©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Preferential Involvement of MEK1 in the Tumor Necrosis Factor--induced Activation of p42 in Mouse Macrophages (*)

(Received for publication, September 5, 1995)

Brent W. Winston (1)(§) Linda K. Remigio (1) David W. H. Riches (1) (2)(¶)

From the  (1)Division of Basic Sciences, Department of Pediatrics, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206 and (2)Department of Biochemistry, Biophysics, and Genetics, Division of Pulmonary Sciences, Department of Medicine and Department of Pharmacology, University of Colorado Health Sciences Center, Denver, Colorado 80262

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The pleiotropic cytokine tumor necrosis factor-alpha (TNFalpha) controls the expression of multiple gene products in macrophages and plays an important role in host defense. TNFalpha is recognized by the receptors, CD120a (p55) and CD120b (p75). Ligation of CD120a (p55) by TNFalpha or by anti-receptor agonistic antibodies initiates signal transduction leading to the activation of mitogen-activated protein kinases (MAPKs) (p42 and p44). Phosphorylation and activation of MAPK are mediated by MAPK kinase (MEK), a family of Thr/Tyr kinases. In this study, we investigated the preferential involvement of the MEK isoforms MEK1 and MEK2 in the activation of p42 in mouse macrophages stimulated with TNFalpha. Exposure of macrophages to TNFalpha stimulated a time-dependent increase in the activity of MEK1 as measured by an in vitro kinase assay using kinase-inactive p42 (rMAPK) as substrate in the presence of -[P]ATP. Maximal activation of MEK1 was detected at 10 min poststimulation and coincided with maximal transphosphorylation of Tyr and Thr residues of rMAPK. By contrast, there was no evidence of MEK2 activation in macrophages in response to TNFalpha. These data suggest that MEK1 is the preferred substrate for MEK kinase, the upstream kinase implicated in activation of the MAPK pathway in macrophages by TNFalpha.


INTRODUCTION

Tumor necrosis factor-alpha (TNFalpha), (^1)a pleiotropic cytokine produced predominantly by macrophages, stimulates the expression of multiple gene products that collectively mediate the role of the macrophage in host defense(1, 2, 3) . TNFalpha is recognized by a binary system of receptors, CD120a (p55) and CD120b (p75), belonging to the TNF/nerve growth factor receptor family, which initiate signal transduction following receptor oligomerization in the plane of the plasma membrane. Although cross-linking of each receptor has been shown to initiate distinct responses in different cell types(4) , a major emphasis has been placed on investigating the functional responses and signaling mechanisms activated by CD120a (p55). Ligation of TNFalpha by CD120a (p55) has been shown to stimulate the formation of several second messengers including, ceramide-1-phosphate (5, 6) and 1, 2-diacylglycerol(7) . However, emerging studies have shown that an important consequence of ligation of TNFalpha is the activation of at least two protein kinase cascades, which result in the activation of mitogen-activated protein kinases (p42 and p44) (6, 8, 9) and c-Jun kinases/stress-activated protein kinases (JNK/SAPK)(10, 11) .

Ligation of CD120a (p55) by TNFalpha or by receptor-specific polyclonal agonistic antibodies results in the transient activation of p42 in mouse macrophages and other cell types with peak tyrosine phosphorylation and catalytic activation occurring 10-15 min poststimulation(6, 8, 9) . Cross-linking of CD120a (p55) is rapidly followed by a transient activation of MEKK, a serine kinase bearing homology to the yeast kinase Ste11, within 30 s of stimulation of TNFalpha in the absence of activation of c-Raf-1(12) . In addition, activation of MEKK is followed by a transient increase in total MEK catalytic activity as measured by fractionation of unstimulated and TNFalpha-stimulated macrophage lysates by ion-exchange chromatography over a mono-S column followed by detection of catalytically active MEK in a coupled assay based on its ability to phosphorylate and activate purified recombinant p42. While these studies have clearly shown MEK to be activated by TNFalpha, MEK represents a family of dual specificity Tyr/Thr kinases that co-elute from mono-S columns, thus raising the question of the specificity of MEK isoform activation by TNFalpha.

