Characterization of homo- and heterodimerization of cardiac Csx/Nkx2.5 homeoprotein.
Csx/Nkx2.5 is an evolutionarily conserved homeodomain (HD)-containing transcription factor that is essential for early cardiac development. We found that the HD of Csx/Nkx2.5 binds as a monomer as well as a dimer to its DNA binding sites in the promoter of the atrial natriuretic factor (ANF) gene, an in vivo target gene of Csx/Nkx2.5. Csx/Nkx2.5 physically interacts with each other in vitro as well as in cells, and the HD is critical for homodimerization. Lys(193) and Arg(194), located at the COOH-terminal end of HD, are essential for dimerization. Lys(193) is also required for a specific interaction with the zinc finger transcription factor GATA4. Csx/Nkx2.5 can heterodimerize with other NK2 homeodomain proteins, Nkx2.3 and Nkx2.6/Tix, with different affinities. A single missense mutation, Ile(183) to Pro in the HD of Csx/Nkx2.5, preserved homodimerization function, but totally abolished DNA binding. Ile(183) --> Pro mutant acts in an inhibitory manner on wild type Csx/Nkx2.5 transcriptional activity through the ANF promoter in 10T1/2 cells. However, Ile(183) --> Pro mutant does not inhibit wild type Csx/Nkx2.5 function on the ANF promoter in cultured neonatal cardiac myocytes, possibly due to failure of dimerization in the presence of the target DNA. These results suggest that complex protein-protein interactions of Csx/Nkx2.5 play a role in its transcriptional regulatory function.
Kasahara
H
H
Cardiovascular Division, Beth Israel Deaconess Medical Center, Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215, USA. hkasahar@caregroup.harvard.edu
Usheva
A
A
Ueyama
T
T
Aoki
H
H
Horikoshi
N
N
Izumo
S
S
D000818:Animals; D000831:Animals, Newborn; D001120:Arginine / Q000378:metabolism; D009320:Atrial Natriuretic Factor / Q000235:genetics; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D019281:Dimerization; D050991:GATA4 Transcription Factor; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D051381:Rats; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D029867:Xenopus Proteins
D004268:DNA-Binding Proteins; D050991:GATA4 Transcription Factor; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins; C095552:NKX2-5 protein, human; C484469:Nkx2-5 protein, mouse; C111285:Nkx2-6 protein, mouse; D014157:Transcription Factors; D029867:Xenopus Proteins; D009320:Atrial Natriuretic Factor; D004247:DNA; D001120:Arginine; D008239:Lysine