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Cloning and Expression of ZAK, a Mixed Lineage Kinase-like Protein Containing a Leucine-Zipper and a Sterile-Alpha Motif
Biochemical and Biophysical Research Communications 274, 811– 816 (2000) doi:10.1006/bbrc.2000.3236, available online at http://www.idealibrary.com on
Cloning and Expression of ZAK, a Mixed Lineage Kinase-like Protein Containing a Leucine-Zipper and a Sterile-Alpha Motif Te-Chung Liu,* Chang-Jen Huang,† Yu-Chuan Chu,‡ Chih-Chang Wei,‡ Chun-Chieh Chou,‡ Ming-Yung Chou,‡ Chen-Kung Chou,§ and Jaw-Ji Yang‡ ,1 *Department of Nutrition, Chung-Shan Medical College, Taichung 402, Taiwan; †Institute of Biological Chemistry, Academia Sinica, Taipei 112, Taiwan; ‡School of Dentistry, Chung-Shan Medical College, Taichung 402, Taiwan; and §Institute of Medical Research, Veterans General Hospital, Taipei 112, Taiwan
Received July 5, 2000
A novel mixed lineage kinase-like protein ZAK, containing a leucine-zipper (LZ) and a sterile-alpha motif (SAM), was cloned. This cDNA has 2456 bp and encodes a protein of 800 amino acids that contains a kinase catalytic domain, a leucine-zipper and a SAM. The molecular weight of this protein is 91kDa. Northern blot analysis revealed that the expression of this ZAK gene is found in various parts of human tissues. We also found that ZAK proteins might form homodimers or oligomers in mammalian cells. MLKs have been proposed to function as mitogen-activated protein kinase kinase kinase in pathways leading to MAPK cascade. The expression of ZAK in mammalian cells specifically leads to the activation of the JNK/SAPK pathway as well as the activation of transcription factor, NF-B. Overexpression of the ZAK gene induces the apoptosis of a hepatoma cell line. © 2000 Academic Press Key Words: leucine-zipper and sterile-alpha motif kinase (ZAK); mixed lineage kinase (MLK); JNK/SAPK; apoptosis.
Protein kinases play important roles in the regulation of many cellular processes, such as the transmission of signals from the growth factor, control of cell growth and differentiation, regulation of cytoskeletal changes, gene expression, and translation and metabolism pathways (1). Among the protein kinases, MAP kinase plays an essential role in the cell signal transduction machinery. Protein kinases of the MAPK family is involved in a variety of cellular responses to Abbreviations used: ZAK, leucine-zipper and sterile-alpha motif kinase; MLK, mixed lineage kinase; MAPK, mitogen-activated protein kinase; JNK/SAPK, c-Jun NH2-terminal kinase/stress-activating protein kinase. 1 To whom correspondence should be addressed. Fax: 886-44759065. E-mail: [email protected].
cytokines, hormones, and stress-inducing reagents. Several mammalian MAP kinases have been cloned, including ERK (2), JNK/SAPK (3, 4) and p38 MAPK (5). JNKs/SAPKs as well as p38Mpk2 are strongly activated in cells exposed to extracellular stress such as UV irradiation, osmotic shock, heat shock (6), and treatment with tumor necrosis factor-␣ (7), interleukin-1 (8, 9) or antibiotics known as the inhibitors of eukaryotic protein synthesis. Accumulating evidence suggests that these MAPK cascades are regulated by small GTPases of the Rho family (10, 11). MEK family protein kinases, MKK4 and MKK7, and the MKK kinases may include TPL2, MLK/DLK, TAK, and ASK activate JNK/SAPK (12, 13). The activated JNKs/ SAPKs are then translocated into the nucleus and activate several transcription factors such as c-jun, ATF2, ELK, and Sap-1 (14, 15). Phosphorylated c-jun, by JNKs/SAPKs, forms homodimers or heterodimers with c-fos, which have potent AP-1 activity and regulate the expression of a number of genes. Recent reports indicate that a newly discovered type of protein kinases is involved in JNK signaling. This family of kinases has been named mixed lineage protein kinase (MLK) (16, 17). MLKs are in turn activated by a number of agonists. For example, MLK-3 has been found to mediate signals from the ste20 homologue germinal center kinase, and hematopoietic kinase, the guaninenucleotide exchange protein C3G, and is a target of the small GTPases Rac and Cdc42 (18). MLKs bear protein-protein interaction domains, which contain two leucine-zippers at the carboxyl-terminal to the catalytic domain. MLK might not only participate in the JNK signaling cascade but also mediate the p38 MAPK cascade (19). MLK has been implicated as a MAP kinase kinase kinase. The five identified members of the MLK family, MLK1, MLK2, MLK3, DLK, and LZK, share two common structure features (17, 20). Each
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has a characteristic kinase catalytic domain with primary structure, which is hybrid between those in serine/threonine and tyrosine protein kinases. Second, closely juxtaposed C-terminal to the kinase domain, each MLK has a domain that is predicted to form two leucine/isoleucine zippers. However, little is to known about the overall biochemical features and functionally roles of MLKs. It has been proposed that MLKs behave functional as MAPK kinase kinase in pathways leading to the activation of JNK/SAPK (11). Furthermore, MLK2 and MLK3 have been shown to phosphorylate and activate MKK4 (21). In this study, we cloned a novel and stress-regulated MLK-like protein kinase, ZAK, which may specifically activate JNK and turn on NF-B transcription factor in several cell lines. The over-expression of this gene induces apoptosis in hepatoma cells. MATERIALS AND METHODS cDNA library screening and 5⬘-end determination of ZAK. An EST clone of ste20-like DNA labeled with [␣- 32P]dCTP and used to screen the human placenta cDNA library. The hybridization conditions for screening this library were 5 ⫻ SSC and 40% formamide at 42°C for 16 h and the final washing condition was 1 ⫻ SSC at 68°C. A human marathon cDNA mix (liver, placenta, thyroid, fetus, and stomach) was employed