At least three MEK isoforms (MEK1, MEK2, and MEK3) have been described (13, 14, 15, 16) . MEK1 and MEK2 are highly conserved, and purified recombinant MEK1 and MEK2 have both been shown to phosphorylate and activate purified recombinant p42 and p44(16) . By contrast, MEK3, an alternatively spliced variant of MEK1, is catalytically inactive with respect to these substrates (16) and does not appear to be important in the activation of the MAPK cascade. The aim of the present study was to investigate the specificity of MEK isoform involvement in the activation of p42 in primary cultures of mouse macrophages stimulated with TNFalpha. Our results show that although both MEK1 and MEK2 isoforms are present in mouse macrophages, TNFalpha preferentially utilizes MEK1 to stimulate the phosphorylation and activation of p42.


EXPERIMENTAL PROCEDURES

Materials

C3H/HeJ mice were bred at the National Jewish Center Biological Resource Center and were used throughout the study to avoid the possibility of stimulation by trace amounts of endotoxin contaminants(17) . Anti-MEK1 and anti-MEK2 monoclonal antibodies and rabbit anti-MEK antibody were purchased from Transduction Laboratories (Lexington, KY). Monoclonal rat anti-mouse IgG directed against an epitope of the T-cell receptor alpha chain was a kind gift from Dr. John Kappler, National Jewish Center, Denver, CO(18) . Anti-rabbit and anti-mouse IgG F(ab`)(2)-horseradish peroxidase-conjugated antibodies and -[P]ATP (Redivue, 3000 Ci/mmol) were purchased from Amersham Life Sciences, Arlington Heights, IL. Histidine-tagged recombinant kinase-inactive p42 (rMAPK), wild type p42 (rMAPK), wild type mouse MEK1 (rMEK1), and wild type MEK2 (rMEK2) were expressed in Escherichia coli and purified as described previously(19, 20) . The plasmid constructs containing these cDNAs were provided by Dr. Gary Johnson, National Jewish Center, Denver, CO. An additional purified kinase-inactive p42 (K52R), previously described(21) , was a kind gift from Dr. Michael Weber, University of Virginia School of Medicine, Charlottesville, VA. Recombinant mouse TNFalpha was generously provided by Genentech Inc., San Francisco, CA.

Macrophage Isolation and Culture

Bone marrow-derived macrophages were cultured from femoral and tibial bone marrow as described previously (22, 23) at a density of 2.4 times 10^5 cells/cm^2 at 37 °C for 5-6 days. Eighteen hours prior to stimulation, the growth medium was changed to one containing 0.1% (v/v) heat-inactivated fetal bovine serum and no L929 cell-conditioned medium since CSF-1 present in L-cell-conditioned medium has been found to activate the MAPK pathway(9) .

Neutrophil Isolation and Preparation

Neutrophils (PMNs) were isolated by the plasma-Percoll method (24) and were resuspended at 25 times 10^6 cells/ml in KRPD containing 0.25% human serum albumin, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. 25 times 10^6 PMNs were preincubated for 30 min at 37 °C, stimulated with 10 ng/ml PMA for 10 min, and centrifuged at 14,000 times g for 20 s to terminate the reaction. Cell pellets were lysed at 4 °C in lysis buffer as described below.

Biosynthetic Labeling with [S]Methionine

Macrophage monolayers were [S]methionine-labeled as previously described (2) . The cells were lysed on ice in 1 ml of modified RIPA buffer, pelleted by centrifugation at 14,000 times g for 10 min at 4 °C, and the supernatants were precleared with 15 µl of protein A-Sepharose. Five µl (1.25 µg) of monoclonal anti-MEK1, anti-MEK2 antibody, or irrelevant monoclonal IgG were added to the precleared lysates and rotated overnight at 4 °C. Thirty µl of protein A-Sepharose were then added to each tube and rotated for 1 h at 4 °C. The immunoprecipitates were washed five times with lysis buffer and boiled in an equal volume of 2 times Laemmli before separating by 10% SDS-PAGE under reducing conditions. The gel was dried and the radioactive bands were localized by fluorography.

