©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Direct Interaction between Ras and the Kinase Domain of Mitogen-activated Protein Kinase Kinase Kinase (MEKK1) (*)

Marijane Russell (1), Carol A. Lange-Carter (1), Gary L. Johnson (1) (2)(§)

From the (1) Division of Basic Sciences, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206 and the (2) Department of Pharmacology, University of Colorado Medical School, Denver, Colorado 80262

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
FOOTNOTES
REFERENCES

ABSTRACT

Mitogen-activated protein kinase kinase kinase (MEKK1) is a serine-threonine kinase that regulates sequential protein kinase pathways involving stress-activated protein kinases and mitogen-activated protein kinases. MEKK1 is activated in response to growth factor stimulation of cells and by expression of activated Ras. We demonstrate that the kinase domain of MEKK1 (MEKK) binds to GST-Ras in a GTP-dependent manner. Purified bacterially expressed MEKK binds to GST-Ras(GTPS) (GTPS is guanosine 5`-3-O-(thio)triphosphate), demonstrating a direct interaction of the two proteins. A Ras effector domain peptide blocks the binding of MEKK to GST-Ras(GTPS). MEKK complexed with GST-Ras(GTPS) is capable of phosphorylating MEK1. These findings indicate that MEKK1 directly binds RasGTP. Thus, Ras interacts with protein kinases of both the Raf and MEKK families.


INTRODUCTION

Ras plays a critical role in multiple signal transduction pathways. Ras is a guanine nucleotide-binding protein, which cycles between an active GTP-bound form and an inactive GDP-bound form (1, 2) . Due to its importance as a mediator of mitogenic stimuli, direct effectors of RasGTP have been searched for other than the GTPase-activating protein (GAP)() that would regulate signal transduction in a Ras-dependent manner. Recently, several groups elegantly demonstrated that the serine-threonine kinase Raf-1 interacts directly with Ha-Ras both in vivo and in vitro. They have mapped the region of interaction to be a portion of the amino-terminal domain of Raf-1 (3-6). An additional effector for RasGTP was recently identified as the catalytic subunit of phosphatidylinositol 3-kinase (PI 3-kinase) (7).

How the RasGTP/Raf-1 interaction participates in the activation of Raf-1 is not clear at this time, but a second regulatory event appears to be required for increased Raf activity. Activated Raf-1 phosphorylates and activates mitogen-activated protein kinase kinase (MEK), which in turn activates mitogen-activated protein kinase (MAPK). Similar to the multiple discreet MAPK pathways that have been identified in yeast (8) , there are also multiple MAPK pathways characterized in mammalian cells (9, 10, 11) . It was due to the homology of several yeast MAPK pathways that the serine/threonine kinase MEK kinase (MEKK1) was originally cloned. This kinase is parallel to Raf-1 in the MAPK cascade in that it acts directly upstream of MEK and is capable of phosphorylating and activating MEK (12) . MEKK1 has also been shown to be the upstream activator of stress-activated protein kinase kinase (SEK), which then phosphorylates and activates stress-activated protein kinase/Jun kinase (SAPK/JNK) family members (13, 14) . SAPK/JNKs are MAPK homologs that have been shown to be activated in response to cellular stress such as heat shock or UV irradiation (15) . While both Raf-1 and MEKK1 are upstream activators of MEK, only MEKK1 has been shown to be an upstream activator of SEK (14) .

An endogenous MEKK1 activity immunoprecipitated from PC12 cells was recently found to be growth factor-regulated in a Ras-dependent manner (16) . The study showed that expression of dominant negative N17Ras inhibited epidermal growth factor-stimulated MEKK1 activity, and that an oncogenic Ras mutant stimulated MEKK1 activity. We were interested to understand if MEKK1 interacts directly with Ras and, if so, which region of MEKK1 is necessary for that interaction. To address these questions, we examined the ability of COS cell-expressed MEKK1 proteins and purified recombinant MEKK1 proteins to bind to GST-Ras.


