Roberts Rhys C, Sutherland-Smith Andrew J, Wheeler Matthew A, Jensen Ole Norregaard, Emerson Lindsay J, Spiliotis Ioannis I, Tate Christopher G, Kendrick-Jones John, Ellis Juliet A
MRC Laboratory of Molecular Biology, Cambridge, UK.
FEBS J. 2006 Oct;273(19):4562-75. doi: 10.1111/j.1742-4658.2006.05464.x.
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.
Emerin是一种广泛表达的内核膜蛋白，其功能未知。其基因突变会导致X连锁的埃默里-德赖富斯肌营养不良症（X-EDMD），这是一种神经肌肉疾病，伴有危及生命的心肌病。我们之前报道过，在人淋巴母细胞系中，Emerin以细胞周期依赖性方式被磷酸化[埃利斯等人（1998年）。Emerin异常的细胞内定位和细胞周期依赖性磷酸化导致了EDMD表型。《细胞科学杂志》111卷，781 - 792页]。最近，利用非洲爪蟾卵有丝分裂胞质溶胶模型系统，在人Emerin中鉴定出五个残基经历细胞周期依赖性磷酸化（平野等人（2005年）。在非洲爪蟾卵无细胞系统中，Emerin从BAF的解离通过Emerin的有丝分裂磷酸化来调节。《生物化学杂志》280卷，39925 - 39933页）。在本文中，重组人Emerin从杆状病毒 - Sf9异源表达系统中纯化出来，通过蛋白质质谱分析，结果表明它至少以四种不同的磷酸化形式存在，每种形式都可以通过用碱性磷酸酶处理而发生去磷酸化。进一步分析鉴定出三种磷酸肽，其m/z值分别为2191.9和2271.7，对应于单磷酸化和双磷酸化的肽段158 - DSAYQSITHYRPVSASRSS - 176，以及一个m/z为2396.9的对应于磷酸肽47 - RLSPPSSSAASSYSFSDLNSTR - 68。序列分析证实残基S49被磷酸化，并且还表明该残基在间期被磷酸化。使用体外蛋白激酶A测定法，我们观察到两种磷酸化的Emerin形式，其中一种在残基S49处被磷酸化。因此，蛋白激酶A是第一个被鉴定出能特异性磷酸化Emerin的激酶。这些结果增进了我们对X - EDMD潜在分子机制的理解，并指出了可能参与调节Emerin功能的信号通路。