In Vitro Kinase Assay and Immunolocalization

Macrophage monolayers were lysed on ice in 1 ml of ice-cold lysis buffer composed of 10 mM Tris/HCl, pH 7.4, containing 1% (v/v) Triton X-100, 5 mM EDTA, 50 mM NaCl, 5 mM NaF, 0.1% (w/v) bovine serum albumin, 20 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 2 mM Na(3)VO(4). Insoluble nuclear material was pelleted by centrifugation at 14,000 times g for 10 min at 4 °C, and the supernatants were precleared with 15 µl of protein A-Sepharose. 5 µl of anti-MEK antibody (1.25 µg) were then added to the precleared lysates along with 12 µl of protein A-Sepharose and rotated for 2 h at 4 °C. The immunoprecipitates were washed twice with lysis buffer and twice with PAN buffer (10 mM PIPES, pH 7.0, containing 100 mM NaCl and 21 µg/ml aprotinin) and resuspended in kinase buffer (20 mM PIPES, pH 7.2, containing 10 mM MnCl(2) and 20 µg of aprotinin) containing 20 µCi of -[P]ATP and 500 ng of rMAPK as substrate in a final volume of 80 µl. The reactions were incubated at 30 °C for 20 min and were terminated by the addition of 20 µl of 5 times Laemmli sample buffer containing 100 mM dithiothreitol, boiled for 5 min, and separated by SDS-PAGE through 12% gels under reducing conditions and transferred to PVDF membranes for autoradiography and Western analysis. P-Labeled phosphoproteins were detected by autoradiography using Kodak X-Omat AR5 film. In some experiments, rMEK1 and rMEK2 were autophosphorylated in vitro by incubating 600 ng of purified recombinant protein in 17.5 µl of PAN buffer (10 mM PIPES, pH 7.0, containing 100 mM NaCl and 21 µg/ml aprotinin), 2.5 µl of 10 times kinase buffer (200 mM PIPES, pH 7.2, containing 100 mM MnCl(2) and 200 mg/ml aprotinin), and 20 µCi of -[P]ATP per 25-µl reaction mixture at 30 °C for 4 h. Immunolocalization of MEK isoforms by Western blotting was as described by Towbin et al.(25) . Bound antibody was detected with anti-rabbit or anti-mouse IgG F(ab`)(2) horseradish peroxidase-conjugated antibody as the secondary antibody. The enhanced chemiluminescence method was used to detect bound conjugated secondary antibody. All experiments were conducted a minimum of three times.

Phosphoamino Acid Analysis of in Vitro Phosphorylated rMAPK

Phosphoproteins were excised from PVDF membranes and subjected to acid hydrolysis in 6 N HCl at 110 °C for 1 h, lyophilized(26, 27) , and reconstituted in 15 µl of pH 1.9 buffer (2.5% v/v concentrated formic acid, 7.8% v/v glacial acetic acid) containing phosphotyrosine, phosphothreonine, and phosphoserine standards at a final concentration of 5 mg/ml. 15 µl of each sample were loaded on a cellulose TLC plate and chromatographed in a solvent system composed of ethanol:butanol:glacial acetic acid:water (1:1:1:1)(28) .


RESULTS AND DISCUSSION

Distribution of MEK Isoforms in Mouse Macrophages

The distribution of MEK1 and MEK2 in mouse macrophages was investigated by immunoprecipitation of S-labeled macrophage lysates with monoclonal anti-MEK1 and anti-MEK2 antibodies. The specificity of the monoclonal antibodies was determined by immunoprecipitating purified P-autophosphorylated rMEK1 and rMEK2 with each antibody as well as with a rabbit polyclonal anti-MEK antibody that reportedly recognizes shared epitopes on both proteins and an irrelevant monoclonal antibody as a negative control. As can be seen in Fig. 1A, P-labeled rMEK1 was immunoprecipitated with anti-MEK1 antibody but not by anti-MEK2 antibody. Conversely, rMEK2 was immunoprecipitated by anti-MEK2 antibody but not by anti-MEK1 antibody (Fig. 1B). Thus, the anti-MEK1 and anti-MEK2 monoclonal antibodies recognized their respective antigens but failed to cross-react with one another to any significant degree. To determine if both MEK1 and MEK2 were present in mouse macrophages MEK1 and MEK2 were immunoprecipitated from [S]methionine-labeled macrophages and analyzed by SDS-PAGE through 10% polyacrylamide gels followed by fluorography. As can be seen in Fig. 1C, S-labeled MEK1 and MEK2, with respective molecular masses of 45 and 46 kDa, were immunoprecipitated from lysates of mouse macrophages confirming the presence of both MEK isoforms in these cells.


