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J. Biol. Chem., Vol. 275, Issue 48, 37303-37306, December 1, 2000

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ACCELERATED PUBLICATION
Uncoupling Raf1 from MEK1/2 Impairs Only a Subset of Cellular Responses to Raf Activation^*

Gray Pearson, Ron Bumeister, Dale O. Henry, Melanie H. Cobb, and Michael A. White^§

From the Departments of Cell Biology and Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390

Received for publication, August 21, 2000, and in revised form, September 21, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The Raf family of serine/threonine protein kinases is intimately involved in the transmission of cell regulatory signals controlling proliferation and differentiation. The best characterized Raf substrates are MEK1 and MEK2. The activation of MEK1/2 by Raf is required to mediate many of the cellular responses to Raf activation, suggesting that MEK1/2 are the dominant Raf effector proteins. However, accumulating evidence suggests that there are additional Raf substrates and that subsets of Raf-induced regulatory events are mediated independently of Raf activation of MEK1/2. To examine the possibility that there is bifurcation at the level of Raf in activation of MEK1/2-dependent and MEK1/2-independent cell regulatory events, we engineered a kinase-active Raf1 variant (RafBXB(T481A)) with an amino acid substitution that disrupts MEK1 binding. We find that disruption of MEK1/2 association uncouples Raf from activation of ERK1/2, induction of serum-response element-dependent gene expression, and induction of growth and morphological transformation. However, activation of NF-B-dependent gene expression and induction of neurite differentiation were unimpaired. In addition, Raf-dependent activation of p90 ribosomal S6 kinase was only slightly impaired. These results support the hypothesis that Raf kinases utilize multiple downstream effectors to regulate distinct cellular activities.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cellular interpretation of growth regulatory signals requires functional grouping of molecules into signal transduction cascades. The Raf family of serine/threonine protein kinases is critically involved in this signal transduction process. Raf kinases were first discovered as gain of function mutants with the ability to induce growth and morphological transformation of established cell lines. Subsequently it was discovered that activation of cellular Raf proteins is a downstream response to growth factors and is required to link growth factor receptor signaling to activation of gene expression (1). Studies of genetic model systems have demonstrated that activation of Raf is an essential step in many growth and developmental programs, and studies of tumor model systems have demonstrated that Raf mediates transformation by many oncogenes (2).

A major substrate of activated Raf is mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK1).¹ Upon activation by Raf, MEK1 can in turn phosphorylate and activate the p42 and p44 MAP kinases (also known as extracellular ligand-regulated kinases, ERK1 and 2) (1). Activated ERKs phosphorylate a number of cytoplasmic and nuclear targets, including transcription factors that mediate growth factor regulation of gene expression. ERK activation is required for cellular transformation induced by oncogenic Ras and Raf (3), and it has been reported that expression of constitutively activated MEK1 is sufficient to induce cellular transformation of immortalized fibroblasts (4, 5).

These studies suggest that activation of MEK1, with subsequent ERK activation, is the primary event mediating cellular responses to activated Raf. However, several cellular responses to activated Raf have been characterized that appear to be independent of MEK or MAP kinase activation. For example, constitutively active variants of Raf but not MEK are sufficient to induce differentiation of hippocampal neuronal cells (6). Expression of mutationally activated Raf1 in CCL 39 cells has been reported to result in an ERK1/2-independent activation of p70 S6 kinase, leading to increased translation of mRNAs with polypyrimidine tracts (7). Raf-mediated activation of NF-B transcription factors may occur independently of MEK1/2 activation in Jurkat T cells (8), and Raf activity promotes, whereas MEK1/2 activity inhibits, atrial natriuretic factor expression in cardiac myocytes (9). Consistent with these observations, candidate Raf substrates in addition to MEK1 have been reported (10-12).

