A Novel Human STE20-related Protein Kinase, HGK, That Specifically Activates the c-Jun N-terminal Kinase Signaling Pathway*

Zhengbin YaoDagger §, Guisheng Zhou§, Xuhong Sunny WangDagger , Amy Brown, Katrina DienerDagger , Hong Gan, and Tse-Hua Tanparallel

From Dagger  Amgen, Inc., Boulder, Colorado 80301 and the  Department of Microbiology and Immunology, Baylor College of Medicine, Houston, Texas 77030

    ABSTRACT
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Abstract
Introduction
References

The yeast serine/threonine kinase STE20 activates a signaling cascade that includes STE11 (mitogen-activated protein kinase kinase kinase), STE7 (mitogen-activated protein kinase kinase), and FUS3/KSS1 (mitogen-activated protein kinase) in response to signals from both Cdc42 and the heterotrimeric G proteins associated with transmembrane pheromone receptors. Using degenerate polymerase chain reaction, we have isolated a human cDNA encoding a protein kinase homologous to STE20. This protein kinase, designated HPK/GCK-like kinase (HGK), has nucleotide sequences that encode an open reading frame of 1165 amino acids with 11 kinase subdomains. HGK was a serine/threonine protein kinase that specifically activated the c-Jun N-terminal kinase (JNK) signaling pathway when transfected into 293T cells, but it did not stimulate either the extracellular signal-regulated kinase or p38 kinase pathway. HGK also increased AP-1-mediated transcriptional activity in vivo. HGK-induced JNK activation was inhibited by the dominant-negative MKK4 and MKK7 mutants. The dominant-negative mutant of TAK1, but not MEKK1 or MAPK upstream kinase (MUK), strongly inhibited HGK-induced JNK activation. TNF-alpha activated HGK in 293T cells, as well as the dominant-negative HGK mutants, inhibited TNF-alpha -induced JNK activation. These results indicate that HGK, a novel activator of the JNK pathway, may function through TAK1, and that the HGK right-arrow TAK1 right-arrow MKK4, MKK7 right-arrow JNK kinase cascade may mediate the TNF-alpha signaling pathway.

    INTRODUCTION
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Abstract
Introduction
References

The c-June N-terminal kinase (JNK)1 family members belong to the mitogen-activated protein kinase (MAPK) superfamily, which also includes extracellular signal-regulated kinases (ERKs) and the p38 family (reviewed in Ref. 1). MAPK superfamily members are serine/threonine-directed kinases that target other kinases, transcription factors, and membrane receptor tails, causing diverse effects such as cell proliferation, transformation, differentiation, and apoptosis. Activation of MAPK members occurs through an assembly of kinase cascades in response to stimuli. The original MAPK cascade (c-Raf right-arrow MEK1/2 right-arrow ERK1/2) includes activation of the serine/threonine kinase c-Raf, which activates a dual-specificity MAPK kinase (MEK1/MEK2). MAPK/ERK is then activated via tyrosine and threonine phosphorylation of the T-X-Y motif on the phosphorylation loop of the enzyme. This phosphorylation event causes local and global conformational changes in the protein, resulting in an active enzyme structure.

The JNK family members were characterized biochemically as two kinases and cloned from human fetal brain and rat brain libraries (2, 3). Presently, the JNK family consists of three genes, JNK1, JNK2, and JNK3, which can be subdivided further into 10 isoforms (4). JNK1 and JNK2 have four isoforms, while JNK3 has two isoforms; these isoforms arise from alternative splicing. Their murine homologues are called stress-activated protein kinases (3). Active JNKs target Ser-Pro and Thr-Pro motifs on their substrates. Substrates for JNK family members include the transcription factors c-Jun, JunD, ATF-2, Elk-1, Sap-1a, and p53; phosphorylation of c-Jun, ATF-2, Elk-1, and Sap-1a increases their transcriptional activity (reviewed in Ref. 1).

JNK plays a crucial role in stress responses, cell proliferation, apoptosis (5-9), and oncogenesis (10). JNK kinase activity can be activated by various agents, including the proinflammatory cytokines (TNF-alpha and interleukin-1), G protein-coupled receptors, lymphocyte co-stimulatory receptors (CD28 and CD40), osmotic shock, heat shock, protein synthesis inhibitors, ceramides, DNA-damaging chemicals, UV irradiation, and ionizing radiation (reviewed in Ref. 1). Activation of JNK involves the upstream kinases MKK4 (also called SEK1) (11, 12) and MEKK1 (13), and together these proteins constitute a kinase cascade (MEKK1 right-arrow MKK4/SEK right-arrow JNK) in which the serine/threonine kinase MEKK1 phosphorylates and activates the dual specificity kinase MKK4/SEK (14, 15). MKK4/SEK then activates JNK via phosphorylation of the T-P-Y motif on its phosphorylation loop (11, 12). Recently, another MKK4/SEK-related kinase, called MKK7, has been cloned and found to specifically activate JNK, but not p38 and ERKs (16, 17).

