Identification of Two Essential Phosphorylated Threonine Residues In the Catalytic Domain of Mekk1
INDIRECT ACTIVATION BY Pak3 AND PROTEIN KINASE C*

(Received for publication, October 7, 1996, and in revised form, December 31, 1996)

Yaw L. Siow §, Gabriel B. Kalmar , Jasbinder S. Sanghera §, Georgia Tai §, Stella S. Oh and Steven L. Pelech §par

From the  Department of Medicine, University of British Columbia, § Kinetek Pharmaceuticals, Inc., Vancouver, British Columbia V5Z 1A1, Canada, and the  Institute of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

The 78-kDa protein kinase Mekk1 plays an important role in the stress response pathway that involves the activation of downstream kinases Sek1 and stress-activated protein kinase/c-Jun NH2-terminal kinase. Conserved serine and threonine residues located between the kinase subdomains VII and VIII of many protein kinases are phosphorylated for maximal kinase activation. Two threonine residues within this region in Mekk1 at positions 560 and 572, but not the serine at 557, were shown to be essential for catalytic activity in this study. When these threonine residues were replaced with alanine, there was a significant loss in phosphotransferase activity toward the primary substrate, Sek1, and a large decrease in autophosphorylation activity. Site-directed mutagenesis demonstrated that these threonine residues cannot be replaced with either serine or glutamic acid for preservation of phosphotransferase activity. Further examination of the Mekk1 mutants isolated from 32P-labeled transfected COS cells showed that Thr-560 and Thr-572 were indeed phosphorylated after two-dimensional tryptic-chymotryptic phosphopeptide analysis. Additional determinants in the NH2-terminal domain of Mekk1 also play a role in the regulation of Mekk1 activity. Although Pak3 and PKC can activate Mekk1 in vivo, this interaction is indirect and independent, since there was no direct phosphorylation of Mekk1 by Pak3 or PKC or of Pak3 by PKC, respectively.


INTRODUCTION

Guanine nucleotide-binding proteins (G proteins)1 and protein kinases are components within the intracellular signaling pathways in diverse eukaryotes. Modules of sequentially activating protein kinases are coupled to hormone receptors through both monomeric and heterotrimeric classes of G proteins. Genetic analysis of yeast mutants that are compromised in various signal transduction pathways has led to the identification of several related protein kinase modules, some of which transduce signals from G proteins (1). For example, budding yeast mating pheromones bind to serpentine seven-transmembrane domain-containing receptors that are coupled through the beta - and gamma -subunits of a trimeric G protein to the activation of a module of protein kinases composed of Ste20, Ste11, Ste7, and Fus3 (2, 3). Mammalian homologs of each of these kinases have been identified, such as p21-activated kinase (Pak), Mek kinase (Mekk), MAP/Erk kinase (Mek), and extracellular signal-regulated kinase (Erk), respectively (4, 5).

A MAP kinase module that is activated by environmental stresses such as heat shock (6), hyperosmotic conditions (7, 8), UV radiation (9, 10), protein synthesis inhibitors such as cycloheximide (11), and anisomysin (12) as well as proinflammatory cytokines like tumor necrosis factor-alpha (6, 8), has been reported recently. The module is proposed to be comprised of the stress-activated protein (SAP) kinases (6) (also known as c-Jun NH2-terminal kinase), SAP/Erk kinase (Sek1) (12, 13) (also known as MKK4), and Mekk (14, 15). However, the intervening steps following surface receptor binding of these agents are still obscure. There are at least three isoforms of Mekk (16). The original 78-kDa form of Mekk (Mekk1) was first identified as a Mek1 kinase (17) but is considered now to target Sek1 (14, 15, 18). Recently, two additional Mekk clones, Mekk2 and Mekk3, which corresponded to proteins of 69.7 and 71 kDa, respectively, were isolated (19). Mekk2 was reported to preferentially activate the downstream SAP kinase, whereas Mekk3 would activate p42-Erk2 (19). Additionally, Lange-Carter and colleagues (20, 21) have identified in rat pheochromocytoma (PC12) cells two immunologically related Mekks of 82 and 98 kDa, where the 98-kDa form was shown to be activated in response to nerve growth factor, epidermal growth factor, phorbol ester, and oncogenic Ras (20, 21). A larger form of Mekk1, with a predicted molecular mass of 161 kDa, has also been cloned (22). Another isoform may correspond to the I-Mekk detected in preadipocyte 3T3-L1 cells by Haystead et al. (23). This 56-kDa kinase is distinct from Raf1 and is rapidly and transiently activated in rat adipocytes treated with insulin. I-Mekk was also not activated in response to phorbol ester and was immunologically distinct from the Mekk1 described by Lange-Carter et al. (17). Another possible member of the Mekk family is the transforming growth factor-beta -activated kinase, which was also stimulated in response to bone morphogenetic protein (24). This kinase, however, has an extremely short amino terminus (22 residues), which appeared to be regulatory for enzyme function.

Enzymological studies of the above mammalian protein kinases should provide further information about their roles in various G protein-dependent signaling pathways. In the present study, we have characterized two threonine residues, Thr-560 and Thr-572, in Mekk1 that are essential for its catalytic function. We have further determined that the NH2-terminal domain of Mekk1 has an additional regulatory role in the enzyme function. In the search for kinases that target Mekk1, in vivo experiments using Mekk1-transfected COS cells and Mekk1 baculovirus-infected insect cells demonstrated that Mekk1 activity can be stimulated by phorbol ester treatment. However, no direct phosphorylation of Mekk1 by PKC could be demonstrated. This implies that while regulation by PKC may be physiologically significant, PKC activation of Mekk1 is achieved indirectly. Co-expression studies with Pak3 baculovirus- and Mekk1 baculovirus-infected insect cells showed an activation of Mekk1. There was, however, no direct phosphorylation of Mekk1 by Pak3.


