|
[PMID]: | 20484411 |
[Au] Autor: | Sierra OL; Towler DA |
[Ad] Endereço: | 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 |
[Ti] Título: | Runx2 trans-activation mediated by the MSX2-interacting nuclear target requires homeodomain interacting protein kinase-3. |
[So] Source: | Mol Endocrinol;24(7):1478-97, 2010 Jul. | [Is] ISSN: | 1944-9917 |
[Cp] País de publicação: | United States |
[La] Idioma: | eng |
[Ab] Resumo: | 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. |
[Mh] Termos MeSH primário: |
Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo Proteínas Nucleares/metabolismo Proteínas Serina-Treonina Quinases/metabolismo
|
[Mh] Termos MeSH secundário: |
Animais Benzimidazóis/farmacologia Linhagem Celular Subunidade alfa 1 de Fator de Ligação ao Core/genética Inativação Gênica Imunoprecipitação Camundongos Microscopia Confocal Proteínas Nucleares/genética Fosforilação Proteínas Quinases Direcionadas a Prolina/antagonistas & inibidores Proteínas Quinases Direcionadas a Prolina/genética Proteínas Quinases Direcionadas a Prolina/metabolismo Ligação Proteica Estrutura Terciária de Proteína/genética Estrutura Terciária de Proteína/fisiologia Proteínas Serina-Treonina Quinases/genética RNA Antissenso RNA Interferente Pequeno Ativação Transcricional
|
[Pt] Tipo de publicação: | JOURNAL ARTICLE; RESEARCH SUPPORT, N.I.H., EXTRAMURAL; RESEARCH SUPPORT, NON-U.S. GOV'T |
[Nm] Nome de substância:
| 0 (2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole); 0 (Benzimidazoles); 0 (Core Binding Factor Alpha 1 Subunit); 0 (Nuclear Proteins); 0 (RNA, Antisense); 0 (RNA, Small Interfering); 0 (Spen protein, mouse); EC 2.7.11.- (Proline-Directed Protein Kinases); EC 2.7.11.1 (Protein-Serine-Threonine Kinases) |
[Em] Mês de entrada: | 1009 |
[Cu] Atualização por classe: | 161019 |
[Lr] Data última revisão:
| 161019 |
[Sb] Subgrupo de revista: | IM |
[Da] Data de entrada para processamento: | 100521 |
[St] Status: | MEDLINE |
[do] DOI: | 10.1210/me.2010-0029 |
|
|
|