The large number and differential nature of the JNK-activating agents suggest multiple pathways leading to JNK activation. Recent work has added complexity to this simple cascade paradigm. There are multiple upstream kinases (MAPK kinase kinases or MAPKKKs), including MEKK1 (13), transforming growth factor beta -activated kinase (TAK1; Ref. 18-20) and MAPK upstream kinase (MUK; Ref. 21), that activate the JNK pathway via MKK4/SEK (reviewed in Ref. 22).

A family of further upstream protein kinases, including germinal center kinase (GCK; Refs. 23 and 24), hematopoietic progenitor kinase (HPK1; Refs. 25 and 26), GCK-like kinase (GLK; Ref. 27), and kinase homologous to STE20 (KHS; Refs. 28 and 29), which have homology to Saccharomyces cerevisiae STE20, have been identified and shown to activate the JNK signaling pathway (reviewed in Ref. 30). The serine/threonine kinase STE20 sequentially activates STE11 and STE7 in the signaling cascade that includes STE11 (MAPKKK), STE7 (MAPKK), and FUS3/KSS1 (MAPK) in the yeast S. cerevisiae pheromone response. STE20 activates this pathway in response to signals from both Cdc42 and the heterotrimeric G proteins associated with transmembrane pheromone receptors (reviewed in Ref. 31). Thus far, p21 (Rac1/Cdc42)-activated kinases (PAKs) and HPK/GCK-related kinases (HPK1, GCK, GLK, and KHS) have been identified as potential upstream kinases for MEKK1. This kinase cascade has been suggested by many laboratories as follows: HPK1, GCK, PAKs right-arrow MEKKs right-arrow MKK4, MKK7 right-arrow JNKs. There is also evidence suggesting that MAPKKKs (e.g. MEKKs and MLK-3) are parallel to, but not downstream of, PAKs and HPK/GCK family kinases (reviewed in Ref. 22). We report here the isolation and characterization of a novel human protein kinase, HGK (HPK/GCK-like kinase), which is homologous to STE20. Expression of HGK specifically activates the JNK signaling pathway.

    EXPERIMENTAL PROCEDURES

cDNA Library Screening and Cloning of HGK-- Degenerate oligonucleotide primers were designed to the conserved regions (kinase subdomains VI and VIII) of the serine/threonine protein kinase and used in a polymerase chain reaction (PCR) as described previously (32). Replicate filters from a human lambda  phage macrophage library were prehybridized for 2 h at 42 °C in 5× standard saline citrate (SSC), 1× Denhardt's solution containing 100 µg/ml salmon sperm DNA, 50% formamide, 0.1% SDS, and then hybridized to the [gamma -32P]dCTP-labeled, 180-base pair probe (overnight in the same solution). After hybridization, the filters were washed twice for 30 min in 0.1% SSC, 0.1% SDS at 55 °C. Positive clones were isolated and sequenced on both strands.

Northern Blot Analysis of HGK-- Filters containing poly(A)+ RNA (2 µg/lane) from various tissues were purchased from CLONTECH. Filters were probed with an HGK probe corresponding to the non-catalytic region of HGK. Hybridization was performed at 68 °C in Express Hybridization Buffer (CLONTECH), followed by three washes in 0.1% SSC, 0.1% SDS at 55 °C. Blots were exposed for 24 h at -70 °C.

Cells, Transfections, Antibodies, and Reagents-- Human embryonic kidney 293T cells were kindly provided by Dr. M. C.-T. Hu (Amgen, Inc., Thousand Oaks, CA). 293T cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 1× penicillin/streptomycin. 293T cells were plated at a density of 1 × 106 cells/100-mm dish (or 1.5 × 105 cells/35-mm plate as indicated) and transfected the next day using the calcium phosphate precipitation protocol (Specialty Media, Inc.) The amounts of DNA used for each transfection were as indicated, and empty vector was used to achieve equal amounts of DNA in each transfection. A monoclonal antibody (12CA5) against the hemagglutinin (HA) epitope was purchased from Boehringer Mannheim. The monoclonal antibody (M2) against the Flag epitope was purchased from Kodak, Inc. To generate polyclonal rabbit anti-HGK antibody, a synthetic peptide (CNPTNTRPQSDTPEIRKYKKRFN) corresponding to the peptide sequence 823-844 of HGK was synthesized, then coupled to keyhole limpet hemocyanin via the cysteine residue at the N terminus, and used to immunize rabbits. Antibody affinity purification was done as described previously (27).

