Identification of Substrates and Regulators of the Mitogen-activated Protein Kinase ERK5 Using Chimeric Protein Kinases*

Jessie M. English, Gray Pearson, Richard BaerDagger , and Melanie H. Cobb§

From the Departments of Pharmacology and Dagger  Microbiology, The University of Texas Southwestern Medical Center, Dallas, Texas 75235-9041

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Extracellular signal-regulated protein kinase 5 (ERK5) is a recently discovered orphan mitogen-activated protein kinase for which no substrates or strong activators have been described. Two ERK5 chimeras were created as a novel approach to discover its substrates and upstream regulators. One chimeric protein contained the N-terminal domain of the ERK5 catalytic core (subdomains I-IV) and the C-terminal domain of the ERK2 catalytic core (subdomains V-XI). This chimera was highly responsive to stimuli that regulate ERK2 in vitro and in cells. A second chimeric protein consisted of the N-terminal domain of ERK2 (subdomains I-IV) and the C-terminal domain of the ERK5 catalytic core (subdomains V-XI). This chimera was activated in bacteria by coexpression with a constitutively active mutant of MEK1. Using the activated chimera, we identified three in vitro substrates of ERK5. Assaying ERK5 activity in immune complexes with one of these substrates, c-Myc, we determined that the ERK5 catalytic domain is activated by V12 H-Ras and to a lesser extent by phorbol ester but not by constitutively active mutants of Raf-1. Thus, ERK5 is a target of a novel Ras effector pathway that may communicate with c-Myc.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Elucidation of signaling pathways from the cell membrane to the nucleus has been furthered by the discovery and analysis of mitogen-activated protein (MAP)1 kinase pathways (1-12). Each of these pathways, which have been found in all eukaryotes from unicellular organisms to plants and animals, contains regulatory molecules upstream of a kinase module consisting of a MAP/ERK kinase kinase (MEKK) that phosphorylates and activates a MEK that, in turn, phosphorylates and activates an ERK (13). These kinase modules are differentially responsive to a variety of cellular signals and contribute to the generation of distinct cellular outcomes. Over a dozen MAP kinase family members have been identified in mammals. Many of these lie in the three best delineated mammalian MAP kinase modules (1-12). These are the mammalian ERK module (consisting of Raf isoforms, MEK1, MEK2, ERK1, and ERK2), which responds strongly to mitogenic signals in most cells and two modules that are often activated by stress stimuli, the c-Jun-N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and the p38 pathways. The ERK and stress-sensitive pathways are transducers of proliferative, differentiating, and inflammatory signals throughout the cytoplasm and in the nucleus.

Analysis of the yeast genome indicates the presence of at least five yeast MAP kinases (14). An extrapolation based on the greater complexity of mammalian genomes suggests that there may be more than 50 MAP kinases in mammals. Identifying novel MAP kinases and delineating their regulation and specificities will provide important information about molecular mechanisms underlying signal transduction and its specificity. Searches for novel MAP kinase cascades using homology cloning techniques, data base analysis, and antibodies have identified several orphan pathways. These include ERK3 kinase/ERK3, ERK4, and MEK5/ERK5 (15-22). Neither the signals activating these orphan pathways nor their substrates and the cellular outcomes of their activation have been determined. Because they were not identified in their natural physiological contexts, a variety of strategies to determine their roles in mammalian signaling are needed. Members of the MAP kinase family require dual phosphorylation for high activity and have not yet been successfully activated by mutagenesis (23-28). Thus, a general method to activate MAP kinases will be of considerable value because in most cases an active upstream MEK with the appropriate specificity will be unavailable.

To discover novel MAP kinase pathways, we used a PCR-based screen to isolate cDNA clones of previously unknown MEKs, the constituents of the module with the greatest substrate specificity. One such clone encoded MEK5 (20, 21). Its putative downstream target ERK5 was cloned both in a two-hybrid screen with MEK5 and in a screen for novel ERKs (21, 22). No substrates for ERK5 have been identified, and no extracellular stimuli have been shown to highly activate ERK5 or MEK5. As a first step in understanding this pathway, we wanted to identify ERK5 substrates and activators upstream of MEK5 in the cascade. Because we were not successful in activating MEK5 either in vitro or in vivo, we chose to exploit our understanding of MAP kinase structure to circumvent this problem.

Herein we describe a novel approach using chimeric proteins consisting of the orphan MAP kinase ERK5 and a well studied MAP kinase, ERK2, the activators of which have been identified. A chimeric protein consisting of the N-terminal domain of ERK2 (subdomains I-IV) and the C-terminal domain of the ERK5 catalytic core (subdomains V-XI) contains the protein substrate binding regions of ERK5. This chimera is expected to display a protein substrate specificity identical to ERK5 itself (29-32). Using an activated form of this chimera, we found that the product of the c-myc proto-oncogene (33, 34) was an ERK5 substrate, which facilitated the further analysis of this orphan MAP kinase module. Potential activators of the pathway were tested using c-Myc as the ERK5 substrate. We demonstrate here that the ERK5 catalytic domain is activated by a constitutively active form of H-Ras but not by constitutively active Raf. These findings indicate that the ERK5 cascade lies in a newly identified Ras effector pathway.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

ERK5 and Mutants-- To construct a truncated form of ERK5 (ERK5kin), the sequence encoding amino acids 1-409 was subcloned into the XbaI and XhoI sites of pGEX-KG using Vent polymerase (New England Biolabs, Beverly, MA). Full-length human ERK5 in pCDNAIII (generously provided by J. D. Lee, Scripps Institute, San Diego, CA) was used as template with primers that inserted XbaI and XhoI sites. The sequence of this clone contained nucleotide differences compared with the previously published sequence (22) that specified valine rather than alanine at residue 23. Full-length ERK5 was subcloned into pGEX-KG by digesting pCDNAIII/ERK5 with XbaI and filling in with Klenow enzyme. After digestion with SphI, the sequence following the catalytic domain of ERK5 (ERK5 tail) was ligated into pGEX-KG/ERK5kin previously digested with SphI and a XhoI site blunt ended with Klenow enzyme. The inactive ERK5kin mutant K84M (25, 30, 35) was also constructed using Vent polymerase. The 3' primer encoded a methionine in place of lysine 84 and spanned a unique BstEII site. The 5' primer added a XhoI site. The resulting PCR product was subcloned into the XhoI and BstEII sites of pGEX-KG/ERK5kin.

