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Mirk Protein Kinase Is Activated by MKK3 and Functions as a Transcriptional Activator of HNF1α

2002; Elsevier BV; Volume: 277; Issue: 28 Linguagem: Inglês

10.1074/jbc.m203257200

1083-351X

Seunghwan Lim, Kideok Jin, Eileen Friedman,

NF-κB Signaling Pathways

Mirk/Dyrk1B is an arginine-directed serine/threonine protein kinase that is expressed at low levels in most normal tissues but at elevated levels in many tumor cell lines and in normal skeletal muscle. Colon carcinoma cell lines stably overexpressing Mirk proliferated in serum-free medium, but the mechanism of Mirk action is unknown. DCoHm(dimerization cofactor of hepatocyte nuclear factor 1α ( HNF1α) from muscle), a novel gene of the DCoH family with 78% amino acid identity to DCoH, was identified as a Mirk-binding protein by yeast two-hybrid analysis and cloned. Mirk co-immunoprecipitated with DCoHm and bound to DCoHm in glutathione S-transferase pull-down assays. DCoH stabilizes HNF1α as a dimer and enhances its transcriptional activity on the β-fibrinogen promoter reporter, and DCoHm had similar activity. Mirk enhanced HNF1α transcriptional activity in a dose-dependent manner, whereas two kinase-inactive Mirk mutants and a Mirk N-terminal deletion mutant did not. Mirk, DCoHm, and HNF1α formed a complex. Mirk bound to a specific region within the CREB-binding protein-binding region of HNF1α and phosphorylated HNF1α at a site adjacent to the Mirk-binding region. Conversely, the HNF1α binding domain was located within the first five conserved kinase subdomains of Mirk. Mirk co-immunoprecipitated with the MAPK kinase MKK3, an upstream activator of p38. MKK3 enhanced Mirk kinase activity and the transcriptional activation of HNF1α by Mirk, suggesting that Mirk, like p38, is activated by certain environmental stress agents. The Mirk-binding protein DCoH has been shown to be selectively expressed in colon carcinomas but not in normal tissue. Mirk may function as an HNF1α transcriptional activator in response to an MKK3-mediated stress signal, and the selective expression of DCoH could restrict the Mirk response to carcinoma cells. Mirk/Dyrk1B is an arginine-directed serine/threonine protein kinase that is expressed at low levels in most normal tissues but at elevated levels in many tumor cell lines and in normal skeletal muscle. Colon carcinoma cell lines stably overexpressing Mirk proliferated in serum-free medium, but the mechanism of Mirk action is unknown. DCoHm(dimerization cofactor of hepatocyte nuclear factor 1α ( HNF1α) from muscle), a novel gene of the DCoH family with 78% amino acid identity to DCoH, was identified as a Mirk-binding protein by yeast two-hybrid analysis and cloned. Mirk co-immunoprecipitated with DCoHm and bound to DCoHm in glutathione S-transferase pull-down assays. DCoH stabilizes HNF1α as a dimer and enhances its transcriptional activity on the β-fibrinogen promoter reporter, and DCoHm had similar activity. Mirk enhanced HNF1α transcriptional activity in a dose-dependent manner, whereas two kinase-inactive Mirk mutants and a Mirk N-terminal deletion mutant did not. Mirk, DCoHm, and HNF1α formed a complex. Mirk bound to a specific region within the CREB-binding protein-binding region of HNF1α and phosphorylated HNF1α at a site adjacent to the Mirk-binding region. Conversely, the HNF1α binding domain was located within the first five conserved kinase subdomains of Mirk. Mirk co-immunoprecipitated with the MAPK kinase MKK3, an upstream activator of p38. MKK3 enhanced Mirk kinase activity and the transcriptional activation of HNF1α by Mirk, suggesting that Mirk, like p38, is activated by certain environmental stress agents. The Mirk-binding protein DCoH has been shown to be selectively expressed in colon carcinomas but not in normal tissue. Mirk may function as an HNF1α transcriptional activator in response to an MKK3-mediated stress signal, and the selective expression of DCoH could restrict the Mirk response to carcinoma cells. minibrain-related kinase dual-specificity Yak-related kinase cAMP-response element CRE-binding protein hepatocyte nuclear factor 1α mitogen-activated protein MAP kinase kinase 3 1 m sorbitol, 0.1 msodium citrate, pH 7.6, 0.06 m EDTA myelin basic protein glutathione S-transferase 20 mm 4-morpholinepropanesulfonic acid (MOPS), pH 7.2, 25 mm β-glycerol phosphate, 5 mm EGTA, 1 mm sodium orthovanadate, and 1 mmdithiothreitol dimerization cofactor of HNF1α from muscle 50 mm Tris-HCl, pH8.0, 120 mm NaCl, 0.5% Nonidet P-40, 1 mm dithiothreitol, and a tablet of protease inhibitor 10 mm Tris-HCL, pH8.0, 150 mmNaCl, and 0.1% Tween 20 CREB-binding protein Mirk1/Dyrk1B is a serine/threonine protein kinase that is expressed at elevated levels in normal skeletal muscle and certain carcinoma cell lines and at low levels in many normal tissues (1Lee K. Deng X. Friedman E. Cancer Res. 2000; 60: 3631-3637PubMed Google Scholar). Colon carcinoma cell lines stably overexpressing Mirk proliferated in serum-free medium (1Lee K. Deng X. Friedman E. Cancer Res. 2000; 60: 3631-3637PubMed Google Scholar), but the mechanism of Mirk action that enabled this survival capacity is unknown. Mirk is a member of the Dyrk/minibrain family of dual specificity, tyrosine-regulated, arginine-directed protein kinases (2Kentrup H. Becker W. Heukelbach J. Wilmes A. Schurman A. Huppertz C. Kainulainen H. Joost H.