Plasmid Construction-- Full-length HGK was cloned into pCR3.1 (Invitrogen) and pCI (Promega, Madison, WI) vectors by PCR using two oligonucleotide primers. An N-terminal Flag epitope sequence was added. A catalytically inactive HGK mutant (pCR-HGK-KR) was created by substituting lysine 54 with an arginine (K54R mutant) by site-directed mutagenesis using the overlapping PCR method as described previously (34). A second catalytically inactive HGK mutant (pCI-HGK-KE) was created by substituting lysine 54 with a glutamic acid (K54E mutant). MKK7-K76E encoding a dominant-negative MKK7 mutant was described previously (17). pUna3-FL-MEKK-KR encoding a dominant-negative MEKK1 mutant was kindly provided by Dr. D. Templeton (Case Western Reserve University, Cleveland, OH). pEBG-SEK1-AL encoding a dominant-negative SEK1 mutant was kindly provided by Dr. L.I. Zon (Children's Hospital, Boston, MA). pHA-p38 and pHA-JNK1 were kindly provided by Dr. J. Woodgett (Ontario Cancer Institute, Toronto, Canada). pEF-TAK1-K63W encoding a dominant-negative TAK1 mutant was kindly given by Dr. K. Matsumoto (Nagoya University, Nagoya, Japan). pT7tag-MUK/KN encoding a dominant-negative MUK mutant was provided by Dr. S. Ohno (Yokohama City University School of Medicine, Yokohama, Japan). GST-ATF2 and pCEV-HA-ERK2 were kindly provided by Dr. J. S. Gutkind (National Institutes of Health, Bethesda, MD). cRaf-BXB and p5xTRE-CAT were kindly provided by Dr. J. Bruder (GenVec, Rockville, MD).

Immunocomplex Kinase Assay and Western Blot Analysis-- Immunocomplex kinase assays were carried out as described previously (33). MAPKs were precipitated by incubation with specific antibodies and protein A-agarose beads (Bio-Rad) in an incubation buffer (20 mM HEPES, pH 7.4, 2 mM EGTA, 50 mM glycerophosphate, 1% Triton X-100, 10% glycerol, 1 mM dithiothreitol, 2 µg/ml leupeptin, 5 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4). The precipitates were washed twice with incubation buffer, twice with LiCl buffer (500 mM LiCl, 100 mM Tris-Cl, pH 7.6, 0.1% Triton X-100), and twice with kinase buffer (20 mM MOPS, pH 7.6, 2 mM EGTA, 10 mM MgCl2, 1 mM dithiothreitol, 0.1% Triton X-100, and 1 mM Na3VO4), then mixed with 5 µg of the indicated substrates, 15 µM ATP, and 10 µCi of [gamma -32P]ATP in 30 µl of kinase buffer. The kinase reaction was performed at 30 °C for 30 min and terminated with an equal volume of SDS sampling buffer. The reaction mixtures were resolved by SDS-PAGE analysis. The Western blot analysis, antibody incubation, and ECL detection were described previously (25).

Phosphoamino Acid Analysis-- After the immunocomplex kinase assay, proteins were separated using a 12% SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane at 40 V overnight in 10 mM Tris, 1 mM glycine, and 20% methanol. The radioactive myelin basic protein (MBP) bands were identified and excised by alignment of the autoradiogram to PVDF membrane. The PVDF membrane was soaked first in methanol and then in deionized water. The proteins on the PVDF membrane were then hydrolyzed in 250 µl of 6 N HCl for 1 h at 110 °C. The acid containing partially hydrolyzed phosphoamino acids were pooled and lyophilized. The lyophilized samples were washed twice with 50 µl of deionized water. The final dried pellets were resuspended in 5 µl of 5.9% glacial acetic acid, 0.8% formic acid, 0.3% pyridine, and 0.3 mM EDTA, pH 2.5, spiked with phosphorylated serine, threonine, and tyrosine standards, and spotted on a 20 × 20-cm TLC plate. The phosphoamino acids were separated in the pH 2.5 buffer at 20 mA for 2 h. The phosphoamino acid standards were visualized by spraying the plate with 0.3% ninhydrin in acetone. The 32P-labeled residues were detected by autoradiography.

    RESULTS

Molecular Cloning of HGK-- In order to identify novel members of the mammalian STE20 family of protein kinases, we employed a degenerate PCR strategy to amplify cDNA from a human macrophage cDNA library. A PCR fragment containing novel sequences with homology to the STE20 family of protein kinases was identified. This cDNA fragment was subsequently used to screen a human macrophage cDNA library. Four cDNA clones of varying sizes were isolated, and the one containing the longest insert (3950 base pairs) was sequenced on both strands. The complete nucleotide sequence of the cDNA predicted an open reading frame of 1165 amino acids with a predicted molecular mass of 133.40 kDa. The deduced amino acid sequence encoded a kinase catalytic domain in the N terminus that contained 11 kinase subdomains (Fig. 1, A and B). Comparison of the amino acid sequence of the putative kinase domain of HGK with other sequences found that the HGK kinase domain was most similar to HPK1, GLK, GCK, KHS, and STE20. Within the kinase domain, HGK displays 47% amino acid identity to the catalytic domain of the HPK1, 52% amino acid identity to the catalytic domain of the human GLK, 48% amino acid identity to the catalytic domain of the GCK, 48% amino acid identity to the catalytic domain of the KHS, and 39% amino acid identity to S. cerevisiae Ste20. The amino acid sequence alignment of the catalytic domain of HGK with that of related kinases is shown in Fig. 1B. Unlike HPK1 and GCK, HGK does not contain proline-rich regions: the putative Src homology 3 domain binding regions. However, one of the HGK cDNA clones contains an additional 77 amino acids with two proline-rich regions (Fig. 1C). To determine whether other forms of HGK exist that contain the proline-rich regions, reverse transcriptase PCR using two primers to flank the region was employed to amplify cDNA from human brain, liver, skeletal muscle, and placenta. Sequencing analysis of the PCR products found that, although the longer form appears to be more abundant in the brain, the short form is the predominant form in human liver, skeletal muscle, and placenta (data not shown).


