title: The transcriptional activator Mirk/Dyrk1B is sequestered by p38alpha/beta MAP kinase.
abstract: Mirk/Dyrk1B protein kinase was shown in an earlier study to function as a transcriptional activator of HNF1alpha, which Mirk phosphorylates at Ser(249) within its CREB (cAMP-response element-binding protein)-binding protein (CBP) binding domain (). The MAPK kinase MKK3 was also shown to activate Mirk as a protein kinase, implicating Mirk in the biological response to certain stress agents. Another MKK3 substrate, p38MAPK, is now shown to inhibit the function of Mirk as a transcriptional activator in a kinase-independent manner. Co-immunoprecipitation experiments demonstrated that kinase-inactive p38AF, as well as wild-type p38, sequestered Mirk and prevented its association with MKK3. Only the p38alpha and p38beta isoforms, but not the gamma or delta isoforms, complexed with Mirk. p38alphaMAPK blocked Mirk activation of HNF1alpha in a dose-dependent manner, with high levels of kinase-inactive p38alphaAF completely suppressing the activity of Mirk. Size fractionation by fast protein liquid chromatography on Superdex 200 demonstrated that Mirk is not found as a monomer in vivo, but is found within 150-700 kDa subnuclear complexes, which co-migrate with the nuclear body scaffolding protein PML. Endogenous Mirk, p38, and MKK3 co-migrate within 500-700-kDa protein complexes, which accumulate when nuclear export is blocked by leptomycin B. Stable overexpression of Mirk increases the fraction of Mirk protein and p38 protein within these 500-700 kDa complexes, suggesting that the complexes act as nuclear depots for Mirk and p38. Sequestration of Mirk by p38 may occur within these subnuclear complexes. Synchronization experiments demonstrated that Mirk levels fluctuate about 10-fold within the cell cycle, while p38 levels do not, leading to the speculation that endogenous p38 could only block Mirk function when Mirk levels were low in S phase and not when Mirk levels were elevated in G(0)/G(1). These data suggest a novel cell cycle-dependent function for p38, suppression of the function of Mirk as a transcriptional activator only when cells are proliferating, and thus limiting Mirk function to growth-arrested cells.
authors.affiliation: Pathology Department, Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210, USA.
authors.forename: Seunghwan
authors.initials: S
authors.lastname: Lim
authors.forename: Yonglong
authors.initials: Y
authors.lastname: Zou
authors.forename: Eileen
authors.initials: E
authors.lastname: Friedman
mesh_terms: D016475:3T3 Cells; D000818:Animals; D015153:Blotting, Western; D002455:Cell Division; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002853:Chromatography, Liquid; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D004789:Enzyme Activation; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D017353:Gene Deletion; D006801:Humans; D048371:MAP Kinase Kinase 3; D051379:Mice; D048290:Mitogen-Activated Protein Kinase 11; D048308:Mitogen-Activated Protein Kinase 14; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D020033:Protein Isoforms; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D013997:Time Factors; D014157:Transcription Factors; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured; D000097603:Dyrk Kinases
chemical_list: D016895:Culture Media, Serum-Free; D005231:Fatty Acids, Unsaturated; D048371:MAP Kinase Kinase 3; D048290:Mitogen-Activated Protein Kinase 11; D048308:Mitogen-Activated Protein Kinase 14; D020929:Mitogen-Activated Protein Kinase Kinases; D020928:Mitogen-Activated Protein Kinases; D020033:Protein Isoforms; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D014157:Transcription Factors; D000097603:Dyrk Kinases; C482530:MAP2K3 protein, human; C482531:Map2k3 protein, mouse; C038753:leptomycin B