-G. J. Biol. Chem. 1996; 271: 3488-3495Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 3Becker W. Weber Y. Wetzel K. Eirmbter K. Tejedor F. Joost H.-G. J. Biol. Chem. 1998; 273: 25893-25902Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 4Himpel S. Tegge W. Frank R. Leder S. Joost H. Becker W. J. Biol. Chem. 2000; 275: 2431-2438Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar) and is identical to Dyrk1B (5Leder S. Weber Y. Altafaj X. Estivill X. Joost H.-G. Becker W. Biochem. Biophys. Res. Commun. 1999; 254: 474-479Crossref PubMed Scopus (57) Google Scholar). Mirk/Dyrk1B and the related kinase Dyrk1A exhibit 54% amino acid identity with 90% identity or homology within the conserved kinase domain. Several lines of evidence indicate that Dyrk1A mediates neuronal differentiation. Dyrk1A has been mapped to the Down's syndrome critical region of chromosome 21, overexpression of Dyrk1A has been found in the Down's syndrome fetal brain (6Guimera J. Casas C. Estivill X. Pritchard M. Genomics. 1999; 57: 407-418Crossref PubMed Scopus (145) Google Scholar), and transgenic mice overexpressing Dyrk1A exhibit cognitive deficits and motor abnormalities characteristic of Down's syndrome (7Altafaj X. Dierssen M. Baamonde C. Marti E. Visa J. Guimera J. Oset M. Gonzalez J. Florez J. Fillat C. Estivill X. Hum. Mol. Genet. 2001; 10: 1915-1923Crossref PubMed Scopus (328) Google Scholar). Dyrk1A has been shown to phosphorylate the cAMP-response element-binding protein (CREB) in vivo, leading to the stimulation of subsequent cAMP response element-mediated transcription during neuronal differentiation in hippocampal progenitor cells (8Yang E. Ahn Y.S. Chung K. J. Biol. Chem. 2001; 276: 39819-39824Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar), an activity consistent with its likely mediation of neuronal maturationin vivo. Because Mirk/Dyrk1B was expressed at elevated levels in normal muscle tissue, we screened a muscle cell cDNA library to detect possible Mirk targets and found that Mirk/Dyrk1B activated the transcription factor HNF1α (hepatocyte nuclear factor 1α) and bound to its cofactor DCoH. Antibodies to MKK3 and HNF1 were from Santa Cruz Biotechnology, and antibodies to the flag-epitope were from Sigma. Rabbit polyclonal antibody to a unique sequence at the C terminus of Mirk was raised as described (1Lee K. Deng X. Friedman E. Cancer Res. 2000; 60: 3631-3637PubMed Google Scholar). Polyvinylidene difluoride transfer paper Immobolin-P was purchased from Millipore. PLUS reagent and LipofectAMINE were from Invitrogen, all radioactive materials were purchased from PerkinElmer Life Sciences, and ECL reagents were from Amersham Biosciences. All other reagents were from Sigma. We thank Stephen E. Mercer of this institution for GST-Mirk preparations. NIH3T3 cells and 293T cells were maintained in Dulbecco's modified Eagle's medium containing 7% fetal bovine serum and modified and supplemented as described (9Hafez M. Hsu S. Yan Z. Winawer S. Friedman E. Cell Growth & Differ. 1992; 3: 753-762PubMed Google Scholar). pHX9F (Mirk) and pHX9F (kinase-inactive YF Mirk) had been previously generated (1Lee K. Deng X. Friedman E. Cancer Res. 2000; 60: 3631-3637PubMed Google Scholar), and all other Mirk expression plasmids, including the pGBKT7-Mirk construct used for the yeast two-hybrid screening, were prepared by Dr. Xiaobing Deng. 2X. Deng, manuscript in preparation. The (β-28)3-luciferase plasmid encoding three tandem repeats of the β-fibrinogen HNF1α binding domain in front of a TATA box promoter and a luciferase reporter gene, the expression plasmid pBJ5-DCoH, and the expression plasmid pBJ5-HNF1α were the kind gifts of Dr. G. Crabtree, Stanford University. FLAG-MKK3b and MKK3b(E), each in pcDNA3, were the kind gifts of Dr. J. Han, Scripps Institute. A human muscle cDNA library subcloned into the pACT2 vector was purchased form CLONTECH. We are grateful to Dr. David Amberg of this institution for help with the two-hybrid screening. For the bait construct, wild-type full-length Mirk cDNA was subcloned into the pGBKT7 vector, because Mirk had no transcriptional activity by itself. The yeast host strain, AH109, contains three different reporter genes that are tightly controlled by the upstream activator sequence, which required GAL4 binding for expression of the reporter gene. AH109 cells were simultaneously co-transformed with pGBKT7-Mirk and the pACT2 muscle cDNA library according to the manufacturer's protocol. The transformants were plated on -His/-Leu/-Trp medium, including 25 mm 3-amino-1,2,4-triasol to screen for the expression of His-3, and incubated at 30 °C for 7–10 days. Subsequently, His+ colonies were replated on -Ade/-His/-Leu/-Trp/X-α-gal medium, and blue colonies were selected as positive candidates. The plasmids