Physical and Functional Interactions between the Prostate Suppressor Homeoprotein NKX3.1 and Serum Response Factor Jeong Ho Ju 1 , Jin-Soo Maeng 2 , Micheas Zemedkun 1 Natalie Ahronovitz 1 , James W. Mack 2 , James A. Ferretti 3 Edward P. Gelmann 1 ⁎ and James M. Gruschus 3 1 Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, 3800 Reservoir Road, NW, Washington, DC 20007, USA 2 Department of Biochemistry and Molecular Biology , College of Medicine, Howard University , 520 W Street, NW, Washington DC 20059, USA 3 Laboratory of Biophysical Chemistry, National Heart, Lung, and Blood Institute, NIH Bldg 50, Room 3513, Bethesda, MD 20892, USA 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 γ-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 home- odomain 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. © 2006 Elsevier Ltd. All rights reserved. *Corresponding author Keywords: NKX3.1; serum response factor; homeodomain; prostate cancer; transcription Introduction Homeobox genes code for a class of transcrip- tional factors that regulate the expression of target genes in a time and space-dependent manner. Homeodomain proteins specify the body plan and regulate development of higher organisms. These proteins share a conserved 60 amino acid home- odomain that forms two parallel and a third perpendicular α-helix that binds in the major groove of the cognate hexanucleotide DNA recog- nition sequences. 1 This class of proteins was first recognized in Drosophila and are now known to exist in all eukaryotes where they perform impor- tant functions during development. 2 In Drosophila the NK homeobox gene family was initially shown to have four members (nk-1 through nk-4) that have Abbreviations used: SRF, serum response factor; SMGA, smooth muscle γ-actin; TN, tinman; EH-1, engrailed homology-1 motif; AD, acidic domain; SI, SRF-interacting motif; CSI, chemical shift index; HSQC, heteronuclear single quantum coherence; NOE, nuclear Overhauser enhancement; NOESY, NOE spectroscopy. E-mail address of the corresponding author: gelmanne@georgetown.edu doi:10.1016/j.jmb.2006.05.064 J. Mol. Biol. (2006) 360, 989–999 0022-2836/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.