EXPERIMENTAL PROCEDURES

Materials

Murine Mekk1 cDNA was kindly provided to us by Drs. Gary Johnson and Carol Lange-Carter of the National Jewish Center for Immunology and Respiratory Medicine in Colorado. The plasmids for GST-Cdc42Hs and murine Pak3 were generously donated by Drs. Subha Bagrodia and Richard Cerione (Cornell University, Ithaca, NY). The plasmids for murine Sek1 and Sek1(K129R/T387A/S390A) were gifts from Dr. Leonard I. Zon (Howard Hughes Medical Institute, Boston, MA). pGEX-Mek1 and pGEX-Mek2 plasmids encoding for the respective human proteins were kindly given by Dr. Kun-Liang Guan (University of Michigan, Ann Arbor, MI). Baculovirus-expressing PKC beta II isoform was a gift from Dr. Daniel E. Koshland (University of California, Berkeley). Purified rat brain protein kinase C (PKC), a mixture of alpha , beta , and gamma  isoforms, was generously provided by Dr. Michael Walsh (University of Calgary, Alberta, Canada).

Construction of Mekk1 Expression Vectors

To express Mekk1 in COS cells, a 2205-base pair (bp) region of Mekk1 containing the entire coding sequence was amplified by PCR using the primers Mekk5-1 (5'-ATG GTT GGC AAG CTC TCT CG) and Mekk3-1 (5'-GTT GCA TAT CCT GTC TCC) with VENT DNA polymerase according to the manufacturer's specifications supplemented with 5% Me2SO. Conditions for amplification of the 2.2-kilobase pair fragment were preincubation at 99 °C for 2 min; 30 cycles of 99 °C for 1 min, 53 °C for 2 min, and 72 °C for 2 min; and then a final extension at 72 °C for 10 min in a Perkin-Elmer 480 thermocycler. The 2.2-kilobase pair PCR product was purified by agarose gel electrophoresis and recovered from the agarose using Qiaex (Qiagen Inc.). The 2.2-kilobase pair Mekk1 fragment was cloned into the mammalian expression vector pEF1 under the control of the human elongation factor-1alpha promoter (25). The pEF1 vector has a simian virus 40 origin that ensures a high copy number of replication for the vector in COS cells. The junction between the Mekk1 PCR fragment and vector pEF1 was sequenced at both the 5'- and 3'-ends of the insert to confirm sequence and orientation. The resulting construct was designated pEF1-Mekk1. Mekk1 with a polyhistidine (His6) tag on the NH2 terminus or COOH terminus was created by PCR mutagenesis using primer Mekk5-H6 (5'-ATG CAC CAT CAC CAT CAC CAT ATG GTT GGC AAG CTC TCT CG) with Mekk3-1 (5'-GTT GCA TAT CCT GTC TCC) or Mekk5-1 (5'-ATG GTT GGC AAG CTC TCT CG) with Mekk-CY (5'-GA GGA TCC CTA GTG ATG ATG GTG GTG GTG CCA CGT GGT ACG GAA GAC CGG), respectively. The truncated kinase was generated using primers Mekk-M353 (5'-ATG GCG ATG TCA GCG TCT CAG) with Mekk3-1. Mutants K432AMekk1, S557AMekk1, T560AMekk1, and T572AMekk1 were also generated by PCR mutagenesis. All mutants were confirmed by dideoxynucleotide sequencing.

Expression of Mekk1 in COS Cells and Fractionation on FPLC

COS-1 (ATCC number CRL 1650) cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum (Life Technologies Ltd.) at 37 °C with 5% CO2. COS cells were transfected with vector, wild type Mekk1, or the various Mekk1 mutants (pEF1, pEF1-Mekk1, pEF1-Mekk1(NT-His6), pEF1-Mekk1(CT-His6), pEF1-K432AMekk1, or pEF1-S557AMekk1 or the threonine mutants, pEF1-T560AMekk1 or pEF1-T572AMekk1) by using DEAE-dextran with a 20% (v/v) glycerol shock for 2 min as described previously (26).

Fifty-two hours after transfection, the cells were washed with DMEM and subsequently incubated in DMEM plus 0.1% bovine serum albumin (w/v). For phorbol 12-myristate 13-acetate (PMA) stimulation, the DMEM plus 0.1% bovine serum albumin was replaced with fresh medium containing 10 nM PMA 18 h later. Unstimulated cells were used as controls. After incubation at 37 °C for the specified period of time, the flask of cells was placed on ice and washed three times with cold phosphate-buffered saline, and the cells were harvested by scrapping in cold phosphate-buffered saline containing 2.5 mM EDTA. COS cells were pelleted by centrifugation and resuspended in homogenization buffer A (10 mM MES (pH 6.0), 50 mM beta -glycerophosphate, 100 µM sodium vanadate, 2 mM MgCl2, 1 mM EGTA, 0.5% Triton X-100, 5 µg/ml leupeptin, 2 µg/ml aprotinin, and 1 mM dithiothreitol) (17). Cells were lysed with a Dounce homogenizer followed by sonication on ice in two 15-s bursts at 80% power output (Branson Sonic Power Company, model 130). A sample of homogenate was removed for kinase assays, and the lysate was centrifuged at 45,000 rpm (TLA45 rotor) for 1 h in a Beckman TL100 ultracentrifuge. The supernatant or cytosolic fraction was collected, and the protein concentration was determined according to the method of Bradford (27) using bovine serum albumin as standard.

For column fractionation, cytosolic protein (1-2 mg) was loaded onto a Mono Q column (1-ml bed volume) previously equilibrated with buffer B containing 10 mM MOPS, pH 7.2, 25 mM beta -glycerol phosphate, 5 mM EGTA, 2 mM EDTA, 0.25 mM dithiothreitol, and 2 mM sodium vanadate. The column was eluted at a flow rate of 0.8 ml/min with a 10-ml linear 0-0.8 M NaCl gradient in buffer B using a Pharmacia FPLC system, and 0.25-ml fractions were collected.

Expression of Mekk1, Pak, and PKC in Insect Cells

Recombinant baculoviruses expressing Mekk1 or Pak3 were isolated using the BaculoGold kit from PharMingen Inc. (San Diego, CA). Virtually 100% of the recombinants were achieved, and one of the plaques was propagated as a recombinant virus stock. Trichoplusia ni cells were grown as monolayers and infected with Mekk1, Pak, and PKC, separately or in combination, as per the manufacturer's instructions at a multiplicity of infection of 10. At 50 h postinfection, cells were harvested as described above for the COS cells.

