©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of an Autoinhibitory Domain in Human Mitogen-activated Protein Kinase-activated Protein Kinase 2 (*)

(Received for publication, September 20, 1994; and in revised form, October 27, 1994)

You-Li Zu Youxi Ai Chi-Kuang Huang (§)

From the Department of Pathology, University of Connecticut Health Center, Farmington, Connecticut 06030

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Mitogen-activated protein (MAP) kinase-activated protein kinase 2, a Ser/Thr kinase, is phosphorylated and activated by MAP kinase. Sequence analysis of a clone isolated from the human HL-60 cell line revealed a 370-amino acid protein with a proline-rich N terminus, a highly conserved catalytic domain, and a C-terminal region containing a MAP kinase phosphorylation site. To better understand how the kinase is regulated, mutation analysis was used to map the functional domain(s). The wild type recombinant kinase had a low basal activity as detected by phosphorylation of a substrate peptide derived from the N terminus of glycogen synthase. Deletion of the proline-rich N terminus showed little effect on the basal activity. Deletion of the C terminus resulted in a marked increase in catalytic activity either with or without the pretreatment of the kinase by MAP kinase. Further analysis indicated that amino acid residues 339-353 in the C-terminal region were acting as an autoinhibitory domain. A synthetic peptide (RVLKEDKERWEDVK-amide) derived from this autoinhibitory domain inhibited the kinase activity in a concentration-dependent manner. These results suggest a regulatory model for the kinase.


INTRODUCTION

A variety of extracellular stimuli activates mitogen-activated protein (MAP) (^1)kinases by an intracellular kinase cascade(1, 2, 3, 4) , which includes MAP kinase kinase kinase(5, 6, 7, 8, 9, 10) and MAP kinase kinase(11, 12) . The activated MAP kinases in turn regulate the phosphorylation and activation of many substrates including cell surface molecules, cytoskeletal proteins, transcription factors, and protein kinases (reviewed in (3) and (4) ). Both ribosomal protein S6 kinase II (13, 14, 15) and MAP kinase-activated protein (MAPKAP) kinase 2 are known as MAP kinase substrates(16) . MAPKAP kinase 2 can be distinguished from the ribosomal protein S6 kinase II by several means including its resistance to the protein kinase inhibitor, H-7, its failure to phosphorylate the peptides derived from the C terminus of ribosomal protein S6, and its partial amino acid sequences(16) . MAPKAP kinase 2 was originally identified by its ability to phosphorylate rabbit skeletal muscle glycogen synthase at Ser-7 as well as the equivalent serine in the synthesized peptide derived from the N terminus of glycogen synthase(16) . MAPKAP kinase 2 also phosphorylates the low molecular weight heat shock proteins (17) and tyrosine hydroxylase(18) .

Partial cDNA sequences of MAPKAP kinase 2 derived from mouse lung(19) , rabbit and human skeletal muscles(20) , and a full-length cDNA sequence derived from human HL-60 cells (21) have been reported. Sequence analysis of the MAPKAP kinase 2 derived from HL-60 cells revealed that it is a 370-amino acid protein containing a proline-rich N terminus, a well conserved catalytic domain in the central area, and a MAP kinase phosphorylation site in the C terminus(21) . In order to understand the possible mechanism of MAPKAP kinase 2 activation we have mapped its functional domains using mutation analysis. An autoinhibitory domain containing 15 amino acids within the C-terminal region was identified, and a regulatory model for the kinase was suggested.


EXPERIMENTAL PROCEDURES

Plasmid Construction

To generate recombinant kinases, a glutathione S-transferase fusion protein system expressed in Escherichia coli was utilized(22, 23) . A cDNA clone isolated from human HL-60 cells was used as a template for the polymerase chain reaction. To obtain the wild type (WT) kinase, an NcoI site (CCATGG) was introduced into the position overlapping the ATG initiation codon of MAPKAP kinase 2, and an XbaI site (TCTAGA) was introduced directly downstream of the stop codon by polymerase chain reaction. The amplified NcoI-XbaI DNA fragment was inserted into the NcoI-XbaI cloning sites of a modified pGEX-2T vector in-frame to express the glutathione S-transferase fusion protein as described previously(24) . Utilizing the same strategy, the plasmids expressing the deletion mutants were constructed: mutants CT327 and CT339 encode amino acid residues 1-326 and 1-338 separately; mutants NCT327, NCT339, NCT354, and NT47 encode amino acid residues 48-326, 48-338, 48-353, and 48-370, respectively. All prepared expression plasmids were confirmed by DNA sequencing.

