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-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-74/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.
Schlosser
Andreas
A
Charité, Institut für Medizinische Immunologie, Berlin, Germany.
Amanchy
Ramars
R
Otto
Henning
H
D000595:Amino Acid Sequence; D000818:Animals; D018929:Cell Culture Techniques; D002460:Cell Line; D002853:Chromatography, Liquid; D006367:HeLa Cells; D006801:Humans; D007553:Isotope Labeling; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D036103:Nanotechnology; D009447:Neuroblastoma / Q000473:pathology; D009685:Nuclear Envelope / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D009687:Nuclear Proteins; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000378:metabolism; D013946:Thymopoietins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism*
D008565:Membrane Proteins; D009687:Nuclear Proteins; D013946:Thymopoietins; C092710:emerin; D014443:Tyrosine