Identification of tyrosine‐phosphorylation sites in the nuclear membrane protein emerin

Schlösser, Andreas; Amanchy, Ramars; Otto, Henning

doi:10.1111/j.1742-4658.2006.05329.x

Cited by 13 publications

(14 citation statements)

References 47 publications

(70 reference statements)

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“…We compiled data from 13 independent proteomic studies (Amanchy et al, 2005;Brill et al, 2004;Cantin et al, 2008;Daub et al, 2008;Guo et al, 2008;Olsen et al, 2006;Pan et al, 2008;Rikova et al, 2007;Rush et al, 2005;Schlosser et al, 2006;Sui et al, 2008;Tao et al, 2005;Tsai et al, 2008), which identified 13 phosphorylated tyrosines in emerin (Fig. 1A).…”

Section: Resultsmentioning

confidence: 99%

Tyrosine phosphorylation of nuclear-membrane protein emerin by Src, Abl and other kinases

Tifft

Bradbury

Wilson

2009

Journal of Cell Science

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X-linked recessive Emery-Dreifuss muscular dystrophy (EDMD) is caused by loss of emerin, a nuclear-membrane protein with roles in nuclear architecture, gene regulation and signaling. Phosphoproteomic studies have identified 13 sites of tyrosine phosphorylation in emerin. We validated one study, confirming that emerin is hyper-tyrosine-phosphorylated in Her2-overexpressing cells. We discovered that non-receptor tyrosine kinases Src and Abl each phosphorylate emerin and a related protein, LAP2, directly. Src phosphorylated emerin specifically at Y59, Y74 and Y95; the corresponding triple Yto-F ('FFF') mutation reduced tyrosine phosphorylation bỹ 70% in vitro and in vivo. Substitutions that removed a single hydroxyl moiety either decreased (Y19F, Y34, Y161F) or increased (Y4F) emerin binding to BAF in cells. Y19F, Y34F, Y161F and the FFF mutant also reduced recombinant emerin binding to BAF from HeLa lysates, demonstrating the involvement of both LEM-domain and distal phosphorylatable tyrosines in binding BAF. We conclude that emerin function is regulated by multiple tyrosine kinases, including Her2, Src and Abl, two of which (Her2, Src) regulate striated muscle. These findings suggest roles for emerin as a downstream effector and 'signal integrator' for tyrosine kinase signaling pathway(s) at the nuclear envelope.Key words: Emerin, LEM domain, Nuclear envelope, Emery-Dreifuss muscular dystrophy, Src, Her2, Abl, Barrier-to-autointegration factor (BAF), Cardiomyopathy, Breast cancer, Laminopathy, Neuromuscular junction Journal of Cell Science 3781 Emerin tyrosine phosphorylation receptor tyrosine kinases that can be activated by Her2 or other signaling pathways (Roskoski, 2005;Srinivasan and Plattner, 2006). We confirm Her2-stimulated emerin tyrosine phosphorylation and report that emerin and LAP2 are phosphorylated directly by Src and Abl. We also show that tyrosine phosphorylation of emerin regulates binding to BAF. ResultsWe compiled data from 13 independent proteomic studies (Amanchy et al., 2005;Brill et al., 2004;Cantin et al., 2008; Daub et al., 2008; Guo et al., 2008;Olsen et al., 2006;Pan et al., 2008;Rikova et al., 2007;Rush et al., 2005;Schlosser et al., 2006;Sui et al., 2008;Tao et al., 2005;Tsai et al., 2008), which identified 13 phosphorylated tyrosines in emerin (Fig. 1A). All sites except Y19 and Y41 were identified more than once. All 13 identified phosphorylated Tyr (Tyr-P) residues in human emerin are conserved in mouse, and six (corresponding to Y4, Y41, Y94, Y95, Y105, Y161) are conserved in Xenopus (Fig. 1B). These studies did not reveal responsible pathways or kinases.We validated tyrosine phosphorylation of emerin by immunoprecipitating endogenous emerin from HeLa cells treated with 1 M pervanadate (PV, a tyrosine phosphatase inhibitor) (Huyer et al., 1997) or buffer (PBS) as control, for 30 minutes before lysing cells. Emerin immunoprecipitated from PV-treated cells was specifically recognized by Tyr-P antibodies (not shown; see Fig. 2B). No other tyrosine-phosphorylated bands were dete...