In Vitro Phosphorylation of Mekk1

To determine whether Mekk1 can be phosphorylated in vitro by PKC or Pak3, Mono-Q-fractionated Mekk1 (wild type and kinase-inactive version, K432AMekk1) was used. Mekk1 (1 µg) was incubated at 30 °C in the presence or absence of PKC (16 ng), 4.5 mM CaCl2, 60 µg/ml phosphatidylserine, 6 µg/ml diolein, and 10 mM MgCl2 in buffer C (12.5 mM MOPS, pH 7.2, 12.5 mM beta -glycerol phosphate, 5 mM EGTA, 7.5 mM MgCl2, 50 mM sodium fluoride, and 250 µM dithiothreitol). The reaction was initiated by the addition of 50 µM [gamma -32P]ATP (~5000 cpm/pmol). After 60 min of incubation, the phosphorylation reaction was terminated by the addition of 5 × SDS sample buffer (250 mM Tris-HCl, pH 6.8, 10% SDS, 25% glycerol, 5% beta -mercaptoethanol, and 0.02% bromphenol blue). Subsequent to SDS-PAGE (28), proteins were transferred to nitrocellulose membrane (pore size, 0.45 µm) as described by Towbin et al. (29), followed by autoradiography.

Pak3 was isolated with glutathione-agarose-immobilized GST-Cdc42Hs (see below) and was mixed with 0.3 µg of Mekk1 in the presence of 10 mM MgCl2 in buffer C. The reaction was initiated by the addition of 50 µM [gamma -32P]ATP (~5000 cpm/pmol) and was incubated for 60 min at 30 °C. An aliquot of the supernatant of the reaction mixture was removed after the reaction, subjected to SDS-PAGE, and then transferred to nitrocellulose membrane followed by autoradiography. Phosphorylated Mekk1 was excised from the membrane, and the radioactivity was determined by liquid scintillation counting. Uninfected insect cell extract was used as control.

Kinase Assays

Mekk1 phosphotransferase activity in COS cells was generally determined using GST-Sek1(K129R/T387A/S390A) as substrate. (Myelin basic protein and histone H1 were also found to be in vitro substrates for Mekk1.) An aliquot of the Mekk1-containing fractions eluted from the Mono Q column or cytosolic cell lysate was added to glutathione-agarose-immobilized GST-Sek1(K129R/T387A/S390A) (1 µg) in the presence of 10 mM MgCl2. The reaction was initiated by the addition of 50 µM [gamma -32P]ATP (~3000 cpm/pmol). After 30 min of incubation at 30 °C, the phosphorylation reaction was terminated by the addition of 1 ml of ice-cold buffer C containing 0.5% Triton X-100 and 150 mM NaCl. The reaction mixture was then centrifuged at maximum speed in a microcentrifuge for 1 min, and the supernatant was discarded. The pellet was washed twice more with buffer C followed by the addition of an aliquot of 5 × SDS sample buffer. Following SDS-PAGE, proteins were transferred to nitrocellulose membrane. GST-Sek1(K129R/T389A/S390A) bands were visualized by Ponceau S staining and autoradiography and then excised for liquid scintillation counting.

Pak activity was measured using MBP as substrate. A 100-µl aliquot of cytosolic cell extract was incubated with 2 µg of glutathione agarose-immobilized GST-Cdc42Hs for 60 min at 30 °C. The preincubation was terminated by the addition of 1 ml of ice-cold buffer C. The reaction mixture was then centrifuged at maximum speed in a microcentrifuge for 1 min, and the supernatant was discarded. The pellet was washed once more with Buffer C followed by the addition of MBP (final concentration: 3 mg/ml) and 10 mM MgCl2. The reaction was initiated by the addition of 70 µM [gamma -32P]ATP (~700 cpm/pmol). After 30 min of incubation at 30 °C, the phosphorylation reaction was terminated by the addition of an aliquot of 5 × SDS sample buffer. Following SDS-PAGE, proteins were transferred to nitrocellulose membrane. Bands corresponding to MBP were visualized by Ponceau S staining and autoradiography and then excised for liquid scintillation counting. The presence of Pak3 in the assay was confirmed by Western immunoblotting using the anti-Pak3-NT polyclonal antibody as described above.

PKC assays were determined using PKC MBP (residues 4-14) substrate peptide, QKRPSQRSKYL (Upstate Biotechnology, Inc., Lake Placid, NY) as substrate.

32P Labeling of Mekk1

COS cells were transfected with COOH terminus polyhistidine-tagged wild type Mekk1 and Mekk1 threonine mutants, T560AMekk1 and T572AMekk1, as described above and subsequently labeled with 0.1 mCi of 32Pi/ml of phosphate-free DMEM for 4 h at 37 °C in the presence of CO2. At the end of the labeling period, the cells were harvested as described above. Cell pellets were lysed in 2 ml of 6 M guanidine HCl, 0.1 M sodium phosphate, 0.01 M Tris-HCl, pH 8.0. This lysate was passed through a 24-gauge syringe needle four times prior to binding to 1 ml of ProBond (Invitrogen). The beads were washed, and polyhistidine-tagged Mekk1 was eluted under denaturing conditions as per the manufacturer's protocol. The eluate was concentrated, exchanged into 2% SDS using Centricon-30 (Amicon), and then boiled in SDS sample buffer. Following SDS-PAGE, proteins were transferred to polyvinylidene difluoride. Mekk1 bands were visualized by autoradiography and excised for tryptic-chymotryptic two-dimensional phosphopeptide mapping (30).