Expression and Purification of the Recombinant Kinases

Expression and purification of the glutathione S-transferase fusion proteins were carried out as described previously (25) with some modifications. Briefly, a 10-ml overnight culture of E. coli Blue XL-1 transformed with expression plasmid was diluted in 1 liter of fresh 2 times YT medium and grown at 37 °C. When the cultures reached an absorption of 0.45 at 600 nm, isopropyl-beta-D-thiogalactopyranoside was added to a final concentration of 1 mM. After another 2-h culture at 37 °C, bacteria were harvested, washed with cold phosphate-buffered saline (PBS) buffer and suspended in 60 ml of PBS containing 1% Triton X-100 (PBS-T) and 1 mM phenylmethylsulfonyl fluoride. The bacteria were then lysed by mild sonication on ice, and the lysate was centrifuged at 10,000 times g for 25 min at 4 °C. The supernatant (20 ml) was loaded onto a glutathione-Sepharose column (about 0.7 ml of bed volume) at 4 °C. The column was washed twice with 4 ml each of PBS-T containing 2 M NaCl, PBS-T once, and a kinase buffer (20 mM Tris-HCl, pH 7.4, 1.0 mM EDTA, 50 mM KCl, and 1.0 mM dithiothreitol) once. Subsequently, the recombinant kinase was eluted from the column with 2 ml of the kinase buffer containing 10 mM reduced glutathione. The eluate was loaded on a DEAE-Sephacel column (0.7-ml bed volume), and the flow-through fraction was collected. Finally, the recombinant kinase was concentrated to 150 µg/ml protein and stored in the kinase buffer containing 50% glycerol at -20 °C.

Kinase Assays

To examine the enzymatic activity of recombinant MAPKAP kinase 2, a synthetic peptide substrate derived from the amino acid residues 1-13 of glycogen synthase N terminus (KKPLNRTLSVASLPG-amide) was used(16) . Kinase assays were initiated by adding 150 ng of the purified kinase to a 40-µl reaction mixture containing 20 mM HEPES, pH 7.3, 10 mM MgCl(2), 1.0 mM EGTA, 20 µM sodium vanadate, 5 µM okadaic acid, 2 mM dithiothreitol, 40 µM [-P]ATP (4.4 times 10^3 cpm/pmol), and 20 µM substrate peptide. The reaction was allowed to proceed for 10 min at 30 °C. The amount of P incorporation into the peptide was analyzed by a liquid scintillation counter as described previously(21) . In order to activate MAPKAP kinase 2, 40 ng of purified MAP kinase (p44, Upstate Biotechnology Inc.) was added into the kinase reaction for 10 min at 30 °C prior to initiation of the kinase assay.

The activities of cAMP-dependent protein kinase (Sigma) and protein kinase C (Calbiochem) were evaluated using a synthetic peptide substrate, RSRKRLSQDAYRRNSVRF-amide, that corresponds to the amino acids 314-331 of p47(26, 27) . In the protein kinase C kinase reaction, additional components (1.6 mM CaCl(2), 20 µg/ml phosphatidylserine, and 2 µg/ml phorbol 12-myristate 13-acetate) were included. MAP kinase activity was assessed using myelin basic protein as a substrate(28, 29) .

Analysis of Inhibitory Domain-derived Peptide, I-pp

A 14-amino acid peptide (RVLKEDKERWEDVK-amide), I-pp, designed from the amino acid residues 340-353 of MAPKAP kinase 2, was synthesized by solid-phase peptide synthesis. The peptide was purified by cation exchange reverse-phase chromatography and characterized by analytical high performance liquid chromatography (Integrated DNA Technologies, Inc.). To detect the inhibitory effect of I-pp, protein kinases were preincubated at 30 °C with various concentrations of I-pp for 1 min. The kinases were then assayed as described above.