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Fig. 1.   Amino acid sequence of human HGK and sequence comparison of HGK with other related kinases. A, the deduced amino acid sequence of HGK is shown. The catalytic domain is underlined. B, the putative catalytic domain of HGK was aligned and compared with human GLK, GCK, HPK1, and STE20 by the PILEUP and PRETTY programs of the University of Wisconsin Genetics Computation Group (GCG). The kinase subdomains are indicated with Roman numerals. The conserved residues are in capital letters, while the non-conserved amino acid residues are shown in lowercase letters. C, sequence comparison between the long and short forms of human HGK. The numbers in parentheses are the amino acid position; dashes are the gaps.

Expression Pattern and Catalytic Activity of HGK-- The expression of HGK was examined in a variety of human tissues by Northern blot analysis using a probe from the non-conserved C-terminal coding region of HGK. The HGK probe hybridized to three transcripts of approximately 4.6, 6.5, and 8.5 kilobase pairs in all tissues examined, suggesting a ubiquitous tissue distribution (Fig. 2A). To determine the expression of endogenous HGK protein, a polyclonal antibody to HGK was generated and purified by affinity chromatography. This antibody specifically recognized a protein of approximately 130 kDa in MCF-7, 293, HeLa, PC3, LNCaP, and Jurkat cells, as shown by Western blot analysis (Fig. 2B). The specificity of the anti-HGK antibody was further confirmed in 293T cells transfected with a Flag-tagged HGK cDNA expression plasmid. Both the anti-HGK antibody and the anti-Flag antibody recognized specifically the Flag-tagged HGK proteins in the HGK-transfected 293T cells (Fig. 2C). HGK phosphorylated the substrate MBP (Fig. 3A). As controls, the HGK catalytically inactive mutants (HGK-KR and HGK-KE, which contain lysine 54 to arginine substitution and lysine 54 to glutamic acid substitution, respectively) did not phosphorylate the substrate MBP (Fig. 3A), indicating that the HGK kinase activity observed was not attributable to coimmunoprecipitated kinases. Furthermore, phosphoamino acid analysis of in vitro phosphorylated MBP detected phosphorylated serines and threonines only (Fig. 3B), establishing HGK as a serine/threonine protein kinase.


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Fig. 2.   Expression of HGK. A, Northern blot analysis of HGK mRNA. Poly(A)+ RNA from the indicated tissues was hybridized with radioactive HGK probes from the 3' non-conserved region. RNA size markers are shown on the left. B, Western blot analysis of HGK protein. 30 µg of cell lysate from several cell lines was resolved by 7% SDS-PAGE and immunoblotted with purified anti-HGK antibody, Ab2228. C, specificity of the anti-HGK antibody (Ab2228). 293T cells (1.5 × 105/35-mm well) were transfected with 2 µg of an empty vector (pCI) or pCI-Flag-HGK. The cells were collected 40 h after transfection. 40 µg of total cellular proteins were resolved by SDS-PAGE electrophoresis and immunoblotted with an anti-HGK antibody (Ab2228; left panel) and an anti-Flag antibody (M2; right panel).


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Fig. 3.   Catalytic activity of HGK. A, 293T cells (1.5 × 105/35-mm well) were transfected with 2 µg of an empty vector (pCI), HGK, HGK-KR, and HGK-KE. The cells were collected 40 h after transfection. Immunocomplex kinase assays were performed with an anti-Flag antibody (M2) using MBP as the substrate. Equivalent levels of wild-type and mutant HGK expression were verified by immunoblotting using an anti-Flag antibody (M2; bottom panel). B, HGK is a serine/threonine protein kinase. 293T cells (1.5 × 105) were transfected with 2 µg of a Flag-HGK plasmid. The cells were harvested at 44 h after transfection. Immunocomplex kinase assays were performed with an anti-Flag antibody (M2) using MBP as a substrate. After electrophoresis in 12% SDS-PAGE, the protein was transferred to PVDF. Phosphorylated MBP was excised from the blot and used to perform phosphoamino acid analysis. The positions of phosphorylated serine (p-Ser), phosphorylated threonine (p-Thr), and phosphorylated tyrosine (p-Tyr) are indicated by arrows.