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Section: Resultsmentioning

confidence: 99%

Tyrosine phosphorylation of nuclear-membrane protein emerin by Src, Abl and other kinases

Tifft

Bradbury

Wilson

2009

Journal of Cell Science

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“…If so, what might be the function of this sumoylated pool of PTP1B? Certain proteins of the INM, including emerin and lamin A/C, are known to be tyrosine phosphorylated Schlosser et al, 2006). Unlike the analogous cell-cycle-dependent serine/threonine phosphorylations (Ellis et al, 1998;Fields and Thompson, 1995), the physiological relevance of these tyrosine phosphorylations are not well understood.…”

Section: Discussionmentioning

confidence: 99%

“…However, there are clues that hint at significant functions for these modifications. For example, Tyr95 in emerin has been mapped as a site of tyrosine phosphorylation (Schlosser et al, 2006). This site lies within a small region (amino acids 95-99) that is deleted in some forms of X-chromosome-linked EDMD (Bengtsson and Wilson, 2004;Ellis et al, 1998).…”

Section: Discussionmentioning

confidence: 99%

“…This same region is required for stable interaction of emerin with lamin, and loss of this region results in weakened association of emerin with the nuclear envelope (Bengtsson and Wilson, 2004;Lee et al, 2001). In addition, emerin has been shown to be tyrosine phosphorylated at multiple sites by Src and Abl (Schlosser et al, 2006;Tifft et al, 2009), and it has been shown that these phosphorylations significantly hinder emerin binding to barrier-to-autointegration factor (BAF), a protein that is crucial for targeting the nuclear membrane proteins to chromatin at the early stage of nuclear assembly (Hirano et al, 2005;Margalit et al, 2007;Margalit et al, 2005). In this setting, sumoylation might be important to temporally inhibit the local INM pool of PTP1B during the mitotic portion of the cell cycle, in order to maintain emerin tyrosine phosphorylation and interactions with lamins and BAF (Fairley et al, 2002).…”

Section: Discussionmentioning

confidence: 99%

“…We focused in particular on emerin, a lamin-binding protein that is associated with the X-chromosome-linked EmeryDreifuss muscular dystrophy (EDMD) (Bione et al, 1994). Several tyrosine phosphorylation sites on emerin have been identified (Schlosser et al, 2006). One tyrosine residue, Tyr95, is Fig.…”

Section: Sumoylated Ptp1b Is Enriched At the Inmmentioning

confidence: 99%

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Sumoylated protein tyrosine phosphatase 1B localizes to the inner nuclear membrane and regulates the tyrosine phosphorylation of emerin

Yip

Cotteret

Chernoff

2012

Journal of Cell Science

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SummaryProtein tyrosine phosphatase (PTP)1B is an abundant non-transmembrane enzyme that plays a major role in regulating insulin and leptin signaling. Recently, we reported that PTP1B is inhibited by sumoylation, and that sumoylated PTP1B accumulates in a perinuclear distribution, consistent with its known localization in the endoplasmic reticulum (ER) and the contiguous outer nuclear membrane. Here, we report that, in addition to its localization at the ER, PTP1B also is found at the inner nuclear membrane, where it is heavily sumoylated. We also find that PTP1B interacts with emerin, an inner nuclear membrane protein that is known to be tyrosine phosphorylated, and that PTP1B expression levels are inversely correlated with tyrosine phosphorylation levels of emerin. PTP1B sumoylation greatly increases as cells approach mitosis, corresponding to the stage where tyrosine phosphorylation of emerin is maximal. In addition, expression of a nonsumoylatable mutant of PTP1B greatly reduced levels of emerin tyrosine phosphorylation. These results suggest that PTP1B regulates the tyrosine phosphorylation of a key inner nuclear membrane protein in a sumoylation-and cell-cycle-dependent manner.

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Mass spectrometry analysis of phosphotyrosine‐containing proteins

Zhan

2023

Mass Spectrometry Reviews

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Tyrosine phosphorylation is a crucial posttranslational modification that is involved in various aspects of cell biology and often has functions in cancers. It is necessary not only to identify the specific phosphorylation sites but also to quantify their phosphorylation levels under specific pathophysiological conditions. Because of its high sensitivity and accuracy, mass spectrometry (MS) has been widely used to identify endogenous and synthetic phosphotyrosine proteins/peptides across a range of biological systems. However, phosphotyrosine‐containing proteins occur in extremely low abundance and they degrade easily, severely challenging the application of MS. This review highlights the advances in both quantitative analysis procedures and enrichment approaches to tyrosine phosphorylation before MS analysis and reviews the differences among phosphorylation, sulfation, and nitration of tyrosine residues in proteins. In‐depth insights into tyrosine phosphorylation in a wide variety of biological systems will offer a deep understanding of how signal transduction regulates cellular physiology and the development of tyrosine phosphorylation‐related drugs as cancer therapeutics.

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Identification of tyrosine‐phosphorylation sites in the nuclear membrane protein emerin

Cited by 13 publications

References 47 publications

Tyrosine phosphorylation of nuclear-membrane protein emerin by Src, Abl and other kinases

Tyrosine phosphorylation of nuclear-membrane protein emerin by Src, Abl and other kinases

Sumoylated protein tyrosine phosphatase 1B localizes to the inner nuclear membrane and regulates the tyrosine phosphorylation of emerin

Mass spectrometry analysis of phosphotyrosine‐containing proteins

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