Protein Phosphorylation

Purified glutathione S-transferase fusion proteins of the recombinant MAPKAP kinase 2 were phosphorylated by MAP kinase (p44, Upstate Biotechnology Inc.), and recombinant p47 (a gift from Dr. Thomas L. Leto) was phosphorylated by cAMP-dependent protein kinase according to the kinase assay procedure described above. Samples were added with SDS buffer (2.3% SDS, 5% 2-mercaptoethanol, 10% glycerol, 62.5 mM Tris-HCl, pH 6.8) and subjected to 10% SDS-PAGE analysis followed by Coomassie Blue staining and autoradiography(30) .


RESULTS

Deletion Analysis

That MAPKAP kinase 2 can be activated by phosphorylation suggests that it may have a regulatory domain(16, 21) . Sequence analysis showed that human MAPKAP kinase 2 has a proline-rich N-terminal region which may mediate protein-protein interaction(31, 32) , a well-conserved catalytic domain, and a C-terminal region containing a MAP kinase phosphorylation site of threonine 334(21) . Fig. 1A shows the strategy for generation of a wild type (WT) kinase, which encodes the full-length MAPKAP kinase 2 (amino acids 1-370), and the C-terminal truncated mutants of the kinase, CT327 and CT339; these encode amino acids 1-326 and 1-338, respectively. The recombinant kinases were expressed as glutathione S-transferase fusion proteins in bacteria, purified by affinity chromatography, and analyzed by SDS-PAGE followed by Coomassie Blue staining (Fig. 1C, lanes 1-3). Enzymatic activities of the WT and mutant kinases were evaluated by examining the phosphorylation of a synthetic peptide substrate (KKPLNRTLSVASLPG-amide)(16) . As shown in Fig. 1B, the WT kinase had a relatively low kinase activity. In contrast, the mutant CT339 had a markedly increased kinase activity (431% of the WT kinase). The mutant CT327, which lacks the MAP kinase phosphorylation site, exhibited a slightly lower kinase activity (70% of the WT kinase). These results indicate that the region comprising amino acids 339-370 may possess an inhibitory domain. They also suggest that the region (amino acids 327-338) containing the MAP kinase phosphorylation site is essential to maintain high enzymatic activity for MAPKAP kinase 2.


Figure 1: Deletion analysis of C terminus. A, construction of the recombinant kinases. MAPKAP kinase 2 has a proline-rich N terminus (amino acids 1-62) containing two SH3 domain-binding motifs (), a well-conserved catalytic domain (hatched box, amino acids 63-326), and a C terminus (amino acids 327-370) containing a MAP kinase phosphorylation site at threonine 334 (T334). The kinases were prepared as glutathione S-transferase fusion proteins as described under ``Experimental Procedures.'' Wild type (WT) kinase expresses the full-length cDNA of human MAPKAP kinase 2 (amino acids 1-370) cloned from HL-60 cells(21) . Mutants CT327 and CT339 are two C-terminal truncated kinases expressing amino acid residues 1-326 and 1-338 of MAPKAP kinase 2, respectively. B, enzymatic analysis of the recombinant kinases. The relative enzymatic activity (counts/min) of the kinases was examined by an in vitro protein kinase assay using a peptide substrate derived from the N terminus of glycogen synthase(16) . Glutathione S-transferase vector protein was used as the kinase(-) background control. In order to activate the kinases, samples were pretreated with or without MAP kinase as indicated. This figure is representative of three separate experiments. C, protein phosphorylation of the recombinant kinases by MAP kinase. Purified recombinant kinases were analyzed by 10% SDS-PAGE with Coomassie Blue staining (lanes 1-6) and autoradiography (lanes 7-12). The kinases were phosphorylated with (lanes 4-6 and 10-12) or without (lanes 1-3 and 7-9) MAP kinase as described in the text. Molecular weight standards are shown on the left. The positions of the recombinant kinases and MAP kinase are indicated on the right.