from candidate colonies were harvested as described (10Amberg D. Zahner J. Mulholland J. Pringle J. Botstein D. Mol. Cell. Biol. 1997; 8: 729-753Crossref Scopus (153) Google Scholar). Briefly, cells were grown in 5 ml of selective media overnight and collected by pelleting. The pellets were resuspended in 200 μl of lysis buffer and then incubated at 37 °C for 30–90 min after brief vortexing. Lysis buffer consisted of a mixture of 5 ml of SCE buffer, 60 μl of 100T zymolyase (10 mg/ml), and 10 μl of β-mercaptoethanol. Following the addition of 400 μl of 0.2 n NaOH/1% SDS solution the lysates were incubated on ice for 5 min and then pelleted, and the supernatants were decanted into fresh tubes and then reclarified by repeated pelleting. The plasmid DNA of each candidate was precipitated and then transformedEscherichia coli DH5α using electroporation. The sequences of candidate DNA were analyzed by using an automatic DNA sequencer (model 3700 BC 1.1.0.0, ABI PRISM). Hexahistidine fusion Mirk was produced by a coupled in vitro transcription and translation system (Promega) using 1 μg of pHX plasmid per reaction. Produced protein was immunoprecipitated overnight by using His6 monoclonal antibody (CLONTECH) and 10 μl of 50% protein A-Sephadex (Sigma). The immunoprecipitate was washed three times with radioimmune precipitation buffer and three times with kinase buffer (10 mm Tris, pH7.4, 150 mm NaCl, 10 mmMgCl2, 0.5 mm dithiothreitol). The kinase activities of Mirk were tested with either the myelin basic protein (MBP) from Upstate Biotechnology, immunoprecipitated HNF1 overexpressed in 293T cells, or recombinant GST-HNF1 proteins as a substrate according to the protocol by Upstate Biotechnology. Briefly, 10 μl of assay dilution buffer (ADB), 10 μl of GST-Mirk (200 ng/1 μg) in ADB, 10 μl of MBP (2 μg/μl) in ADB, and 10 μl of [γ-32P]ATP (0.5 μCi/μl) in a magnesium/ATP mixture (500 μm cold ATP and 75 mm MgCl2in ADB) were added to the reaction mixture. Immunoprecipitated MKK3E or recombinant p38 (Upstate Biotech.) was added in the mixture. The reaction was performed at 30 °C for 15 min with gentle agitation. The reaction was stopped by adding sample buffer and boiling for 5 min. The phospholabeled proteins were separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and detected by autoradiography. 293T cells were transiently transfected by adding a complex of LipofectAMINE (2–4 μg/μg of DNA) in serum-free media for 24 h. For reporter gene assays, NIH3T3 cells were seeded the day before transfection at 0.9 × 105/well and allowed to grow overnight in 7% serum-containing media. Cells were transfected by incubating with a complex of PLUS reagent (3 μl/μg DNA) and LipofectAMINE (2 μl/μg DNA) in serum-free media for 18–24 h in a CO2incubator. The amount of total DNA used was kept constant by the addition of empty vector DNA, and luciferase activities were calibrated by co-transfected β-galactosidase activity to normalize the transfection efficiency. These assays were carried out in triplicate, and the data shown are representative of three independent experiments. [35S]-Met-labeled Mirk and DCoHm produced by using the TnT transcription and translation system (Promega) were co-immunoprecipitated in vitro. TnT products (5 μl each) were mixed, and 5 μl of rabbit polyclonal antibody to the unique C terminus of Mirk or preimmune serum was added with rocking overnight at 4 °C. 20 μl of a slurry of protein A-agarose conjugates (Santa Cruz Biotechnology) was then added and incubated for an additional 2 h at 4 °C. The agarose beads in each tube were extensively washed five times, followed by SDS-PAGE and autoradiography. 293T cells in multiple 60-mm dishes were co-transfected with 1 μg of the DNA of either MKK3 or MKK3E together with expression plasmids for either Mirk, kinase-inactive YF-Mirk, or pHisA vector (1 μg/well), allowed to express for 24 h, and then each dish was lysed in 0.25 ml of EBC buffer (Roche Molecular Biochemicals). An aliquot of total lysate of 300 μg was immunoprecipitated with 5 μl of an anti-C2 Mirk anti-peptide rabbit polyclonal antibody overnight at 4 °C; the complexes were then collected by the addition of 20 μl of protein A-agarose, incubated for 2 h at 4 °C, washed three times with EBC buffer, and separated by SDS-PAGE. Following treatment as indicated and washing with cold phosphate-buffered saline, cells were lysed in EBC or radioimmune precipitation buffer (1× phosphate-buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and the following protease inhibitors: 20 μg/ml leupeptin, 20 μg/ml aprotinin, 0.5 mm phenylmethylsulfonyl fluoride, 200 μmsodium orthovanadate, and 20 mm sodium fluoride). Lysates were pelleted in a microfuge for 15 min to remove insoluble material. Depending upon the experiment, 10–50 μg of cell lysate were blotted onto polyvinylidene difluoride membranes after separation on SDS-PAGE. The blots were blocked in 5% milk in TBST for 1 h at room temperature, incubated for 1 h at room temperature with primary antibody in TBST buffer-3% milk, and proteins were subsequently detected by enhanced