Chimeras-- To create chimeras between human ERK5 and rat ERK2 (Fig. 1A), the four PCR products encoding the residues indicated below were generated using Vent polymerase and oligomers that added the appropriate restriction enzyme sites for subcloning into the following sites of pGEX-KG: 1) N terminus of ERK5 (amino acids 1-139), XbaI and SacI; 2) N terminus of ERK2 (amino acids 1-105), NcoI and SacI; 3) C terminus of ERK5kin (amino acids 140-409), SacI and HindIII; and 4) C terminus of ERK2 (amino acids 106-358) SacI-HindIII. In each case, a SacI site was introduced that resulted in a conservative Asp right-arrow Glu mutation at amino acid 138 of ERK5 and amino acid 104 of ERK2 (Fig. 1B). pGEX-KG/ERK5kin and NpT75/ERK2 were used as the templates for the PCRs. The chimeras were prepared by digesting the N-terminal constructs with SacI and HindIII. The PCR products encoding the C-terminal domains were first digested with HindIII followed by a partial digestion with SacI. The appropriate SacI-HindIII fragment was then ligated into pGEX-KG containing the appropriate N-terminal domain. The sequences of the junctions were VQELMESDLH for ERK2/5 and VLELMETDLY for ERK5/2 (Fig. 1B). The ERK5/2 chimera was digested with NheI and XhoI (these sites were added by the original PCR oligomers) and subcloned into the NheI and SalI sites of pCEP4HAB.

Expression and Purification of Recombinant Proteins-- All constructs derived from PCR were sequenced to confirm that no changes in coding sequence had been introduced. Bacteria were grown at 30 °C, and 40 µM isopropyl-1-thio-beta -D-galactopyranoside was used to induce expression of all proteins. Expression of glutathione S-transferase (GST)-ERK5kin and GST-ERK5kin K84M was induced for 8 h; GST-ERK5, GST-ERK5/2, GST-ERK2/5, and GST-ERK2/5-P (the phosphorylated form from coexpression with MEK1R4F, a constitutively active mutant of MEK1) were induced for 18 h. The GST fusion proteins were purified on glutathione-agarose using standard protocols (36). Histidine-tagged (His6) ERK2 (25), MEK1, MEK1R4F (kindly provided by Natalie Ahn, Univ of Colorado, Boulder, CO), and GST substrates were expressed as described (37). DNAs encoding the protein kinase Mnk-1 and the ternary complex factor Elk-1 were obtained from Tony Hunter (38) and Peter Shaw (39). Coexpression of the GST-ERK2/ERK5 chimera with untagged MEK1R4F in pBB131 employed the two-plasmid system for bacterial expression described previously (40). Active ERK2, p38, and SAPKalpha were prepared as described (40); these were provided by Andrei Khokhlatchev. cDNAs encoding SAPKalpha and p38 were originally obtained from J. Kyriakis (Harvard) and J. Han (Scripps).

Protein Kinase Assays-- Protein kinases and substrates were incubated in 30 µl of kinase buffer (10 mM Hepes, pH 8.0, 10 mM MgCl2, 1 mM benzamidine, 50 µM ATP, and [gamma -32P]ATP (10-30 cpm/fmol)) for 30 min at 30 °C. Reactions were stopped by addition of 7 µl of 5× electrophoresis sample buffer and heated for 2 min at 100 °C. Substrates used were myelin basic protein (MBP), GST-Mnk-1, and the following fusion fragments: GST-Myc-(1-143), GST-ATF2-(1-254), GST-Elk-1-(1-205), and GST-Tal2-(53-108). Substrate protein concentrations were equalized by Coomassie Blue staining of the correct band in the recombinant protein preparation and were approximately 0.1 mg/ml in the kinase reactions. MBP was used as the screening substrate, because it is commonly phosphorylated by MAP kinase family members. However, because many other kinases phosphorylate MBP, c-Myc fragments were used as the ERK5 substrate in subsequent assays. Significantly less Elk-1 was assayed than other protein substrates. Thus, its capacity to serve as a substrate for a given MAP kinase may be underestimated.

Mammalian Cell Culture and Transfection-- ERK5kin and ERK5 were subcloned from the corresponding pGEX-KG constructs into pCEP4HAB to incorporate the hemagglutinin (HA) epitope tag. Both were subcloned into the NheI and XhoI sites using restriction sites that had been added by the PCR primers with blunt ending with Klenow enzyme for ERK5. pCMV5/V12Ras, pCMV5/Raf-1, pCMV5/Raf-1S259D, and pCMV5/Raf BXB were as described (41) and were kindly provided by Jeff Frost. pCMV5/MEKK1 was provided by Shuichan Xu (42).

293 human kidney cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 1% L-glutamine. Cells were transfected at 50-80% confluency using calcium phosphate DNA coprecipitation (43) and harvested 48 h after transfection; the last 12-18 h of transfection were in the absence of serum. Cell lysates were prepared in buffer containing 50 mM Hepes, pH 8.0, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 100 mM NaF, 0.2 mM sodium orthovanadate, 1% phenylmethylsulfonyl fluoride, 1 mM benzamidine, 1 µg/ml pepstatin, and 1 µg/ml leupeptin. For assays of ERK5kin activity, 293 cells were cotransfected with 2 µg of pCMV/V12 H-Ras and 5 µg of pCEP4HA/ERK5kin, and HA-ERK5kin was harvested by immunoprecipitation.

Immunoblotting-- A synthetic peptide (RISAAAALRHPFLAKY) derived from subdomain XI of ERK5 was conjugated to Limulus polyphemus hemocyanin and injected into rabbits as described (19) generating anti-ERK5 antiserum S741. Equal amounts of protein were loaded on 10% polyacrylamide gels in sodium dodecyl sulfate for immunoblotting with S741 or Y691 as described (19, 42).

Transfected V12 H-Ras was detected using a mouse monoclonal antibody purchased from CalBiochem (OP40). Raf-1 and Raf mutants were detected using an antibody from Santa Cruz (SC133). Dually phosphorylated active endogenous ERK1 and ERK2 were detected using a rabbit antibody from Promega (V6671) as described previously (44).

Immunoprecipitation-- Lysates from transfected cells were probed with a mouse monoclonal antibody to the HA epitope tag (12CA5, Babco, Richmond, CA) to estimate the amount of HA-ERK5 in each lysate. Lysate volumes containing equal amounts of HA-ERK5 were incubated with the anti-HA antibody and protein A-Sepharose at 4 °C for 2 h with rotation. The beads were washed once with lysis buffer; three times with 0.25 M Tris, pH 8.0, 0.3 M NaCl; and once with 10 mM Hepes, pH 8.0. Kinase assays were performed with 20 µl of beads in a 50-µl reaction. Reaction conditions were the same as described above except that 100 nM staurosporine was added. Reactions were stopped by the addition of 13 µl of 5× electrophoresis sample buffer.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Expression of ERK5 Chimeras-- To probe the function and regulation of ERK5, we utilized chimeric proteins fusing individual domains of ERK2, the activators and substrates of which have been identified, to the complementary domains of the orphan MAP kinase ERK5. A region that connects the N-terminal to the C-terminal domain of ERK2 and ERK5, in the vicinity of kinase subdomain V, was selected as the site to fuse the chimeras (Fig. 1, A and B). A consideration of sequences and structures of protein kinases indicates that this is a variable region and that chimeras formed at this point between the two domains are unlikely to contain disrupted tertiary structures (29, 32, 45, 46). The ERK2/ERK5 chimera contained residues 1-105 of ERK2 and 140-409 of ERK5. ERK5 contains an extension of approximately 400 residues C-terminal to the conserved MAP kinase core sequence; these residues were truncated from the ERK2/5 chimera so that each chimera could be compared with wild type ERK2. The ERK5/ERK2 chimera contained residues 1-139 of ERK5 and 106-358 of ERK2. An ERK5 mutant, ERK5kin, was truncated after residue 409 of ERK5 as a control for the chimeras and for comparison to ERK2. This mutant contains the complete kinase domain of ERK5 and is roughly equal in length to ERK2.


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 1.   Chimeric proteins. A, diagram of ERK5/2 and ERK2/5 chimeric proteins. The phosphorylation lip contains the activating phosphorylation sites threonine and tyrosine with an intervening glutamate, and the substrate binding site is encoded by the C-terminal portion of each chimera (shaded). ERK5/2 contains residues 1-139 of ERK5 linked to residues 106-358 of ERK2. ERK2/5 contains residues 1-105 of ERK2 linked to residues 140-409 of ERK5. B, the junctions occur between subdomains IV and V and have the indicated sequences. C, duplicate Western blots of recombinant GST-ERK2, GST-ERK5kin, GST-ERK5, and GST-tagged chimeras. ERK2/5-P refers to GST-ERK2/5 purified from bacteria coexpressing untagged MEK1R4F. Equal amounts of protein were loaded, and the blots were probed with antisera raised against peptides contained in kinase subdomain XI. Top, blot probed with a 1/1000 dilution of anti-ERK5 antiserum S741; bottom, blot probed with a 1/5000 dilution of anti-ERK1/2 antiserum Y691.

Characterization and Activation of the ERK5 Chimeras-- The chimeras were expressed as GST fusion proteins, and the presence of the appropriate C-terminal domain in each chimera was confirmed by Western blotting with antipeptide antibodies (Fig. 1C). Full-length ERK5 sometimes contains breakdown products as revealed by smaller immunoreactive bands. Without activation, the protein kinase activity of the ERK5/2 chimera strongly resembled wild type ERK2, with a specific activity toward MBP in the low nmol/min/mg range (Fig. 2A). On the other hand, the activity of the ERK2/5 chimera, like that of ERK5 itself, was below the limit of detection under the same conditions. Based on findings described below, this is most likely due to a very low basal activity and not to an inability to recognize MBP.


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 2.   Activities of ERK5, ERK2, and chimeras in vitro. A, GST-ERK5kin, GST-ERK5/2, GST-ERK2, and GST-ERK2/5 were incubated with and without MEK1R4F under phosphorylating conditions, and the resulting MBP kinase activity in a 5-µl aliquot was measured. An autoradiogram is shown. B, diagram of coexpression strategy for activating GST-ERK2/5. A plasmid containing GST-ERK2/5 and conferring ampicillin resistance was cotransformed in the BL21DE3 strain of E. coli with a plasmid containing untagged MEK1R4F and conferring kanamacin resistance. Bacteria were grown under dual antibiotic selection to induce production of active GST-ERK2/5. C, autoradiogram of MBP kinase assay with GST-ERK2/5, GST-ERK2/5 purified from bacteria coexpressing untagged MEK1R4F (ERK2/5-P), GST-ERK5kin, and GST-ERK5. Four other protein preparations in which ERK2/5-P was isolated from cultures originating from four independent bacterial colonies gave similar increases in ERK2/5 MBP kinase activity.

Previously published results with p38/ERK1 chimeras suggested that the N-terminal domain of a MAP kinase family member would determine which regulatory events would activate the chimeric enzyme (32). If this is generally correct for other family members, then the ERK5/2 chimera should fail to be activated by MEK1 or MEK2 of the ERK2 pathway. We therefore determined whether the constitutively active MEK1 mutant MEK1R4F (47) phosphorylated and activated the ERK5/2 chimera in vitro. The activity of the ERK5/2 chimera increased significantly upon treatment with MEK1R4F (Fig. 2A), suggesting that it is recognized as a consequence of the C-terminal domain of ERK2. To confirm that activation of the chimera was not due to the ability of MEK1 to recognize ERK5, ERK5kin was also tested as a MEK1 substrate; phosphorylation of MBP by ERK5kin was not enhanced by prior incubation with MEK1R4F (Fig. 2A). In keeping with the activity data, ERK5/2 was phosphorylated weakly by MEK1R4F, whereas ERK5 was not (data not shown). These results indicate that the C-terminal domain also contributes to recognition of MAP kinases by MEKs and, further, that the ERK5/2 chimera will not selectively respond to ERK5 activators.