To examine MAP kinase-induced activation of MAPKAP kinase 2, the recombinant kinases were pretreated with MAP kinase prior to initiation of the kinase assay. Fig. 1B shows that MAP kinase treatment increased the enzymatic activity of the WT kinase 249%, but had little effect on either mutant CT339 (115%) or CT327 (82%). Phosphorylation of MAPKAP kinase 2 (with or without MAP kinase pretreatment) was analyzed by SDS-PAGE and autoradiography. As shown in Fig. 1C (lanes 7-12), MAP kinase phosphorylated both the WT kinase and the mutant CT339 to an approximately equivalent extent but not the mutant CT327 which lacked a MAP kinase phosphorylation site.

Human MAPKAP kinase 2 has two proline-rich regions in the N terminus (Fig. 1A). To examine the role of the N terminus in regulating the enzymatic activity, an N-terminal truncated mutant, NT47 (amino acids 48-370) which lacked the proline-rich regions was generated (Fig. 2A). Deletion of the N terminus resulted in a lower kinase activity (68% of the WT kinase). The result suggests that this region may have a role in maintaining the kinase activity but not in the autoinhibitory function (Fig. 2C).


Figure 2: Identification of an autoinhibitory domain. A, construction of mutant kinases. Mutants of the kinase were generated using the same strategy as described in Fig. 1. Mutant NT47 is an N-terminal truncated kinase encoding amino acid residues 48-370, which lacks the proline-rich region. Mutants NCT327, NCT339, and NCT354 are both N- and C-terminal truncated mutants of the kinase expressing amino acid residues 48-326, 48-338, and 48-353 of MAPKAP kinase 2, respectively. B, preparation of the recombinant kinases. The kinase were expressed in E. coli, purified on a glutathione-Sepharose column, and analyzed by 10% SDS-PAGE with Coomassie Blue staining. C, analysis of enzymatic activity. Relative kinase activities of mutants were examined by in vitro kinase assay as described in Fig. 1, and the percentages of enzymatic activity as compared to the wild type kinase (100%) are shown in the graph. This is representative of three separate experiments.



Identification of the Autoinhibitory Domain

To localize the inhibitory domain within the C-terminal region of the kinase, a more detailed mutational analysis was carried out using the mutants NCT327 (amino acids 48-326), NCT339 (amino acids 48-338), NCT354 (amino acids 48-353), and NT47 (amino acids 48-370) (Fig. 2, A and B). Deletion of the 17-amino acid residues from the C terminus 354-370 caused a slightly higher kinase activity (140% of the WT kinase). Further deletion of the 15-amino acid residues 339-353 resulted in a marked increase in kinase activity (360% of the WT kinase). Deletion of the amino acid residues 327-338 which contain the MAP kinase phosphorylation site resulted in a slight decrease of the kinase activity (70% of the WT kinase) (Fig. 2C). These results indicate that the region comprising amino acids 339-353 of MAPKAP kinase 2 may serve as an autoinhibitory domain.

Characterization of the Inhibitory Domain-derived Peptide, I-pp

Based on the sequence of the autoinhibitory domain of MAPKAP kinase 2, I-pp (RVLKEDKERWEDVK-amide) was prepared (Fig. 3A). Fig. 3B shows that I-pp inhibited the activity of MAPKAP kinase 2 in a concentration-dependent manner. A similar result was observed with MAP kinase-pretreated MAPKAP kinase 2 (data not shown). To test whether I-pp inhibits the activity of other protein kinases, cAMP-dependent protein kinase, MAP kinase, and protein kinase C were utilized. I-pp also inhibited the enzymatic activity of cAMPdependent protein kinase and protein kinase C toward a synthetic peptide derived from p47 (Fig. 3B), as well as the phosphorylation of intact p47 by cAMP-dependent protein kinase (Fig. 3C). However, I-pp had no effect on the enzymatic activity of MAP kinase (Fig. 3B).