chemiluminescence. All Mirk blots used an affinity-purified polyclonal antibody directed to the Mirk-unique C terminus. Band density in autoradiograms was measured using a Lacie Silverscanner and Silverscanner III software and analyzed by the IP LabGel program. Mirk and DCoHm were producedin vitro by a coupled transcription and translation system (Promega) using 1 μg of plasmid per reaction. Translation took place in the presence of 2 μl of 10 μCi/ml [35S]methionine. The labeled TnT proteins and the GST-fusion proteins were incubated together in binding buffer (20 mm Tris-HCl, pH 8.0, 100 mm NaCl, 1 mm EDTA, pH 8.0, and 0.1% Nonidet P-40) overnight and washed six times in the same buffer before analysis by SDS-PAGE and autoradiography. To discover the function of Mirk, we used the yeast two-hybrid assay to find Mirk-binding proteins. We isolated a novel gene of the DCoH (11Mendel D. Khavari P. Conley P. Graves M. Hansen L. Admon A. Crabtree G. Science. 1991; 254: 1762-1767Crossref PubMed Scopus (189) Google Scholar) family, DCoHm, by yeast two-hybrid analysis using full-length Mirk as bait. Yeast two-hybrid could be used because Mirk alone does not transactivate in the yeast two-hybrid assay. 3.5 × 106 clones of a human skeletal muscle cDNA library were screened to obtain 17 candidate genes, one of which wasDCoHm. HNF1α is a transcription factor found in endoderm-derived tissues, including kidney, liver, intestine and pancreas, where it may function in maintaining the differentiated phenotype (12Erickson R. Lai R. Kim Y. Biochem. Biophys. Res. Commun. 2000; 270: 235-239Crossref PubMed Scopus (27) Google Scholar). HNF1α is also a tissue-specific transcription factor regulating glucose metabolism-related genes in the liver (11Mendel D. Khavari P. Conley P. Graves M. Hansen L. Admon A. Crabtree G. Science. 1991; 254: 1762-1767Crossref PubMed Scopus (189) Google Scholar), insulin and insulin-like growth factor 1 (IGF-1) (13Nolten L. Steenberg P. Sussenbach J. Mol. Endocrinol. 1995; 9: 1488-1499Crossref PubMed Google Scholar) in the pancreatic-beta cells, and the protooncogene c-Src in the intestine (14Bonham K. Ritchie S. Dehm S. Snyder K. Boyd F. J. Biol. Chem. 2000; 275: 37604-37611Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). DCoH binds as a dimer to the unstable HNF1α dimer and enables effective binding of the tetrameric complex to DNA (15Rhee K. Steir G. Becker P. Suck D. Sandaltzopoulos J. Mol. Biol. 1997; 265: 20-29Crossref PubMed Scopus (34) Google Scholar). DCoH enhances HNF1α transcription activity 2–3-fold by stabilizing this complex (11Mendel D. Khavari P. Conley P. Graves M. Hansen L. Admon A. Crabtree G. Science. 1991; 254: 1762-1767Crossref PubMed Scopus (189) Google Scholar). The DCoH family is highly conserved. DCoHm has 78 and 88% amino acid identity to human and chicken DCoH, respectively (Fig.1). DCoHm was considered likely to have activity similar to other members of the DCoH family, because it retained the amino acids necessary for transcriptional activation of HNF1α, His-62, His-63, and His-80 as well as amino acid Phe-67, which mediates binding to HNF1α (16Johnen G. Kaufman S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13469-13474Crossref PubMed Scopus (24) Google Scholar). The interaction of Mirk and DCoHm was confirmed in yeast AH109 cells by a 1.5–2-fold activation of a β-galactosidase reporter gene localized downstream of DCoHm (data not shown). Protein interaction was also demonstrated by co-immunoprecipitation of in vitro 35S-labeled DCoHm with Mirk using antibody to the unique C terminus of Mirk (Fig. 2) with preimmune serum (P) used in the control. Thelast lane shows 30% of input DCoHm. The physical association between DCoHm and Mirk shown by co-immunoprecipitation confirms the binding between DCoHmand Mirk in the yeast two-hybrid analysis.Figure 2DCoHm is found associated with immunoprecipitated Mirk. Both Mirk and DCoHm (Dm) were [35S]methionine-labeled during in vitrocoupled transcription and translation, immunoprecipitated with the anti-peptide antibody to Mirk, and the immunoprecipitates were resolved by SDS-PAGE and autoradiography. P, preimmune serum used in control immunoprecipitation. One of two replicate experiments is shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because DCoH functions to stabilize HNF1α and increase its transcriptional capacity, we next tested whether Mirk altered the activity of HNF1α. HNF1α has been shown to increase by 60-fold the activity of the reporter construct (β-28)3-Luc, which consists of three copies of the HNF1α binding element, whereas DCoH further enhanced this activation about 2-fold (16Johnen G. Kaufman S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13469-13474Crossref PubMed Scopus (24) Google Scholar). We assayed for the effects of Mirk on HNF1α transcriptional activation of (β-28)3-Luc in transient transfection assays in NIH3T3 cells, which exhibit low levels of endogenous DCoH. DCoH alone, Mirk alone, or DCoH plus Mirk were unable to activate (β-28)3-Luc. In contrast, the expression of HNF1α alone was enough to