To search for ERK5 substrates using the ERK2/5 chimera, attempts were made to activate the ERK2/5 chimera with MEK1R4F in vitro. The ERK2/5 chimera was a poor in vitro substrate MEK1. Thus, to activate the ERK2/5 chimera, we utilized the bacterial coexpression system that we had previously developed to produce other purified phosphorylated MAP kinases (40). GST-ERK2/5 was expressed from one plasmid in bacteria either with or without coexpression of MEK1R4F from a second plasmid (Fig. 2B). Coexpression of ERK2/5 with MEK1R4F resulted in activation of GST-ERK2/5, as assessed by its enhanced ability to phosphorylate MBP (Fig. 2C), even though it was not activated by MEK1R4F in vitro.

Identification of Substrates-- Compared with three other purified active MAP kinase family members examined, the activity of the activated ERK2/5 chimera was lower, and its substrate specificity was distinct (Fig. 3, A and B). Under a defined set of conditions, the chimera phosphorylated MBP and an N-terminal fragment of c-Myc to a greater extent than the other MAP kinase substrates tested. The chimera did not phosphorylate the ternary complex factor Elk-1, the transcriptional regulators ATF2 and Tal2, or the protein kinase Mnk-1 well (Fig. 3, A and B) (39, 7, 48, 38). In contrast, p38, which had approximately the same relative activity toward MBP and c-Myc, also phosphorylated ATF2 and Mnk-1 equivalently, whereas ERK2 phosphorylated Mnk-1 and Elk-1. SAPKalpha phosphorylated ATF2 strongly but MBP to a much lesser extent than the ERK5/2 chimera. Both SAPKalpha and ERK2 phosphorylated Elk-1 sufficiently to induce an electrophoretic mobility shift, whereas p38 did not (Fig. 3A). In addition, the chimera phosphorylated its putative upstream regulator MEK5 (Fig. 4A). In contrast, MEK5 was not a substrate for active ERK2, p38, SAPKalpha , or the ERK5/2 chimera.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 3.   Substrate specificity of MAP kinases. A, purified active ERK2, p38, SAPKalpha , and ERK2/5-P derived from coexpression in bacteria with upstream activators (40) and ERK2/5 and ERK5kin were assayed in vitro with equal amounts of the substrates, except for Elk-1, which was present at a concentration of 10%. Autoradiograms show a representative experiment measuring substrate phosphorylation, and longer exposures are shown for TAL2 and Elk-1. B, the substrate specificities of purified recombinant active ERK2, p38, SAPKalpha , and ERK2/5 compared are quantitated. For each kinase, the greatest incorporation into a substrate was assigned a value of 100, and incorporation by that kinase into the other substrates is represented as a percentage. C, HA-ERK5kin from lysates of cells cotransfected with V12H-Ras was immunoprecipitated with the antibody to the epitope tag and assayed as above. A shorter exposure of the autoradiogram is shown for c-Myc and MBP.


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 4.   MEK5 is a substrate for ERK5. A, autoradiogram of kinase assays performed with recombinant ERK2/5-P, ERK5kin, and ERK5/2 (left) or ERK2, p38, and SAPKalpha (right) using GST-MEK5 as the substrate. B, autoradiogram of immune complex kinase assay from 293 cells transfected with V12 H-Ras alone or cotransfected with HA-ERK5kin and assayed using GST-MEK5 as the substrate.

To determine whether the substrate specificity of the ERK2/5 chimera correctly mirrored that of ERK5, the specificity of active ERK5kin immunoprecipitated from cells was examined (Figs. 3C and 4B; see below for activating conditions). c-Myc, MBP, and MEK5 were phosphorylated by immunoprecipitated ERK5kin, whereas Tal2, Elk-1, ATF2, and Mnk-1 were not. ERK5kin phosphorylated Myc fragments encompassing residues 1-103 and 41-143; this suggests that the phosphorylation sites are contained in the serine-rich region from residue 41 to residue 103.

Activation of ERK5kin in Cells-- To identify potential activators of ERK5, cells were cotransfected with plasmids expressing ERK5kin and various potential regulators. When ERK5kin was cotransfected with the constitutively activated H-Ras mutant, V12 H-Ras, increased kinase activity toward GST-Myc was detected (Fig. 5A, lanes 4 and 6). A kinase-defective ERK5kin mutant, in which the lysine conserved among protein kinases because of its role in catalysis was mutated to methionine, did not have detectable Ras-dependent activity (data not shown). ERK5kin activity was further increased when cells cotransfected with ERK5kin and V12 H-Ras were treated with phorbol ester (Fig. 5B, lanes 5 and 6). Phorbol ester treatment of cells transfected with ERK5kin alone resulted in a small but reproducible enhancement of ERK5kin activity (Fig. 5B, lanes 3 and 4). Treatment of cells with serum, H2O2, or anisomycin (49) did not enhance ERK5kin activity in transfected 293 cells (data not shown).


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 5.   Effects of V12 H-Ras and PMA on activity of ERK5kin. A, top, 293 cells were transfected with pCMV5/V12 H-Ras, pCMV5/MEKK1, or empty vector plus pCEP4HA encoding one of the following kinases: ERK5kin, ERK5/2, or ERK2, as indicated. Immune complex kinase assays used GST-Myc-(1-143) as the substrate. To ensure that equivalent amounts of HA-tagged protein were used in immunoprecipitation assays, immunoprecipitation was performed on quantities of lysates containing equal amounts of kinase proteins, as assessed by Western analysis using the HA antibody. Bottom, graph showing activation of ERK5kin by V12 H-Ras (black bars) and ERK5/2 activated by V12 H-Ras and MEKK1 (shaded bars); n = 3. B, 293 cells expressing ERK5kin, alone or with V12 H-Ras or MEKK1, were untreated or treated with 100 nM PMA (+) for 30 min prior to harvest. ERK5kin activity in immune complexes was measured with GST-Myc-(1-143) as the substrate. Left, autoradiogram of c-Myc phosphorylation. Right, graph showing ERK5kin activation by phorbol ester and V12 H-Ras but not MEKK1. C, HA-ERK5 was coexpressed with V12 H-Ras, immunoprecipitated, and allowed to autophosphorylate in vitro. Autoradiogram is shown. D, active ERK2/5-P was incubated alone or with either purified GST-ERK5 tail or GST under phosphorylating conditions. Lanes 2, 5, 7, and 9 have double the amount of GST-ERK5 tail or GST. Autoradiogram is shown.