Figure 3: Characterization of an autoinhibitory domain-derived peptide, I-pp. A, design of I-pp. Based on the sequence of the autoinhibitory domain (amino acids 339-353), a 14-amino acid peptide (underline) was synthesized and designated as I-pp. B, effects of I-pp on protein kinases. Various concentrations of I-pp were incubated with the kinases for 1 min prior to the initiation of kinase reaction as indicated. Kinase assay was started by the addition of substrate peptides and ATP. For cAMP-dependent protein kinase and protein kinase C, a synthetic substrate peptide derived from the amino acid residues 314-331 of p47 was used. For MAP kinase, myelin basic protein was used as a substrate. C, inhibition of p47phosphorylation by I-pp. p47 (from left to right) was phosphorylated by cAMP-dependent protein kinase in the presence of 0, 12.5, 25, 50, and 100 µM (final concentrations) I-pp, respectively. Phosphorylated proteins were analyzed by 10% SDS-PAGE followed by Coomassie Blue staining (upper panel) and autoradiography (lower panel).



Fig. 4shows the sequence alignment of I-pp with the substrates and pseudosubstrates derived from glycogen synthase(16) , cAMP-dependent protein kinase(33) , and calcium/calmodulin-dependent protein kinase II (CaM kinase IIalpha)(34) . The alignment indicates that the phosphorylated serine residue of the peptide substrate for MAPKAP kinase 2 is substituted with an aspartic acid in I-pp (pointed arrow). A serine residue of the cAMP-dependent protein kinase substrate is similarly replaced with an alanine in the pseudosubstrate, the cAMP-dependentprotein kinase inhibitor. It is noteworthy that all of the amino acid residues important for peptide recognition, which have been identified for both the cAMP-dependent protein kinase inhibitor and the pseudosubstrate of CaM kinase IIalpha, are conserved in I-pp (bold type).


Figure 4: Sequence alignment of substrates and inhibitory domains of protein kinases. The sequence of the autoinhibitory domain of MAPKAP kinase 2 (amino acid residues 339-353) is listed based on the present study. The sequence of the substrate peptide of MAPKAP kinase 2 is the amino acid residues 1-13 of glycogen synthase(16) . The sequence of the pseudosubstrate of cAMP-dependent protein kinase is a segment of the cAMP-dependent protein kinase inhibitor PKI(43) . The sequence of the substrate of cAMP-dependent protein kinase is the in vivo phosphorylation site of pyruvate kinase(44) . The sequence of the autoinhibitory peptide of CaM kinase IIalpha is the amino acid residues 288-306 of CaM kinase IIalpha (34) . The alignment was made based on the substrate phosphorylation sites (pointed arrow) and the amino acid residues important for peptide recognition (bold type)(33) .




DISCUSSION

Using mutational analysis we have studied the roles of the N- and C-terminal regions of MAPKAP kinase 2 in regulating its enzymatic activity. Stepwise deletion of the C terminus revealed an autoinhibitory domain. Deletion of this domain resulted in a marked increase in kinase activity (431% of the wild type kinase). In contrast, deletion of the N terminus resulted in a slight decrease in kinase activity. In addition, the detailed mutational analysis demonstrated that the autoinhibitory domain was located within amino acids 339-353. An autoinhibitory domain-derived peptide (I-pp) inhibited the activity of MAPKAP kinase 2.

Based on the results of this study, we suggest the following regulatory model for MAPKAP kinase 2 (Fig. 5): first, extracellular stimuli induce the activation of the MAP kinase cascade; in turn, activated MAP kinases phosphorylate MAPKAP kinase 2 at Thr-334; the phosphorylation of Thr-334 results in a conformational change and subsequent release of MAPKAP kinase 2 from the repression of its autoinhibitory domain; and finally, MAPKAP kinase 2 becomes activated. Competing with the phosphorylation are protein phosphatases, which dephosphorylate MAPKAP kinase 2 at Thr-334 and return the kinase to its inactive state.


