RESUMO
The NKX3.1 transcription factor is an NK family homeodomain protein and a tumor suppressor gene that is haploinsufficient and down-regulated in the early phases of prostate cancer. Like its cardiac homolog, NKX2.5, NKX3.1 acts synergistically with serum response factor (SRF) to activate expression from the smooth muscle gamma-actin (SMGA) gene promoter. Using NMR spectroscopy, three conserved motifs in a construct containing the N-terminal region and homeodomain of NKX3.1 were observed to interact with the MADS box domain of SRF. These motifs interacted both in the absence of DNA and when both proteins were bound to a SMGA promoter DNA sequence. No significant interaction was seen between the homeodomain and SRF MADS box. One of the SRF-interacting regions was the tinman (TN) or engrailed homology-1 motif (EH-1), residues 29-35 (FLIQDIL), which for other NK proteins is the site of interaction with the repressor protein Groucho. A second hydrophobic interacting region was designated the SRF-interacting (SI) motif and included residues 99-105 (LGSYLLD). A third interacting motif was the acidic region adjacent to the SI motif including residues 88-96 (ETLAETEPE). The acidic domain (AD) motif signals also showed strengthening upon the NKX3.1 homeodomain binding to DNA in the absence of SRF, consistent with the acidic region weakly interacting with the homeodomain in the unbound state. The importance of these linear motifs in the transcriptional interaction of NKX3.1 and SRF was demonstrated by targeted mutagenesis of an NKX3.1 expression vector in a SMGA reporter assay. The results implicate the NKX3.1 N-terminal region in regulation of transcriptional activity of this tumor suppressor.
Assuntos
Genes Supressores de Tumor , Proteínas de Homeodomínio/metabolismo , Modelos Moleculares , Próstata/metabolismo , Fator de Resposta Sérica/metabolismo , Fatores de Transcrição/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência Conservada , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Humanos , Interações Hidrofóbicas e Hidrofílicas , Masculino , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Ressonância Magnética Nuclear Biomolecular , Regiões Promotoras Genéticas , Ligação Proteica , Fator de Resposta Sérica/química , Fator de Resposta Sérica/genética , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
NKX3.1, a member of the NK class of homeodomain proteins, is expressed primarily in the adult prostate and has growth suppression and differentiating effects in prostate epithelial cells. A C-->T polymorphism at nucleotide 154 (NKX3.1 C154T) is present in approximately 11% of healthy men with equal distribution among whites and blacks. In a cohort of 1253 prostate cancer patients and age-matched controls, the presence of the polymorphism was associated with a 1.8-fold risk of having stage C or D prostate cancer or Gleason score > or =7 (confidence interval, 1.01-3.22). The NKX3.1 C154T polymorphism codes for a variant protein that contains an arginine-to-cysteine substitution at amino acid 52 (R52C) adjacent to a protein kinase C phosphorylation site at serine 48. Substitution of cysteine for arginine 52 or of alanine for serine 48 (S48A) reduced phosphorylation at serine 48 in vitro and in vivo. Phosphorylation of wild-type NKX3.1, but not of NKX3.1 R52C or NKX3.1 S48A, diminished binding in vitro to a high-affinity DNA binding sequence. NKX3.1 also serves as a transcriptional coactivator of serum response factor. Treatment of cells with 12-O-tetradecanoylphorbol-13-acetate to phosphorylate NKX3.1 had no effect on NKX3.1 coactivation of serum response factor. Neither the R52C nor the S48A substitution affected serum response factor coactivation by NKX3.1 We conclude that the polymorphic NKX3.1 allele codes for a variant protein with altered DNA binding activity that may affect prostate cancer risk.