Figure 1: Immunoprecipitation of autophosphorylated rMEK1 and rMEK2 and S-labeled macrophages to investigate MEK antibody specificity. Panel A, autoradiograph of autophosphorylated rMEK1 immunoprecipitated with 1) monoclonal anti-MEK1 antibody, 2) monoclonal anti-MEK2 antibody, 3) polyclonal anti-MEK antibody, and 4) monoclonal IgG antibody. Panel B, autoradiograph of autophosphorylated rMEK2 immunoprecipitated with 1) monoclonal anti-MEK1 antibody, 2) monoclonal anti-MEK2 antibody, 3) polyclonal anti-MEK antibody, and 4) monoclonal IgG antibody. Panel C, autoradiograph of S-labeled macrophages immunoprecipitated with 1) monoclonal anti-MEK1 antibody, 2) monoclonal anti-MEK2 antibody, and 3) monoclonal IgG antibody.



TNFalpha-induced Activation of MEK1

To determine which MEK isoform was activated by TNFalpha, macrophage monolayers were stimulated with an optimal concentration of TNFalpha (40 ng/ml) for time intervals up to 30 min. The cells were then lysed and immunoprecipitated with monoclonal anti-MEK1 or anti-MEK2 antibodies, and the immunoprecipitates were subjected to an in vitro kinase assay using purified rMAPK as substrate in the presence of -[P]ATP. The reaction mixtures were separated by SDS-PAGE, transferred to PVDF membranes, and analyzed by autoradiography and immunoblotting. As shown in Fig. 2A, there was a marked increase in phosphorylation of rMAPK in response to stimulation with TNFalpha. The TNFalpha-stimulated increase in activity of MEK1 was initially detected at 5 min, peaked at 10 min, and diminished to basal levels by 30 min (Fig. 2A). A maximal 5-6-fold increase in phosphorylation of the rMAPK was seen following stimulation with TNFalpha for 10 min. Western blotting of the immunoprecipitates with anti-MEK1 antibody revealed that equivalent amounts of MEK1 were immunoprecipitated at each time point both in the presence and absence of TNFalpha (Fig. 2B). In addition to its ability to transphosphorylate rMAPK, basal autophosphorylation of MEK1 was detected (i) in unstimulated macrophages and (ii) appeared to increase in response to both a medium change and, additionally, in response to TNFalpha (Fig. 2A). Autophosphorylation of MEK1 has also been observed by Gardner and colleagues(29) . We also observed a mobility shift in MEK1 to a higher apparent molecular weight at 10 and 15 min (Fig. 2B). However, by 30 min poststimulation, the mobility shift in MEK1 was no longer apparent. Thus, exposure of macrophages to TNFalpha was associated with the activation of MEK1 with a time course that paralleled the previously reported total catalytic activity of MEK measured by a coupled peptide phosphorylation assay(12) .


Figure 2: In vitro kinase time course and immunoblot of MEK1-immunoprecipitated macrophage lysates. Panel A, autoradiograph of MEK1 activity time course. MEK1 was immunoprecipitated from unstimulated and TNFalpha-stimulated (40 ng/ml) murine macrophage lysates at 2, 5, 10, 15, and 30 min, and immunoprecipitated MEK1 was then subjected to in vitro kinase assay using recombinant kinase-inactive p42(rMAPK) as substrate. Monoclonal IgG is used as a negative control. Panel B, anti-MEK1 immunoblot of the samples shown in panel A above. No Subst., no substrate; Autophos., autophosphorylation of the rMAPK substrate in the absence of cell lysate.