To further explore the potential of Raf kinases to modulate cell regulatory pathways independently of MEK1/2 activation, we isolated a kinase active, MEK1/2 binding-defective Raf variant, RafBXB(T481A). RafBXB(T481A) can efficiently stimulate morphological changes in PC12 cells indicative of differentiation events, NF-B-dependent gene expression, and activation of p90 ribosomal S6 kinase (RSK), despite severely impaired ERK1/2 activation.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Plasmids, Reagents, and Expression of Recombinant Proteins-- 3× SRE-Luc, pCEP4-GFP, pCH110Gal (30), 2× NF-B-Luc (31), heparin-binding epidermal growth factor-like growth factor (HB-EGF)-Luc (32), and pCMV5-MEK1R4F (33) are as described elsewhere. pCEP4HA-RSK expresses full-length avian RSK with an amino-terminal HA tag (gift from Megan Robinson). MycPCDNA3-rafBXB and MycPCDNA3-rafBXB(T481A) contain Raf1 cDNAs encoding amino acids 330-648 inserted as EcoRI/BamHI fragments into the EcoRI/BamHI sites of MycPCDNA3. For protein expression analysis, mouse anti-Myc (Cell Culture Center), mouse anti-HA (Berkeley Antibody Company), rabbit anti-Raf-1 (sc-133; Santa Cruz Biotechnology), rabbit anti-ERK1/2 (sc-93; Santa Cruz Biotechnology), and rabbit anti-active ERK1/2 (44-680; QCB/BIOSOURCE International) were used. Expression and purification of recombinant GST-MEK1K-M and histone 7 S were performed by standard methods.

Library Construction and Screening-- A library of randomly mutated cDNAs encoding RafBXB with an amino-terminal fusion to the LexA DNA binding domain was constructed in the yeast expression vector pBTM116 using methods previously described (34). This library was screened for clones encoding fusion proteins that did not interact with a GAL4 activation domain/MEK1 fusion in the yeast reporter strain L40 (35) by standard techniques.

Cell Culture and Transfection Assays-- HEK 293 cells and NIH 3T3 cells were maintained as described (36, 37). Transfections were performed using calcium phosphate precipitation in 60-mm plates. Lysates were assayed for Firefly luciferase and Firefly renilla activity using a Dual Luciferase Assay kit (Promega) and the Turner Designs luminometer. Levels of reporter gene induction were calculated by normalizing luciferase activity to either renilla or -galactosidase activity. Focus assays were performed as described (37). PC12 cells were maintained in RPMI 1640 with 5% horse serum and 10% fetal bovine serum. Transfections were performed with calcium phosphate precipitates. To detect levels of active ERK1/2, 24 h post-transfection cells were washed and incubated for an additional 16 h in serum-free medium and then lysed in sample buffer. Neurite extensions were visualized 72 h post-transfection.

Immunoprecipitations and Kinase Assays-- Immunoprecipitations were performed as described (36) using either anti-myc (myc-rafBXB and myc-rafBXB(T481A) or anti-HA (HA-RSK) antibodies. Kinase assays were performed as described (36) using either GST-MEK1K-M or histone 7 S subunit as indicated.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Isolation of a MEK Association-defective Raf Variant-- We used the yeast two-hybrid protein interaction detection system to isolate Raf variants that are uncoupled from MEK1 and MEK2. cDNA encoding RafBXB, a constitutively active Raf1 variant with a deletion in the amino-terminal regulatory domain, was randomly mutagenized along its entire length by a polymerase chain reaction employing low fidelity Taq polymerase (13). The polymerase chain reaction product was used to generate a yeast expression library encoding fusions of RafBXB to the LexA DNA binding domain. The resulting library was introduced into a yeast two-hybrid reporter strain, together with MEK1 expressed as a GAL4 activation domain fusion, to isolate RafBXB variants that fail to associate with MEK1. In addition to various alterations that lead to expression of truncated products, we identified a substitution of alanine for threonine at position 481 as an alteration that inhibits association with MEK1 and MEK2 (Fig. 1A).