HGK Specifically Activates JNK, but Not ERK or p38 Kinase, in Transfected 293T Cells-- To determine whether HGK can activate JNK, 293T cells were co-transfected with mammalian expression vectors encoding HGK and an HA epitope-tagged JNK1. Recombinant JNK was then immunoprecipitated from cell lysates and used in a protein kinase assay with GST-c-Jun-(1-79) as a substrate. Transfection with HGK resulted in strong JNK1 activation (Fig. 4A, lane 3), while cells transfected with vector showed little JNK1 activation (lanes 1 and 2). Transfection of a kinase-inactive form of HGK, in which lysine 54 in the ATP binding domain was mutated to a glutamic acid, did not result in JNK activation (Fig. 4A, lane 4). Thus, the kinase activity of HGK is required for JNK activation.


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Fig. 4.   Activation of JNK, but not p38 or ERK, by HGK in transfected 293T cells. A, 293T cells (1 × 106 cells/100-mm dish) were transfected with 15 µg of the empty vector pCI alone (lane 1), 5 µg of HA epitope-tagged JNK1 plasmid (pHA-JNK1) plus 10 µg of the empty vector pCI (lane 2), 10 µg of pCI-HGK (lane 3), or 10 µg of kinase-dead mutant pCI-HGK-KE (lane 4). The cells were collected 48 h later, and immunocomplex kinase assays were performed with anti-HA antibody (12CA5) using GST-c-Jun-(1-79) as the substrate. B, 293T cells were transfected with 15 µg of the empty vector pCI alone (lane 1), 5 µg of HA epitope-tagged p38 (pHA-p38) plus 10 µg of the empty vector pCI (lane 2), 10 µg of pCI-HGK (lane 3), or 10 µg of pCI-MKK6 (lane 4). Immunocomplex kinase assays were performed with anti-HA antibody (12CA5) using GST-ATF2-(1-96) as the substrate. C, 293T cells were transfected with 20 µg of the empty vector pCI (lane 1), 10 µg of HA epitope-tagged ERK2 (pHA-ERK2) plus either 10 µg of the empty vector pCI (lane 2), 10 µg of pCI-HGK (lane 3), or 10 µg of pcRaf-BXB (lane 4). Immunocomplex kinase assays were performed with anti-HA antibody, using MBP as the substrate. Equivalent expression levels of HA-JNK1, HA-p38, and HA-ERK2 were verified by immunoblotting using an anti-HA antibody (12CA5; bottom panels). D, activation of AP-1 activity by HGK. 293T cells were transfected with 1 µg of 5xTRE-CAT reporter construct plus empty vectors (lane 1), 2 µg of pHA-JNK1 (lane 2), 4 µg of pCI-HGK (lane 3), 4 µg of pCI-HGK-KE (lane 4), 2 µg of pHA-JNK1 and 4 µg of pCI-HGK (lane 5), or 2 µg of pHA-JNK1 and 4 µg of pCI-HGK-KE (lane 6). Empty vector was used to normalize the amount of transfected DNA. The cells were harvested 44 h after transfection, and the CAT activity was measured and compared with that of the empty vector. The results are expressed as -fold induction.

To determine whether HGK can also activate the p38 kinase or ERK, 293T cells were transiently transfected with HGK along with HA-tagged p38 or HA-tagged ERK2. p38 kinase and ERK2 were then immunoprecipitated and their activities assayed using ATF-2 or MBP peptides as substrates. Even though MKK6 activated p38 (Fig. 4B, lane 4) and the activated c-Raf1, Raf-BXB, strongly activated ERK2 (Fig. 4C, lane 4), neither p38 or ERK kinase activities were increased when HGK was overexpressed in 293T cells (Fig. 4, B and C, lane 3). These data suggest that the activation of JNK by HGK is specific.

HGK Activates c-Jun Transcriptional Activity-- Because c-Jun is a substrate of JNK and the expression of HGK can activate the JNK signaling pathway, we tested whether the expression of HGK can result in the activation of c-Jun transcriptional activity in vivo. Cells were transfected with an HGK expression plasmid and a chloramphenicol acetyltransferase (CAT) reporter construct (5xTRE-CAT) containing five copies of the c-Jun-binding sites (AP1-binding sites). CAT assays were performed 44 h after transfection. Co-transfection of HGK plus JNK1 stimulated the transcriptional activity mediated by AP-1 (about 7-fold) as compared with cells transfected with vector control (Fig. 4D). JNK1 or HGK alone showed minimal AP1-CAT induction (about 1.5-fold). Due to the low levels of JNK expression in 293T cells, inclusion of the JNK plasmid in the transfection assay was necessary in order to detect optimal AP-1 induction by HGK via the HGK right-arrow TAK1 right-arrow MKK4,7 right-arrow JNK kinase cascade. Co-transfection of cells with JNK1 and a kinase-negative mutant of HGK did not increase the transcriptional activity of AP-1. This result indicates that HGK can activate JNK, which in turn stimulates the transcriptional activity of c-Jun in vivo.