to amplify the 5⬘ end of this gene. Antisense gene-specific primers (AGSPs), AGSP1 5⬘-GAGAGGCACCAAAGTCACAGAT and AGSP2 5⬘-CACCTTGACAGGAGCCTCCATATGTAA, were designed and adaptor primers (AP1 and AP2) (Clontech) used to perform the 5⬘-end RACE. The first-PCR was performed by using the cDNA mix and AGSP1 and AP1 and conditions were as follows: denaturation at 94°C for 1 min, annealing at 65°C for 1 min, and extension at 72°C for 1 min, for 35 cycles, followed by a 7 min extension at 72°C. The result of the first PCR was then used as a template to perform secondary-PCR with AGSP2 and AP2 and PCR conditions as previously. A band about 400 bp was amplified and subcloned into the pGEM-T vector and sequenced. The additional 5⬘-end DNA fragment of 303bp was then added to the original clone to make up the full-length of this gene with a putative open reading frame of 800 amino acids. Analysis of ZAK transcript. mRNA from various human tissues (Clontech) was probed with 3⬘-end of ZAK cDNA labeled by random priming method and hybridization was performed as described (22). Immunoprecipitation and Western blot analysis. Cell lysates were prepared in IP buffer (40 mM Tris–HCl (pH 7.5), 1% NP40, 150 mM NaCl, 5 mM EGTA, 1 mM DTT, 1 mM PMSF, 20 mM NaF, proteinase inhibitors, and 1 mM sodium vanadate). Cell extracts (600 g) were incubated with 5 g of anti GFP mAb (Clontech) for 6 h at 4°C, mixed with 20 l protein-A sepharose suspension, and incubated for an additional hour. Immunoprecipitates were collected by centrifugation, washed three times with IP buffer plus 0.5% deoxycholate, five times with IP buffer alone, and subjected to SDS–PAGE. Immunoblot analysis was performed with the anti-FLAG (Sigma). ZAK- or empty vector-expressed cells were harvested in lysis buffer (50 mM Tris–HCl, pH 8.0/250 mM NaCl/1% NP-40, 2 mM EDTA) containing 1 mM PMSF, 10 ng/ml leupeptin, 50 mM NaF, and 1 mM sodium orthovanadate. Total proteins were then separated on SDS– PAGE and specific protein bands visualized with an ECL chemiluminescent detection system (Amersham) was performed as described (23). Detection of the JNK/SAPK activities. Protein kinase assays were carried out using a fusion protein between glutathione S-transferase
(GST) and c-Jun (amino acids 1–79) as a substrate. The GST-Jun fusion proteins were bound to glutathione sepharose beads and incubated for 15 min on ice with the cellular extract that contains JNK, in the presence of kinase buffer (20 mM Hepes, pH 7.6, 1 mM EGTA, 1 mM dithiothreitol, 2 mM MgCl, 2 mM MnCl, 5 mM NaF, 1 mM NaVO, 50 mM NaCl). The beads were pelleted and thoroughly washed with PBST (150 mM NaCl, 16 mM sodium phosphate, pH 7.5, 1% Triton X-100, 2 mM EDTA, 0.1% MeOH, 0.2 mM phenylmethylsulfonyl fluoride, and 5 mM benzamidine), before they were incubated with [␥- 32P]ATP (50 cpm/fmol) in the presence of kinase buffer. These steps were undertaken to ensure that c-Jun phosphorylation was carried out by JNK, which is known to exhibit high affinity to this portion of c-Jun under these conditions. Following extensive washing, the phosphorylated GST-Jun was boiled in SDS sample buffer, and the eluted proteins were run on a 15% SDS– PAGE. The gel was dried, and phosphorylation of the Jun substrate was determined by autoradiography. Microscopic observation of apoptosis cells. Hep3B cells growing on glass coverslips were transfected 1 g of empty or ZAK expressing plasmid using calcium phosphate method. Twenty-four hours after transfection, apoptotic cells were detected with an in situ cells detection kit as instructed by the manufacturer (Roche; in situ cell death detection kit, Fluorescein). Cells were examined photographed in a Zesis Axioskop microscope.
RESULTS Isolation of a ZAK cDNA and its deduced amino acid sequence. The DNA fragment was released from an EST clone of the ste 20-like plasmid. This fragment was then used as a probe to screen the human placenta cDNA library (Stratagene) by a low stringency hybridization method. A positive clone was found and sequenced. This cDNA extends 2179 nucleotide bases with a poly-A tail and contains a continuous open reading frame of 2097 bp. In order to obtain the 5⬘-end of this gene, a mixture (liver, placenta, thyroid, fetus, and stomach; Stratagene) of the human 5⬘-RACE cDNA library was employed and a further upstream DNA fragment of 303 bp was then found. Furthermore, this DNA fragment was sequenced and ligated to the previous cDNA clone to fill up the full-length of this gene (GenBank Accession No. AF238255). The open reading frame of this cDNA encodes a putative polypeptide of 800 amino acids, with a calculated molecular mass of 91 kDa (Fig. 1A). Surprisingly, a comparison of the amino acid sequence of this cDNA encoded protein with Genbank revealed it to be a novel gene belonging to the MLK family based on the similarity of their sequences. However, the cDNA is not a ste-20 homologue. Hydrophobicity analysis revealed that this protein contains no obvious signal sequence or transmembrane domain. Comparison of the sequence with other known proteins revealed that the protein could be divided into at least three structural domains: a kinase catalytic domain, a leucine-zipper motif and a sterile-alpha motif (SAM) (Fig. 1B). The kinase domain extends 245 amino acids from amino acid 16 through 260. This kinase catalytic domain has the features of both serine/threonine kinases and tyrosine kinases, where Trp197-Glu198, Pro205-
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Tissue distribution of ZAK mRNA. To ascertain the expression pattern of the ZAK gene, we studied the endogenous expression of ZAK mRNA in various human tissues. Northern blot analysis of various human tissues demonstrated that the mRNA for ZAK is 3.0 kb. The expression of the ZAK gene could be detected in many tissues, including the heart, placenta, lung liver, and pancreas (Fig. 2).