RESULTS

Substrate Specificity of Mekk1

The 78-kDa protein kinase Mekk1 was originally identified as a Mek1 kinase (17), but recent expression studies in COS cells (14) and NIH-3T3 cells (15) have instead pointed to Sek1 as its physiological target. Low levels of Mekk1 lead to increased activation of SAP kinases without stimulation of Erk1 or Erk2. We have found that recombinant Mekk1 expressed in COS cells (Fig. 1B) catalyzes the phosphorylation of a GST fusion protein of Sek1 only about 3 times better than GST-Mek1 and twice as well as GST-Mek2 in vitro (Fig. 1D). Furthermore, when phosphate incorporation caused by autophosphotransferase activity of the substrates was eliminated, their phosphorylation by Mekk1 was quite similar. This was accomplished by using kinase-inactive versions of GST-Sek1 (Sek1(K129R/T387A/S390A)) and GST-Mek1 (Mek1(K97A)) as substrates, resulting in Mekk1 activities of 1.9 and 1.8 pmol/min/ml, respectively. Mekk1 phosphorylation of GST-Sek1 has been demonstrated on Ser-220 and Thr-224 just upstream of the kinase subdomain VIII region (15). The phosphorylation of Mek1 by Mekk1 has also been shown to occur at residues Ser-218 and Ser-222 in a homologous kinase subdomain to that of Sek1 (31-34). All of the following studies were conducted using the kinase-inactive version of Sek1, which also had its two potential MAP kinase phosphorylation sites at Thr-387 and Ser-390 mutated to alanine. These MAP kinase sites can be phosphorylated in vitro by Erk2, but the functional effect of this is unclear (13).


Fig. 1. Substrate specificity of Mekk1. Cytosols from vector-only and Mekk1-transfected cells were fractionated on FPLC, and the Mekk-containing fractions were identified by Western immunoblotting using anti-Mekk-CT (Upstate Biotechnology). Panel A shows the expression of endogenous Mekk in vector-alone transfected COS cells, whereas panel B shows an immunoblot of recombinant Mekk1 expression in COS cells. The Mono Q fractions that contained Mekk1 in panels A and B were assayed for phosphotransferase activity toward recombinant GST-Mek1 as described for GST-Sek1(K129R/T387A/S390A) under "Experimental Procedures" and then subjected to SDS-10% PAGE and transferred to nitrocellulose membranes. Panel C shows the corresponding autoradiogram. Panel D depicts the average incorporation of 32P associated with GST-Mek1, GST-Mek2, and GST-Sek1 when they were used as substrates for the pooled Mono Q fractions that contained Mekk1. After the excision of the GST fusion protein substrates from the nitrocellulose membrane, they were counted in a liquid scintillation counter. Values are from two separate experiments (± range) and have been corrected for differences in protein concentration.
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The NH2-terminal Domain of Mekk1 Is Regulatory

The NH2-terminal domain of Mekk1 has been suggested to play a regulatory role in its phosphotransferase activity (17). Interestingly, when a polyhistidine sequence was tagged onto the NH2 terminus of Mekk1, the enzyme was inactive (Fig. 2). A bacterially expressed GST-Mekk1 (GST tagged at the NH2 terminus) fusion protein was also inactive (data not shown). However, when the polyhistidine was tagged onto the C terminus of Mekk1, it did not affect its phosphotransferase activity. A catalytically active Mekk1 was also generated when the NH2-terminal domain (residues 1-352) was deleted from the protein (Fig. 2). As reported by Minden et al. (14), this truncated form of Mekk1 was expressed at much higher levels than all other forms of Mekk1.


Fig. 2. Regulation of Mekk1 activity by NH2-terminal domain. COS cells were transfected with vector, wild type Mekk1, NH2-terminal truncated Mekk1, or NH2 terminus or COOH terminus polyhistidine-tagged Mekk1, grown, and harvested as described under "Experimental Procedures." Mekk1 activity was assayed using GST-Sek1(K129R/T387A/S390A) as substrate and analyzed as described in the legend to Fig. 1. Figure shows the average incorporation of 32P into GST-Sek1(K129R/T387A/S390A) from two separate experiments (± range).
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Essential Threonine Residues in the COOH-terminal Catalytic Domain

We next assessed whether the COOH-terminal catalytic kinase domain contains regulatory residues. Of particular interest was the loop 12 region between conserved kinase subdomains VII (DFG) and VIII (APE) (35), where activating serine, threonine, and tyrosine residues for many protein kinases have been reported (9, 12, 13, 15, 29, 36-46). In the homologous region of Mekk1, there are three potential serine/threonine residues, namely Ser-557, Thr-560, and Thr-572 (Fig. 3). In the present study, these residues were mutated to alanine, and the autophosphotransferase activity was determined. Fig. 4A showed that the two threonine mutants had substantially reduced autophosphotransferase activities: 36 and 12% of wild type for T560AMekk1 and T572AMekk1 mutants, respectively. In contrast, the S557AMekk1 mutant did not exhibit any difference in autophosphotransferase activity. Similar patterns were obtained when the enzyme activity was determined using GST-Sek1(K129R/T387A/S390A) as substrate (Fig. 4B). Both T560AMekk1 and T572AMekk1 mutants were reduced to 8% of wild type Mekk1 activity, while S557AMekk1 was essentially unchanged.


Fig. 3. Activating serine/threonine residues between kinase subdomains VII and VIII. The amino acid sequence between conserved kinase subdomain VII (DFG) and subdomain VIII (APE) of several protein kinases is shown. Asterisks denote residues shown to be phosphorylated and/or implicated in the activation of the kinases. One-letter abbreviations for the amino acid residues are used.
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Fig. 4. Analysis of wild type Mekk1 and phosphorylation site mutants. Three potential phosphorylation sites between kinase subdomains VII and VIII of Mekk1 were individually mutated to alanine using PCR mutagenesis. A kinase-inactive version of Mekk1, K432AMekk1, was also generated for use as a control. COS cells were then transfected with vector, wild type, or mutant Mekk1, and 1 mg of cytosolic protein was fractionated on a Mono Q column as described under "Experimental Procedures." Peak fractions (as confirmed by Western blotting) from vector or Mekk1-transfected cells were pooled, assayed, and then subjected to SDS-10% PAGE and electrophoretic transfer to nitrocellulose membrane. Figure depicts the mean incorporation (± S.D., n = 3-5) of 32P associated with the respective proteins after it was excised from the nitrocellulose membrane and counted in a liquid scintillation counter. Panel A shows Mekk1 autophosphorylation activity, whereas panel B shows Mekk1 phosphotransferase activity toward GST-Sek1(K129R/T387A/S390A). Values are expressed as a percentage of wild type Mekk1 autophosphorylation activity (1.07 ± 0.06 pmol/min/ml) and phosphotransferase activity toward GST-Sek1(K129R/T387A/S390A) (1.56 ± 0.10 pmol/min/ml).
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To determine whether these two threonine sites are phosphorylated in vivo, COOH-terminal polyhistidine-tagged Mekk1 mutants were isolated from 32P-labeled transfected COS cells and analyzed by two-dimensional tryptic-chymotryptic phosphopeptide mapping. The wild type Mekk1 generated at least 12 phosphopeptides (Fig. 5A). When the transfected COS cells were transfected with T560AMekk1 mutant (Fig. 5B) or with T572AMekk1 mutant (Fig. 5C), there was a disappearance of several phosphopeptides. In each of the phosphopeptide maps of the threonine mutants, a unique phosphopeptide (labeled a and b for T560AMekk1 and T572AMekk1, respectively) was missing compared with that of the wild type. Therefore, it appears that both Thr-560 and Thr-572 of Mekk1 were phosphorylated in vivo and probably corresponded to phosphopeptide a and b, respectively.