MATERIALS AND METHODS

COS Cell Expression of MEKK1 Constructs

COS cells were transiently transfected by the DEAE-dextran protocol as described previously (17) . MEKK1 encodes the original full-length MEKK1 described previously (12) , which is actually a partial cDNA of the full-length MEKK1 gene.() MEKK encodes a 1270-base pair fragment of the amino terminus constructed by an EcoRI(322)-PstI(1592) restriction digest. MEKK encodes a 1435-base pair fragment encoding the entire kinase domain constructed by an NcoI(1541)-SspI(2976) restriction digest. The different MEKK1 constructs were ligated into the plasmid pCMV5 for expression studies.

Binding of Expressed MEKK1 to GST-Ras

COS cells expressing various MEKK1 proteins were lysed in EB (1% Triton X-100, 10 mM Tris HCl, pH 7.4, 5 mM EDTA, 50 mM NaCl, 50 mM NaF, 0.1% bovine serum albumin, 0.2 unit/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 2 mM NaVO). Lysates were separated into two equal parts for separate binding reactions. Half of the lysate was incubated with GST-agarose (1.5 µg), while half of the lysate was incubated with GST-Ras-agarose (1.5 µg, Upstate Biotechnology Inc.) for 1 h at 4 °C. The GST-Ras was preincubated at 30 °C for 30 min with 1 mM nucleotide (GDP or GTPS). The nucleotide binding reaction was stopped by adding MgCl to a final concentration of 20 mM. After the 1-h binding reaction, the agarose beads were pelleted at 2000 rpm for 2 min and washed three times with phosphate-buffered saline + 1.0% Triton X-100. The washed agarose beads were boiled in Laemmli SDS sample buffer and the proteins resolved by SDS-polyacrylamide gel electrophoresis. Proteins were transferred onto nitrocellulose for immunoblotting with MEKK1 antibodies. Both MEKK antibody raised against an NH-terminal fusion protein (16) and MEKK antibody raised against a COOH-terminal peptide were used (12) .

In all of the binding studies using COS cell lysates, a significant fraction of the expressed MEKK1 and C4Raf remained in the unbound fraction (data not shown). This was due to a very abundant expression of MEKK1 and C4Raf proteins and the use of limiting GST-Ras-agarose per binding reaction.

Binding of recombinant MEKKto GST-Ras

A construct encoding the kinase domain of a Rat MEKK1 cDNA (95% identical to mouse MEKK1) with a NH-terminal hexahistidine tag was kindly provided by Dr. Melanie Cobb. MEKK was expressed in bacteria, and soluble active enzyme was purified on Ni-NTA-agarose (18). Purified recombinant MEKK was incubated with either GST or GST-Ras in PAN (10 mM PIPES, pH 7.0, 100 mM NaCl, 0.2 unit/ml aprotinin) for 1 h at 4 °C. The agarose beads were pelleted and washed three times in PAN. The washed agarose beads were then incubated in kinase buffer (20 mM PIPES, pH 7.0, 10 mM MnCl, 40 µCi of [-P]ATP, 20 µg/ml aprotinin, and 100 ng of recombinant kinase-inactive MEK1 (KMMEK1) as substrate in a final volume of 150 µl at 30 °C for 20 min. Reactions were terminated by addition of 5 Laemmli SDS sample buffer, boiled, and resolved by SDS-PAGE.

RESULTS

To validate our Ras binding assay, we tested the ability of GST-Ras to bind to expressed C4Raf protein (19) . C4Raf encodes the amino terminus of Raf-1, which has been shown to interact with RasGTP (3, 4, 5, 6) . As seen in Fig. 1A, C4Raf bound to GST-Ras(GTPS)-agarose but not to the GST-agarose control. Additionally, no Raf immunoreactive proteins were detected bound to Ras from control transfected cells (pCMV5).


