©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Components of a New Human Protein Kinase Signal Transduction Pathway (*)

Gaochao Zhou, Zhao Qin Bao, and Jack E. Dixon (§)

From the (1) Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0606

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have identified two components of a new protein kinase signaling cascade, MAPK/ERK kinase 5 (MEK5) and extracellular signal-regulated kinase 5 (ERK5). The MEK5 cDNA was isolated by degenerate PCR and encodes a 444-amino acid protein, which has approximately 40% identity to known MEKs. ERK5 was identified by a specific interaction with the MEK5 mutants S311A/T315A and K195M in the yeast two-hybrid system. The proteins were found to interact in an in vitro binding assay as well. ERK5 did not interact with MEK1 or MEK2. ERK5 is predicted to contain 815 amino acids and is approximately twice the size of all known ERKs. The C terminus of ERK5 has sequences which suggest that it may be targeted to the cytoskeleton. Sequences located in the N terminus of MEK5 may be important in coupling GTPase signaling molecules to the MEK5 protein kinase cascade. Both MEK5 and ERK5 are expressed in many adult tissue and are abundant in heart and skeletal muscle. A recombinant GST-ERK5 kinase domain displays autophosphorylation on Ser/Thr and Tyr residues.


INTRODUCTION

Some cell surface receptors respond to extracellular stimuli via a sequential protein kinase cascade (1, 2) . The kinase signaling cascade involves multiple protein kinases, two of which are extracellular signal-regulated kinase (ERK() ; Refs. 3 and 4)() and MAPK/ERK kinase (MEK; Refs. 5 and 6).() Several ERK related proteins have been identified in vertebrates including c-Jun N-terminal kinase (JNK; Refs. 7 and 8)() and p38 (9, 10, 11) . A variety of similar proteins have also been identified in yeast (12, 13, 14) . All of the ERK kinases are activated by phosphorylation on Tyr and Thr residues within a TXY phosphorylation motif, where ``X'' can be Glu, Pro, or Gly (1). There are also a growing number of MEKs (15, 16) . In several cases, specific MEKs have been shown to phosphorylate specific ERKs in a given pathway. MEK1 and MEK2 phosphorylate ERK1 and ERK2 in a mitogenesis or differentiation response; MEK3 and p38 show coordinate activation in the cytokine response, as do MEK4 (i.e. SEK1, the murine homolog of MEK4) and the JNKs in the stress response. Sequential protein kinase cascades have been studied extensively in yeast, where genetic tools have been invaluable in assigning kinase cascades to specific signal transduction pathways (12, 13, 14) . In higher eukaryotic systems, less is known about the specific stimuli responsible for activation of individual signal transduction pathways. Although there are clearly multiple genes encoding ERKs and MEKs, we have no good idea about how many pathways there are in eukaryotic cells.

We have identified a new MEK, which we will refer to as MEK5. When MEK5 was employed in the yeast two-hybrid system, we identified a new member of the ERK family of protein kinases, which we have named ERK5. MEK5 and ERK5 interacted specifically with one another and did not interact with kinases in the MEK1/ERK1 signaling pathway, suggesting that the MEK5/ERK5 protein cascade represents a novel signaling pathway in higher eukaryotes. MEK5 contains unique amino acid sequences in its N terminus, which suggest that this molecule may interact with GTPases such as CDC42. In addition, the C terminus of ERK5 also contains sequences which may target this kinase to the cytoskeletal elements within the cell.


MATERIALS AND METHODS

PCR and cDNA Screening

Two degenerate PCR primers were synthesized based on the conserved amino acid sequences present in MEKs from mammals and yeast (6) . The sense primer was 5`-CTTGGATCCTA(C/T)ATA(C/T)GTNGGNTT(C/T)TA-3` and the antisense primer was 5`-CTTGGATCC(G/T)TCNGGN(C/G)(A/T)CAT(A/G)TA-3`, which corresponds to amino acid sequences YIVGFY and YMSPER, respectively. The underlined BamHI sites were used for cloning the PCR product. Plasmid DNA from a ZAP human fetal brain cDNA library was used as template. The PCR was performed for 35 cycles at 94 °C for 1 min, 50 °C for 2 min, and 72 °C for 2 min. The resultant 0.35-kb PCR products were resolved by 3% Nusieve-agarose gel electrophoresis and subcloned into a M13 vector and sequenced.