View larger version (63K): [in this window] [in a new window]   Fig. 1.   A single amino acid substitution in Raf1 disrupts association with MEK1. A, L40 cells expressing the indicated fusion proteins were tested for the ability to grow on medium lacking histidine. Growth on the selective plate indicates a positive two-hybrid interaction. B, HEK 293 cells were transiently transfected with the indicated constructs expressing myc-tagged RafBXB or myc-tagged RafBXB(T481A) or vector (V). Lysates from serum-starved cells were separated by SDS polyacrylamide gel electrophoresis and immunoblotted with the indicated antibodies to detect dually phosphorylated ERK1/2 (p-Erk1/2), total ERK1/2, and the expressed RafBXB variants. Similar results were obtained in repeated experiments. C, RafBXB and RafBXB(T481A) were immunoprecipitated from serum-starved transiently transfected HEK 293 cells using anti-myc monoclonal antibodies. Following extensive washing, the precipitates were added to in vitro kinase reactions with recombinant GST-MEK1K-M as substrate. A representative autoradiogram of one of three immune complex kinase assays performed in duplicate is shown (top panel). A portion of the precipitates were immunoblotted with anti-Raf antibodies to confirm that equivalent levels of Raf kinases were present in the reactions (bottom panel).

Thr⁴⁸¹ is located in a loop (14, 15) between conserved kinase subdomains VIB and VII as defined by Hanks et al. (16). All known Raf kinase genes encode a threonine at this position except for Drosophila raf (pole hole), which encodes a serine. Expression of RafBXB but not RafBXB(T481A) in serum-starved HEK 293 cell culture results in detectable activation of endogenous Erk1 and Erk2 proteins suggesting an uncoupling of RafBXB(T481A) from endogenous MEK proteins (Fig. 1B). The T481A mutation does not appear to affect activity of the Raf kinase domain, as immunoprecipitated RafBXB and RafBXB(T481A) showed equivalent phosphorylation activity on saturating amounts of GST-MEK1K-M in vitro (Fig. 1C).

T481A Uncouples Activities That Mediate RafBXB Stimulation of Neurite Differentiation versus Cellular Transformation-- The use of dominant interfering MEK variants has defined MEK activation as a crucial step mediating oncogene induction of cellular transformation (4, 17). Not surprisingly, the T481A substitution virtually eliminates RafBXB focus-forming activity in NIH 3T3 cells (Fig. 2). As in HEK 293 cells, RafBXB(T481A) expression does not result in detectable activation of ERK1/2 in NIH3T3 cells (data not shown). In contrast, despite defective ERK1/2 activation, RafBXB(T481A) retains the ability to induce formation of neurite-like extensions in PC12 cells (Fig. 3) to a similar extent as observed with RafBXB. Kinase-inactive RafBXB had no effect (data not shown). These observations suggest that Raf can positively modulate differentiation through a MEK-independent pathway.

View larger version (59K): [in this window] [in a new window]   Fig. 2.   RafBXB(T481A) has severely impaired transformation activity. NIH 3T3 cells were transfected with constructs expressing the indicated proteins. Following 14 days of incubation in 5% serum, foci of growth and morphologically transformed cells were counted by microscopic observation. Transfections with MycPCDNA3-RafBXB typically resulted in 160 foci/µg of DNA. Relative focus-forming activity was determined from values obtained from three independent experiments performed in duplicate. Error bars represent S.E. Focus assay plates fixed and stained with Giemsa, to reveal foci, are shown from a representative experiment.

View larger version (68K): [in this window] [in a new window]   Fig. 3.   RafBXB(T481A) can stimulate PC12 cell differentiation in the absence of detectable ERK1/2 activation. A, PC12 cells were transfected with the indicated expression vectors together with pCEP4-GFP. 72 h post-transfection, cells were fixed and visualized by GFP autofluorescence. Representative cells are shown in each panel. The ratio of transfected cells to transfected cells with "neurites" was 0.37 ± 0.04 and 0.43 ± 0.05 for rafBXB and rafBXB(T481A), respectively. No "neurite-like" cells were observed in empty vector transfections. B, levels of active ERK1 and ERK2 were assayed in serum-starved transiently transfected PC12 cells. Methods are as described in the legend to Fig. 1B.