In contrast to these findings, there was no detectable basal or TNFalpha-stimulated activation of MEK2 (Fig. 3A) at either 5 or 10 min. To verify that the inability to detect activation of MEK2 was not due to an inability of the assay procedure to detect activation of the kinase, macrophages were stimulated with a variety of well characterized stimuli including: PMA (10 ng/ml), ATP (100 µM), calcium ionophore A23187 (1 µM), platelet-activating factor (1 µM), and CSF-1 (1000 units/ml). None of these stimuli were capable of activating MEK2 in this assay although MEK2 protein was detected in immunoblots of these immunoprecipitates. However, as shown in Fig. 3A and in marked contrast to macrophages, basal MEK2 activity was detected in unstimulated neutrophils, and a modest increase in activity associated with a decrease in the electrophoretic mobility of rMAPK was detected following stimulation of neutrophil suspensions with PMA (10 ng/ml) for 10 min. Fig. 3B shows an immunoblot of the anti-MEK2 immunoprecipitates confirming that equivalent amounts of MEK2 antigen were immunoprecipitated from unstimulated and stimulated cells. These data thus indicate that stimulation of mouse macrophages with TNFalpha resulted in a selective activation of MEK1 in the absence of a detectable increase in MEK2 catalytic activity. In addition, and in contrast to MEK1, MEK2 appeared to be catalytically silent in unstimulated mouse macrophages.


Figure 3: In vitro kinase time course and immunoblot of MEK2-immunoprecipitated macrophage lysates. Panel A, autoradiograph of MEK2 activity time course. MEK2 was immunoprecipitated from unstimulated and TNFalpha-stimulated (40 ng/ml) murine macrophage lysates at 5 and 10 min and unstimulated (U) and PMA-stimulated (P) (10 ng/ml) human neutrophil (PMN) lysates; the immunoprecipitated MEK2 was then subjected to an in vitro kinase assay using rMAPK as substrate. Autophosphorylated rMAPK is used to localize rMAPK. Monoclonal IgG is used as a negative control. Panel B, anti-MEK2 immunoblot of the samples shown in panel A above. hc, IgG heavy chain.



MEK1 Activation Parallels the Tyr and Thr Phosphorylation of rMAPK

In previously reported work(12) , minimal catalytic activity of MEK was detected in lysates of unstimulated macrophages, and thus the finding of autophosphorylation of MEK1 in anti-MEK1 immunoprecipitates of unstimulated macrophages was somewhat unexpected. To further investigate this finding, we analyzed the phosphoamino acid composition of the MEK1 in vitro kinase reaction mixtures under unstimulated conditions and in response to TNFalpha. Macrophage monolayers were stimulated with TNFalpha (40 ng/ml) for 2-30 min or were incubated in medium alone, lysed, and immunoprecipitated with anti-MEK1, and the immunoprecipitates were subjected to an in vitro kinase assay in the presence of rMAPK and -[P]ATP. The reaction mixtures were then separated by SDS-PAGE, transferred to PVDF membranes, and localized by autoradiography, and the P-labeled rMAPK bands and MEK1 bands were excised and subjected to phosphoamino acid analysis. As can be seen in Fig. 4, in the absence of stimulation, there was minimal phosphorylation of rMAPK on tyrosine and threonine residues. However, in response to stimulation with TNFalpha an increase in phosphorylation of threonine and tyrosine residues was detected that peaked at 10 min. Of note, there was also an absence of phosphorylation of serine residues at each time point in the excised rMAPK bands. However, when the MEK1 band was excised and subjected to phosphoamino acid analysis, there was detectable phosphorylation on serine residues (data not shown). These findings are compatible with the known ability of MEK1 to undergo autophosphorylation on serine residues in vitro(16, 29) . Collectively, these findings indicate that (i) peak activation of MEK1 detected by in vitro phosphorylation of rMAPK coincided with the peak level of Thr/Tyr phosphorylation of rMAPK and (ii) the basal autophosphorylation of MEK1 detected in the absence (or presence) of stimulation was due to phosphorylation on serine residues.


Figure 4: Phosphoamino acid analysis of phosphorylated rMAPK acting as substrate for the in vitro kinase reaction of anti-MEK1 immunoprecipitates of unstimulated and TNFalpha-stimulated (40 ng/ml) macrophage lysates at 2-, 5-, 10-, and 15-min time points. Autoradiograph of phosphoamino acids separated by TLC on cellulose plate is shown. Phosphorylated bands are compared with phosphotyrosine (P-Tyr), phosphothreonine (P-Thr), and phosphoserine (P-Ser) standards visualized on TLC plates by ninhydrin development.