A human fetal brain cDNA library was screened (Stratagene), using the cloned MEK5 PCR fragment labeled with a multiprimer DNA labeling kit. Hybridization was carried out at 42 °C in 6 SSC (1 SSC is 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), 1 Denhardt's solution (1 Denhardt's solution is 0.02% Ficoll 400, 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone), 0.1 mg/ml denatured salmon sperm DNA, 50% formamide. Both strands of the DNA corresponding to the clone designated pBluescriptSK MEK5 were sequenced.

The Yeast Two-hybrid Screen

The yeast strain Y190 (MATa, leu2-3, 112, ura3-52 trp1-901 his3-200 ade2-101 gal4 gal 80 URA 3 GAL-lacZ LYS GAL-HIS3 cyh) was a generous gift of Stephen Elledge, Departments of Biochemistry and Human and Molecular Genetics, Baylor College of Medicine. The NcoI fragment encoding amino acid 12 through the stop codon of MEK5 were subcloned into pAS1-CYH2 vector. Y190 was transformed with the pAS1-CYH2-MEK5, pAS1-CYH2 S311A/T315A MEK5 mutant, or pAS1-CYH2 K195M MEK5 mutant using the lithium acetate procedure (17) . Plasmid DNA from a HeLa cell Match Maker cDNA library was then transformed into the yeast strain containing each ``bait'' plasmid. Transformants were selected on SC yeast medium without Leu, Trp, His, and with 35 mM 3-amino-1,2,4-triazole at 30 °C for 7-10 days (17) . The transformants were lifted onto nitrocellulose membrane and assayed for -galactosidase (17) . Plasmids were recovered from -galactosidase positive clones and transformed into Escherichia coli strain DH5F` by electroporation. The clone containing the longest ERK5 open reading frame was named A1.

In Vitro Binding Assay

A full-length cDNA encoding the ERK5 protein sequence was constructed from two partial cDNAs into pBluescriptSK (pBluescriptSK ERK5). This plasmid was used as template for in vitro transcription/translation employing T3 polymerase and a TNT kit (Promega). The in vitro synthesized ERK5 was labeled with [S]Met, and incubated with glutathione-agarose-immobilized GST, GST-MEK1, or GST-MEK5 at 4 °C for 1-2 h in PBST (4 mM NaHPO, 16 mM NaHPO, 150 mM NaCl, 0.5% Triton X-100, pH 7.4), 0.05% -mercaptoethanol, 10% glycerol, 0.2 mM phenymethylsulfonyl fluoride. Following SDS-PAGE, the gel was treated with Amplify (Amersham Corp.) and autoradiographed.

Recombinant Proteins and Kinase Assays

GST-MEK5 was constructed by ligating a 2-kb NcoI fragment encoding amino acid 12 through the stop codon of MEK5 into pGEX-KG (18) . GST-ERK5 was constructed by ligating the NcoI-BstEII fragment of the cDNA clone 3-1 and the BstEII-XhoI fragment of clone A1 (the XhoI site is in the vector) into pGEX-KG digested with NcoI and SalI. GST-ERK5 (residues 1-431) was constructed by subcloning a 1.1-kb NcoI fragment from pBluescriptSK ERK5 (encoding ERK5 amino acids 1-431) into pGEX-KG. GST-fusion proteins were affinity-purified as described (18) . The kinase assays were performed at 30 °C for 10-40 min in a buffer containing 18 mM Hepes (pH 7.5), 10 mM magnesium acetate, 50 µM ATP, and 10 µCi of [-P]ATP. Phosphoamino acid analysis was carried out as described previously (6) .


