| Emery-Dreifuss muscular dystrophy (EDMD) is characterized by weakening of selected skeletal muscles, contractures, and cardiac conduction system defects. X-linked recessive EDMD is caused by loss of emerin, a nuclear inner membrane protein involved in nuclear structure, gene expression and cell signaling. The LEM-domain of emerin mediates binding to Barrier-to-Autointegration Factor (BAF), an essential chromatin protein conserved in metazoans. BAF has roles in nuclear assembly, chromatin organization, and gene expression and is required to maintain specific muscles in C. elegans. BAF forms stable dimers that bind two dsDNA molecules, plus histones, lamins and homeodomain transcription factors. We discovered BAF-Like (BAF-L), a vertebrate protein 40% identical to BAF. Both are predominantly nuclear proteins. Recombinant BAF-L forms stable homodimers, but interestingly can heterodimerize with BAF in vitro and interacts with BAF in vivo. BAF-L does not bind directly to DNA, LAP2beta or emerin. However, it formed ternary complexes in vitro with BAF (as a presumed heterodimer) plus either DNA or LAP2beta. BAF-L mRNA was detected in 14 of 16 tested tissues: exceptions were heart and skeletal muscle. We proposed that BAF-L regulates BAF, and speculate that tissues lacking BAF-L are susceptible to EDMD disease. Emerin binds many partners directly including transcription and splicing factors, signaling proteins and architectural proteins. To understand these interactions we hypothesized that emerin is differentially regulated by tyrosine phosphorylation. Our compilation of proteomic studies revealed 12 sites of tyrosine phosphorylation in emerin. We validated emerin as a target of Her2-dependent Tyr phosphorylation in vivo, and identified two tyrosine kinases that phosphorylate emerin (and LAP2beta) directly: Src and Abl. Three residues (Y59, Y74, Y95) phosphorylated by Src in vitro were identified by LC-MS/MS, and their in vivo relevance demonstrated using the triple Y-to-F emerin mutant. Further analysis identified single Tyr residues, including Src phosphorylated residues, that are important to bind BAF supporting the hypothesis that tyrosine phosphorylation of emerin regulates binding partners. We conclude that emerin activity is regulated by at least three signaling pathways: Her2, Src-family kinases and Abl. These findings support an emerging model for EDMD disease: defective signaling. |