T481A Uncouples RafBXB Regulation of SRE- but Not NF-Bdependent Gene Expression-- RafBXB activates both ternary complex factor and NF-B family transcription factors (2). Raf activation of TCF is ERK1/2-dependent (18), whereas some studies suggest that vRAF can activate NF-B through MEK1/2- and ERK1/2-independent pathways (8). Expression of RafBXB in quiescent NIH 3T3 cells is sufficient to induce both SRE- and NF-B-coupled luciferase reporter constructs (Fig. 4). Consistent with defective ERK1/2 activation, RafBXB(T481A) expression results in poor activation of 3× SRE-Luc (Fig. 4A). In contrast, RafBXB(T481A) induces 2× NF-B-Luc to a similar if not higher level than as observed with RafBXB (Fig. 4B).

View larger version (11K): [in this window] [in a new window]   Fig. 4.   RafBXB(T481A) is uncoupled from regulation of SRE-dependent but not NF-B-dependent gene expression. NIH 3T3 cells were transfected with mycPCDNA3, mycPCDNA3-RafBXB, or mycPCDNA3-RafBXB(T481A) together with luciferase reporter constructs driven by three tandem copies of the c-Fos SRE (3× SRE-Luc; panel A) or two tandem copies of the NF-B-binding site from the B promoter (2× NF-B-Luc; panel B). Relative luciferase activities were calculated by normalizing the -fold reporter gene induction above empty vector to the values obtained with RafBXB, which were arbitrarily set at 100. RafBXB expression typically resulted in a more that 100-fold activation of 3× SRE-Luc and a 4-fold activation of 2× NF-B-Luc above empty vector controls. Error bars are the S.E. from three independent experiments performed in duplicate.

The activation of NF-B-dependent gene expression by Raf in HEK 293 cells has been reported to occur downstream of activation of HB-EGF expression (19). Raf activation of the HB-EGF promoter appears to occur through activation of MEK and ERK1/2 (20). However, results using Jurkat T-cells suggest that Raf activation of NF-B can also occur through more direct mechanisms independent of ERK1/2 activation (8). As shown in Fig. 5A, the T481A mutation eliminates the ability of RafBXB to stimulate gene expression from the HB-EGF promoter. This observation suggests that Raf can activate NF-B in NIH 3T3 cells, as in Jurkat cells, through an HB-EGF-independent pathway.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   RafBXB(T481A) is uncoupled from regulation of the HB-EGF promoter but can activate RSK. A, experiments were performed as in Fig. 4 except that a luciferase reporter driven by a single copy of the murine HB-EGF promoter was used. RafBXB expression typically resulted in a 25-fold activation of the HB-EGF promoter above values obtained with empty vector. B, HA-RSK was immunoprecipitated from lysates from NIH3T3 cells expressing the indicated constructs. Immune complex kinase assays with equal amounts of HA-RSK were performed using purified histone 7 S. Kinase reactions were quantitated by measuring p32 incorporation into histone 7 S and normalized to the activity observed with RafBXB. RafBXB expression typically resulted in a 10-fold elevation in RSK activity above that observed with empty vector. Bars represent the S.E. of the average values of three independent experiments performed in duplicate (left panel). Anti-HA and anti-Raf1 immunoblots from a representative experiment are shown below the graph.

p90 RSK is activated in response to active Raf and in some cell types RSK can phosphorylate inhibitor of B on serine 32 (21, 22). This raises the possibility that RSK can contribute to Raf-mediated NF-B activation. Interestingly, RafBXB(T481A) retains the ability to activate p90 RSK in NIH 3T3 cells (Fig. 5B). This results suggest that Raf may regulate p90 RSK through both MEK-dependent and MEK-independent pathways.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Although it is clear that MEK1 and MEK2 are major substrates of Raf, accumulating observations suggest that Raf may regulate important cellular events independently of, or in parallel with, MEK1/2 activation (see the Introduction). To begin to explore this concept in detail, we have engineered a kinase-active Raf1 variant that is uncoupled from MEK1/2-dependent signaling events because of an amino acid substitution (T481A of human Raf1) that interferes with MEK1/2 binding. Expression of this Raf variant (RafBXB(T481A)) in cells revealed that it is uncoupled from activation of ERK1 and ERK2, cannot induce growth and morphological transformation, and cannot activate transcription from serum response elements or the HB-EGF promoter. In contrast RafBXB(T481A) retained the ability to induce neurite-like outgrowths in PC12 cells, activate transcription from NF-B-dependent promoters, and induce the kinase activity of p90 RSK.