Figure 1: A, C4Raf binds GST-Ras(GTPS). Lysates of either empty vector (pCMV5) or C4Raf transiently expressed in COS cells, as described under ``Materials and Methods,'' were incubated with GST-agarose (1.5 µg) or GST-Ras(GTPS)-agarose (1.5 µg) for 1 h at 4 °C. The agarose was pelleted and washed three times with lysis buffer and resuspended in sample buffer, and proteins were resolved by SDS-PAGE. Proteins were transferred onto nitrocellulose and immunoblotted with Raf-1 antibody. Similar results were obtained in three independent experiments. B, MEKK1 binds GST-Ras in a GTP-dependent manner. Lysates of either empty vector (pCMV5) or MEKK1 transiently expressed in COS cells, as described under ``Materials and Methods,'' were incubated with GST-agarose (1.5 µg), GST-Ras(GDP)-agarose (1.5 µg), or GST-Ras(GTPS)-agarose (1.5 µg) for 1 h at 4 °C. The agarose was pelleted, washed three times with lysis buffer, and resuspended in sample buffer, and proteins were resolved by SDS-PAGE. Proteins were transferred onto nitrocellulose and immunoblotted with MEKK antibody. A triplet of MEKK1 immunoreactive bands is created by phosphorylation of MEKK1 at multiple sites within the NH terminus (20). The faster migrating MEKK1 immunoreactive species is derived from MEKK1 (12) and is created by initiation of translation at an internal methionine within the MEKK1 open reading frame (C. Lange-Carter, unpublished results). Similar results were obtained in three independent experiments.



MEKK1 Binds GST-Ras in a GTP-dependent Manner

An endogenous MEKK1 activity immunoprecipitated from PC12 cells was recently shown to be growth factor-regulated in a Ras-dependent manner (16) . In order to determine if MEKK1 could bind directly to Ras in vitro, we designed experiments in which various MEKK1 proteins were expressed in COS cells and then incubated with GST-Ras-agarose beads that had been preloaded with GDP or GTPS. MEKK1 transiently expressed in COS cells was capable of binding GST-Ras in a GTP-stimulated manner (Fig. 1B). The predominant triplet of MEKK1 immunoreactive bands is created by phosphorylation of MEKK1 at multiple sites within the NH terminus (20) . The faster migrating MEKK1 immunoreactive species is derived from MEKK1 (12) and is created by initiation of translation at an internal methionine within the MEKK1 open reading frame. MEKK1 from COS cell lysates bound to GST-Ras(GTPS), while very little binding to GST-Ras(GDP) was detectable. With the conditions used MEKK1 binding to GST-Ras(GTPS) was at least 5-fold greater than the binding to GST-Ras(GDP). No detectable MEKK1 was bound to GST.

MEKK1 Binds GST-Ras through Its Kinase Domain

In order to map which domain of MEKK1 is critical for binding to Ras, we expressed different domains of the MEKK1 protein in COS cells. The COOH-terminal kinase domain, MEKK, of MEKK1 binds Ras. MEKK bound to GST-Ras in a GTP-stimulated manner (Fig. 2). Little MEKK bound to GST-Ras(GDP). Thus the GTPS-dependent binding to GST-Ras is encoded within the COOH-terminal catalytic domain of MEKK1. No detectable MEKK was bound to GST. Interestingly, when a MEKK protein was expressed that encodes a 858-base pair fragment of the amino terminus no binding to Ras was detected. Fig. 3shows that in contrast to the ability of Raf-1 to bind to Ras through its amino terminus (Fig. 1A), MEKK failed to bind GST-Ras(GTPS) even though the protein was expressed to levels similar to that of MEKK1 in the same experiment.


Figure 2: MEKK binds GST-Ras(GTPS). Lysates of either empty vector (pCMV5) or MEKK transiently expressed in COS cells, as described under ``Materials and Methods,'' were incubated with GST-agarose (1.5 µg), GST-Ras(GDP)-agarose (1.5 µg), or GST-Ras(GTPS)-agarose (1.5 µg) for 1 h at 4 °C. The agarose was pelleted, washed three times with lysis buffer, and resuspended in sample buffer, and proteins were resolved by SDS-PAGE. Proteins were transferred onto nitrocellulose and immunoblotted with MEKK antibody. Similar results were obtained in three independent experiments.