RESULTS AND DISCUSSION

Isolation and Characterization of MEK5 cDNA

We utilized PCR to identify additional proteins with sequence similarity to MEK. Oligonucleotide primers for PCR amplification were based on the conserved sequences from the yeast STE7 and byr1 genes and the partial amino acid sequence of the human MEK1 (6) . A population of 0.35-kb fragments were produced by PCR using human fetal brain cDNA as a template. The PCR products were subcloned into an M13 vector. MEK1 cDNA was used subsequently as a hybridization probe to eliminate MEK1 and MEK2 from the PCR clones. The non-hybridizing clones were sequenced. Sequence analysis revealed clones encoding MEK3 and MEK4 (16) as well as a novel MEK homolog. We shall refer to the novel sequence as MEK5. The PCR fragment of MEK5 was used to screen a human fetal brain cDNA library. Twenty-four positive clones were analyzed by restriction mapping. The longest insert (2.5 kb) was sequenced and yielded a predicted protein sequence of 444 amino acids (Fig. 1). As shown in Fig. 1, all 11 kinase subdomains are conserved for MEK5 (16) . The amino acid sequence identity of MEK5 with known MEKs is approximately 40%. The Raf-1 phosphorylation and activation motif of MEK1, SXXXS(19, 20) , was also conserved as SXXXT in MEK5, suggesting a similar regulatory mechanism. Like MEK3 and MEK4 (15) , there was an amino acid sequence gap between domains IX and X, resulting in deletion of the proline-rich region seen in MEK1 and MEK2. This region contains phosphorylation sites and negative regulatory sequences (5, 21) . The most divergent region among the MEKs is upstream of kinase subdomain I. MEK5 is distinct from other MEKs in that it contains a long N-terminal sequence. A data bank search with the first 150 residues of MEK5 revealed sequence identity with two proteins that have important roles in cell division, mating and morphogenesis, namely the yeast cell division protein 24 (encoded by Saccharomyces cerevisiaCDC24 gene (22) and the homologous protein encoded by the scd1 gene from Schizosaccharomyces pombe(23) . The amino acid sequence alignment of MEK5 with the encoded proteins of the scd1 gene and CDC24 gene is shown in Fig. 2.


Figure 1: Primary structure of MEK5. The primary sequence of MEK5 was deduced from the sequence of cDNA clones isolated from human fetal brain library. The PILEUP program (Wisconsin Genetics Computation Group) was used to align the amino acid sequence of MEK5 with that of MEK1 and MEK2 (6), MKK3 and MKK4 (16), and SEK1 (15). Romannumerals designate protein kinase domains; *, conserved lysine in the ATP binding site and the activating phosphorylation sites of MEK1; shading indicates identity of all six sequences.




Figure 2: Sequence alignment of MEK5 with the scd1 gene and CDC24 gene encoded proteins.



Genetic and biochemical studies have demonstrated that the CDC24 encoded protein has GDP release activity. When the CDC24 encoded protein binds to the GTPase encoded by CDC42, it enhances the GTP/GDP exchange (24) . In a similar fashion, the scd1 encoded protein enhances the nucleotide exchange of the corresponding S. pombe encoded protein (cdc42sp). The binding domain of scd1 that recognizes cdc42sp is located C-terminal to amino acid 671 of scd1 (23). It is this region of scd1 that shows sequence identity to MEK5 as well as CDC24. Although the function of this sequence is unknown, it is possible that the N terminus of MEK5 interacts with the mammalian equivalent of CDC42. The mammalian equivalent of yeast CDC42 is Rac (25). The amino acid sequences in question may provide a mechanism for coupling GTPases (i.e. CDC42 and Rac) to downstream protein kinase signaling cascades.