HGK Functions Upstream of MKK4 and MKK7-- MKK4 is an upstream activator of JNK which phosphorylates and activates JNK. Recently, another MAPKK/MKK, called MKK7, has been cloned and found to specifically activate JNK, but not p38 and ERK. To determine whether HGK activates JNK through MKK4 and MKK7, we co-transfected cells with an expression plasmid expressing HGK and a dominant-negative mutant of MKK4/SEK1 (SEK-AL) or a dominant-negative mutant of MKK7 (MKK7-K76E) to determine whether they can inhibit HGK-induced JNK activation. The expression of a dominant-negative form of MKK4 or MKK7 inhibited JNK activity induced by HGK (Fig. 5A) and GLK (Fig. 5B), indicating that HGK functions upstream of MKK4 and/or MKK7.


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Fig. 5.   HGK-induced JNK activation is blocked by either SEK-AL or MKK7-K76E. A, 293T cells (1.5 × 105/35-mm cell well) were transfected with 1 µg of pHA-JNK1 plus 4 µg of the empty vector pCI (lane 1), 2 µg of pCI-HGK (lane 2), 2 µg of pCI-HGK and 2 µg of dominant-negative pEBG-SEK-AL (lane 3), or 2 µg of pCI-HGK and 2 µg of dominant-negative MKK7-K764 (lane 4). B, for comparison, 293T cells were transfected with 1 µg of pHA-JNK1 plus 4 µg of the empty vector pCI (lane 1), 2 µg of pCI-GLK (lane 2), 2 µg of pCI-GLK and 2 µg of pEBG-SEK-AL (lane 3), or 2 µg of pCI-GLK and 2 µg of MKK7-K764 (lane 4). The empty vector pCI was used to normalize the amount of transfected DNA. Immunocomplex kinase assays were performed 44 h after transfection with anti-HA (12CA5) antibody using GST-c-Jun-(1-79) as the substrate. Equivalent expression levels of HA-JNK1, Flag-HGK, and Flag-GLK were verified by immunoblotting using anti-HA (12CA5) and anti-Flag (M2) antibodies, respectively (bottom panels).

HGK-Induced JNK Activation Is Blocked by a Dominant-negative Mutant of TAK1, but Not MEKK1 or MUK-- The MAPK kinase kinase MEKK1 is a direct activator of MKK4, which in turn phosphorylates and activates JNK. In addition, MEKK1 was shown to function downstream of HPK1 and GLK (25, 27), two kinases that are highly homologous to HGK. To determine whether HGK also activates JNK through MEKK1, we analyzed the effect of the kinase-inactive mutant MEKK1 (MEKK-KR) on HGK-induced JNK activation. In repeated experiments, we found that, although the MEKK1-KR mutant strongly inhibited HPK-induced JNK activation (Fig. 6B), it did not efficiently block HGK-stimulated JNK activity (Fig. 6A). These results suggest that HGK utilizes different downstream kinases for signaling. TAK1 was shown to function downstream of HPK1 (20); hence, we tested whether HGK activates JNK through TAK1. We co-transfected cells with HGK plus the dominant-negative mutant of either TAK1 (TAK-K63W) or MUK (MUK-KN), and determined their effects on HGK-induced JNK activation. We found that the kinase-negative TAK1 mutant (TAK-K63W), but not the MUK mutant, significantly inhibited HGK-induced JNK activation (Fig. 7). This result strongly suggests that TAK1 is a downstream target for HGK activity in the JNK signaling cascade.


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Fig. 6.   HGK-induced JNK activation is not blocked by dominant-negative mutant of MEKK1. A, 293T cells (1 × 106 cells/100-mm dish) were transfected with the empty vector pCI alone (lane 1), 1 µg of pHA-JNK1 plus empty vector (pCI) (lane 2), 10 µg of pCI-HGK (lane 3), or 10 µg of pCI-HGK and dominant-negative pUna3-FL-MEKK1-KR (lane 4). The empty vector pCI was used to normalize the amount of transfected DNA. The cells were collected 48 h later, and immunocomplex kinase assays were performed with an anti-HA antibody using GST-c-Jun-(1-79) as the substrate. Equivalent expression levels of HA-JNK1 and Flag-HGK were verified by immunoblotting using anti-HA (12CA5) and anti-Flag (M2) antibodies, respectively (bottom panels). B, for comparison, the same procedures were performed on 293T cells transfected with empty vector (pCI) alone (lane 1), 1 µg of pHA-JNK1 plus empty vector (lane 2), 10 µg of pCI-HPK1 (lane 3), or 10 µg of pCI-HPK1 and dominant-negative pUna3-FL-MEKK-KR (lane 4). Equivalent expression levels of HA-JNK1 and Flag-HPK were verified by immunoblotting using anti-HA (12CA5) and anti-Flag (M2) antibodies, respectively (bottom panels).