The above results suggest that RafBXB(T481A) can be used to define subsets of Raf-induced responses that are MEK-independent. Our observation that Raf activation of NF-B can be uncoupled from MEK1/2 regulation is consistent with recent data examining NF-B regulation in Jurkat T cells (8). Our observations that RafBXB(T481A) mimics RafBXB effects on PC12 cell neurite differentiation and p90 RSK activation were more unexpected. Experiments using dominant inhibitory variants of MEK1 and the pharmacological inhibitor PD98059 suggest that MEK activation is required for Raf-induced neurite differentiation and p90 RSK activation (4, 21-24). Our results suggest that although some extent of MEK1/2 activation may be required for these responses, activated Raf can contribute to neurite differentiation and RSK activation independently of MEK1/2 activation. It is important to note that dominant interfering MEK1/2 variants retain the ability to associate with Raf and therefore may block both MEK1/2-dependent and MEK1/2-independent Raf functions (25). In addition, both of the widely used chemical inhibitors of MEK1/2, PD98059 and U0126, also inhibit at least one other MEK family member involved with Raf signaling, MEK5 (26).

The epistatic relationships of proteins functioning in mammalian signal transduction cascades are most often explored using combinations of constitutively active and dominant interfering variants of the components that contribute to regulation of the cascade. This approach has been highly successful in ordering the components of the Raf/MEK/ERK protein kinase cascade and for assessing the importance of this cascade in mediating mitogenic signals (27). However, as a more sophisticated understanding of the molecular mechanisms of cell regulation develops, it is becoming clear that many signal transduction proteins are multifunctional, introducing branch-points into what were once considered to be simple linear pathways of information flow (28, 29). In the context of complex regulatory networks, phenotypes observed with dominant interfering variants of signaling proteins can often be difficult to interpret because of association of the variants with multiple regulatory and effector proteins.

As all of the components that mediate Raf action in cells have not been identified, we currently can not rule out the formal possibility that RafBXB(T481A) regulation of neurite induction, NFB, and p90 RSK is mediated by activation of ERK1/2 to levels that are below the threshold of detection. Nevertheless, the dramatic uncoupling of RafBXB(T481A) from ERK1/2 activation, SRE activation, and focus formation is in stark contrast to the intact regulation of NFB, neurite formation, and p90 RSK activation. Further characterization of the mechanism of action of RafBXB(T481A) in cells, as well as the isolation of additional Raf variants that differentially uncouple association with Raf-binding proteins, will contribute to a better understanding of the complexities of cellular Raf kinase function.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants CA71443 (to M. A. W.) and DK34128 (to M. H. C.) and by the Welch Foundation (to M. A. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by Pharmacological Sciences Training Grant G1907062- 25.

^§ To whom correspondence should be addressed. Tel.: 214-648-2861; Fax: 214-648-8694; E-mail: white08@utsw.swmed.edu.

Published, JBC Papers in Press, October 3, 2000, DOI 10.1074/jbc.C000570200

	ABBREVIATIONS

The abbreviations used are: MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; MAP, mitogen-activated protein; ERK, extracellular ligand-regulated kinases; NF-B, nuclear factor B; RSK, ribosomal S6 kinase; Luc, luciferase; GFP, green fluorescent protein; HB-EGF, heparin-binding epidermal growth factor-like growth factor; HA, hemagglutinin; GST, glutathione S-transferase; HEK, human embryonic kidney; SRE, serum-response element.

	REFERENCES

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