activate the reporter construct 20-fold, and co-expression of HNF1α and DCoH activated the reporter 30-fold (Fig. 3 A,first six lanes). Increasing amounts of Mirk were co-transfected with expression plasmids for HNF1α and DCoH, which resulted in a dose-dependent activation of HNF1α, up to five times the activation induced by HNF1α and DCoH and 7.5 times the activation by HNF1α alone (Fig. 3 A,last four lanes). Therefore, Mirk can substantially increase the transcriptional activity of HNF1α and may function as a co-activator of HNF1α in vivo. What is its significance? Mirk/Dyrk1B was cloned from colon carcinoma cells, where it is likely to interact with DCoH in vivo. Immunohistochemistry studies demonstrated that DCoH is expressed in each of 20 colon carcinomas independent of Dukes' stage and in each of two colon carcinoma cell lines but not in normal tissue (17Eskinazi R. Thony B. Svoboda M. Robberecht P. Dassesse D. Heizmann C. Van Laethem J.-L. Resibois A. Am. J. Pathol. 1999; 155: 1105-1113Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar), so the presence of DCoH selectively in tumor tissue may allow Mirk through DCoH to activate HNF1α in cancer cells. We next tested whether Mirk might be able to activate HNF1α without DCoH. To determine whether DCoH could potentiate the co-activating effect of Mirk, HNF1α was transfected at a suboptimal level, one-half of that used in the previous experiment. A 50-fold range of Mirk concentrations was co-transfected with HNF1α with or without DCoH (Fig. 3 B). Mirk was more effective as a co-activator of HNF1α in the presence of DCoH. The highest Mirk concentration tested induced over twice as much reporter gene activity in the presence of DCoH as in its absence. Mirk, like DCoH and HNF1α, forms dimers (data not shown). These data together suggest that HNF1α, DCoH, and Mirk could act as a heterohexamer, with dimers of DCoH stabilizing the HNF1α dimer and serving as an attachment factor for dimerized Mirk. Mirk is a serine/threonine protein kinase activated by autophosphorylation on tyrosine (1Lee K. Deng X. Friedman E. Cancer Res. 2000; 60: 3631-3637PubMed Google Scholar). Was the capacity to phosphorylate HNF1α necessary for the activation of this transcription factor by Mirk? Wild-type Mirk increased HNF1α activation 3-fold when co-transfected with DCoH and HNF1α, whereas the co-transfection of two Mirk kinase-inactive mutant constructs, Mirk-YF and Mirk-KR (1Lee K. Deng X. Friedman E. Cancer Res. 2000; 60: 3631-3637PubMed Google Scholar), had no effect (Fig. 4 A), demonstrating that the kinase activity of Mirk is essential for its ability to increase HNF1α function. Further mutational analysis of Mirk was performed. Mirk is composed of a central conserved kinase domain flanked by non-conserved N-terminal and C-terminal sequences (1Lee K. Deng X. Friedman E. Cancer Res. 2000; 60: 3631-3637PubMed Google Scholar). Mirk deletion constructs were made; ΔNMirk consists of amino acids 110–629, and ΔCMirk consists of amino acids 1–435. The deletion of the N-terminal region of Mirk completely suppressed the activation of HNF1α by Mirk, whereas the deletion of the Mirk C terminus had little effect (Fig. 4 A). We next tested whether a double mutant, ΔN-KR, would act as a dominant negative, but the double mutant did not completely suppress HNF1α activity and simply maintained it at background levels. These co-transfection experiments (Fig. 4 A) had included DCoH. We next compared the effect of wild-type and mutant Mirk on HNF1α activation in the absence of DCoH. Wild-type Mirk enhanced HNF1α transcriptional activation in a dose-dependent manner in the absence of DCoH, whereas kinase-inactive YF-Mirk and the Mirk deletion mutant ΔNMirk had no activity (Fig. 4 B). These data indicate that Mirk kinase activity was essential for Mirk function as a transcriptional co-activator of HNF1. Transcription factors are often activated through a kinase cascade such as the well known MAP kinase superfamily. Mirk was shown to be a substrate of extracellular signal-related kinase 2 (ERK2), c-Jun NH2-terminal kinase (JNK1), and p38 in vitro (1Lee K. Deng X. Friedman E. Cancer Res. 2000; 60: 3631-3637PubMed Google Scholar), suggesting that one or more MAP kinases might activate Mirk in vivo, allowing Mirk to then activate HNF1α. We screened a series of MAP kinase kinases and their activators for their ability to substitute for Mirk in HNF1α activation using the β-fibrinogen promoter reporter construct (β-28)3-Luc in transient transfection experiments. The MAP kinase kinase MKK3, an activator of p38 (18Lin A. Minden A. Martinetto H. Claret F.-X. Lange-Carter C. Mercurio F. Johnson G. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (714) Google Scholar, 19Cuenda A. Alonso G. Morrice N. Jones M. Meier R. Cohen P. Nebreda A. EMBO J. 1996; 15: 4156-4164Crossref PubMed Scopus (115) Google Scholar, 20Derijard B. Raingeaud J. Barret T.T., Wu, I. Han J. Ulevitch R. Davis R. Science. 