A Partnership between MEK5 and ERK5

Mutations in activating phosphorylation sites and the conserved lysine residue in the ATP binding site of MEK1 result in dominant negative mutants that interfere with the kinase function (26, 27, 28) . We anticipated that similar modifications in MEK5 might enhance its interaction with upstream and/or downstream signaling molecules. To enhance the enzyme-substrate interaction, mutations that led to the dominant negative phenotype of MEK1 were made in MEK5, i.e. S311A/T315A and K195M (20) . Using the yeast two-hybrid system, three ``bait'' plasmids containing sequences encoding wild-type MEK5, MEK5 S311A/T315A, or MEK5 K195M were used to screen a HeLa cell library. From a pool of 4.2 10 transformants (2 10 from MEK5, 2 10 from S311A/T315A mutant, and 0.2 10 from K195M mutant), 6 clones were identified that encoded a novel ERK, which we shall refer to as ERK5. Five clones in this pool were obtained with the S311A/T315A mutant of MEK5 and one with the K195M mutant. All clones contained the TEY activation motif. The product encoded by the longest ERK5 clone, A1, interacted with MEK5 in the yeast two-hybrid system, but not with MEK1 or MEK2 (Fig. 3, A-C). In addition, MEK5 does not interact with p38 or Raf A, B, or C in the yeast two-hybrid system. This result supports the highly specific nature of the interactions between MEK5 and ERK5. It should be noted that wild-type MEK5 also interacted with ERK5, although we did not isolate any ERK5 clones from the initial yeast two-hybrid screen. We anticipate that dominant negative MEKs may be useful in the identification of additional upstream as well as downstream signaling proteins in similar cascades.


Figure 3: Specific interaction between MEK5 and ERK5. The ERK5-containing plasmid A1 (ERK5 fusion with gal4 activation domain) was cotransformed into yeast strain Y190 with vector pAS1-CYH2 or ``bait'' plasmids of MEK1, MEK2, MEK5, MEK5 S311A/T315A, and MEK5 K195M. A, transformants were restreaked to SC medium minus Leu, Trp, and His, plus 3-amino-1,2,4-triazole (35 mM) to select for interaction. B, the transformants were also restreaked to SC medium minus Leu and Trp and screened for -galactosidase activity. C, diagram showing the orientation of each transformant. D, in vitro translated ERK5 (1) was incubated with glutathione-agarose-immobilized GST (2), GST-MEK1 (3), GST-MEK5 (4), and GST-MEK5(K195M) (5) followed by washing and SDS-PAGE. After treatment with Amplify, the SDS gel was autoradiographed with Kodak x-ray film.



To obtain the full-length clone for ERK5, the 2.5-kb insert of A1 was used to screen a human fetal brain cDNA library. From 60 positive clones, two were used to complete sequence analysis. The 2828-base pair sequence contained an open reading frame of 2445 nucleotides encoding 815 amino acids (Fig. 4). A number of residues important for the kinase activity of ERKs are conserved, including the TEY activation motif (1) . ERK5 contains a 400-amino acid C-terminal domain, which houses two proline-rich regions. The first proline-rich region, consisting of 32 amino acids, contains 16 prolines. The second proline-rich region (124 amino acids with 44 prolines) contained several small Pro-Ala repeats, (PA), followed by Pro-Thr repeats, (PT), and three repeats of PPGP. The (PA) repeat is present in myosin light chain kinase, and this sequence has been shown to directly interact with actin, targeting the kinase to a specific location in the cell (29) . The unique ERK5 C-terminal sequence may serve as a localization domain and/or a regulatory domain. It is interesting to speculate that the (PA) sequences which target myosin light chain kinase to actin may also target ERK5 to a similar location. Northern blot analysis of ERK5 mRNA (3.1 kb) and MEK5 mRNA (2.7 kb) demonstrated they are expressed in many human tissues and that the RNAs are most abundant in heart and skeletal muscle (data not shown).


Figure 4: The primary structure of ERK5. A, the deduced amino acid sequence of ERK5 was aligned with that of ERK1 and ERK2, JNK1 and human p38.*, lysine residue in the conserved ATP binding site and important catalytic amino acid residues; &cjs1219;, activating phosphorylation motif. Proline-rich region 1 and proline-rich region 2 are underlined.



To further document the specific interaction between MEK5 and ERK5, in vitro binding assays were conducted (Fig. 3D). In this experiment, full-length ERK5 was synthesized in an in vitro transcription/translation system. As shown in Fig. 3D, ERK5 binds to GST-MEK5 and the GST-MEK5 mutant K195M, but not to GST alone or to GST-MEK1. Thus, MEK5 specifically interacted with ERK5 in both our in vitro system and in the yeast two-hybrid system.

