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

Abstract

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...

O-Linked β-N-Acetylglucosamine (O-GlcNAc) Regulates Emerin Binding to Barrier to Autointegration Factor (BAF) in a Chromatin- and Lamin B-enriched “Niche”

“…Third, Ser-173 O-GlcNAcylation might promote BAF association by blocking an alternative fate in the sonication-dependent niche: Ser-173 phosphorylation and subsequent hyperphosphorylation. Our model that Ser-173 modifications, far from the LEM domain, somehow control binding to BAF is supported by previous evidence that the Y161F and S175A mutations also influence BAF association in whole cell lysates (62,80). These findings collectively define emerin residues 161-175 as a nexus for complex posttranslational regulation of emerin binding to BAF.…”

“…GST-emerin 1-222 polypeptides were purified as described (62). To purify other GSTtagged polypeptides, bacterial pellets were resuspended in icecold resuspension buffer (PBS containing 0.05% (v/v) Tween 20, 2 mM EDTA, pH 8.0, 0.1% (v/v) ␤-mercaptoethanol (BME), 0.1 mM PMSF), sonicated on ice (5 ϫ 30 s; 10-s rests), and centrifuged (20,000 ϫ g, 30 min).…”

“…7A) was conservative, because it did not include potentially phosphorylated molecules in the main emerin band (62). We conclude that emerin Ser-173 phosphorylation, mimicked by S173D, increases emerin phosphorylation in vivo in different ways, depending on whether emerin occupies the easy or sonication-dependent niche.…”

“…5B (GFP blot) and 6A (lane 17)). Furthermore, previous studies showed that emerin-BAF association in human cell lysates or mitotic Xenopus cell lysates was inexplicably affected by the Y161F or S175A mutations, respectively (62,80), near Ser-173. We therefore examined potential posttranslational consequences of emerin Ser-173 mutations.…”

“…Emerin migrates slowly in SDS gels when hyperphosphorylated (62,81,82), and slowly migrating bands were obvious in the endogenous easy emerin population (e.g. Figs.…”