1995; 267: 682-685Crossref PubMed Scopus (1415) Google Scholar), was capable of activating HNF1α (Fig. 5). The constitutively activated form of MKK3, doubly mutated at its activation domain MKK3E (S189E/T193E) (21Raingeaud J. Whitmarsh A. Barrett T. Derijard B. Davis R. Mol. Cell. Biol. 1996; 16: 1247-1255Crossref PubMed Scopus (1150) Google Scholar), was used in place of MKK3 because it activates its downstream kinase targets without itself requiring activation by upstream signals. Both Mirk and MKK3E activated HNF1α 3–5-fold. When Mirk and MKK3E were added together, they induced a synergistic increase of 15-fold HNF1α activation. Activation of HNF1α as a transcription factor, potentiated by MKK3E, also required the same Mirk functions as described earlier (Fig. 4), i.e.Mirk kinase activity, the nonconserved N terminus of Mirk, but not the nonconserved C terminus of Mirk. Wild-type Mirk could not be replaced by either kinase-inactive Mirk or ΔNMirk but could be replaced to some extent by ΔCMirk (Fig. 5). MKK3E is a potent activator of p38 (21Raingeaud J. Whitmarsh A. Barrett T. Derijard B. Davis R. Mol. Cell. Biol. 1996; 16: 1247-1255Crossref PubMed Scopus (1150) Google Scholar). These data suggested three models. 1) Mirk and MKK3E activated HNF1α independently. 2) MKK3E directly activated Mirk by phosphorylation. 3) MKK3E activated p38, which in turn phosphorylated and activated Mirk. To test this hypothesis, we initially determined whether Mirk and MKK3 interacted directly by co-immunoprecipitation experiments. 293T cells were co-transfected with either MKK3 or MKK3E together with either wild-type Mirk, kinase-inactive YF-Mirk, or epitope-tagged vector, and Mirk was immunoprecipitated after expression (Fig.6 A). A distinct band of MKK3E was observed in both the immunoprecipitates of wild-type Mirk and kinase-inactive Mirk. Much less (6–7%) wild-type MKK3 associated with Mirk, although similar amounts of MKK3E, MKK3, and Mirk were synthesized in the lysates (Western blot of total lysates shown inlower two panels, Fig. 6 A). Therefore, MKK3E associated directly with Mirk, and any effect MKK3E exerted on HNF1α could be mediated through exogenous or endogenous Mirk (Fig. 5, lanes 2 and 3, respectively). Because MKK3 is a potent activator of p38 (21Raingeaud J. Whitmarsh A. Barrett T. Derijard B. Davis R. Mol. Cell. Biol. 1996; 16: 1247-1255Crossref PubMed Scopus (1150) Google Scholar), it was also possible that MKK3 activates Mirk. MKK3E was expressed in 293T cells, immunoprecipitated, and added to in vitro kinase reaction mixtures containing recombinant purified Mirk or recombinant purified p38, as indicated, with [γ-32P]-ATP and MBP as substrates. The reaction mixtures containing [γ-32P]-ATP were analyzed by SDS-PAGE and autoradiography (Fig. 6 B). Mirk phosphorylation of MBP was increased ∼50% in the presence of MKK3E, although MKK3E itself did not phosphorylate MBP. As a control, MKK3E was shown to increase the activity of a small concentration of p38 as an MBP kinase ∼50% (Fig.6 B, last two lanes). Therefore, MKK3 binds to Mirk and increases Mirk kinase activity. Because MKK3 mediates various environmental stress signals, it is possible that MKK3 increases the function of Mirk as a transcriptional activator of HNF1α in response to certain stresses. Stably overexpressed Mirk enables cells to proliferate and remain viable in serum-free conditions (1Lee K. Deng X. Friedman E. Cancer Res. 2000; 60: 3631-3637PubMed Google Scholar), possibly because Mirk activates the transcription of survival factors such as insulin-like growth factor 1 through the activation of HNF1α. We next determined the regions of Mirk and HNF1α necessary for their interaction. Glutathione S-transferase pull-down assays confirmed a three-part association betweenDCoHm, Mirk, and HNF1 (Fig. 7 A, lanes 4–6). Both DCoHm and Mirk were 35S-labeled by coupled transcription and translation reactions, incubated with either GST-HNF1α or GST coupled to beads, and the bead eluates were examined by PAGE followed by autoradiography. DCoHm and Mirk, when added individually or added together, bound to HNF1α (Fig.7 A). DCoHm bound more strongly to HNF1α than did Mirk when the bound fraction to total input was compared. There was no additive effect of DCoHm and Mirk in multiple experiments. Neither DCoHm or HNF1α bound to GST. Next, a series of deletion mutants of HNF1α were generated, and GST-pull down assays were repeated with 35S-labeled Mirk (Fig. 7 B). Mirk bound to HNF1α fragments encompassing amino acids 1–203 and 1–283 but not to fragments of the N terminus (amino acids 1–111) or C-terminal regions (amino acids 283–481 and 481 to 628). Therefore, the minimal region of Mirk/HNF1α binding encompassed HNF1α amino acids 112–203. This region was similar to the minimal region for HNF1α interaction with the co-activator CBP (22Soutoglou E. Papafotiou G. Katrakili N. Talianidis I. J. Biol. Chem. 2000; 275: 12515-12520Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), amino acids 95–295. We next determined which region of Mirk was necessary for binding to HNF1α by performing GST pull-down assays with deletion constructs of Mirk (Fig.8). The constructs consisted of ΔNMirk (amino acids 110–629) cleaved