Protein Kinase Activity of MEK5 and ERK5

GST fusion protein containing full-length MEK5 could not be overexpressed in E. coli. However, elimination of the N-terminal 11 residues produced a MEK5 fusion protein which could be overexpressed and affinity-purified. The purified protein showed no protein kinase activity toward ERK1, JNK1, or GST-ERK5. However, a weak autophosphorylation activity could be detected with the wild-type enzyme but not with the MEK5 K195M mutant (data not shown). These experiments suggested that additional modifications are required for full kinase activity.

Two GST fusion proteins of ERK5 were created for expression in E. coli, a full-length GST-ERK5 and a truncated GST-ERK5 with the C-terminal 384 residues removed. This truncated ERK5 corresponds to the kinase domain of the protein, i.e. GST-ERK5(1-431). Both fusion proteins were purified on glutathione-agarose affinity columns. The full-length GST-ERK5 protein had no detectable protein kinase activity (Fig. 5A and data not shown). The purified GST-ERK5(1-431) showed basal autophosphorylation activity, as was evident from the radioactive band of the predicted molecular weight following SDS-PAGE analysis (Fig. 5A). Phosphoamino acid analysis of the radioactive protein revealed phosphotyrosine, phosphothreonine, and phosphoserine (Fig. 5B). These results suggest that the kinase domain of the full-length ERK5 is rendered inactive by the presence of the C-terminal 384 residues. Removal of the C-terminal domain results in the ability of the functional ERK5 kinase domain to display its kinase activity. The fact that ERK5, like ERK1, has a TEY motif and that phospho-Ser/Thr and Tyr are observed in the analysis of labeled ERK5, suggests that the catalytic properties of the two kinases may be similar.


Figure 5: Autophosphorylation of recombinant GST-ERK5 (1-431). A, autophosphorylation of purified full-length GST-ERK5 and GST-ERK5 (1-431). The samples were resolved on a SDS-PAGE gel followed by autoradiography. The arrows indicate the position of the purified GST-ERK5 and GST-ERK5 (1-431). B, phosphoamino acid analysis of autophosphorylated GST-ERK5 (1-431 shown in A. P-S, phosphoserine; P-T, phosphothreonine; P-Y, phosphotyrosine.



In summary, we have identified two novel kinases that interact selectively with one another in what appears to represent a new mammalian signal transduction pathway. Our results suggest that there are no interactions between the proteins in the MEK1/ERK1 pathway and the MEK5/ERK5 pathway, suggesting that the two signaling pathways have either different or complementary functions. Our results also suggest that the two different signaling pathways most likely do not have overlapping functions in the cell. We are currently attempting to identify both upstream activators of MEK5 as well as downstream substrates of ERK5. The unusual length of the C-domain of ERK5 suggests that the sequences located here may undergo additional post-translational modifications as well as play roles in regulating or targeting of ERK5.


FOOTNOTES

*
This work was supported by National Institutes of Health NIDDKD Grant 18024 and a postdoctoral fellowship from the American Diabetes Association (to G. Z.). 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) U25265 and U25278.

§
To whom correspondence should be addressed: Dept. of Biological Chemistry, University of Michigan Medical School, Rm. 5416, Medical Science I, Ann Arbor, MI 48109-0606. Tel.: 313-764-8192; Fax: 313-763-4581.

The abbreviations used are: ERK, extracellular-regulated kinase; MAP, mitogen-activated protein; MAPK, MAP kinase; MAPKK or MKK, MAP kinase kinase; MEK, MAPK/ERK kinase; JNK, c-Jun N-terminal kinase; kb, kilobase pair(s); PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase.

ERK is also referred to as mitogen-activated protein kinase (MAPK) in the literature. We will employ the ERK nomenclature.

MEK is also referred to as mitogen-activated protein kinase kinase (MAPKK or MKK) in the literature. We will employ the MEK nomenclature.

JNK is also referred to as stress-activated protein kinase (SAPK) in the literature.


ACKNOWLEDGEMENTS

We thank K.-L. Guan for plasmids encoding MEK1, MEK2 and ERK1. We also thank B. Yashar and R. Stone for critical reading of the manuscript. We also acknowledge the help of J. Clemens, M. Wishart, and C. F. Zheng with the yeast two-hybrid system.


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