Fig. 5. Autoradiographs showing two-dimensional phosphopeptide map of metabolically labeled wild type and mutant Mekk1. COS cells transfected with polyhistidine-tagged wild type or mutant Mekk1 were labeled with 32Pi and isolated as described under "Experimental Procedures." After affinity chromatography under denaturing conditions, the labeled proteins were subjected to SDS-PAGE, transferred to polyvinylidene difluoride membrane, visualized by autoradiography, and excised. Phosphorylated Mekk1 bands were dissected and subjected to phosphopeptide mapping (30). After exhaustive sequential digestion with chymotrypsin and then trypsin, phosphopeptides were applied to cellulose thin layer chromatography sheets (O represents origin) and were separated in two dimensions, first by electrophoresis in the horizontal dimension and then by ascending chromatography in the vertical dimension. Panel A corresponds to the phosphopeptide map obtained with wild type Mekk1, whereas panels B and C show the phosphopeptide maps obtained from T560AMekk1 and T572AMekk1 mutants, respectively. Broken circles and arrows point toward missing phosphopeptides in the threonine mutants, and their corresponding positions are also marked in the phosphopeptide map obtained from the wild type.
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To investigate further the significance of the contribution of Thr-560 and Thr-572 to the enzyme activity of Mekk1, we mutated these residues to serine and to glutamic acid residues separately. Fig. 6A shows that when the two threonine residues were separately mutated to serine, the autophosphotransferase activities were reduced to 86 and 37% of wild type Mekk1 for T560SMekk1 and T572SMekk1 mutants, respectively. Although these autokinase activities were higher than that of the alanine mutants (T560AMekk1 and T572AMekk1), their catalytic activities toward Sek1 were still significantly low compared with that of wild type (Fig. 6B). The T560SMekk1 and T572SMekk1 mutants had only 26 and 15% catalytic activity, respectively, compared with that of wild type. Fig. 6, A and B, also indicate that the two threonine residues cannot be substituted with glutamic acid residues, which can partially mimic the introduction of phosphorylated residues (47).


Fig. 6. Analysis of wild type Mekk1 and threonine site mutants. The threonines at positions 560 and 572 of Mekk1 were mutated to alanine, serine, or glutamic acid residues using PCR mutagenesis. COS cells were then transfected with vector, wild type, or mutant Mekk1, and the cytosolic cell lysate was assayed and analyzed as described in the legend to Fig. 4. Panel A shows Mekk1 autophosphorylation activity, whereas panel B shows Mekk1 phosphotransferase activity toward GST-Sek1(K129R/T387A/S390A). Values are expressed as a percentage of wild type Mekk1 autophosphorylation activity (1.07 ± 0.06 pmol/min/ml) and phosphotransferase activity toward GST-Sek1(K129R/T387A/S390A) (1.07 ± 0.06 pmol/min/ml). T560A, T572A, T560S, T572S, T560E, and T572E denote single mutations at Thr-560 and Thr-572 to alanine, serine, and glutamic acid, respectively, while T560E/T572E, T560E/T572A, and T560A/T572E denote double mutations at Thr-560 and Thr-572.
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Mekk1 Is Activated in Phorbol Ester-stimulated COS Cells

We next sought to identify the activating kinase that targets the threonine sites. Since the primary structure of the 78-kDa Mekk1 features several potential PKC phosphorylation sites (17), we proceeded to explore the regulation of Mekk1 by PKC. Mekk1 was expressed in COS cells and stimulated in vivo by phorbol PMA, a known activator of PKC. Cytosolic extracts from the stimulated and unstimulated cells were fractionated by Mono Q chromatography. Western immunoblotting confirmed that Mekk1 was appreciably expressed in COS cells transfected with the Mekk1 plasmid and eluted from the anion exchange column at a NaCl concentration of 0.4-0.45 M (data not shown). The kinase was also very active, since it phosphorylated GST-Sek1 (Fig. 7) and autophosphorylated (data not shown). Fig. 7 shows that PMA further stimulated, approximately 2.5-fold, Mekk1 phosphotransferase activity toward GST-Sek1. In these PMA-stimulated cells, PKC activity was also stimulated by approximately 2-fold (data not shown). Mekk1 was stimulated by as low as 1 nM PMA and optimally with 10 nM (data not shown). Higher concentrations of PMA did not produce detectable activation of Mekk1. Additionally, the maximal stimulatory effect with PMA was within 10 min. This phorbol ester-stimulated increase in 32P incorporation into Sek1 is the first indication that PKC may be involved in the activation of Mekk1. This activation of Mekk1 by phorbol ester could also be blocked by incubation of the transfected COS cells with 10 µM of the PKC inhibitor Roche compound 3 for 1 h prior to harvest of the cells for determination of Mekk1 phosphotransferase activity toward Sek1 (data not shown).


