown). Thus, even though a fibroblast protein immunologically related to c-Mos was detected in these cells, we obtained no evidence for its regulation as a MEK activator.

Identification of sites of activation of MEK1 clarifies the role of MEK activators like MEKK and c-Raf-1 in transduction of cellular growth signals. Recently, two serine residues (Ser-218 and -222 in rat MEK1) of MEK-1 have been identified that are phosphorylated during activation by c-Raf-1 and MEKK(17) . Partially purified B-Raf from bovine brain-phosphorylated MEK1 on residues tentatively identified as serine 218 and 222(36) . In addition, glutamic or aspartic acid substitutions of these residues constitutively activate MEK, stimulate PC12 cell neuronal differentiation, and transform NIH3T3 cells(90, 91) . MEK1 mutants in which these residues are substituted by alanine are unable to become activated by c-Raf-1 or MEKK and therefore block MAP kinase activation as dominant negative mutants(17, 23, 89) . Since phosphorylation of Ser-218 and -222 are necessary and sufficient for activation of MEK1 by c-Raf-1, B-Raf, and MEKK, it is likely, but still unproven, that the novel activators described here function in the same manner.

The biochemical pathways linking surface tyrosine kinases through Ras to activation of cytosolic kinases are still incompletely defined (92, 93, 94) . Although most reports on proliferating fibroblasts place c-Raf-1 downstream of Ras and upstream of MAP kinase and RSK kinases (92, 93, 95) , multiple lines of evidence suggest that c-Raf-1 independent pathways of MAP kinase activation exist depending on the cell type or the agonist(94) . Our results indicate that B-Raf is a major MEK activator in fibroblasts and that two other novel activators appear as well. Whereas c-Raf-1 responds to PDGF and phorbol ester, B-Raf and a novel 40-50-kDa activator appear to be the major MEK activators in response to serum. It remains to be determined what the functional significance is for such a multitude of MEK activators. Although these activators function in parallel with respect to activation of the MAP kinase pathway, it is possible that they each display, in addition, unique signaling properties which have not yet been identified.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant GM47332 and United States Public Health Service Grant CA39076 (to M. J. W.). 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 postdoctoral fellowship from the Deutsche Forschungsgemeinschaft (Re-864/2-1).

To whom correspondence should be addressed: Dept. of Microbiology and Cancer Center, University of Virginia Health Sciences Center, School of Medicine, Box 441, Charlottesville, VA 22908. Fax: 804-982-0689.

(^1)
The abbreviations used are: MAP kinase, mitogen-activated protein kinase; MEK, MAP kinase or ERK kinase; PDGF, platelet-derived growth factor; PDBu, phorbol dibutyrate; CR, conserved region; PAGE, polyacrylamide gel electrophoresis; FCS, fetal calf serum; GMP-PNP, guanosine 5`-(beta,-imino)triphosphate.


ACKNOWLEDGEMENTS

We thank Rukmini Reddy, Sheri Meyer, and Vicki Gordon for excellent technical assistance, and Hans Schaeffer for discussions.


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