Figure 5: Regulatory model for MAPKAP kinase 2. An extracellular signal triggers a kinase cascade leading to the phosphorylation of MAPKAP kinase 2 at Thr-334. This phosphorylation event may result in a conformational change of MAPKAP kinase 2 and may release it from the repression of its autoinhibitory domain located in the C-terminal region. The activated MAPKAP kinase 2 regulates the activities of its substrates by phosphorylation. To restore the balance, protein phosphatases may inactivate MAPKAP kinase 2 by dephosphorylation of Thr-334.



Kinetic studies of the substrate specificity of MAPKAP kinase 2 indicate that the minimum sequence required for efficient phosphorylation is X-X-Hyd-X-Arg-X-X-Ser/Thr-X-X(20) . A hydrophobic residue (Hyd) at position n - 5 is required for efficient binding. In contrast, the residue at n - 5 of I-pp derived from the autoinhibitory domain of MAPKAP kinase 2 is lysine (K), a basic residue. This residue is conserved in the pseudosubstrates of cAMP-dependent protein kinase, CaM kinase IIalpha (Fig. 4), and MAPKAP kinase 1(20) . The existence of this basic residue at n - 5 of I-pp may explain why I-pp also inhibited cAMP-dependent protein kinase and protein kinase C (Fig. 3B). At present, we are not sure that I-pp is a true pseudosubstrate of MAPKAP kinase 2.

To date, only a few proteins, such as glycogen synthase(16) , low molecular weight heat shock proteins (17, 19, 35, and 36), and tyrosine hydroxylase (18) have been reported to be substrates for MAPKAP kinase 2. The active mutants of MAPKAP kinase 2 reported in this work should facilitate the search for new substrates. Using the active mutants, we have demonstrated that the major substrate for MAPKAP kinase 2 in an HL-60 cell lysate is a 27-kDa protein with mobility in SDS-PAGE similar to that of the heat shock protein, Hsp27 (data not shown).

The proline-rich N terminus of MAPKAP kinase 2 may interact with proteins that contain SH3 domain(s)(31, 32) . The protein-protein interaction between the SH3 domain and the proline-rich domain may play an important role in signal transduction(37, 38) . Our results indicate that the N terminus of MAPKAP kinase 2 is not involved in the autoinhibitory event. Identification of the proteins whose SH3 domain can bind specifically to the proline-rich domain of MAPKAP kinase 2 remains to be done.

While this paper was under review, Rouse et al. (39) and Freshney et al. (40) reported a novel protein kinase cascade triggered by interleukin-1, stress, and heat shock that stimulates MAPKAP kinase 2 and phosphorylation of the small heat shock protein. In intact cells, the kinase cascade that leads to the activation of MAPKAP kinase 2 is distinct from the classical pathway leading to the activation of MAP kinase. The upstream kinase which regulates MAPKAP kinase 2 was identified as a novel kinase termed RK (39) or p40(40) . RK is related to the yeast kinase HOG1 and the mammalian kinases JnK1, p38, and SAPKs(41, 42) . Further studies on the signal transduction pathway leading to the activation of MAPKAP kinase 2 should provide important insights into the intracellular action of interleukin-1, endotoxin, stress, and heat shock as well as the mechanism of cell volume control.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant AI 20943. 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(TM)/EMBL Data Bank with accession number(s) U12779[GenBank].

§
To whom correspondence and reprint requests should be addressed. Fax: 203-679-2936.

(^1)
The abbreviations used are: MAP kinases, mitogen-activated protein kinases; MAPKAP kinase 2, MAP kinase-activated protein kinase 2; I-pp, inhibitory domain-derived peptide; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; CaM kinase IIalpha, calcium/calmodulin-dependent protein kinase IIalpha; SH3, src homology 3.


ACKNOWLEDGEMENTS

We thank Dr. Annette Gilchrist, Dr. Mark E. Labadia, and Dr. Elmer L. Becker (University of Connecticut Health Center) for helpful discussions and review of the manuscript and Dr. Thomas L. Leto (NIAID, National Institutes of Health) for providing the recombinant p47 protein.