FIG. 1. Amino acid sequence and the possible structure of the ZAK protein. (A) Conceptually translated amino acid sequence of the ZAK protein. Bold letters denote the kinase catalytic domain (16 – 260) and the SAM domain (336 – 410). The leucines/isoleucines in the putative leucine-zipper motif are underlined. (B) Schematic representation of the primary structure of ZAK. The numbers in the diagram represent amino acid residue numbers and delineate the boundaries of the indicated domains. Besides the regions of kinase catalytic domain and leucine/isoleucine zippers, ZAK does not show similarity with other protein kinases of the MLK family.
Phe206 and Trp172 (GTFPWMAPE) are characteristic for tyrosine kinase. However, the presence of Lys134 (HRDLKSRN) and Thr169 (GTFPWMAPE) indicate that this kinase would have serine/threonine specificity, and Trp172-Met173 (GTFPWMAPE) is often found in Raf family proteins (25). Following the kinase catalytic domain is a single leucine-zipper motif and a sterile-alpha motif, which may be involved in the protein-protein interaction. The amino acid sequences of the kinase catalytic domain and leucine-zipper motif of this protein have about 40% identity and are about 60% positive to the sequences of previously reported protein kinases, such as MLK2, MUK, ZPK, and LZK, suggesting that this protein kinase belongs to the MLK family (Fig. 1B). In contrast to most MLK family protein kinases, this protein kinase bears a single leucinezipper motif. Moreover, there is no homology with other reported proteins beyond the leucine-zipper motif. We named this protein kinase ZAK (leucine-zipper and sterile-alpha motif protein kinase).
ZAK forms oligomers. Since ZAK contains a leucine-zipper motif and a SAM, which have been implicated in protein-protein interaction (24), it is reasonable to suspect that ZAK can form homodimers in cells. This interaction was investigated by using in vivo coprecipitation. HepG2 cells were co-transfected with plasmids that express a GFP-tagged ZAK construct and a FLAG-tagged ZAK construct. Protein complexes were immunoprecipitated using anti-GFP antibody, and co-precipitated FLAG-ZAK was probed by M2 monoclonal antibody. As shown in Fig. 3, FLAG-ZAK could be detected only when cells expressed both FLAG-ZAK and GFP-ZAK plasmids. This result demonstrates that ZAK proteins might form homodimers or oligomers in mammalian cells. Moreover, the double negative controls when cells were expressed both pFLAG and pGFP demonstrated that FLAG and GFP do not interact directly (data not shown). However, it is possible that ZAK might also form complexes with other proteins in cells. It is under determination that the oligomerization of ZAK proteins is mediate through the leucine-zipper motif or SAM domain or may be both. Expression of ZAK proteins activates the JNK/SAPK and NF-B pathways. Recently, reports have accumulated that MLK family protein kinases activate the JNK/SAPK pathway, raising the intriguing possibility that ZAK might activate the JNK/SAPK pathway or other MAPK pathways. We test whether the transient expression of ZAK could activate JNK/SAPK pathways. An in vitro kinase assay for JNK/SAPK was performed. As shown in Fig. 4A, the induction of JNK/ SAPK activity is associated with the expression of ZAK in Rat6, Hep3B and HepG2 cells. This result indicates that ZAK might activate the JNK/SAPK pathway in mammalian cells. We examined whether other signal transduction pathways besides the JNK/SAPK cascade are activated by ZAK using the “Mercury Pathway Profiling System” (Clontech). This system contains eight SEAP (secreted alkaline phosphatase) reporter vectors, which represent eight response elements, AP1, CRE, MYC, GRE, HSE, NFAT, NF-B, and SRE, to monitor the activation of transcription factors that represent the response to different signaling pathways. Media from HepG2 cells expressing ZAK with an indicated reporter vector were collected 48 h after transfection and SEAP activity was then measured. Levels of SEAP
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FIG. 2. Expression of ZAK mRNA as shown by Northern blot analysis from various human tissues using ZAK cDNA as a probe. A human multiple tissue Northern blot containing poly (A) ⫹ RNA was probed with a 3⬘-end 1.4 kb DNA fragment of ZAK and a human -actin cDNA.
activity driven by the AP1 response element were increased when ZAK was expressed (Fig. 4B). This result is consistent with the finding that expression of ZAK in mammalian cells induces the activation of JNK/SAPK. No activity could be detected in cells expressing ZAK and CRE or SRE reporter vector, which represent the activity of p38 MAPK and ERKs, respectively (Fig. 4B). Furthermore, ZAK stimulates the activity of NF-B transcription factor (Fig. 4B). We concluded that, therefore, the expression of ZAK in mammalian cells not only stimulates the activation of the JNK/SAPK pathway, but also activates the transcription factor, NF-B. Expression of ZAK is cytotoxic and induces apoptosis. We then tested the biological outcome when ZAK is over expressed in a hepatoma cell line. Over expression of ZAK reduced the number of viable Hep3B cells by 70% compared to control cells transfected with the empty expression vector (Fig. 5A). To examine whether this cell growth suppression is due to apoptosis, Hep3B cells were transiently transfected with a ZAK expression plasmid. After 24 h, apoptotic cells were detected in situ under a fluorescence microscope by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) technique (Fig. 5B). The apoptotic cells were detected with the association of cells transfected with ZAK cDNA. Thus, expression of ZAK is cytotoxic and is by a mechanism due to the apoptosis.