Fig. 7. Phorbol ester stimulation of Mekk1 activity in COS cells. COS cells were stimulated with 10 nM PMA for 10 min, and 1 mg of cytosolic protein was fractionated on a Mono Q column as described under "Experimental Procedures." Peak fractions (as confirmed by Western blotting) from stimulated and unstimulated vector or Mekk1-transfected cells were assayed for Mekk1 activity using GST-Sek1(K129R/T387A/S390A) and then subjected to SDS-10% PAGE, transferred to nitrocellulose membrane. Figure depicts the mean incorporation (± S.D., n = 3) of 32P associated with GST-Sek1(K129R/T387A/S390A) after it was excised from the nitrocellulose membrane and counted in a liquid scintillation counter. Values are expressed as a percentage of unstimulated Mekk1 activity (1.42 ± 0.26 fmol/min/ml) and have been adjusted for differences in protein concentration.
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Mekk1 Is Activated when Co-expressed with PKC in Insect Cells

We next tested whether Mekk1 could be further activated by co-expression with PKC in insect cells. Recombinant baculoviruses encoding Mekk1 and PKCbeta II were used individually or in combination to infect the T. ni insect cells. When expressed alone, Mekk1 was very active, but it could again be further stimulated about 2-fold by PMA (Fig. 8, A and C). When co-expressed with PKC, Mekk1 phosphotransferase activity toward GST-Sek1 was increased to levels similar to that observed when stimulated by PMA. The level of PKC protein and enzymatic activity was reduced about 50% when the PKC was co-expressed with Mekk1 in the same experiments, although the expression of Mekk1 was unaffected (Fig. 8B). The addition of PMA to insect cells expressing both PKC and Mekk1 did not further stimulate Mekk1 activity. PKC could not be further stimulated by the addition of PMA to these cells (Fig. 8B). Apparently, co-infection of PKC and Mekk1 resulted in the maximal activation of Mekk1.


Fig. 8. Effect of co-expression of PKC and Mekk1 in insect cells. Insect cells (1 × 106) were infected with Mekk1 and/or PKC baculoviruses and incubated with and without with 10 nM PMA for 10 min prior to harvest as described under "Experimental Procedures." For panel A, Mekk1 activity was assayed and analyzed after MonoQ fractionation as described in the legend to Fig. 1. The background autophosphorylation of GST-Sek1(K129R/T387A/S390A) was approximately 15 pmol/min/ml, and this has not been substracted from the values shown in panel A. For panel B, PKC activity was determined as indicated under "Experimental Procedures." Panel C depicts mean incorporation (± S.D.) of 32P incorporated into GST-Sek1(K129R/T387A/S390A) from three separate experiments. Values are expressed as a percentage of unstimulated Mekk1 activity (4.0 pmol/min/ml).
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Mekk1 Cannot Be Directly Phosphorylated by PKC

The ability of highly purified preparations of PKC (alpha , beta , and gamma ) from rat brain to phosphorylate recombinant Mekk1 in vitro was tested. No cross-phosphorylation of Mekk1, isolated from transiently transfected COS cells and Mekk1 baculovirus-infected insect cells, could be demonstrated (data not shown). A kinase-inactive form of Mekk1 (K432AMekk1), which was expressed in COS cells, was also not a substrate of PKC (data not shown). However, PKC could directly phosphorylate bacterially expressed GST-Mekk1 in vitro, and three sites of phosphorylation (Ser-194, Ser-504, and Ser-557) were identified using coupled high pressure liquid chromatography-electrospray ionization mass spectrometry. Nevertheless, in vivo labeling of the mutated forms of Mekk1, in which each of these serine residues was replaced by alanine, followed by two-dimensional phosphopeptide analysis showed that these sites were not phosphorylated in response to PMA stimulation of COS cells.

Co-expression of Mekk1 with Pak3 or Cdc42Hs in Insect Cells

Recent studies (15, 48) have indicated that Pak may be the upstream kinase that targets Mekk1. Additionally, two small GTP-binding proteins, Rac1 and Cdc42Hs, have been shown to activate the c-Jun NH2-terminal kinase/SAP kinase signaling pathway (48, 49). In an attempt to ascertain whether Pak is the kinase responsible for phosphorylating the two threonine residues in vivo, we tested whether Mekk1 is activated when co-expressed with Pak3 or Cdc42Hs in insect cells. In cells expressing Pak3 alone, there was a 3-fold higher Mekk1 phosphotransferase activity toward GST-Sek1 compared with that expressing Mekk1 alone (Fig. 9). When Pak3 was co-expressed with Mekk1, the Mekk1 activity was increased 8.4-fold over that expressing Mekk1 alone (Fig. 9). This Mekk1 activity was attenuated when these cells were co-infected with Cdc42Hs. However, when Cdc42Hs was co-infected with Mekk1, the Mekk1 activity was increased 4-fold.


Fig. 9. Effect of co-expression of Mekk1, Pak3, and Cdc42Hs in insect cells. Insect cells (1 × 106) were infected with Mekk1, Pak3, or Cdc42Hs baculoviruses, individually or in combination, and harvested as described under "Experimental Procedures." Mekk1 activity was assayed and analyzed after Mono Q fractionation as described in the legend to Fig. 1. Figure depicts the mean incorporation (± S.D.) of 32P incorporated into GST-Sek1(K129R/T387A/S390A) from three separate experiments. Values are expressed as a percentage of unstimulated Mekk1 activity.
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Mekk1 Cannot Be Phosphorylated by Pak3 Alone

As with PKC, we also used active Pak3 to phosphorylate recombinant Mekk1 in vitro. Similar to the findings with PKC, Pak3 was unable to cross-phosphorylate recombinant Mekk1 expressed in baculovirus-infected insect cells or transiently transfected COS cells (data not shown). The recombinant Pak3 was nevertheless very active, since it could catalyze the phosphorylation of myelin basic protein.

Pak3 Is Not Targeted by PKC

Although Pak3 and PKC did not directly phosphorylate Mekk1, we were interested in determining whether the activations of Mekk1 by these kinases were independent. We thus measured Pak activity in PMA-stimulated Mekk1-transfected COS cell lysates. Using MBP as substrate, Pak activity was not significantly elevated in these cells as compared with those that were unstimulated (data not shown). To determine whether PKC directly activates Pak, insect cells were infected with baculoviruses encoding Pak3 and PKCbeta II either individually or in combination. When co-expressed with PKC, Pak3 phosphotransferase activity toward MBP was not substantially different from that which expressed Pak3 alone (Fig. 10). When stimulated with PMA, there was no difference in the levels of Pak3 activity (Fig. 10).