Cotransfection with MEKK1 did not activate ERK5kin (Fig. 5A, lanes 4 and 5), even though either endogenous or cotransfected ERK2 was strongly activated under these conditions, as assessed by kinase assay or immunoblotting with an antibody that selectively recognizes the active form of ERK2 (Fig. 5A, lanes 10 and 11, and data not shown). This is consistent with the previous finding that MEKK1 did not phosphorylate MEK5 in vitro (20). This observation is striking given that MEKK1 will phosphorylate and activate MEKs 1-4 and MEK6 in vitro and that when overexpressed in cells, it will activate ERK1, and ERK2, JNK/SAPK, and p38 (37, 40, 42). The activation of ERK5kin by V12 H-Ras but not MEKK1 indicates that the upstream regulation of ERK5 is distinct from other MAP kinase family members. Consistent with our in vitro data, the ERK5/2 chimera was activated by both V12 H-Ras and MEKK1 (Fig. 5A, lanes 7-9). This finding confirmed our earlier in vitro result that the ERK5/2 chimera did not sense upstream activators of ERK5 selectively.

Autophosphorylation of ERK5 on its C-Terminal Noncatalytic Domain-- ERK5 contains a 396-amino acid C-terminal extension following the conserved MAP kinase catalytic domain. Although the Myc kinase activity of ERK5kin was increased up to 65-fold in cells cotransfected with V12 H-Ras, the same was not true for full-length ERK5. Full-length ERK5 was expressed at levels similar to those of its kinase domain, as assessed by Western blotting of lysates with an HA antibody. In addition, a Coomassie Blue-stainable band of the correct size was detected in immunoprecipitates from these lysates. Although V12 H-Ras or phorbol ester treatment of V12 H-Ras cotransfected cells was not able to increase the activity of full-length ERK5 toward GST-Myc, an increase in ERK5 autophosphorylation was detected (Fig. 5C, lanes 2 and 3). In some experiments, ERK5 underwent a shift in electrophoretic mobility, consistent with modification of all of the precipitated protein. This indicates that ERK5 senses the signal from Ras and suggests that additional signals are needed for its activation.

Autophosphorylation of ERK1 and ERK2 occurs in the kinase domain and is not enhanced by activation (50, 51). Because ERK5 autophosphorylation was stimulated by coexpression with V12 H-Ras, we considered the possibility that one or more sites of ERK5 autophosphorylation may lie C-terminal to the conserved catalytic domain. The ERK5 tail contains 10 potential MAP kinase phosphorylation sites, 5 serine and 5 threonine residues. To determine whether the tail domain of ERK5 contains autophosphorylation sites, we expressed residues 339-816 (ERK5 tail) separately as a fusion protein and tested the fusion protein as a substrate for the ERK2/5 chimera. The ERK5 tail was phosphorylated by ERK2/5 (Fig. 5D, lanes 4 and 5) in a concentration-dependent manner.

Raf Does Not Activate ERK5kin in Cotransfected Cells-- Because V12 H-Ras activated ERK5kin, we wanted to determine whether other components of the ERK/MAP kinase pathway downstream of Ras also activated ERK5kin. ERK5kin was coexpressed with either wild type Raf-1, activated mutant Raf-1 S259D, or activated mutant Raf BXB, a constitutively active truncation mutant of Raf that contains the kinase domain but lacks the Ras binding domain (41). Neither Raf-1 S259D nor Raf BXB activated ERK5kin (Fig. 6A, top panel). However, both caused the expected activation of endogenous ERK2 measured in the same lysates (Fig. 6B).


View larger version (48K):
[in this window]
[in a new window]
 
Fig. 6.   Constitutively active Raf mutants do not activate ERK5kin. A, 293 cells were cotransfected with pCEP4HA/ERK5kin plus pCMV5 without insert, with V12 H-Ras, or with the indicated form of Raf-1. HA-ERK5kin immune complex kinase assays used GST-Myc-(1-143) as the substrate. Top, autoradiogram; middle, anti-HA immunoblot of lysates; bottom, anti-Raf-1 immunoblots of lysates. B, immunoblots of the same lysates examined in A probed with antibody recognizing the dually phosphorylated active forms of ERK1 and ERK2.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

MAP kinase modules mediate responses to a diverse array of cellular stimuli (1-12). Studies in both yeast and mammals have demonstrated that activation of distinct MAP kinase modules alone or in combination results in distinct cellular outcomes. Thus, studies of novel mammalian MAP kinase modules will further our understanding of the molecular mechanisms underlying signaling specificity.

We devised a strategy for activating the orphan MAP kinase ERK5 in the absence of known activators employing enzyme chimeras. From structural and mutagenesis studies of several protein kinases, it is clear that substrate specificity is determined primarily, if not entirely, by the C-terminal domain of the kinase core structure (29-32, 46). Therefore, we constructed a chimeric protein containing the N-terminal domain of ERK2 and the C-terminal domain of the kinase core of ERK5. The protein kinase activity of this chimera was increased to a readily detectable level by coexpression with the constitutively active ERK2 activator, MEK1R4F, despite the fact that ERK2 and ERK5 share a sequence identity of less than 50%. This strategy should prove generally applicable to other orphan kinases for which related family members and appropriate activating enzymes have already been identified. Even in the absence of candidate substrates, purified active chimeric kinases can be used to identify potential substrates using broad-based methods such as peptide, cDNA library, or phage display screening (38, 52).