which bear two leucine-zippers. Follow the leucinezipper is a SAM domain. Both leucine-zipper and SAM domains potentially function as protein interaction modules. However, there is a long stretch of the C-terminal for which the function is uncertain. Since ZAK contains a leucine/isoleucine and SAM regions, we first asked whether ZAK forms homodimers or not. When ZAK proteins are expressed in cells, they form protein complexes, which are typically homodimers. It is under determination now that the transition from monomer to dimer plays a role in the regulation of ZAK activation. Our results also indicated that ZAK might activate MAPK pathways especially activate the JNK/ SAPK pathway. Moreover, we found that ZAK might also activate the NF-B pathway. ZAK belongs to a new family of protein kinases, MLKs, which share several distinctive structural features. ZAK has a characteristic kinase catalytic domain with primary sequence contains motifs conserved
DISCUSSION A novel kinase, ZAK, was cloned and this new kinase belongs to the mixed lineage kinase family. However, the role of MLK family proteins remains obscure. ZAK is expressed in most tissues and this expression is regulated by environmental stresses. The kinase domain is located at the beginning of the N-terminal and following the kinase domain is a single leucine-zipper motif, which is distinct from most of the MLK proteins,
FIG. 3. GFP- and FLAG epitope-tagged forms of ZAK were transiently expressed in HepG2 cells either alone or together as indicated. Anti-GFP antibody was used for immunoprecipitation and anti-FLAG antibody for the Western blot. The middle and bottom panel are Western blots, which demonstrate that either GFP- or FLAG-ZAK were expressed in both the single and double transfections.
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in both serine/threonine and tyrosine protein kinases. In addition, immediately C-terminal to the kinase catalytic domain is a predicted single leucine/isoleucine zipper domain, which is a clear distinct from all members of MLK. Furthermore, the SAM domain located in ZAK is the first SAM domain ever discovered in MLK family. Moreover, ZAK protein contains an extremely
FIG. 5. Over expression of ZAK proteins is cytotoxic. (A) Expression of ZAK proteins suppresses cell growth. Hep3B cells (5 ⫻ 10 5 cells) were transfected with 20 g of either the empty expression vector pcDNA3-HA (Control) or pcDNA3-HA-ZAK or -T-ZAK. After 48 h, transfected cells were subjected to G418 selection on 10 cm dishes. After 2 weeks, the plates were fixed and stained with Giemsa solution. (B) Hep3B cells growing on coverslips were transfected with 1 g of either pFLAG expression vector, pFLAF-ZAK or -T-ZAK. Twenty-four hours after transfection, DNA fragments were examined using an in situ cell detection assay (TUNEL).
FIG. 4. JNK and NF-B activation followed by cells transfected with ZAK expression plasmid. (A) Solid phase in vitro activation of JNK/SAPK by ZAK. Bacterial expressed GST-c-jun (1–79) proteins were immobilized through binding to Glutathione Sepharose 4B. This substrate was incubated with the indicated cell lysate (20 g) with or without expression of ZAK in the presence of [␥- 32P]ATP. The kinase reaction was carried out at 25°C for 15 min and the phosphorylated substrates were separated by SDS-PAGE and detected by autoradiography. (B) ZAK mediated the induction of transcription factors in HepG2 cells. HepG2 cells were seeded in six-well plates and transfected with 4 g of expressed ZAK and 1 g of the indicated “mercury reporter vector” plasmids. Samples were collected from the media 48 hours after transfection and measured for SEAP activity using the Great EscAPe chemiluminescence assay. AP1 and NF-B signaling pathways were induced when cells expressed ZAK compared to the empty expression vector. These experiments were performed four times; representative data are shown.
large C-terminal. The presence of the large C-terminal domain raises the possibility that ZAK may play a role in as yet underdetermined signal transduction pathways. The precise biological functions of MLKs are not yet quite understood. We found that the over expression of ZAK induces apoptosis of a hepatoma cell line. Furthermore, our results indicate that ZAK might share an apoptotic signal, but it might also activate additional pathways including one that activates NFB, which provides a survival signal (25). The finding supports this result that TRAIL induces apoptosis, NF-B activation, and JNK/SAPK activation through distinct pathways (26). These findings demonstrate that NF-B activation is not sufficient for protecting cells from entering apoptosis. ACKNOWLEDGMENTS We would like to thank Drs. Robert S. Krauss, D. C. Chang, T. J. Chang, Y. C. Hsu, David D.-W. Kuo, and P. P. Lin for encouragement
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and discussion of the manuscript. This work was supported by grants to J.J.Y. from the National Science Council (NSC) (NSC88-2314-B040-022 and NSC89-2311-B-040-004), to C.J.H. from the National Science Council (NSC) (NSC88-2318-B-001-005-M51), Taiwan.
