The Emery-Dreifuss muscular dystrophy associated-protein emerin is phosphorylated on serine 49 by protein kinase A.
Emerin is a ubiquitously expressed inner nuclear membrane protein of unknown function. Mutations in its gene give rise to X-linked Emery-Dreifuss muscular dystrophy (X-EDMD), a neuromuscular condition with an associated life-threatening cardiomyopathy. We have previously reported that emerin is phosphorylated in a cell cycle-dependent manner in human lymphoblastoid cell lines [Ellis et al. (1998) Aberrant intracellular targeting and cell cycle-dependent phosphorylation of emerin contribute to the EDMD phenotype. J. Cell Sci. 111, 781-792]. Recently, five residues in human emerin were identified as undergoing cell cycle-dependent phosphorylation using a Xenopus egg mitotic cytosol model system (Hirano et al. (2005) Dissociation of emerin from BAF is regulated through mitotic phosphorylation of emerin in a Xenopus egg cell-free system. J. Biol. Chem.280, 39 925-39 933). In the present paper, recombinant human emerin was purified from a baculovirus-Sf9 heterogeneous expression system, analyzed by protein mass spectrometry and shown to exist in at least four different phosphorylated species, each of which could be dephosphorylated by treatment with alkaline phosphatase. Further analysis identified three phosphopeptides with m/z values of 2191.9 and 2271.7 corresponding to the singly and doubly phosphorylated peptide 158-DSAYQSITHYRPVSASRSS-176, and a m/z of 2396.9 corresponding to the phosphopeptide 47-RLSPPSSSAASSYSFSDLNSTR-68. Sequence analysis confirmed that residue S49 was phosphorylated and also demonstrated that this residue was phosphorylated in interphase. Using an in vitro protein kinase A assay, we observed two phospho-emerin species, one of which was phosphorylated at residue S49. Protein kinase A is thus the first kinase that has been identified to specifically phosphorylate emerin. These results improve our understanding of the molecular mechanisms underlying X-EDMD and point towards possible signalling pathways involved in regulating emerin's functions.
Roberts
Rhys C
RC
MRC Laboratory of Molecular Biology, Cambridge, UK.
Sutherland-Smith
Andrew J
AJ
Wheeler
Matthew A
MA
Jensen
Ole Norregaard
ON
Emerson
Lindsay J
LJ
Spiliotis
Ioannis I
II
Tate
Christopher G
CG
Kendrick-Jones
John
J
Ellis
Juliet A
JA
D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000595:Amino Acid Sequence; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009685:Nuclear Envelope / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012694:Serine; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D054758:A Kinase Anchor Proteins; C105137:AKAP1 protein, human; D048868:Adaptor Proteins, Signal Transducing; D008565:Membrane Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; C092710:emerin; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases