Abstract
LIM-domain only protein 4 (LMO4) is a widely expressed protein with important roles in embryonic development and breast cancer. It has been reported to bind many partners, including the transcription factor Deformed epidermal autoregulatory factor-1 (DEAF1), with which LMO4 shares many biological parallels. We used yeast two-hybrid assays to show that DEAF1 binds both LIM domains of LMO4 and that DEAF1 binds the same face on LMO4 as two other LMO4-binding partners, namely LIM domain binding protein 1 (LDB1) and C-terminal binding protein interacting protein (CtIP/RBBP8). Mutagenic screening analysed by the same method, indicates that the key residues in the interaction lie in LMO4LIM2 and the N-terminal half of the LMO4-binding domain in DEAF1. We generated a stable LMO4LIM2-DEAF1 complex and determined the solution structure of that complex. Although the LMO4-binding domain from DEAF1 is intrinsically disordered, it becomes structured on binding. The structure confirms that LDB1, CtIP and DEAF1 all bind to the same face on LMO4. LMO4 appears to form a hub in protein-protein interaction networks, linking numerous pathways within cells. Competitive binding for LMO4 therefore most likely provides a level of regulation between those different pathways.
Highlights
LIM-domain only protein 4 (LMO4) is a member of the LIM-only protein family (LMO1–4) of metazoan transcriptional co-regulators, and has important roles in neural and skeletal development [1,2,3]
An interaction between LMO4 and Deformed epidermal autoregulatory factor-1 (DEAF1) was only observed in the presence of both LIM domains and in the absence of LDB1LID (Fig. 1a)
These data suggest that both LIM domains are involved in binding DEAF1 and that the presence of LDB1LID prevents DEAF1 from binding to LMO4
Summary
LMO4 is a member of the LIM-only protein family (LMO1–4) of metazoan transcriptional co-regulators, and has important roles in neural and skeletal development [1,2,3]. It is overexpressed in greater than 50% of sporadic breast cancers and overexpression is correlated with a poor outcome [4,5]. LMO proteins are known to regulate gene transcription, they do not bind DNA directly Rather, they use their protein-interacting LIM (Lin-11/ Isl1/Mec-3) domains to make simultaneous contacts with two or more other proteins that do bind to DNA, such as GATA1 and basic helix-loop-helix (bHLH) proteins [6,7]. Reported binding partners include LDB1 [16,17], GATA6 [18], the tumour suppressor CtIP/RBBP8
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