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12. Hirai, S., Katoh, M., Terada, M., Kyriakis, J. M., Zon, L. I., Rana, A., Avruch, J., and Ohno, S. (1997) J. Biol. Chem. 272, 15167– 15173. 13. Ichijo, H., Nishida, E., Irie, K., Ten Dijke, P., Saitoh, M., Moriguchi, T., Takagi, M., Matsumoto, K., Miyazano, K., and Gotch, Y. (1997) Science 275, 90 –94. 14. Gupta, S., Campbell, D., Derijard, B., and Davis, R. J. (1995) Science 267, 389 –393. 15. Janknecht, R., and Hunter, T. (1997) EMBO J. 16, 1620 –1627. 16. Leung, I. W.-L., and Lassam, N. (1998) J. Biol. Chem. 273, 32408 –32415. 17. Sakuma, H., Ikeda, A., Oka, S., Kozutsumi, Y., Zanetta, J.-P., and Kawasaki, T. (1997) J. Biol. Chem. 272, 28622–28629. 18. Tanaka, S., and Hanafusa, H. (1998) J. Biol. Chem. 273, 1281– 1284. 19. Fan, G., Merritt, S. E., Kortenjann, M., Shaw, P. E., and Holzman, L. B. (1996) J. Biol. Chem. 271, 24788 –24793. 20. Mata, M., Merritt, S. E., Fan, G., Yu, G. G., and Holzman, L. B. (1996) J. Biol. Chem. 271, 16888 –16896. 21. Merritt, S. P., Mata, M., Nihalani, D., Zhu, C., Hu, X., and Holzman, L. B. (1999) J. Biol. Chem. 274, 10195–10202. 22. Yang, J.-J., and Krauss, R. S. (1997) J. Biol. Chem. 272, 3103– 3108. 23. Yang, J.-J., Kang, J.-S., and Krauss, R. S. (1998) Mol. Cell. Biol. 18, 2586 –2595. 24. Thanos, C. D., Goodwill, K. E., and Bowie, J. U. (1999) Science 283, 833– 836. 25. Mayo, M. W., Wang, C.-Y., Cogswell, P. C., Rogers-Graham, K. S., Lowe, S. W., Der, C. J., and Baldwin, A. S., Jr. (1997) Science 278, 1812–1815. 26. Hu, W.-H., Johnson, H., and Shu, H.-B. (1999) J. Biol. Chem. 274, 30603–30610.
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Cloning and Expression of ZAK, a Mixed Lineage Kinase-like Protein Containing a Leucine-Zipper and a Sterile-Alpha Motif Te-Chung Liu,* Chang-Jen Huang,† Yu-Chuan Chu,‡ Chih-Chang Wei,‡ Chun-Chieh Chou,‡ Ming-Yung Chou,‡ Chen-Kung Chou,§ and Jaw-Ji Yang‡ ,1 *Department of Nutrition, Chung-Shan Medical College, Taichung 402, Taiwan; †Institute of Biological Chemistry, Academia Sinica, Taipei 112, Taiwan; ‡School of Dentistry, Chung-Shan Medical College, Taichung 402, Taiwan; and §Institute of Medical Research, Veterans General Hospital, Taipei 112, Taiwan
Received July 5, 2000
A novel mixed lineage kinase-like protein ZAK, containing a leucine-zipper (LZ) and a sterile-alpha motif (SAM), was cloned. This cDNA has 2456 bp and encodes a protein of 800 amino acids that contains a kinase catalytic domain, a leucine-zipper and a SAM. The molecular weight of this protein is 91kDa. Northern blot analysis revealed that the expression of this ZAK gene is found in various parts of human tissues. We also found that ZAK proteins might form homodimers or oligomers in mammalian cells. MLKs have been proposed to function as mitogen-activated protein kinase kinase kinase in pathways leading to MAPK cascade. The expression of ZAK in mammalian cells specifically leads to the activation of the JNK/SAPK pathway as well as the activation of transcription factor, NF-B. Overexpression of the ZAK gene induces the apoptosis of a hepatoma cell line. © 2000 Academic Press Key Words: leucine-zipper and sterile-alpha motif kinase (ZAK); mixed lineage kinase (MLK); JNK/SAPK; apoptosis.
Protein kinases play important roles in the regulation of many cellular processes, such as the transmission of signals from the growth factor, control of cell growth and differentiation, regulation of cytoskeletal changes, gene expression, and translation and metabolism pathways (1). Among the protein kinases, MAP kinase plays an essential role in the cell signal transduction machinery. Protein kinases of the MAPK family is involved in a variety of cellular responses to Abbreviations used: ZAK, leucine-zipper and sterile-alpha motif kinase; MLK, mixed lineage kinase; MAPK, mitogen-activated protein kinase; JNK/SAPK, c-Jun NH2-terminal kinase/stress-activating protein kinase. 1 To whom correspondence should be addressed. Fax: 886-44759065. E-mail: [email protected].
cytokines, hormones, and stress-inducing reagents. Several mammalian MAP kinases have been cloned, including ERK (2), JNK/SAPK (3, 4) and p38 MAPK (5). JNKs/SAPKs as well as p38Mpk2 are strongly activated in cells exposed to extracellular stress such as UV irradiation, osmotic shock, heat shock (6), and treatment with tumor necrosis factor-␣ (7), interleukin-1 (8, 9) or antibiotics known as the inhibitors of eukaryotic protein synthesis. Accumulating evidence suggests that these MAPK cascades are regulated by small GTPases of the Rho family (10, 11). MEK family protein kinases, MKK4 and MKK7, and the MKK kinases may include TPL2, MLK/DLK, TAK, and ASK activate JNK/SAPK (12, 13). The activated JNKs/ SAPKs are then translocated into the nucleus and activate several transcription factors such as c-jun, ATF2, ELK, and Sap-1 (14, 15). Phosphorylated c-jun, by JNKs/SAPKs, forms homodimers or heterodimers with c-fos, which have potent AP-1 activity and regulate the expression of a number of genes. Recent reports indicate that a newly discovered type of protein kinases is involved in JNK signaling. This family of kinases has been named mixed lineage protein kinase (MLK) (16, 17). MLKs are in turn activated by a number of agonists. For example, MLK-3 has been found to mediate signals from the ste20 homologue germinal center kinase, and hematopoietic kinase, the guaninenucleotide exchange protein C3G, and is a target of the small GTPases Rac and Cdc42 (18). MLKs bear protein-protein interaction domains, which contain two leucine-zippers at the carboxyl-terminal to the catalytic domain. MLK might not only participate in the JNK signaling cascade but also mediate the p38 MAPK cascade (19). MLK has been implicated as a MAP kinase kinase kinase. The five identified members of the MLK family, MLK1, MLK2, MLK3, DLK, and LZK, share two common structure features (17, 20). Each
