Direct Binding of Nuclear Membrane Protein MAN1 to Emerin in Vitro and Two Modes of Binding to Barrier-to-Autointegration Factor

Abstract

MAN1 is a vertebrate nuclear inner membrane protein that inhibits Smad signaling downstream of transforming growth factor ␤. MAN1 has an exposed LEM domaincontaining N-terminal region ("MAN1-N"), two transmembrane domains, and an exposed C-terminal domain ("MAN1-C"). Many regions of human MAN1 are homologous to emerin, a LEM domain nuclear protein, loss of which causes Emery-Dreifuss muscular dystrophy (EDMD). To test the hypothesis that MAN1 function might overlap with emerin, we tested different polypeptide fragments of MAN1 for binding to selected partners of emerin. Our findings support this hypothesis. Blot overlay assays and co-immunoprecipitation studies showed that MAN1-C binds the transcription regulators GCL, Btf, and barrier-to-autointegration factor (BAF). BAF binding to this region, which has no LEM domain, was notable. Sequence alignments identified a potential BAF-binding motif, characterized by the conserved residues Ser-Arg-Val, in MAN1-C and two other BAF-binding proteins. The other region, MAN1-N, bound directly to BAF, lamin A, and lamin B1, supporting functional overlap with emerin. Unexpectedly, three independent assays showed that MAN1-N also bound directly to emerin. Proposed MAN1-emerin complexes are discussed in the context of EDMD disease mechanisms and potential in vivo functions.

Tissue-Specific Defects Are Caused by Loss of the Drosophila MAN1 LEM Domain Protein

“…First, individual domains of dMAN1 were tested for interaction with nuclear lamina proteins (Table 1). We found that the amino-terminal domain located between the LEM and first transmembrane domain (NTDDLEM) associated with Bocksbeutel, lamin Dm 0 , and lamin C, mirroring domain interactions seen with hMAN1 (Mansharamani and Wilson 2005). Our studies showed that dBAF interactions were limited to the LEM-D, unlike BAF association with hMAN1 that occurs with both the LEM-D and amino acids in the carboxyl terminus (Mansharamani and Wilson 2005).…”

“…The amino terminus of dMAN1 is shorter than that of hMAN1 and similar in size to that of LEM2. The carboxyl terminus of dMAN1 contains a UHM/RRM domain, a domain present in hMAN1 but absent in LEM2 (Mansharamani and Wilson 2005). We compared the amino acid sequences of dMAN1 with MAN1 from several invertebrate and vertebrate species, to gain insights into the degree of conservation of the structural domains.…”

“…These proteins associate directly with lamins (Holaska et al 2003) and are required for nuclear envelope reformation during mitosis (Ashery- Padan et al 1997a). LEM-D proteins interact with transcriptional repressors, such as germ cell-less and Bcl-2-associated transcription factor (Haraguchi et al 2004;Mansharamani and Wilson 2005). In addition, interactions with the downstream transcriptional effectors of multiple signal transduction pathways have been observed, such as Smads [receptor-regulated (R)-Smads], retinoblastoma protein, and b-catenin (Markiewicz et al 2002(Markiewicz et al , 2006Osada et al 2003;Raju et al 2003;Lin et al 2005;Pan et al 2005;Jiang et al 2008).…”

Inner nuclear membrane and signal transduction

“…This domain interacts with lamin A, lamin B1 and emerin [Mansharamani and Wilson, 2005]. MAN1 can diffuse relatively freely in the endoplasmic reticulum membrane but its lateral diffusion is decreased in the inner nuclear membrane [Wu et al, 2002].…”

“…and Wilson, 2005]. As a result of these interactions, mutations in emerin and lamins could therefore alter the localization of function of MAN1, consequently affecting regulation of Smads.…”