Contribution of DEAF1 Structural Domains to the Interaction with the Breast Cancer Oncogene LMO4

Abstract

The proteins LMO4 and DEAF1 contribute to the proliferation of mammary epithelial cells. During breast cancer LMO4 is upregulated, affecting its interaction with other protein partners. This may set cells on a path to tumour formation. LMO4 and DEAF1 interact, but it is unknown how they cooperate to regulate cell proliferation. In this study, we identify a specific LMO4-binding domain in DEAF1. This domain contains an unstructured region that directly contacts LMO4, and a coiled coil that contains the DEAF1 nuclear export signal (NES). The coiled coil region can form tetramers and has the typical properties of a coiled coil domain. Using a simple cell-based assay, we show that LMO4 modulates the activity of the DEAF NES, causing nuclear accumulation of a construct containing the LMO4-interaction region of DEAF1.

The Structure of an LIM-Only Protein 4 (LMO4) and Deformed Epidermal Autoregulatory Factor-1 (DEAF1) Complex Reveals a Common Mode of Binding to LMO4

“…We previously defined the LMO4-interaction domain in DEAF1 as DEAF1 404–438 [52] , which is predicted to be unstructured in the context of the full-length protein [52] , [53] by sequence analysis programs that predict order/disorder, PONDR [61] and IUPred [62] , and lacks substantial levels of secondary structure by JPred3 (which predicts secondary structure based on sequence) [63] . To identify residues of DEAF1 that are important for binding LMO4, sequential sets of three residues in DEAF1 404–438 were mutated to alanine (or glycine if the original residue was alanine) and screened for an interaction with LMO4 by yeast two-hybrid analysis ( Fig.…”

“…Vectors containing inserts encoding surface mutants of LMO4 [19] were sub-cloned into pGAD10. DEAF1 mutants were generated using overlap extension PCR on the background template DEAF1 404–438_457–479 [52] . L4-DEAF1 (DEAF1 404–438 containing a T435D mutation and a polyproline C-terminal tail) was cloned into the plasmid pRSET-A.…”

“…We previously reported that LMO4 binds a putatively disordered region in DEAF1 (DEAF1 404–438 ). This region of DEAF1 lies close to a coiled-coil domain that forms tetramers in vitro [52] , and encompasses the nuclear export signal (NES) [53] . In addition, we conducted cell-based localisation studies using constructs spanning the LMO4-interacting domain, NES and coiled-coil domains of DEAF1 to show that LMO4 likely modulates the sub-cellular localisation of DEAF1.…”

DEAF1 Is a Pellino1-interacting Protein Required for Interferon Production by Sendai Virus and Double-stranded RNA*

“…Interestingly, LMO4 interacts with amino acid residues 404–479 of DEAF1, which includes the NES. The interaction of DEAF1 with LMO4 was found to stimulate the nuclear accumulation of DEAF1 in overexpression studies, presumably because the NES was masked (38). In contrast to LMO4, we were unable to detect an interaction between Pellino1 and amino acid residues 404–479 of DEAF1 4 …”

A Quantitative Fluorescence‐Based Assay for Assessing LIM Domain–Peptide Interactions

“…However, tethered complexes are not amenable to standard quantitative binding studies. Yeast‐two hybrid (Y2 H) assays suggest that the interactions of LMO4 with CtIP and Deaf1 are significantly weaker than with LDB1 ,. Homologous competition ELISA experiments using complexes in which the tether has been cut by a protease yielded estimates of LDB1 LID :LMO2/4 LIM1+2 affinity ( K d =20 and 10 n m respectively), but are not suited to weaker interactions due to multiple washing steps.…”