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has a characteristic kinase catalytic domain with primary structure, which is hybrid between those in serine/threonine and tyrosine protein kinases. Second, closely juxtaposed C-terminal to the kinase domain, each MLK has a domain that is predicted to form two leucine/isoleucine zippers. However, little is to known about the overall biochemical features and functionally roles of MLKs. It has been proposed that MLKs behave functional as MAPK kinase kinase in pathways leading to the activation of JNK/SAPK (11). Furthermore, MLK2 and MLK3 have been shown to phosphorylate and activate MKK4 (21). In this study, we cloned a novel and stress-regulated MLK-like protein kinase, ZAK, which may specifically activate JNK and turn on NF-B transcription factor in several cell lines. The over-expression of this gene induces apoptosis in hepatoma cells. MATERIALS AND METHODS cDNA library screening and 5⬘-end determination of ZAK. An EST clone of ste20-like DNA labeled with [␣- 32P]dCTP and used to screen the human placenta cDNA library. The hybridization conditions for screening this library were 5 ⫻ SSC and 40% formamide at 42°C for 16 h and the final washing condition was 1 ⫻ SSC at 68°C. A human marathon cDNA mix (liver, placenta, thyroid, fetus, and stomach) was employed to amplify the 5⬘ end of this gene. Antisense gene-specific primers (AGSPs), AGSP1 5⬘-GAGAGGCACCAAAGTCACAGAT and AGSP2 5⬘-CACCTTGACAGGAGCCTCCATATGTAA, were designed and adaptor primers (AP1 and AP2) (Clontech) used to perform the 5⬘-end RACE. The first-PCR was performed by using the cDNA mix and AGSP1 and AP1 and conditions were as follows: denaturation at 94°C for 1 min, annealing at 65°C for 1 min, and extension at 72°C for 1 min, for 35 cycles, followed by a 7 min extension at 72°C. The result of the first PCR was then used as a template to perform secondary-PCR with AGSP2 and AP2 and PCR conditions as previously. A band about 400 bp was amplified and subcloned into the pGEM-T vector and sequenced. The additional 5⬘-end DNA fragment of 303bp was then added to the original clone to make up the full-length of this gene with a putative open reading frame of 800 amino acids. Analysis of ZAK transcript. mRNA from various human tissues (Clontech) was probed with 3⬘-end of ZAK cDNA labeled by random priming method and hybridization was performed as described (22). Immunoprecipitation and Western blot analysis. Cell lysates were prepared in IP buffer (40 mM Tris–HCl (pH 7.5), 1% NP40, 150 mM NaCl, 5 mM EGTA, 1 mM DTT, 1 mM PMSF, 20 mM NaF, proteinase inhibitors, and 1 mM sodium vanadate). Cell extracts (600 g) were incubated with 5 g of anti GFP mAb (Clontech) for 6 h at 4°C, mixed with 20 l protein-A sepharose suspension, and incubated for an additional hour. Immunoprecipitates were collected by centrifugation, washed three times with IP buffer plus 0.5% deoxycholate, five times with IP buffer alone, and subjected to SDS–PAGE. Immunoblot analysis was performed with the anti-FLAG (Sigma). ZAK- or empty vector-expressed cells were harvested in lysis buffer (50 mM Tris–HCl, pH 8.0/250 mM NaCl/1% NP-40, 2 mM EDTA) containing 1 mM PMSF, 10 ng/ml leupeptin, 50 mM NaF, and 1 mM sodium orthovanadate. Total proteins were then separated on SDS– PAGE and specific protein bands visualized with an ECL chemiluminescent detection system (Amersham) was performed as described (23). Detection of the JNK/SAPK activities. Protein kinase assays were carried out using a fusion protein between glutathione S-transferase
(GST) and c-Jun (amino acids 1–79) as a substrate. The GST-Jun fusion proteins were bound to glutathione sepharose beads and incubated for 15 min on ice with the cellular extract that contains JNK, in the presence of kinase buffer (20 mM Hepes, pH 7.6, 1 mM EGTA, 1 mM dithiothreitol, 2 mM MgCl, 2 mM MnCl, 5 mM NaF, 1 mM NaVO, 50 mM NaCl). The beads were pelleted and thoroughly washed with PBST (150 mM NaCl, 16 mM sodium phosphate, pH 7.5, 1% Triton X-100, 2 mM EDTA, 0.1% MeOH, 0.2 mM phenylmethylsulfonyl fluoride, and 5 mM benzamidine), before they were incubated with [␥- 32P]ATP (50 cpm/fmol) in the presence of kinase buffer. These steps were undertaken to ensure that c-Jun phosphorylation was carried out by JNK, which is known to exhibit high affinity to this portion of c-Jun under these conditions. Following extensive washing, the phosphorylated GST-Jun was boiled in SDS sample buffer, and the eluted proteins were run on a 15% SDS– PAGE. The gel was dried, and phosphorylation of the Jun substrate was determined by autoradiography. Microscopic observation of apoptosis cells. Hep3B cells growing on glass coverslips were transfected 1 g of empty or ZAK expressing plasmid using calcium phosphate method. Twenty-four hours after transfection, apoptotic cells were detected with an in situ cells detection kit as instructed by the manufacturer (Roche; in situ cell death detection kit, Fluorescein). Cells were examined photographed in a Zesis Axioskop microscope.