Work reported by Zheng and Guan (16) has shown that autophosphorylation of GST-MEK1 and GST-MEK2 on Ser and Thr residues is sufficient to activate MEK activity in a transphosphorylation assay using ERK1 and ERK2 as substrates although full activation of ERK required upstream activators present in cytosolic extracts of epidermal growth factor-stimulated Swiss 3T3 cells. The results of the present study indicate that while native MEK1 undergoes autophosphorylation in the presence of -[P]ATP in vitro, this was not associated with an increase in the Thr and Tyr phosphorylation of p42 nor, as we have previously shown(12) , is there detectable total MEK catalytic activity in lysates of unstimulated macrophages. In addition, when p42 was immunoprecipitated from lysates of unstimulated and TNFalpha-stimulated [P]orthophosphate-labeled mouse macrophages, radioactivity was detected in p42 following stimulation with TNFalpha but not in lysates of unstimulated macrophages (9) . Similar findings have been reported in human fibroblasts(8) . These findings thus suggest that the constitutive autophosphorylation on Ser residues of MEK1 is insufficient for activation of p42.

Although this report has focused on the activation of MEK1 (and thus the MAPK/ERK pathway) by TNFalpha, recent studies have also shown that TNFalpha activates the JNK/SAPK pathway resulting in the phosphorylation of c-Jun(10, 11) . Indeed, it has been suggested that the SAPK pathway may be the primary pathway of activation by TNFalpha(30) . These reports, however, were conducted predominantly in transformed fibroblast cell lines and in PC12 cells. In recent work (^2)we have confirmed that JNK is activated in macrophages in response to TNFalpha. The significance of the activation of these different MAPK pathways in the regulation of macrophage functions is largely unknown. However, unlike the situation in Swiss 3T3 cells, macrophages do not undergo programmed cell death in response to TNFalpha, and thus the activation of other MAPK/ERK pathways such as p42 may be an important determinant in cell survival and differentiation in response to TNFalpha(2, 22) .

In conclusion, the findings of this and previous work (9, 12) support the concept of TNFalpha activation of the MAPK/ERK pathway in macrophages; specifically, TNFalpha causes aggregation of CD120a (p55), which initiates the rapid and transient activation of an MEKK followed by the selective and sequential activation of MEK1 and p42 in the absence of activation of c-Raf-1 and MEK2. Moreover, the specificity of MEK1 activation by TNFalpha suggests a substrate preference by MEKK for this MEK isoform.


FOOTNOTES

*
This work was supported in part by Public Health Service Specialized Center for Research Grant HL27353 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a Medical Research Council of Canada fellowship grant.

To whom correspondence should be addressed: Dept. of Pediatrics, Neustadt Rm. D 405, National Jewish Center for Immunology and Respiratory Medicine, 1400 Jackson St., Denver, CO 80206. Tel.: 303-398-1188; Fax: 303-398-1851.

(^1)
The abbreviations used are: TNFalpha, tumor necrosis factor-alpha; JNK, c-Jun kinase; SAPK, stress-activated protein kinase; MAPK, mitogen-activated protein kinase; p42, p42 mitogen-activated protein kinase; rMAPK, recombinant wild type or kinase-active MAPK; rMAPK, recombinant kinase dead MAPK; MEK, MAPK kinase; MEKK, MEK kinase or MAPK kinase kinase; CSF-1, colony-stimulating factor-1; PMA, phorbol myristate acetate; PVDF, polyvinyldifluoride; PAGE, polyacrylamide gel electrophoresis; PIPES, 1,4-piperazinediethanesulfonic acid; PMN, neutrophil; rMEK1, recombinant wild type MEK1; rMEK2, recombinant wild type MEK2.

(^2)
B. W. Winston, E. D. Chan, and D. W. H. Riches, unpublished observations.


ACKNOWLEDGEMENTS

We are indebted to Dr. Gary Johnson for providing the kinase or plasmid constructs and for constructive discussion. We also thank Dr. Michael Weber, University of Virginia School of Medicine, Charlottesville, VA for generously providing the kinase-inactive p42 protein K52R.


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