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Fig. 7.   HGK-induced JNK activation is specifically blocked by the dominant-negative TAK1 mutant, TAK-K63W. 293T cells (1.5 × 105/35-mm cell wells) were transfected with 0.1 µg of pHA-JNK1 alone (lane 1), 0.1 µg of pHA-JNK1 and 2 µg of pCI-HGK plus empty vector (lane 2), 2 µg of dominant-negative pEF-TAK1-K63W (lane 3), and 2 µg of dominant-negative pT7tag-MUK-KN (lane 4). Empty vector (pCI) was used to normalize the amount of transfected DNA. Immunocomplex kinase assays were done 44 h after transfection with an anti-HA antibody (12CA5) using GST-c-Jun as the substrate (top panel). Equivalent expression levels of HA-JNK1 and Flag-HGK were verified by immunoblotting using anti-HA (12CA5) and anti-Flag (M2) antibodies, respectively (bottom panels).

Activation of HGK by TNF-alpha -- Since TNF-alpha stimulates TAK1 and JNK kinase activity (19) and HGK was found to be an upstream kinase of TAK1 and JNK (this study), we tested whether TNF-alpha can also regulate HGK kinase activity. To study the activation of HGK, an expression construct encoding the Flag epitope-tagged HGK was transiently transfected into 293T cells. The HGK-transfected 293T cells were treated with TNF-alpha , and the cell lysates were prepared and subjected to in vitro immune complex kinase assays. After TNF-alpha stimulation, HGK kinase activity increased within 1 min and peaked at 5 min (Fig. 8A). Equivalent levels of overexpressed HGK were verified by immunoblotting with anti-Flag (M2) antibody (bottom panel). This result indicates that TNF-alpha stimulated HGK kinase activity.


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Fig. 8.   HGK is activated by TNF-alpha and the TNF-alpha -induced JNK activation is blocked by dominant-negative HGK mutants. A, stimulation of the HGK kinase activity by TNF-alpha . 293T cells (1.5 × 105) were transfected with 50 ng of Flag-tagged HGK. After 44 h, the cells were treated with TNF-alpha (100 ng/ml) for various periods of time as indicated. The cell lysates were prepared and immunocomplex kinase assays were performed with an anti-Flag antibody (M2) using MBP as the substrate (top panel). Equivalent levels of HGK expression were verified by immunoblotting using an anti-Flag antibody (M2; bottom panel). B, TNF-alpha -induced JNK activation is blocked by HGK mutants (HGK-KR and HGK-KE). 293T cells (1.5 × 105) were transfected with HA-JNK (0.1 µg) alone, HA-JNK1 plus HGK-KR (2 µg), or HA-JNK1 plus HGK-KE (2 µg), as indicated. The empty vector pCI was used to normalize the total amount of transfected DNA. The cells were treated with TNF-alpha (100 ng/ml) for 10 min at 44 h after transfection. The cell lysates were prepared, and immunocomplex kinase assays were performed with an anti-HA antibody (12CA5) using GST-c-Jun as the substrate (top panel). Equivalent levels of HA-JNK1 expression were verified by immunoblotting using an anti-HA antibody (12CA5; bottom panel).

TNF-alpha -induced JNK Activation Is Blocked by the Dominant-negative HGK Mutants-- Like many other STE20-like and MEKK-like kinases, HGK kinase regulation using in vitro kinase assays was extremely difficult to detect (see above). Hence, we used the dominant-negative HGK mutants to further address the role of HGK in the TNF-alpha -mediated JNK activation (Fig. 8B, top panel). 293T cells were transfected with HA-JNK plus empty vector, HA-JNK plus the dominant-negative HGK mutant HGK-KR, or HA-JNK plus the the dominant-negative HGK mutant HGK-KE. Transfected cells were then treated with TNF-alpha , followed by immunocomplex JNK kinase assays. We found that the dominant-negative HGK mutants (HGK-KR or HGK-KE) inhibited TNF-alpha -stimulated JNK activation (Fig. 8B, top panel, lanes 3 and 4). Equivalent levels of HA-JNK1 expression were verified by immunoblotting using anti-HA (12CA5) antibody (bottom panel). Taken together, our results indicate that HGK is involved in the TNF-alpha -induced signaling pathway.