RESULTS Isolation of a ZAK cDNA and its deduced amino acid sequence. The DNA fragment was released from an EST clone of the ste 20-like plasmid. This fragment was then used as a probe to screen the human placenta cDNA library (Stratagene) by a low stringency hybridization method. A positive clone was found and sequenced. This cDNA extends 2179 nucleotide bases with a poly-A tail and contains a continuous open reading frame of 2097 bp. In order to obtain the 5⬘-end of this gene, a mixture (liver, placenta, thyroid, fetus, and stomach; Stratagene) of the human 5⬘-RACE cDNA library was employed and a further upstream DNA fragment of 303 bp was then found. Furthermore, this DNA fragment was sequenced and ligated to the previous cDNA clone to fill up the full-length of this gene (GenBank Accession No. AF238255). The open reading frame of this cDNA encodes a putative polypeptide of 800 amino acids, with a calculated molecular mass of 91 kDa (Fig. 1A). Surprisingly, a comparison of the amino acid sequence of this cDNA encoded protein with Genbank revealed it to be a novel gene belonging to the MLK family based on the similarity of their sequences. However, the cDNA is not a ste-20 homologue. Hydrophobicity analysis revealed that this protein contains no obvious signal sequence or transmembrane domain. Comparison of the sequence with other known proteins revealed that the protein could be divided into at least three structural domains: a kinase catalytic domain, a leucine-zipper motif and a sterile-alpha motif (SAM) (Fig. 1B). The kinase domain extends 245 amino acids from amino acid 16 through 260. This kinase catalytic domain has the features of both serine/threonine kinases and tyrosine kinases, where Trp197-Glu198, Pro205-
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Tissue distribution of ZAK mRNA. To ascertain the expression pattern of the ZAK gene, we studied the endogenous expression of ZAK mRNA in various human tissues. Northern blot analysis of various human tissues demonstrated that the mRNA for ZAK is 3.0 kb. The expression of the ZAK gene could be detected in many tissues, including the heart, placenta, lung liver, and pancreas (Fig. 2).
FIG. 1. Amino acid sequence and the possible structure of the ZAK protein. (A) Conceptually translated amino acid sequence of the ZAK protein. Bold letters denote the kinase catalytic domain (16 – 260) and the SAM domain (336 – 410). The leucines/isoleucines in the putative leucine-zipper motif are underlined. (B) Schematic representation of the primary structure of ZAK. The numbers in the diagram represent amino acid residue numbers and delineate the boundaries of the indicated domains. Besides the regions of kinase catalytic domain and leucine/isoleucine zippers, ZAK does not show similarity with other protein kinases of the MLK family.
Phe206 and Trp172 (GTFPWMAPE) are characteristic for tyrosine kinase. However, the presence of Lys134 (HRDLKSRN) and Thr169 (GTFPWMAPE) indicate that this kinase would have serine/threonine specificity, and Trp172-Met173 (GTFPWMAPE) is often found in Raf family proteins (25). Following the kinase catalytic domain is a single leucine-zipper motif and a sterile-alpha motif, which may be involved in the protein-protein interaction. The amino acid sequences of the kinase catalytic domain and leucine-zipper motif of this protein have about 40% identity and are about 60% positive to the sequences of previously reported protein kinases, such as MLK2, MUK, ZPK, and LZK, suggesting that this protein kinase belongs to the MLK family (Fig. 1B). In contrast to most MLK family protein kinases, this protein kinase bears a single leucinezipper motif. Moreover, there is no homology with other reported proteins beyond the leucine-zipper motif. We named this protein kinase ZAK (leucine-zipper and sterile-alpha motif protein kinase).
ZAK forms oligomers. Since ZAK contains a leucine-zipper motif and a SAM, which have been implicated in protein-protein interaction (24), it is reasonable to suspect that ZAK can form homodimers in cells. This interaction was investigated by using in vivo coprecipitation. HepG2 cells were co-transfected with plasmids that express a GFP-tagged ZAK construct and a FLAG-tagged ZAK construct. Protein complexes were immunoprecipitated using anti-GFP antibody, and co-precipitated FLAG-ZAK was probed by M2 monoclonal antibody. As shown in Fig. 3, FLAG-ZAK could be detected only when cells expressed both FLAG-ZAK and GFP-ZAK plasmids. This result demonstrates that ZAK proteins might form homodimers or oligomers in mammalian cells. Moreover, the double negative controls when cells were expressed both pFLAG and pGFP demonstrated that FLAG and GFP do not interact directly (data not shown). However, it is possible that ZAK might also form complexes with other proteins in cells. It is under determination that the oligomerization of ZAK proteins is mediate through the leucine-zipper motif or SAM domain or may be both. Expression of ZAK proteins activates the JNK/SAPK and NF-B pathways. Recently, reports have accumulated that MLK family protein kinases activate the JNK/SAPK pathway, raising the intriguing possibility that ZAK might activate the JNK/SAPK pathway or other MAPK pathways. We test whether the transient expression of ZAK could activate JNK/SAPK pathways. An in vitro kinase assay for JNK/SAPK was performed. As shown in Fig. 4A, the induction of JNK/ SAPK activity is associated with the expression of ZAK in Rat6, Hep3B and HepG2 cells. This result indicates that ZAK might activate the JNK/SAPK pathway in mammalian cells. We examined whether other signal transduction pathways besides the JNK/SAPK cascade are activated by ZAK using the “Mercury Pathway Profiling System” (Clontech). This system contains eight SEAP (secreted alkaline phosphatase) reporter vectors, which represent eight response elements, AP1, CRE, MYC, GRE, HSE, NFAT, NF-B, and SRE, to monitor the activation of transcription factors that represent the response to different signaling pathways. Media from HepG2 cells expressing ZAK with an indicated reporter vector were collected 48 h after transfection and SEAP activity was then measured. Levels of SEAP
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FIG. 2. Expression of ZAK mRNA as shown by Northern blot analysis from various human tissues using ZAK cDNA as a probe. A human multiple tissue Northern blot containing poly (A) ⫹ RNA was probed with a 3⬘-end 1.4 kb DNA fragment of ZAK and a human -actin cDNA.