Figure 3: MEKK does not bind GST-Ras(GTPS). Lysates of empty vector (pCMV5), MEKK, or MEKK1 transiently expressed in COS cells, as described under ``Materials and Methods,'' were incubated with GST-agarose (1.5 µg) or GST-Ras(GTPS)-agarose (1.5 µg) for 1 h at 4 °C. The agarose was pelleted, washed three times with lysis buffer, and resuspended in sample buffer, and proteins were resolved by SDS-PAGE. Proteins were transferred onto nitrocellulose and immunoblotted with MEKK antibody. Similar results were obtained in three independent experiments.



Direct Interaction between Ras and the Kinase Domain of MEKK1

A question that cannot be addressed by the COS cell expression binding studies was: is the interaction between MEKK1 and Ras direct or mediated through other MEKK1-associated proteins present in the COS cell lysate? In order to address this question, we bacterially expressed recombinant MEKK protein. This protein was isolated from bacterial lysates using Ni-NTA-agarose. The soluble protein was an active kinase capable of phosphorylating one of its potential substrates, kinase-inactive MEK1 (KMMEK1) (Fig. 4A). MEKK1 has been shown to phosphorylate and activate either MEK1 or SEK in vitro(12, 14) . In addition the purified MEKK protein was recognized by MEKK antibody when immunoblotted (data not shown). We used the recombinant MEKK protein to determine whether the interaction between MEKK and Ras was direct. Binding of MEKK recombinant protein to GST-Ras(GTPS) was assayed by an in vitro kinase assay on washed beads with kinase-inactive MEK1 as substrate. Fig. 4A demonstrates that there was indeed direct binding of RasGTPS to purified MEKK. The interaction between Ras and MEKK was GTP-stimulated as measured by the increased phosphorylation of KMMEK1 using GST-Ras(GTPS) beads incubated with recombinant MEKK. Little or no KMMEK1 phosphorylation could be detected with GST-Ras(GDP) beads incubated with recombinant MEKK.


Figure 4: A, recombinant MEKK binds GST-Ras(GTPS). Purified recombinant MEKK was incubated with GST-agarose (1.5 µg), GST-Ras(GDP)-agarose (1.5 µg), or GST-Ras(GTPS) (1.5 µg) for 1 h at 4 °C in PAN as described under ``Materials and Methods.'' The agarose beads were pelleted and washed three times in PAN. The washed agarose beads were then incubated in kinase buffer with 100 ng of recombinant kinase-inactive MEK1 (KMMEK1) as substrate in a final volume of 150 µl at 30 °C for 20 min. A control reaction containing wild-type MEK1 (WTMEK1), which autophosphorylates, serves as a marker for the KMMEK1 substrate. Reactions were terminated by addition of 5 Laemmli SDS sample buffer, boiled, and resolved by SDS-PAGE. The autoradiogram is shown. Similar results were obtained in three independent experiments. B, Ras effector domain peptide blocks binding of MEKK to GST-Ras(GTPS). GST-Ras(GTPS)-agarose was preincubated with a Ras effector domain peptide (peptide encodes residues 17-42 of Ha-Ras) or a control peptide ([D-Arg,D-Phe,D-Trp,Leu]substance P peptide) (21) 100 µM of each for 1 h at 4 °C. The agarose was then incubated with MEKK and assayed as described for panelA. The autoradiogram is shown. Similar results were obtained in three independent experiments.



Ras Effector Domain Peptide Inhibits Binding of MEKKto GST-Ras

We also utilized this recombinant binding system to determine which region of Ras was involved in binding MEKK. The binding of MEKK to GST-Ras was blocked by preincubating the GST-Ras(GTPS) beads with a Ras effector domain peptide. The Ras effector peptide encodes residues 17-42 of Ha-Ras and has been shown to block the binding of Ras to other effectors including GAP, Raf-1, and PI 3-kinase (5, 7, 21) . GST-Ras(GTPS)-agarose, preincubated with Ras effector peptide for 1 h prior to incubation with MEKK, was unable to bind MEKK (Fig. 4B); GST-Ras(GTPS) incubated with buffer alone or in the presence of a control peptide ([D-Arg,D-Phe,D-Trp,Leu]substance P peptide) (22) , which does not encode the Ras effector domain, bound MEKK (Fig. 4B). These studies clearly show that MEKK1 interacts directly with Ras in vitro in a GTP-dependent manner via the COOH-terminal region of MEKK1 that encodes the catalytic kinase domain. MEKK1, Raf-1, PI 3-kinase, and GAP all bind to the Ras effector domain.