    DISCUSSION

A human kinase homologous to yeast STE20, called HGK, was cloned. HGK shares both sequence and structural homology with several known members of the mammalian STE20 family of kinases, including HPK1, GLK, GCK, and KHS. HGK has a catalytic domain in its N terminus and a long putative regulatory region. The C terminus of HGK does not appear to contain the Cdc42/Rac1 binding motifs found in the PAK family of protein kinases (35). Like HPK1, GLK, GCK, and KHS, overexpression of HGK in 293T cells strongly activates the JNK pathway, but has no effect on either the ERK or p38 signaling pathways, suggesting that HGK-induced JNK activation is specific. Although the C-terminal domain of HGK does not contain the Cdc42/Rac1 binding motifs, it appears to act as a dominant-negative mutant. Therefore, the C-terminal domain may interact with other signaling molecules that are important for HGK kinase activation. Interestingly, HGK-mediated JNK activation was not affected by the dominant-negative mutant of MEKK1, which is a potent JNK activator. In contrast, JNK activation stimulated by the highly homologous kinase, HPK1, can be efficiently blocked by this mutant. Similarly, our previous studies found that GLK-induced JNK activation is also inhibited by a dominant-negative mutant of MEKK1 (27). These data suggest that HGK may function independently of MEKK1 and that different STE20-like protein kinases may signal through different downstream MAPKKKs. At the MAPKKK level, there are several kinases that can activate the JNK pathway. Among several MAPKKKs tested, we found that only the dominant-negative TAK1 mutant strongly inhibited HGK-induced JNK activation. TAK1 was originally identified as a kinase that can activate the p38 kinase signaling pathway. More recent data found that TAK1 can also potently activate the JNK pathway (19, 20). Thus, HGK likely utilizes TAK1 as its downstream kinase.

Two isoforms of human HGK were detected, one of which has a deletion in the region that contains two proline-rich domains. The proline-rich region, contained in the long isoform of HGK, may mediate its interaction with other Src homology 3 domain-containing adaptors. The fact that human HGK that is missing this region can activate the JNK pathway indicates that this region is not essential for JNK activation in transfected 293T cells. It appears that the isoform that contained the proline-rich region was predominantly expressed in the brain, while the shorter form was more abundantly expressed in other tissues such as human liver, skeletal muscle, and placenta (data not shown). The differential expression of different isoforms of HGK suggests that these different isoforms might have tissue- and/or cell-specific functions and may be regulated by different mechanisms.

The specificity of JNK activation is not well understood at the present time. Given that the JNK pathway is activated by a variety of extracellular stimuli including UV irradiation, ionizing radiation, heat shock, osmotic stress, protein synthesis inhibitors, apoptosis-inducing agents, and proinflammatory cytokines, it should not be surprising that multiple mammalian STE20-like protein kinases can mediate JNK activation. One plausible mechanism of controlling the specificity of JNK activation is to have different upstream kinases, each responding to distinct stimuli. In addition, different mammalian STE20-like protein kinases might have cell-type specific functions; for example, HPK1 may have a specific function in hematopoietic progenitor cells, as its transcripts are preferentially expressed in hematopoietic organs and cell lines (25, 26). This study indicates that HGK is involved in the TNF-alpha -mediated signaling pathway. Our preliminary result also suggests that HGK is involved in the UV-C-mediated signaling pathway. Current studies are focused on elucidating the underlying mechanisms of the HGK right-arrow TAK1 right-arrow MKK4, MKK7 right-arrow JNK kinase cascade in UV-C and TNF-alpha signaling pathways.

    ACKNOWLEDGEMENTS

We thank Dean Jannuzi for the DNA sequencing; Marynette Rihanek for generating the HGK antibodies; Drs. J. Bruder, R. J. Davis, J. S. Gutkind, S.-i. Hirai, M.C.-T. Hu, K. Matsumoto, S. Ohno, D. Templeton, J. Woodgett, and L. I. Zon for providing valuable reagents; members of the Tan laboratory for the helpful discussions; Susan Lee and Roshi Afshar for technical assistance; and M. Lowe for secretarial assistance.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants RO1-AI38649 and RO1-AI42532 (to T.-H. T.).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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF096300.

§ The first two authors contributed equally to this work.

parallel A Scholar of the Leukemia Society of America. To whom correspondence should be addressed: Dept. of Microbiology and Immunology, Baylor College of Medicine, M929, One Baylor Plaza, Houston, TX 77030. Tel: 713-798-4665; Fax: 713-798-3700. E-mail: ttan{at}bcm.tmc.edu.

The abbreviations used are: JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MKK, MAPKK, or MEK, MAPK kinase; SEK, stress-activated protein kinase/ERK kinase; GCK, germinal center kinase; GLK, GCK-like kinase; KHS, kinase homologous to STE20; HPK1, hematopoietic progenitor kinase-1; MAPKKK or MEKK, MAPK kinase kinase; TAK1, transforming growth factor beta -activated kinase; MUK, MAPK-upstream kinase; TNF-alpha , tumor necrosis factor alpha ; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin; PAK, p21-activated kinase; GST, glutathione S-transferase; CAT, chloramphenicol acetyltransferase; PVDF, polyvinylidene difluoride; MOPS, 4-morpholinepropanesulfonic acid; MBP, myelin basic protein; HGK, HPK/GCK-like kinase.
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