of the nonconserved N terminus; ΔN2Mirk (amino acids 233–629) cleaved of the nonconserved N terminus and the first five subdomains of the conserved kinase domain and the ATP binding site K140; Δ233–435, a partial kinase-domain deletion of amino acids 233–435 that retained the N and C termini; ΔCMirk (amino acids 1–435 with the nonconserved C terminus deleted); and C, an unrelated control protein p53. There were equivalent amounts of Mirk deletion products added to the GST pull-downs (Fig. 8, lower panel). However, ΔN2 Mirk bound very poorly to GST-HNF1α-(1–203) compared with the other deletion constructs and to full-length Mirk (Fig. 8, lane 1). Therefore, the Mirk region of amino acids 111–233 encompassing the first five conserved kinase subdomains was essential for Mirk binding to HNF1α. A direct interaction between Mirk and HNF1α occurred in the absence of DCoHm, shown in the GST pull-down assays (Fig. 7 A, lane 5). These results suggested that DCoHm simply facilitated Mirk association with HNF1α and that HNF1α might be the immediate target of Mirk. This was shown to be the case, because Mirk phosphorylated GST-HNF1α in vitro in the absence of DCoHm while exhibiting no kinase activity on GST itself (Fig. 9 A) or on DCoHm (data not shown). Therefore, Mirk binds to DCoHm in the DCoHm/HNF1α tetramer and is thus able to directly bind and then phosphorylate HNF1α. There was no canonical arginine-directed Dyrk1A phosphorylation site (4Himpel S. Tegge W. Frank R. Leder S. Joost H. Becker W. J. Biol. Chem. 2000; 275: 2431-2438Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar) within the region of HNF1α that binds to Mirk (HNF1α, amino acids 112–203). However, a perfect Dyrk1A phosphorylation site was seen at Ser-249, which is still within the CBP binding domain (22Soutoglou E. Papafotiou G. Katrakili N. Talianidis I. J. Biol. Chem. 2000; 275: 12515-12520Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Mutation of this site decreased phosphorylation by Mirk 2.5-fold (Fig. 9 B). Mirk binds to a region within the N terminus of HNF1α and then phosphorylates an amino acid just distal to the binding region but still within the CBP binding domain. Additional studies will determine whether phosphorylation at this site increases CBP binding and HNF1α activation in vivo. Mirk/Dyrk1B, like its family member Dyrk1A, is now shown to function as a transcription factor activator. There is strong evidence that Dyrk1A functions in neurogenesis and brain development (6Guimera J. Casas C. Estivill X. Pritchard M. Genomics. 1999; 57: 407-418Crossref PubMed Scopus (145) Google Scholar, 7Altafaj X. Dierssen M. Baamonde C. Marti E. Visa J. Guimera J. Oset M. Gonzalez J. Florez J. Fillat C. Estivill X. Hum. Mol. Genet. 2001; 10: 1915-1923Crossref PubMed Scopus (328) Google Scholar, 23Tejedor F. Zhu X. Kaltenbach E. Ackermann A. Baumann A. Canal I. Heisenberg M. Fischbach K. Pongs O. Neuron. 1995; 14: 287-301Abstract Full Text PDF PubMed Scopus (313) Google Scholar,24Song W.-J. Sternberg L. Kasten-Sportes C. Van Keuren M. Chung S.-H. Slack A. Miller D. Glover T. Chiang P.-W. Lou L. Kurnit D. Genomics. 1996; 38: 331-339Crossref PubMed Scopus (149) Google Scholar). Dyrk1A activity was induced during the differentiation of immortalized hippocampal progenitor cells, and the addition of the neurogenic factor basic fibroblast growth factor to these cells resulted in the specific binding of Dyrk1A to CREB, the phosphorylation of CREB by Dyrk1A, and the stimulation of CRE-mediated gene transcription (8Yang E. Ahn Y.S. Chung K. J. Biol. Chem. 2001; 276: 39819-39824Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Mirk/Dyrk1B is also expressed in the brain, but its highest expression is in skeletal muscle (1Lee K. Deng X. Friedman E. Cancer Res. 2000; 60: 3631-3637PubMed Google Scholar). Screening a normal skeletal muscle cDNA library led us to discover that Mirk binds to a novel member of the highly conserved DCoH family, DCoHm. DCoH binds as a dimer to the unstable HNF1α dimer and enables effective binding of the tetrameric complex to DNA (15Rhee K. Steir G. Becker P. Suck D. Sandaltzopoulos J. Mol. Biol. 1997; 265: 20-29Crossref PubMed Scopus (34) Google Scholar). DCoH enhances HNF1α transcription activity 2–3-fold by stabilizing this complex (11Mendel D. Khavari P. Conley P. Graves M. Hansen L. Admon A. Crabtree G. Science. 