We also made a chimeric protein containing the N-terminal domain of ERK5 and the C-terminal domain of ERK2. Previous work by Brunet and Pouysségur (32) had suggested to us that this might be a viable approach to identify activators of ERK5 in the absence of known substrates, making use of the well described substrate specificity of ERK2. Using chimeras of p38 and ERK1, they reported that a portion of the N-terminal domain of p38 was sufficient to enable a chimera to respond to stress stimuli characteristic of p38 and not to serum, an ERK1 stimulus. In contrast to the conclusion drawn from p38/ERK1 chimeras, our data in vitro and in cells indicate that the C-terminal domain of ERK2 contributes significantly to recognition of ERK2 by upstream activators. MEK1R4F activated the ERK5/2 chimera, even though it did not activate ERK5kin. In cotransfected cells, MEKK1, which did not cause activation of ERK5 kin, was able to activate the ERK5/2 chimera. These findings demonstrate that ERK5/2 was not activated by mechanisms that are coupled only to ERK5. The presence of the ERK2 C-terminal domain conferred upon the chimera the ability to be activated by agents that turn on ERK2 but not ERK5 and to be recognized by the ERK1/2-specific MEKs. These results are not entirely surprising given that the activation loop, between subdomains VII and VIII, of MAP kinases is located in the C-terminal portion of the kinase core and contains the residues that are phosphorylated by MEKs (24, 46). It is possible that our results more clearly demonstrate the contribution of the C-terminal domain to pathway recognition because of the inherently lower basal activity of ERK5. However, results with the ERK2/5 chimera, which is not a good MEK1 substrate in vitro, and other chimeras2 generally support our findings with the ERK5/2 chimera.

Based on two lines of evidence, we hypothesize that the C-terminal tail of ERK5 contains an autoinhibitory domain. First, the kinase activity of the catalytic domain of ERK5 toward exogenous substrates is increased as much as 65-fold by V12 H-Ras, although the activity of the full-length kinase toward exogenous substrates is not detectably increased. Second, the ERK5 tail contains MAP kinase phosphorylation sites and, when expressed as a separate polypeptide, is phosphorylated by ERK5. Perhaps the ERK5 tail inhibits ERK5 kinase activity by orienting a substrate-like domain in the tail so that it interferes with productive binding of exogenous substrates or orientation of the catalytic residues (53). Consistent with this idea, V12 H-Ras increases the ability of full-length ERK5 to autophosphorylate. The significant increase in ERK5 autophosphorylation when ERK5 is coexpressed with V12 H-Ras suggests that the activating signal is being received by ERK5 but is insufficient to overcome the inhibition derived from the C-terminal domain. Supporting data also come from very recent studies on the activation of ERK5 by Src (54). Activation was reported as an increase in autophosphorylation because it was measured more readily than MBP kinase activity. Validation of this hypothesis will require a more detailed kinetic characterization of the kinase and its C terminus or the delineation of the mechanism of ERK5 activation.

The ERK2/5 chimera has a substrate specificity profile distinct from ERK2, indicating that the substrate specificity of the chimera reflects that of ERK5. This was confirmed by comparing chimera specificity to that of the immunoprecipitated, activated kinase domain of ERK5 itself, once conditions to activate it had been ascertained. Further comparison of the substrate specificity profile of the other MAP kinase family members p38 and JNK/SAPK indicated that ERK5 recognizes an overlapping set of substrates; however, ERK5 specificity is easily distinguished from that of other members of the MAP kinase family. The phosphorylation of MEK5 by ERK2/5 but not by ERK2, p38, or JNK/SAPK demonstrates the success of this strategy for identifying unique substrates for protein kinases.

Consistent with the predicted specificity of distinct MAP kinase pathways, each kinase examined had a unique profile of substrate phosphorylation. Of the proteins tested as substrates, c-Myc and MEK5 are the most likely to be physiologically significant ERK5 substrates. Other MAP kinase family members phosphorylate their upstream activating MEK (55-58). Such phosphorylation may be involved in feedback inhibition, cellular localization, or complex formation with other proteins (59, 41). The phosphorylation of MEK5 by ERK5 and not by ERK2, p38, or JNK/SAPK suggests that this phosphorylation is specific and provides additional evidence that MEK5 is a relevant ERK5 kinase.

The transcription factor c-Myc is involved in processes ranging from mitogenesis to apoptosis (33, 34, 60-64). Mutations and translocations within the coding sequence of c-Myc occur in a number of cancers, such as Burkitt's lymphoma (33, 34). Residues that are frequent sites of mutations include Ser-58 and Thr-62 within the N terminus. These sites are phosphorylated in vivo in response to mitogens, suggesting that phosphorylaton of c-Myc may be a significant mechanism regulating its function (60). The kinases that phosphorylate these residues in vivo have not been established; however, preliminary data suggest that Ser-62 is phosphorylated by ERK5, indicating that ERK5 is a candidate c-Myc kinase.

We demonstrated that V12 H-Ras, a constitutively activated Ras mutant, activates ERK5kin in cotransfected cells. However, ERK5kin is not responsive to activated forms of Raf-1. The current findings are consistent with earlier data that indicated that immunoprecipitated Raf-1 did not phosphorylate MEK5 in vitro (20). Despite the similarity of ERK5 to ERK1/2, the intermediates in the ERK5 cascade are apparently unique. Thus, the pathway culminating in ERK5 represents a newly identified Ras effector pathway.

In conclusion, our understanding of the ERK5/MAP kinase module is in its infancy. However, tantalizing clues to its function are beginning to emerge. Activation by H-Ras and Src and phosphorylation of c-Myc all suggest a kinase cascade likely to be involved in normal and pathological cellular processes, such as cancer (this work and Ref. 54). Future efforts are being directed toward characterizing its effects on Myc function and its contribution to Ras-mediated cell transformation.

    ACKNOWLEDGEMENTS

We thank John Minna, Lori Christerson, and Kevin Berman for comments concerning the manuscript; Colleen Vanderbilt and Quynh Do for technical assistance; Don Arnette and Shilpi Wadhwani for recombinant proteins; and Andrei Khokhlatchev for active MAP kinases. We also thank Tina Arikan for preparation of the manuscript.

    FOOTNOTES

* This work was supported by Research Grant DK34128 and Postdoctoral Training Fellowship NIH-T32-CA66187 (to J. M. E.) from the National Institutes of Health and by Research Grant I1243 from the Welch Foundation.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.

§ To whom correspondence should be addressed: Department of Pharmacology, The University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9041. Tel.: 214-648-3627; Fax: 214-648-3811.

