tailieunhanh - Báo cáo khoa học: Identification of tyrosine-phosphorylation sites in the nuclear membrane protein emerin

Although several proteins undergo tyrosine phosphorylation at the nuclear envelope, we achieved, for the first time, the identification of tyrosine-phos-phorylation sites of a nuclear-membrane protein, emerin, by applying two mass spectrometry-based techniques. | iFEBS Journal Identification of tyrosine-phosphorylation sites in the nuclear membrane protein emerin Andreas Schlosser1 Ramars Amanchy2 and Henning Otto3 1 Charite Institut fur Medizinische Immunologie Berlin Germany 2 McKusick-Nathans Institute for Genetic Medicine and the Department of BiologicalChemistry Johns Hopkins University Baltimore MD USA 3 Freie Universitat Berlin Institut fur Chemie und Biochemie Germany Keywords Emerin Emery-Dreifuss muscular dystrophy nuclear envelope phosphorylation proteomics Correspondence H. Otto Freie Universitat Berlin Institut fur Chemie und Biochemie Thielallee 63 D-14195 Berlin Germany Fax 49 30 83853753 Tel 49 30 83856425 E-mail hotto@ These authors contributed equally to this work Received 17 January 2006 revised 27 April 2006 accepted 18 May 2006 Although several proteins undergo tyrosine phosphorylation at the nuclear envelope we achieved for the first time the identification of tyrosine-phosphorylation sites of a nuclear-membrane protein emerin by applying two mass spectrometry-based techniques. With a multiprotease approach combined with highly specific phosphopeptide enrichment and nano liquid chromatography tandem mass spectrometry analysis we identified three tyrosine-phosphorylation sites Y-75 Y-95 and Y-106 in mouse emerin. Stable isotope labeling with amino acids in cell culture revealed phosphotyrosines at Y-59 Y-74 Y-86 Y-161 and Y-167 of human emerin. The phosphorylation sites Y-7Y Y-75 human mouse emerin Y-85 Y-86 Y-94 Y-95. and Y-105 Y-106 are located in regions previously shown to be critical for interactions of emerin with lamin A actin or the transcriptional regulators GCL and Btf while the residues Y-161 and Y-167 are in a region linked to binding lamin-A or actin. Tyrosine Y-94 Y-95 is located adjacent to a five-residue motif in human emerin whose deletion has been associated with X-linked Emery-Dreifuss muscle dystrophy. doi The nuclear envelope .