REFERENCES

  1. Lange-Carter, C. A., Pleiman, C. M., Gardner, A. M., Blumer, K. J., and Johnson, G. L. (1993) Science 260, 315-319 [Medline] [Order article via Infotrieve]
  2. Ruderman, J. V. (1993) Curr. Opin. Cell Biol. 5, 207-213 [Medline] [Order article via Infotrieve]
  3. Davis, R. J. (1993) J. Biol. Chem. 268, 14553-14556 [Free Full Text]
  4. Blenis, J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5889-5892 [Abstract]
  5. Kyriakis, J. M., App, H., Zhang, X. F., Banerjee, P., Brautigan, D. L., Rapp, U. R., and Avruch, J. (1992) Nature 358, 417-421 [CrossRef][Medline] [Order article via Infotrieve]
  6. Howe, L. R., Leevers, S. J., Gomez, N., Nakielny, S., Cohen, P., and Marshall, C. J. (1992) Cell 71, 335-342 [Medline] [Order article via Infotrieve]
  7. Dent, P., Haser, W., Haystead, T. A., Vincent, L. A., Roberts, T. M., and Sturgill, T. W. (1992) Science 257, 1404-1407 [Medline] [Order article via Infotrieve]
  8. Posada, J., Yew, N., Ahu, N., Vande Woude, G. F., and Cooper, J. A. (1993) Mol. Cell. Biol. 13, 2546-2553 [Abstract]
  9. Haccard, O., Sarcevic, B., Lewellyn, A., Hartley, R., Roy, L., Izumi, T., Erikson, E., and Maller, J. L. (1993) Science 262, 1262-1265 [Medline] [Order article via Infotrieve]
  10. Matsuda, S., Kosako, H., Takenaka, K., Moriyama, K., Sakai, H., Akiyama, T., Gotoh, Y., and Nishda, E. (1992) EMBO J. 11, 973-982 [Abstract]
  11. Ahn, N. G., Seger, R., and Krebs, E. G. (1992) Curr. Opin. Cell Biol. 4, 992-999 [Medline] [Order article via Infotrieve]
  12. Cobb, M. H., Robbins, D. J., and Boulton, T. G. (1991) Curr. Opin. Cell Biol. 3, 1025-1032 [Medline] [Order article via Infotrieve]
  13. Sturgill, T. W., Ray, L. B., Erikson, E., and Maller, J. L. (1988) Nature 334, 715-718 [CrossRef][Medline] [Order article via Infotrieve]
  14. Lavoinne, A., Erikson, E., Maller, J. L., Price, D. J., Avruch, J., and Cohen, P. (1991) Eur. J. Biochem. 199, 723-728 [Abstract]
  15. Sutherland, C., Campbell, D. G., and Cohen, P. (1993) Eur. J. Biochem. 212, 581-588 [Abstract]
  16. Stokoe, D., Campbell, D. G., Nakielny, S., Hidaka, H., Leevers, S. J., Marshall, C., and Cohen, P. (1992) EMBO J. 11, 3985-3994 [Abstract]
  17. Stokoe, D., Engel, K., Campbell, D. G., Cohen, P., and Gaestel, M. (1992) FEBS Lett. 313, 307-313 [CrossRef][Medline] [Order article via Infotrieve]
  18. Sutherland, C., Alterio, J., Campbell, D. G., Le Bourdelles, B., Mallet, J., Haavik, J., and Cohen, P. (1993) Eur. J. Biochem. 217, 715-722 [Abstract]
  19. Engel, K., Plath, K., and Gaestel, M. (1993) FEBS Lett. 336, 143-147 [CrossRef][Medline] [Order article via Infotrieve]
  20. Stokoe, D., Caudwell, B., Cohen, P. T. W., and Cohen, P. (1993) Biochem. J. 296, 843-849 [Medline] [Order article via Infotrieve]
  21. Zu, Y.-L., Wu, F., Gilchrist, A., Ai, Y., Labadia, M. E., and Huang, C.-K. (1994) Biochem. Biophys. Res. Commun. 200, 1118-1124 [CrossRef][Medline] [Order article via Infotrieve]
  22. Smith, D. B., and Johnson, K. S. (1988) Gene (Amst.) 67, 31-40 [CrossRef][Medline] [Order article via Infotrieve]