activity driven by the AP1 response element were increased when ZAK was expressed (Fig. 4B). This result is consistent with the finding that expression of ZAK in mammalian cells induces the activation of JNK/SAPK. No activity could be detected in cells expressing ZAK and CRE or SRE reporter vector, which represent the activity of p38 MAPK and ERKs, respectively (Fig. 4B). Furthermore, ZAK stimulates the activity of NF-B transcription factor (Fig. 4B). We concluded that, therefore, the expression of ZAK in mammalian cells not only stimulates the activation of the JNK/SAPK pathway, but also activates the transcription factor, NF-B. Expression of ZAK is cytotoxic and induces apoptosis. We then tested the biological outcome when ZAK is over expressed in a hepatoma cell line. Over expression of ZAK reduced the number of viable Hep3B cells by 70% compared to control cells transfected with the empty expression vector (Fig. 5A). To examine whether this cell growth suppression is due to apoptosis, Hep3B cells were transiently transfected with a ZAK expression plasmid. After 24 h, apoptotic cells were detected in situ under a fluorescence microscope by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) technique (Fig. 5B). The apoptotic cells were detected with the association of cells transfected with ZAK cDNA. Thus, expression of ZAK is cytotoxic and is by a mechanism due to the apoptosis.
which bear two leucine-zippers. Follow the leucinezipper is a SAM domain. Both leucine-zipper and SAM domains potentially function as protein interaction modules. However, there is a long stretch of the C-terminal for which the function is uncertain. Since ZAK contains a leucine/isoleucine and SAM regions, we first asked whether ZAK forms homodimers or not. When ZAK proteins are expressed in cells, they form protein complexes, which are typically homodimers. It is under determination now that the transition from monomer to dimer plays a role in the regulation of ZAK activation. Our results also indicated that ZAK might activate MAPK pathways especially activate the JNK/ SAPK pathway. Moreover, we found that ZAK might also activate the NF-B pathway. ZAK belongs to a new family of protein kinases, MLKs, which share several distinctive structural features. ZAK has a characteristic kinase catalytic domain with primary sequence contains motifs conserved
DISCUSSION A novel kinase, ZAK, was cloned and this new kinase belongs to the mixed lineage kinase family. However, the role of MLK family proteins remains obscure. ZAK is expressed in most tissues and this expression is regulated by environmental stresses. The kinase domain is located at the beginning of the N-terminal and following the kinase domain is a single leucine-zipper motif, which is distinct from most of the MLK proteins,
FIG. 3. GFP- and FLAG epitope-tagged forms of ZAK were transiently expressed in HepG2 cells either alone or together as indicated. Anti-GFP antibody was used for immunoprecipitation and anti-FLAG antibody for the Western blot. The middle and bottom panel are Western blots, which demonstrate that either GFP- or FLAG-ZAK were expressed in both the single and double transfections.
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in both serine/threonine and tyrosine protein kinases. In addition, immediately C-terminal to the kinase catalytic domain is a predicted single leucine/isoleucine zipper domain, which is a clear distinct from all members of MLK. Furthermore, the SAM domain located in ZAK is the first SAM domain ever discovered in MLK family. Moreover, ZAK protein contains an extremely
FIG. 5. Over expression of ZAK proteins is cytotoxic. (A) Expression of ZAK proteins suppresses cell growth. Hep3B cells (5 ⫻ 10 5 cells) were transfected with 20 g of either the empty expression vector pcDNA3-HA (Control) or pcDNA3-HA-ZAK or -T-ZAK. After 48 h, transfected cells were subjected to G418 selection on 10 cm dishes. After 2 weeks, the plates were fixed and stained with Giemsa solution. (B) Hep3B cells growing on coverslips were transfected with 1 g of either pFLAG expression vector, pFLAF-ZAK or -T-ZAK. Twenty-four hours after transfection, DNA fragments were examined using an in situ cell detection assay (TUNEL).
FIG. 4. JNK and NF-B activation followed by cells transfected with ZAK expression plasmid. (A) Solid phase in vitro activation of JNK/SAPK by ZAK. Bacterial expressed GST-c-jun (1–79) proteins were immobilized through binding to Glutathione Sepharose 4B. This substrate was incubated with the indicated cell lysate (20 g) with or without expression of ZAK in the presence of [␥- 32P]ATP. The kinase reaction was carried out at 25°C for 15 min and the phosphorylated substrates were separated by SDS-PAGE and detected by autoradiography. (B) ZAK mediated the induction of transcription factors in HepG2 cells. HepG2 cells were seeded in six-well plates and transfected with 4 g of expressed ZAK and 1 g of the indicated “mercury reporter vector” plasmids. Samples were collected from the media 48 hours after transfection and measured for SEAP activity using the Great EscAPe chemiluminescence assay. AP1 and NF-B signaling pathways were induced when cells expressed ZAK compared to the empty expression vector. These experiments were performed four times; representative data are shown.
large C-terminal. The presence of the large C-terminal domain raises the possibility that ZAK may play a role in as yet underdetermined signal transduction pathways. The precise biological functions of MLKs are not yet quite understood. We found that the over expression of ZAK induces apoptosis of a hepatoma cell line. Furthermore, our results indicate that ZAK might share an apoptotic signal, but it might also activate additional pathways including one that activates NFB, which provides a survival signal (25). The finding supports this result that TRAIL induces apoptosis, NF-B activation, and JNK/SAPK activation through distinct pathways (26). These findings demonstrate that NF-B activation is not sufficient for protecting cells from entering apoptosis. ACKNOWLEDGMENTS We would like to thank Drs. Robert S. Krauss, D. C. Chang, T. J. Chang, Y. C. Hsu, David D.-W. Kuo, and P. P. Lin for encouragement
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and discussion of the manuscript. This work was supported by grants to J.J.Y. from the National Science Council (NSC) (NSC88-2314-B040-022 and NSC89-2311-B-040-004), to C.J.H. from the National Science Council (NSC) (NSC88-2318-B-001-005-M51), Taiwan.
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