Fig. 10. Effect of co-expression of Pak3 and PKC on Pak activity. Insect cells (1 × 106) were infected with Pak3 and/or PKC baculoviruses and harvested as described under "Experimental Procedures." The cytosolic cell lysate was assayed for Pak activity as described under "Experimental Procedures." Figure depicts mean incorporation (± S.D.) of 32P incorporated into myelin basic protein from three separate experiments.
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DISCUSSION

In mammalian cells, many modules of protein kinases transduce signals from receptors that are coupled to both monomeric and heterotrimeric G proteins. The kinases in these modules may be assembled into complexes to prevent cross-talk with related kinase modules in parallel pathways, to increase the efficiency of signal transmission, and to accept multiple negative and positive inputs from other signaling pathways. Protein phosphorylation has been shown to be critical for the activation of MAP kinases, SAP kinases, Mek, and Sek (5), which exhibit strong homology with Mekk1 in their catalytic domains. It was anticipated that the conserved serine and threonine residues located just before the subdomain VIII region of the MAP kinase/Ste-related kinase superfamily need to be phosphorylated for maximal kinase activation. The results in this study indicate that two threonine residues at positions 560 and 572 in the sequence of Mekk1 are essential for catalytic activity. First, when these threonine residues were substituted with alanine, there was a significant loss in phosphotransferase activity toward the primary substrate, Sek1, and a near complete loss in autophosphorylation activity. Second, the phosphopeptide pattern after metabolic labeling showed that there was a disappearance of major spots in the T560AMekk1 and T572AMekk1 mutants compared with that of wild type Mekk1, indicating that these two threonine residues are indeed phosphorylated in vivo. Furthermore, results here also support the notion that the NH2-terminal domain of Mekk1 has a regulatory function.

As has been suggested by Rhodes et al. (50) for Ste11 and Lange-Carter et al. (17) for Mekk1, the NH2-terminal domain of these protein kinases participates in the regulation of their activities. When amino acid residues 1-352 were deleted from Mekk1 and expressed in COS cells, there was an increase in phosphotransferase activity toward Sek1 for the truncated mutant. These results corroborate the published observations for truncated Mekk1 (15, 32, 34). Additionally, it is intriguing that Mekk1 was completely inactivated when a polyhistidine tag was fused to the NH2 terminus of the kinase but exhibited no difference in activity when the tag was fused to the COOH terminus. It is tempting to suggest that the NH2 terminus of the kinase is embedded within the protein molecule but not the COOH terminus. Thus, the addition of a peptide sequence, especially a charged one, to the NH2 terminus may disrupt the proper folding of the molecule. Further investigation will be necessary to establish the precise residue(s) or peptide region in Mekk1 that is involved in regulation.

In addition to the regulatory NH2-terminal domain, there are additional determinants of enzyme activity present in the COOH-terminal domain of Mekk1. As previously mentioned, there are two essential threonine residues in Mekk1, Thr-560 and Thr-572, which are located in a region analogous to the phosphorylation lip of MAP kinases and cyclin-dependent kinases 2 and 4 (42, 47), i.e. between the conserved kinase subdomain VII and VIII region. In the present study, when Thr-560 and Thr-572 were individually mutated to alanine residues, Mekk1 lost a significant portion of its catalytic activity both in terms of autophosphorylation and phosphotransferase activity toward Sek1. The effect was much more pronounced with the Thr-572 mutation, which is located just before kinase subdomain VIII. Wu and associates (51) recently reported that in yeast Ste20p, Thr-777 could also autophosphorylate and was crucial for its catalytic activity. In Ste20p, Thr-777 lies in a position equivalent to that of Thr-572 in Mekk1. Furthermore, the catalytic subunit of cAMP-dependent protein kinase can also autophosphorylate at Thr-197, which is located in an equivalent kinase region (41). The autophosphorylation at that threonine residue has been shown to be sufficient to activate the cAMP-dependent protein kinase. In an attempt to distinguish whether autophosphorylation or cross-phosphorylation by another kinase is required for Mekk1 activity, both Thr-560 and Thr-572 were individually mutated to serine, which was a conservative amino acid substitution. This partially restored the autophosphorylation activity about 2- (Thr-560) to 6-fold (Thr-572) for the respective Mekk1 mutant. With a serine at position 572, the mutant Mekk1 did not recover much phosphotransferase activity toward Sek1. This implies that a fully autophosphorylated Thr-572 may be crucial for catalytic activity. Despite restoration of up to 86% of wild type Mekk1 autophosphorylation activity, the T560SMekk1 mutant only had about 26% of wild type phosphotransferase activity toward Sek1. Thus, the presence of a threonine residue at position 560 may be important for the specific interaction or recognition by a regulatory protein or an activating kinase. This is based on the recent suggestion that the phosphothreonine (Thr-197) region of the cAMP-dependent protein kinase may serve as a docking surface for the regulatory subunit of the holoenzyme complex (52). Further, it has been reported that the calcium/calmodulin-dependent protein kinase I was phosphorylated on Thr-177 and that its activity was enhanced 25-fold by calcium/calmodulin-dependent protein kinase I kinase (46). Ribosomal S6 kinase (Rsk1) was also activated by MAP kinase when it was phosphorylated at Thr-562 (53). Both activating threonine residues of calcium/calmodulin-dependent protein kinase I and Rsk1 are located at equivalent positions just before the catalytic subdomain VIII region.