DISCUSSION

Ras is a critical component of tyrosine kinase growth factor receptor and G-protein-coupled receptor regulation of signal transduction pathways controlling mitogenesis and differentiation (23, 24) . Raf-1 and the p110 catalytic subunit of PI 3-kinase have been shown to directly interact with Ras in a GTP-dependent manner (3, 4, 5, 6, 7) . In this report we have demonstrated that MEKK1 is also a potential Ras effector and selectively binds to Ras in a GTP-stimulated manner. This finding supports at a biochemical level the observation that MEKK1 is activated in a Ras-dependent manner in response to growth factors in PC12 pheochromocytoma cells (16) .

At present, five different proteins have been shown to interact with Ras in a GTP-dependent manner. Two of these proteins (Raf-1 and MEKK1) are protein serine-threonine kinases, one is a lipid kinase (PI 3-kinase), and two additional regulatory proteins (Ras-GAP and neurofibromin) function to regulate Ras GTPase activity (3, 4, 5, 6, 25, 26) . Raf-1, MEKK1, and PI 3-kinase have each been shown to have increased activity in cells expressing GTPase-deficient Ras (7, 16) , consistent with their interaction with RasGTP being involved in their regulation. The ability of Ras to regulate multiple effector proteins is consistent with the diversity of signal transduction pathways controlled by cell surface receptors in a Ras-dependent manner.

A consensus sequence for effector binding to Ras has not been identified. It is also apparent that different functional domains of Ras effectors bind to Ras in a GTP-dependent manner. The Ras binding domain for Raf-1 is encoded in the extreme NH-terminal regulatory domain of Raf-1 (3, 4, 5, 6) , while the Ras binding domain is encoded within the catalytic domain of MEKK1. Both Raf-1 and MEKK1 binding to Ras is blocked by a Ras effector domain peptide. The prediction from these findings is that Raf-1, MEKK1, and other Ras effectors would compete for interaction with RasGTP presumably at the Ras effector domain. The relative abundance and affinity of each Ras effector in different cells may influence the magnitude, onset, and duration of each effector response. Secondary inputs such as phosphorylation of the different Ras effectors may also influence their interaction with RasGTP. It is now possible to begin to define the kinetic properties of Ras effector activation in cells relative to effector affinity for RasGTP. In this regard MEKK1 has been shown to preferentially regulate the SEK/Jun kinase pathways relative to MAPK (14) . Activation of the SEK/Jun kinase pathway is generally slower in onset and maintained at maximal activity longer than the activation of MAPK, consistent with the finding that MEKK1 is persistently activated compared to Raf in PC12 cells (16) . As additional MEKKs are characterized, it will be important to characterize their regulation and interaction with RasGTP. Undoubtedly additional Ras effectors will be identified in the near future.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants GM30324, DK37871, CA09313, and CA58187.The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Division of Basic Sciences, National Jewish Center for Immunology and Respiratory Medicine, 1400 Jackson St., Denver, CO 80206. Tel.: 303-398-1504; Fax: 303-398-1225.

The abbreviations used are: GAP, GTPase-activating protein; PI, phosphatidylinositol; MAP, mitogen-activated protein; MAPK, mitogen-activated protein kinase; MEK, MAP kinase kinase; MEKK, MAP kinase kinase kinase; SEK, stress-activated protein kinase kinase; GTPS, guanosine 5`-3-O-(thio)triphosphate; PIPES, 1,4-piperazinediethanesulfonic acid; KMMEK, kinase-inactive MEK; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase.

C. Lange-Carter, unpublished observation.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.