Interactions of the acidic domain and SRF interacting motifs with the NKX3.1 homeodomain.
NKX3.1 is a prostate tumor suppressor belonging to the NK-2 family of homeodomain (HD) transcription factors. NK-2 family members often possess a stretch of 10-15 residues enriched in acidic amino acids, the acidic domain (AD), in the flexible, disordered region N-terminal to the HD. Interactions between the N-terminal region of NKX3.1 and its homeodomain affect protein stability and DNA binding. CD spectroscopy measuring the thermal unfolding of NKX3.1 constructs showed a 2 degrees C intramolecular stabilization of the HD by the N-terminal region containing the acidic domain (residues 85-96). CD of mixtures of various N-terminal peptides with a construct containing just the HD showed that the acidic domain and the following region, the SRF interacting (SI) motif (residues 99-105), was necessary for this stabilization. Phosphorylation of the acidic domain is known to slow proteasomal degradation of NKX3.1 in prostate cells, and NMR spectroscopy was used to measure and map the interaction of the HD with phosphorylated and nonphosphorylated forms of the AD peptide. The interaction with the phosphorylated AD peptide was considerably stronger (K(d) = 0.5 +/- 0.2 mM), resulting in large chemical shift perturbations for residues Ser150 and Arg175 in the HD, as well as a 2 degrees C increase in the HD thermal stability compared to that of the nonphosphorylated form. NKX3.1 constructs with AD phosphorylation site threonine residues (89 and 93) mutated to glutamate were 4 degrees C more stable than HD alone. Using polymer theory, effective concentrations for interactions between domains connected by flexible linkers are predicted to be in the millimolar range, and thus, the weak intramolecular interactions observed here could conceivably modulate or compete with stronger, intermolecular interactions with the NKX3.1 HD.
Division of Hematology/Oncology, Herbert Irving Comprehensive Cancer Center, Columbia University, New York, New York 10032, USA.
Jeong Ho
JH
Ju
Jin-Soo
JS
Maeng
Duck-Yeon
DY
Lee
Grzegorz
G
Piszczek
Edward P
EP
Gelmann
James M
JM
Gruschus
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D002942:Circular Dichroism; D016147:Genes, Tumor Suppressor; D018398:Homeodomain Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010766:Phosphorylation; D011485:Protein Binding; D017510:Protein Folding; D026362:Serum Response Factor / Q000737:chemistry / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*
D018398:Homeodomain Proteins; C491178:NKX3-1 protein, human; C476858:SRF protein, human; D026362:Serum Response Factor; D014157:Transcription Factors