title: Runx2 trans-activation mediated by the MSX2-interacting nuclear target requires homeodomain interacting protein kinase-3.
abstract: Runt-related transcription factor 2 (Runx2) and muscle segment homeobox homolog 2-interacting nuclear target (MINT) (Spen homolog) are transcriptional regulators critical for mammalian development. MINT enhances Runx2 activation of osteocalcin (OC) fibroblast growth factor (FGF) response element in an FGF2-dependent fashion in C3H10T1/2 cells. Although the MINT N-terminal RNA recognition motif domain contributes, the muscle segment homeobox homolog 2-interacting domain is sufficient for Runx2 activation. Intriguingly, Runx1 cannot replace Runx2 in this assay. To better understand this Runx2 signaling cascade, we performed structure-function analysis of the Runx2-MINT trans-activation relationship. Systematic truncation and domain swapping in Runx1:Runx2 chimeras identified that the unique Runx2 activation domain 3 (AD3), encompassed by residues 316-421, conveys MINT+FGF2 trans-activation in transfection assays. Ala mutagenesis of Runx2 Ser/Thr residues identified that S301 and T326 in AD3 are necessary for full MINT+FGF2 trans-activation. Conversely, phosphomimetic Asp substitution of these AD3 Ser/Thr residues enhanced activation by MINT. Adjacent Pro residues implicated regulation by a proline-directed protein kinase (PDPK). Systematic screening with PDPK inhibitors identified that the casein kinase-2/homeodomain-interacting protein kinase (HIPK)/dual specificity tyrosine phosphorylation regulated kinase inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT), but not ERK, c-Jun N-terminal kinase, p38MAPK, or other casein kinase-2 inhibitors, abrogated Runx2-, MINT-, and FGF2-activation. Systematic small interfering RNA-mediated silencing of DMAT-inhibited PDPKs revealed that HIPK3 depletion reduced MINT+FGF2-dependent activation of Runx2. HIPK3 and Runx2 coprecipitate after in vitro transcription-translation, and recombinant HIPK3 recognizes Runx2 AD3 as kinase substrate. Furthermore, DMAT treatment and HIPK3 RNAi inhibited MINT+FGF2 activation of Runx2 AD3, and nuclear HIPK3 colocalized with MINT. HIPK3 antisense oligodeoxynucleotide selectively reduced Runx2 protein accumulation and OC gene expression in C3H10T1/2 cells. Thus, HIPK3 participates in MINT+FGF2 regulation of Runx2 AD3 activity and controls Runx2-dependent OC expression.
authors.affiliation: Washington University School of Medicine, Internal Medicine-Endocrinology/Metabolism, Campus Box 8301, 660 South Euclid Avenue, St. Louis, Missouri 63110, USA. towler@im.wustl.edu
authors.forename: Oscar L
authors.initials: OL
authors.lastname: Sierra
authors.forename: Dwight A
authors.initials: DA
authors.lastname: Towler
mesh_terms: D000818:Animals; D001562:Benzimidazoles / Q000494:pharmacology; D002460:Cell Line; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins; D020868:Gene Silencing; D047468:Immunoprecipitation; D051379:Mice; D018613:Microscopy, Confocal; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D038461:Proline-Directed Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D016372:RNA, Antisense; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D015533:Transcriptional Activation
chemical_list: C492322:2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole; D001562:Benzimidazoles; D050659:Core Binding Factor Alpha 1 Subunit; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D016372:RNA, Antisense; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; C121253:Spen protein, mouse; D038461:Proline-Directed Protein Kinases; D017346:Protein Serine-Threonine Kinases