1 The abbreviations used are: MAP, mitogen-activated protein; ERK, extracellular signal-regulated protein kinase; MEK, MAP kinase/ERK kinase (also called MAP kinase kinase or MKK); MEKK, MEK kinase; JNK, Jun-N-terminal kinase; SAPK, stress-activated protein kinase; GST, glutathione S-transferase; MBP, myelin basic protein; Mnk-1, MAP kinase-interacting kinase; ERK5kin, catalytic domain of ERK5; HA, hemagglutinin; PCR, polymerase chain reaction.

2 M. Robinson, J. Wilsbacher, L. Christerson, and M. Cobb, unpublished data.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

  1. Robinson, M. J., and Cobb, M. H. (1997) Curr. Opin. Cell Biol. 9:, 180-186[CrossRef][Medline] [Order article via Infotrieve]
  2. Herskowitz, I. (1995) Cell 80:, 187-197[Medline] [Order article via Infotrieve]
  3. Campbell, J. S., Seger, R., Graves, J. D., Graves, L. M., Jensen, A. M., Krebs, E. G. (1994) Recent Prog. Horm. Res. 50:, 131-159
  4. Waskiewicz, A. J., and Cooper, J. A. (1995) Curr. Opin. Cell Biol. 7:, 798-805[CrossRef][Medline] [Order article via Infotrieve]
  5. Treisman, R. (1996) Curr. Opin. Cell Biol. 8:, 205-215[CrossRef][Medline] [Order article via Infotrieve]
  6. Lewis, T. S., Shapiro, P. S., and Ahn, N. G. Adv. Cancer Res., in press
  7. Kyriakis, J. M., and Avruch, J. (1996) J. Biol. Chem. 271:, 24313-24316[Free Full Text]
  8. Hunter, T. (1995) Cell 80:, 225-236[Medline] [Order article via Infotrieve]
  9. Karin, M. (1995) J. Biol. Chem. 270:, 16483-16486[Free Full Text]
  10. Cobb, M. H., and Goldsmith, E. (1995) J. Biol. Chem. 270:, 14843-14846[Free Full Text]
  11. Davis, R. (1994) Trends Biochem. Sci. 19, 470-473[CrossRef][Medline] [Order article via Infotrieve]
  12. Moriguchi, T., Gotoh, Y., and Nishida, E. (1996) Adv. Pharmacol. 36:, 121-137[Medline] [Order article via Infotrieve]
  13. Neiman, A. M., Stevenson, B. J., Xu, H.-P., Sprague, G. F., Jr., Herskowitz, I., Wigler, M., Marcus, S. (1993) Mol. Biol. Cell 4:, 107-120[Abstract]
  14. Hunter, T., and Plowman, G. D. (1997) Trends Biochem. Sci. 22:, 18-22[CrossRef][Medline] [Order article via Infotrieve]
  15. Boulton, T. G., Nye, S. H., Robbins, D. J., Ip, N. Y., Radziejewska, E., Morgenbesser, S. D., DePinho, R. A., Panayotatos, N., Cobb, M. H., Yancopoulos, G. D. (1991) Cell 65:, 663-675[Medline] [Order article via Infotrieve]
  16. Gonzalez, F. A., Raden, D. L., Rigby, M. R., Davis, R. J. (1992) FEBS Lett. 304:, 170-178[CrossRef][Medline] [Order article via Infotrieve]
  17. Cheng, M., Boulton, T. G., and Cobb, M. H. (1996) J. Biol. Chem. 271:, 8951-8958[Abstract/Free Full Text]
  18. Cheng, M., Zhen, E., Robinson, M. J., Ebert, D., Goldsmith, E., Cobb, M. H. (1996) J. Biol. Chem. 271:, 12057-12062[Abstract/Free Full Text]
  19. Boulton, T. G., and Cobb, M. H. (1991) Cell Regul. 2:, 357-371[Medline] [Order article via Infotrieve]
  20. English, J. M., Vanderbilt, C. A., Xu, S., Marcus, S., and Cobb, M. H. (1995) J. Biol. Chem. 270:, 28897-28902[Abstract/Free Full Text]
  21. Zhou, G., Bao, Z. Q., and Dixon, J. E. (1995) J. Biol. Chem. 270:, 12665-12669[Abstract/Free Full Text]
  22. Lee, J., Ulevitch, R. J., and Han, J. (1995) Biochem. Biophys. Res. Comm. 213:, 715-724[CrossRef][Medline] [Order article via Infotrieve]
  23. Payne, D. M., Rossomando, A. J., Martino, P., Erickson, A. K., Her, J.-H., Shananowitz, J., Hunt, D. F., Weber, M. J., Sturgill, T. W. (1991) EMBO J. 10:, 885-892[Abstract]
  24. Robbins, D. J., and Cobb, M. H. (1992) Mol. Biol. Cell 3:, 299-308[Abstract]
  25. Robbins, D. J., Zhen, E., Owaki, H., Vanderbilt, C. A., Ebert, D., Geppert, T. D., Cobb, M. H. (1993) J. Biol. Chem. 268:, 5097-5106[Abstract/Free Full Text]
  26. Brunner, D., Oellers, N., Szabad, J., Biggs, W. H., III, Zipursky, S. L., Hafen, E. (1994) Cell 76:, 1025-1037[Medline] [Order article via Infotrieve]
  27. Brill, J. A., Elion, E. A., and Fink, G. R. (1994) Mol. Cell. Biol. 5:, 297-312
  28. Zhang, J., Zhang, F., Ebert, D., Cobb, M. H., Goldsmith, E. J. (1995) Structure 3:, 299-307[Abstract]
  29. Knighton, D. R., Zheng, J., Ten Eyck, L. F., Xuong, N.-H., Taylor, S. S., Sowadski, J. M. (1991) Science 253:, 414-429[Medline] [Order article via Infotrieve]
  30. Gibbs, C. S., and Zoller, M. J. (1991) J. Biol. Chem. 266:, 8923-8931[Abstract/Free Full Text]
  31. Kallunki, T., Su, B., Tsigelny, I., Sluss, H. K., Dérijard, B., Moore, G., Davis, R., Karin, M. (1994) Genes Dev. 8:, 2996-3007[Abstract]