  23. Neiman, A. M., and Herskowitz, I. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 3398-3402 [Abstract]
  24. Nomura, N., Zu, Y., Maekawa, T., Tabata, S., Akiyama, T., and Ishii, S. (1993) J. Biol. Chem. 268, 4259-4266 [Abstract/Free Full Text]
  25. Zu, Y., Takamatsu, Y., Zhao, M., Maekawa, T., Handa, H., and Ishii, S. (1992) J. Biol. Chem. 267, 20180-20187
  26. Volpp, B. D., Nauseef, W. M., Donelson, J. E., Moser, D. R., and Clark, R. A. (1989) Proc. Natl. Acid. Sci. U. S. A. 86, 9563
  27. Lomax, K. J., Leto, T. L., Nunol, H., Gallin, J. I., and Malech, H. L. (1989) Science 245, 409-412 [Medline] [Order article via Infotrieve]
  28. Boulton, T. G., and Cobb, M. H. (1991) Cell Regul. 2, 357-371 [Medline] [Order article via Infotrieve]
  29. Clark-Lewis, I., Sanghera, J. S., and Pelech, S. (1991) J. Biol. Chem. 266, 15180-15184 [Abstract/Free Full Text]
  30. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  31. Ren, R., Mayer, B. J., Cicchetti, P., and Baltimore, D. (1993) Science 259, 1157-1161 [Medline] [Order article via Infotrieve]
  32. Birge, R. B., and Hanafusa, H. (1993) Science 262, 1522-1524 [Medline] [Order article via Infotrieve]
  33. Knighton, D. R., Zheng, J., Ten Eyck, L. F., Xuong, N.-H., Taylor, S. S., and Sowadski, J. M. (1991) Science 253, 414-420 [Medline] [Order article via Infotrieve]
  34. Cruzalegui, F. H., Kapiloff, M. S., Morfin, J.-P., Kemp, B. E., Rosenfeld, M. G., and Means, A. R. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 12127-12131 [Abstract]
  35. Zhou, M., Lambert, H., and Landry, J. (1993) J. Biol. Chem. 268, 35-43 [Abstract/Free Full Text]
  36. Guesdon, F., Freshney, N., Waller, R. J., Rawlinson, L., and Saklatvala, J. (1993) J. Biol. Chem. 268, 4236-4243 [Abstract/Free Full Text]
  37. Olivier, J. P., Raabe, T., Henkemeyer, M., Dickson, B., Mbamalu, G., Margolis, B., Schlessinger, J., Hafen, E., and Pawson, T. (1993) Cell 73, 179-191 [Medline] [Order article via Infotrieve]
  38. Cicchetti, P., Mayer, D. J., Thiel, G., and Baltimore, D. (1992) Science 257, 803-806 [Medline] [Order article via Infotrieve]
  39. Rouse, J., Cohen, P., Trigon, S., Morange, M., Alonso-Liamazares, A., Zamanillo, D., Hunt, T., and Nebreda, A. R. (1994) Cell 78, 1027-1037 [Medline] [Order article via Infotrieve]
  40. Freshney, N. W., Rawlinson, L., Guesdon, F., Jones, E., Cowley, S., Hsuan, J., and Saklatvala, J. (1994) Cell 78, 1039-1049 [Medline] [Order article via Infotrieve]
  41. Han, J., Lee, J.-D., Bibbs, L., and Ulevitch, R. J. (1994) Science 265, 808-811 [Medline] [Order article via Infotrieve]
  42. Galcheva-Gorgova, Z., Derjard, B., Wu, I.-H., and Davis, R. J. (1994) Science 265, 806-808 [Medline] [Order article via Infotrieve]
  43. Cheng, H.-C., van Patten, S. M., Smith, A. J., and Walsh, D. A. (1985) Biochem. J. 231, 655-661 [Medline] [Order article via Infotrieve]
  44. Hjelmquist, G., Anderson, J., Edlund, B., and Engstrom, L. (1974) Biochem. Biophys. Res. Commun. 61, 559-563 [Medline] [Order article via Infotrieve]

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