Efforts were also made to change the two putative threonine phosphorylation sites to glutamic acid residues to mimic the introduction of negative charges due to phosphorylation. Such mutations have been reported to produce constitutively active Mek1 (54-56). Moreover, it has been proposed that a negative charge on this loop is important for the correct alignment of the residues participating in catalysis (44). In the present study, however, these glutamic acid replacements in Mekk1 did not translate into elevated Mekk1 activity toward Sek1 nor increased autophosphorylation activity. Similar observations have been reported for analogous mutants in two other Mek1 homologs, i.e. Mek5 and MKK4 (Sek1) (57). This further demonstrates that the environment of the activating Thr-560 and Thr-572 in Mekk1 is more involved than the requirement of a negatively charged residue. Precise determination of the molecular environment of these residues will require further structural studies.

In an attempt to identify the kinase(s) that targets Mekk1, we demonstrated that both Pak3 and PKC can activate Mekk1 in vivo. Mekk1 phosphotransferase activity was stimulated 2-fold in response to PMA when expressed either in simian COS cells or in T. ni insect cells, and this could be blocked by preincubation of the cells with the PKC inhibitor Roche compound 3. When co-expressed with PKC in insect cells, Mekk1 activity was elevated to levels similar to that observed in the presence of PMA. We also tested whether the Cdc42/Rac-activated kinase, Pak, which is suggested to be upstream of Mekk1 (15, 48), can activate Mekk1. When Mekk1 was co-expressed with Pak3, Mekk1 activity was stimulated 8.4-fold. However, direct phosphorylation by Pak3 or by PKC could not be demonstrated. Prior treatment of the Mekk1 with phosphatases followed by the addition of Pak3 or PKC also did not result in any increase in 32P incorporation into Mekk1.2 Hence, activation of Mekk1 in vivo does not appear to be a consequence of direct phosphorylation by Pak3 or by PKC. Furthermore, the activation of Mekk1 by Pak3 is independent of the activation by PKC. It is still possible that Mekk1 is phosphorylated and activated by another Ste20-like member of the family of p21 G protein-activated kinases (58-61). Likewise, Pak3 may target other mammalian Mekk homologs. In Saccharomyces cerevisiae, it has been demonstrated that Ste20p can directly phosphorylate but not activate Ste11p (51).

Although Mekk1 could phosphorylate Mek1, Mek2, Sek1, and MKK3, when it was expressed at physiological levels, only SAP kinase was markedly activated without Hog (13) or Erk (34) stimulation. Because of the promiscuity of enzymes like Mekk1, the question of how the cell maintains specificity in intracellular signaling begs further investigation. As reported for the mating pheromone signaling cascade in S. cerevisiae, a plausible alternative may be the mammalian homolog of Ste5p, which is suggested to be the scaffolding protein involved in coordinating the Ste20 right-arrow Ste11 right-arrow Ste7 right-arrow Fus3 protein kinases (2, 3, 51, 62). The mammalian Ste5-like homolog may interact with Mekk1 in a manner that orientates Mekk1 in a conformation that allows only limited and more specific access to activating kinases. A more recent report suggests that extracellular stress stimuli and Mekk may result in the induction of MAP kinase phosphatase 1 gene expression that can inactivate Erk and thus convey specificity to the stress-activated pathway (63). Since Mekk1 is demonstrated here to be phosphorylated in vivo, one explanation for the inability of Pak3 to phosphorylate Mekk1 may be that transphosphorylation of the expressed Mekk1 may already have occurred in vivo and thus renders it catalytically competent. The autophosphorylation at the two threonine sites may be a consequence of an already active Mekk1. This, nonetheless, does not diminish the significance of the finding that there is a loss of function when either Thr-560 or Thr-572 is replaced with another amino acid. These threonine residues may be involved in substrate recognition, and their phosphorylation may change the conformation of the substrate binding cleft, thus allowing access only to a specific substrate. The results of the present study, nevertheless, cannot differentiate the order of phosphorylation for Thr-560 and Thr-572. It should be reiterated that there are additional determinants present in the NH2-terminal domain of Mekk1, which may be key to understanding the regulation of this enzyme. Regions of the NH2-terminal domain may fold over and interact with the active site, thus restricting access to a substrate. Resolution of this awaits the three-dimensional structure determination of Mekk1.


FOOTNOTES

*   These studies are supported by operating grants from the National Cancer Institute of Canada.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.
par    Recipient of a Medical Research Council of Canada Scientist Award. To whom correspondence should be addressed: Kinetek Pharmaceuticals, Inc., Suite 150, 520 W. 6th Ave., Vancouver, BC, Canada V5Z 1A1. Tel.: 604-876-5420 (ext. 129); Fax: 604-876-5498; E-mail: spelech{at}kinetekpharm.com.
1   The abbreviations used are: G protein, guanine nucleotide-binding protein; bp, base pair(s); DMEM, Dulbecco's modified Eagle medium; Erk, extracellular signal-regulated kinase; Erk1, extracellular signal-regulated kinase 1, 44-kDa mitogen-activated protein kinase encoded by the erk1 gene; GST, glutathione S-transferase; His6, polyhistidine tag with six residues; MAP kinase, mitogen-activated protein kinase; MBP, myelin basic protein; Mek1, 46-kDa MAP kinase kinase-1; Mekk1, 78-kDa Mek kinase-1; Mekk1Delta N, Mekk1 with NH2-terminal residues 1-352 deleted; Mekk1(CT-His6), Mekk1 with a polyhistidine tag at the COOH terminus; Mekk1(NT-His6), Mekk1 with a polyhistidine tag at the NH2 terminus; MOPS, 4-morpholinepropanesulfonic acid; Pak, p21-activated kinase; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; PMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C; SAP kinase, stress-activated protein kinase; Sek, SAP/Erk kinase-1; Sek1(K129R/T387A/S390A), kinase-inactive version of Sek1 that has potential MAP kinase phosphorylation sites at Thr-387 and Ser-390 mutated; MES, 2-(N-morpholino)ethanesulfonic acid; FPLC, fast protein liquid chromatography.
2   Y. L. Siow, G. B. Kalmar, J. S. Sanghera, G. Tai, S. S. Oh, and S. L. Pelech, unpublished observations.

Acknowledgments

We are grateful to Drs. Subha Bagrodia, Richard Cerione, Kun-Liang Guan, Gary Johnson, Carol Lange-Carter, and Leonard Zon for plasmids and to Monika Ahluwalia, Gloria Leung, and Edward Lynn for technical assistance.


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