  32. Brunet, A., and Pouysségur, J. (1996) Science 272:, 1653-1655
  33. Rabbitts, T. H., Hamlyn, P. H., and Baer, R. (1983) Nature 306:, 760-765[Medline] [Order article via Infotrieve]
  34. Rabbitts, T. H., Forster, A., Hamlyn, P. H., Baer, R. (1984) Nature 309:, 592-597[Medline] [Order article via Infotrieve]
  35. Taylor, S. S. (1989) J. Biol. Chem. 264:, 8443-8446[Free Full Text]
  36. Guan, K. L., and Dixon, J. E. (1991) Anal. Biochem. 192:, 262-267[Medline] [Order article via Infotrieve]
  37. Robinson, M. J., Cheng, M., Khokhlatchev, A., Ebert, D., Ahn, N., Guan, K.-L., Stein, B., Goldsmith, E., and Cobb, M. H. (1996) J. Biol. Chem. 271:, 29734-29739[Abstract/Free Full Text]
  38. Fukunaga, R., and Hunter, T. (1997) EMBO J. 16:, 1921-1933[Abstract/Free Full Text]
  39. Gille, H., Kortenjann, M., Thomae, O., Moomaw, C., Slaughter, C., Cobb, M. H., Shaw, P. E. (1995) EMBO J. 14:, 951-962[Abstract]
  40. Khokhlatchev, A., Xu, S., English, J., Wu, P., Schaefer, E., and Cobb, M. H. (1997) J. Biol. Chem. 272:, 11057-11062[Abstract/Free Full Text]
  41. Frost, J. A., Steen, H., Shapiro, P., Lewis, T., Ahn, N., Shaw, P., and Cobb, M. H. (1997) EMBO J. 16:, 6426-6438[Abstract/Free Full Text]
  42. Xu, S., Robbins, D. J., Christerson, L. B., English, J. M., Vanderbilt, C. A., Cobb, M. H. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5291-5295[Abstract/Free Full Text]
  43. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  44. Khoo, S., and Cobb, M. H. (1997) Proc. Natl. Acad. Sci. U. S. A. 94:, 5599-5604[Abstract/Free Full Text]
  45. Hanks, S. K., Quinn, A. M., and Hunter, T. (1988) Science 241:, 42-52[Medline] [Order article via Infotrieve]
  46. Zhang, F., Strand, A., Robbins, D., Cobb, M. H., Goldsmith, E. J. (1994) Nature 367:, 704-710[CrossRef][Medline] [Order article via Infotrieve]
  47. Mansour, S. J., Matten, W. T., Hermann, A. S., Candia, J. M., Rong, S., Fukasawa, K., Vande Woude, G. F., Ahn, N. G. (1994) Science 265:, 966-970[Medline] [Order article via Infotrieve]
  48. Xia, Y., Hwang, L.-Y., Cobb, M. H., Baer, R. (1994) Oncogene 9:, 1437-1446[Medline] [Order article via Infotrieve]
  49. Abe, J., Kusuhara, M., Ulevitch, R. J., Berk, B. C., Lee, J.-D. (1996) J. Biol. Chem. 271:, 16586-16590[Abstract/Free Full Text]
  50. Seger, R., Ahn, N. G., Boulton, T. G., Yancopoulos, G. D., Panayotatos, N., Radziejewska, E., Ericsson, L., Bratlien, R. L., Cobb, M. H., Krebs, E. G. (1991) Proc. Natl. Acad. Sci. U. S. A. 88:, 6142-6146[Abstract]
  51. Boulton, T. G., Gregory, J. S., and Cobb, M. H. (1991) Biochemistry 30:, 278-286[Medline] [Order article via Infotrieve]
  52. Songyang, Z., Blechner, S., Hoagland, N., Hoekstra, M. F., Piwnica-Worms, H., Cantley, L. C. (1994) Curr. Biol. 4:, 973-982[Medline] [Order article via Infotrieve]
  53. Knighton, D. R., Pearson, R. B., Sowadski, J. M., Means, A. R., Ten Eyck, L. F., Taylor, S. S., Kemp, B. E. (1992) Science 258:, 130-135[Medline] [Order article via Infotrieve]
  54. Abe, J., Takahashi, M., Ishida, M., Lee, J.-D., and Berk, B. C. (1997) J. Biol. Chem. 272:, 20389-20394[Abstract/Free Full Text]
  55. Matsuda, S., Gotoh, Y., and Nishida, E. (1993) J. Biol. Chem. 268:, 3277-3281[Abstract/Free Full Text]
  56. Mansour, S. J., Resing, K. A., Candia, J. M., Hermann, A. S., Gloor, J. W., Herskind, K. R., Wartmann, M., Davis, R. J., Ahn, N. G. (1994) J. Biochem. (Tokyo) 116:, 304-314[Abstract]
  57. Dérijard, B., Raingeaud, J., Barrett, T., Wu, I., Han, J., Ulevitch, R. J., Davis, R. J. (1995) Science 267:, 682-685[Medline] [Order article via Infotrieve]
  58. Tournier, C., Whitmarsh, A. J., Cavanagh, J., Barrett, T., and Davis, R. J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94:, 7337-7342[Abstract/Free Full Text]
  59. Jelinek, T., Catling, A. D., Reuter, C. W. M., Moodie, S. A., Wolfman, A., Weber, M. J. (1994) Mol. Cell. Biol. 14:, 8212-8218[Abstract]
  60. Lutterbach, B., and Hann, S. R. (1994) Mol. Cell. Biol. 14:, 5510-5522[Abstract]
  61. Henriksson, M., Bakardjiev, A., Klein, G., and Luscher, B. (1993) Oncogene 3199-3209
  62. Kato, G., and Dang, C. (1992) FASEB J. 6:, 3065-3072[Abstract/Free Full Text]
  63. Luscher, B., and Eisenman, R. (1990) Genes Dev. 4:, 2025-2035[CrossRef][Medline] [Order article via Infotrieve]
  64. Marcu, K., Bossone, S., and Patel, A. (1992) Annu. Rev. Biochem. 61:, 809-860[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.