1991; 254: 1762-1767Crossref PubMed Scopus (189) Google Scholar), and Mirk increased this activity a further 5-fold. The interaction between DCoHm and Mirk was confirmed by co-immunoprecipitation studies and GST-pull down assays. Mirk was shown to substantially increase the transcription activity of HNF1α in transient transfection assays and may function as a co-activator of HNF1α in vivo. Mirk did not require DCoH to active HNF1α. Mirk, through the N terminus of its conserved kinase domain, bound to HNF1α at a site within its CBP binding domain and then directly phosphorylated HNF1α adjacent to the binding site but still within the CBP binding domain. Mirk kinase activity was required for transcriptional activation of HNF1α, because kinase-inactive mutants were also unable to activate HNF1α. HNF1α mediates the transcription of several genes that potentially could contribute to the differentiation and growth of normal muscle cells through Mirk interaction with DCoHm. However, Mirk is expressed in several cell lines established from solid tumors, including colon, lung, and ovarian, with much less expression in leukemia and lymphoma-derived cell lines (1Lee K. Deng X. Friedman E. Cancer Res. 2000; 60: 3631-3637PubMed Google Scholar). DCoH and Mirk are both expressed in colon carcinomas. DCoH was not found in normal colon tissue but was found in every colon carcinoma examined by immunohistochemistry (17Eskinazi R. Thony B. Svoboda M. Robberecht P. Dassesse D. Heizmann C. Van Laethem J.-L. Resibois A. Am. J. Pathol. 1999; 155: 1105-1113Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar), and Mirk was found in each of seven colon carcinoma cell lines (1Lee K. Deng X. Friedman E. Cancer Res. 2000; 60: 3631-3637PubMed Google Scholar). Mirk, DCoH, and HNF1α were found to form a complex in vitro, thus Mirk may complex with the DCoH/HNF1α tetramerin vivo. Therefore, in many colon carcinomas Mirk and DCoH are co-expressed and may function as an activating complex for HNF1α to induce ectopic gene expression. The expression of some of these genes may contribute to the ability of cell lines with stably overexpressed Mirk to maintain serum-free proliferation (1Lee K. Deng X. Friedman E. Cancer Res. 2000; 60: 3631-3637PubMed Google Scholar). Recombinant Mirk is a constitutively active kinase. However, its kinase activity on MBP and its transcriptional activation of HNF1α was enhanced by MKK3, an upstream activator of the stress-activated MAP kinase p38 (21Raingeaud J. Whitmarsh A. Barrett T. Derijard B. Davis R. Mol. Cell. Biol. 1996; 16: 1247-1255Crossref PubMed Scopus (1150) Google Scholar). MKK3 also directly bound to Mirk in vivo as demonstrated in co-immunoprecipitation experiments. MKK3 is activated by phosphorylation at Ser-189 and Thr-193 within a P-activation loop of its conserved kinase subdomain VIII (21Raingeaud J. Whitmarsh A. Barrett T. Derijard B. Davis R. Mol. Cell. Biol. 1996; 16: 1247-1255Crossref PubMed Scopus (1150) Google Scholar) by upstream kinases in response to stress agents. p38, in turn, is activated by dual phosphorylation at threonine and tyrosine within the TPY motif in conserved kinase subdomain VIII (25Han J. Lee J. Bibbs L. Ulevitch R. Science. 1994; 265: 808-811Crossref PubMed Scopus (2420) Google Scholar, 26Lee J. Laydon J. McDonnell P. Gallagher T. Kumar S. Green D. McNulty D. Blumenthal M. Heys J. Landvatter S. Strickler M. McLaughlin I. Siemens I. Fisher S. Livi G. White J. Adams J. Young P. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3147) Google Scholar, 27Rouse J. Cohen P. Trigon S. Morange M. Alonso-Llamazares A. Zamanillo D. Hunt T. Nebreda A. Cell. 1994; 78: 1027-1037Abstract Full Text PDF PubMed Scopus (1507) Google Scholar). However, Mirk/Dyrk1B's activation domain is YQY, which is at a position located within conserved subdomains VII and VIII of the catalytic domain homologous to the p38 activation domain (1Lee K. Deng X. Friedman E. Cancer Res. 2000; 60: 3631-3637PubMed Google Scholar). Recent studies of the related kinase Dyrk1A have shown that only the second tyrosine in the activation domain, Tyr-321 and not Tyr-319, was autophosphorylated, and the mutation of Tyr-319 to Phe failed to reduce kinase activity (28Himpel S. Panzer P. Eirmbter K. Czajkowska H. Sayed M. Packman L. Blundell T. Kentrup H. Grotzinger J. Joost H. Becker W. Biochem. J. 2001; 359: 497-505Crossref PubMed Scopus (139) Google Scholar). MKK3 possibly activates Mirk by phosphorylating it at the second tyrosine in its activation domain. Autophosphorylated tyrosine residues were also found in the N terminus of Dyrk1A, and deletion of the N terminus suppressed kinase activity (28Himpel S. Panzer P. Eirmbter K. Czajkowska H. Sayed M. Packman L. Blundell T. Kentrup H. Grotzinger J. Joost H. Becker W. Biochem. J. 2001; 359: 497-505Crossref PubMed Scopus (139) Google Scholar) as deletion of the N terminus of Mirk suppressed its transactivator activity (Fig. 4). Dyrk1A is an arginine-directed kinase (4Himpel S. Tegge W. Frank R. Leder S. Joost H. Becker W. J. Biol. Chem. 2000; 275: 2431-2438Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar), and comparison of the Dyrk1A and ERK2 catalytic cores revealed that Tyr-321 of Dyrk1A can interact with Arg-325 and Arg-328 (28Himpel S. Panzer P. Eirmbter K. Czajkowska H. Sayed M. Packman L. Blundell T. Kentrup H. Grotzinger J. Joost H. Becker W. Biochem. J. 2001; 359: 497-505Crossref PubMed Scopus (139) Google Scholar). There are also two comparable arginine residues upstream of the YQY activation motif of Mirk. Although Dyrk1A and Mirk/Dyrk1B can autophosphorylate in bacteria, greater phosphorylation was seen when both proteins were expressed in mammalian cells. MKK3 binding sites and phosphorylation sites on Mirk must be mapped to uncover the precise relationship between MKK3 and Mirk.