Emerin tyrosine phosphorylation by Her2, Src and Abl. (A) Her2 overexpression induces emerin tyrosine phosphorylation. Endogenous emerin was immunoprecipitated (NCL-emerin antibody) from NIH-3T3 cells that stably expressed either control vector or a Her2 overexpression vector (Bose et al., 2006). Antibody was omitted from controls. Immunoprecipitates were analyzed by western blotting with antibodies against emerin (αemr; sc-15378) and Tyr-P (αpTyr). A representative blot is shown (n=3). (B) PV-induced emerin tyrosine phosphorylation is reduced by Src-specific inhibitors. HeLa cells were treated with either DMSO, DMSO and 10 μM PP2 (SFK inhibitor), DMSO and 1 nM SU6656 (SFK inhibitor) or DMSO and 10 μM PP3 (inactive analog of PP2) for 30 minutes before treatment with PBS or 1 μM PV for 30 minutes. Endogenous emerin was then immunoprecipitated (serum 2999), resolved by SDS-PAGE and western blotted for emerin (NCL-emerin) and Tyr-P. Representative blot is shown (n=3). (C) HeLa cells were transiently transfected to express GFP or GFP-emerin (GFP-emr), plus either wild-type Src (WT) or kinase-dead Src (KD). Lysates (1%) were western blotted for Src and actin as controls (inputs). GFP proteins were immunoprecipitated with GFP antibody (GFP IP), resolved by SDS-PAGE and western blotted for GFP and Tyr-P. A representative autoradiograph is shown (n=3). (D) In vitro Src phosphorylation using purified recombinant Src, purified recombinant untagged emerin (residues 1-222) and [³³P]ATP. Samples were resolved by SDS-PAGE and analyzed by autoradiography. A representative blot is shown (n=3). (E) Same in vitro Src phosphorylation assay as D, but incubated for 10 minutes with recombinant His-emerin (residues 1-176) or His-LAP2β (residues 1-408) as substrates, either with (+) or without (–) Src. Samples were resolved by SDS-PAGE and western blotted with antibodies against the His-tag and Tyr-P. A representative blot is shown (n=3). (F) Recombinant emerin (residues 1-176) and LAP2β (residues 1-408) were incubated in kinase buffer alone or with purified recombinant Abl kinase domain (wild type or mutant R367A). Reactions were resolved by SDS-PAGE and western blotted for His and Tyr-P (n=3); representative blots are shown.

Characterization of emerin Y-to-F missense mutations at identified Src phosphorylation sites. (A,B) In vitro phosphorylation assay with recombinant purified Src, recombinant purified emerin (residues 1-176; wild type or triple mutant Y59F, Y74F, Y95F; `FFF') and incubated with ATP for 10 minutes, then resolved by SDS-PAGE and western blotted with antibodies against His tag or Tyr-P (A), and quantified (B) as the Tyr-P-to-His signal expressed as a percentage normalized to the wild type. Reduced tyrosine phosphorylation of FFF was significant; error bars show s.e.m.; ^*P<0.05 (n=3). (C,D) Same in vitro Src phosphorylation assay as A but using as substrates either wild-type emerin residues 1-176, the corresponding FFF mutant, or each of three double mutant (FF) combinations: Y59F/Y74F, Y59F/Y95F or Y74F/Y95F. Reactions were resolved by SDS-PAGE and western blotted with antibodies to the His-tag or Tyr-P (C), then quantified (D) as described for B. Error bars show s.e.m.; ^*P<0.05, ^**P<0.01, ^***P<0.005 (n=3). (E) Fluorescence micrographs of HeLa cells that transiently expressed GFP-emerin (wild type or FFF) for 24 hours, fixed, permeabilized, stained with DAPI and visualized by direct GFP fluorescence. (F) GFP-antibody immunoprecipitation of GFP-emerin (wild type or FFF mutant) after 24 hours co-expression in HeLa cells with either wild-type Src (WT) or kinase-dead Src (KD), resolved by SDS-PAGE and western blotted with antibodies against GFP or Tyr-P. As controls, input lysates (1%) were western blotted for Src and actin. (G) Results from F for wild-type Src were quantified as described in B. The reduced Tyr-P signal in the FFF mutant emerin was significant; ^**P<0.01; error bars show s.e.m. (n=4). (H) HeLa cells that transiently expressed GFP or GFP-emerin (wild type or FFF mutant) for 24 hours were treated 30 minutes with PBS or 1 μM PV, then lysed, immunoprecipitated with GFP antibodies, resolved by 16% Tris-Tricine SDS-PAGE and western blotted for GFP or Tyr-P. (I) Results from H were quantified as described in B. The reduced Tyr-P signal in the FFF mutant emerin was significant; ^*P<0.05; error bars show s.e.m. (n=3). (J) Reduced Abl phosphorylation of FFF-mutant emerin. Recombinant emerin 1-176 (wild type or FFF mutant) polypeptides were incubated alone or with the Abl kinase domain (wild type or R367A). (K) Results from J for wild-type Abl were quantified as described in B. Error bars show s.e.m.; ^*P<0.05 (n=3).

Emerin is tyrosine phosphorylated. (A) Emerin Tyr-P sites identified in published proteomic mass spectrometry studies. (B) Amino acid sequences of emerin from human (Acc. no. NP_000108), mouse (NP_031953) and Xenopus laevis (NP_001089081). Conserved residues are light gray. All tyrosines are dark gray and numbered; black dots indicate those reported to be phosphorylated in A. The LEM domain is boxed and transmembrane domain (TMD) is double underlined.

LC-MS/MS identification of emerin tyrosine residues phosphorylated by Src and Abl. (A) Summary of emerin peptides identified by mass spectrometry. (B-D) Mass spectra of emerin peptides that demonstrated Src phosphorylation shown for Y59 (B; ion score=113; expect value=1.3×10⁸), Y74 (C; ion score=53; expect value=0.0096) and Y95 (D; ion score=79; expect value=3.2×10^–5). Inserts in B-E show the predicted c-ion and z-ion masses of each identified sequence. Ions used for identification are underlined and corresponding peaks are labeled. (E) Mass spectrum demonstrating Abl phosphorylation of emerin Y167 (ion score=47; expect value=2.1).

Effects of tyrosine mutations and tyrosine phosphorylation on emerin binding to BAF. (A) Functional map based on mutations in emerin that disrupt binding to each indicated partner (Holaska et al., 2004; Holaska et al., 2006; Mansharamani and Wilson, 2005) and evidence that the APC-like (`A') region (residues 169-180) is sufficient to bind β-catenin (β-cat) (Markiewicz et al., 2006). The nesprin-binding region (residues 140-176) is not shown (Wheeler et al., 2007). LEM, LEM domain; TM, transmembrane. All Tyr residues are labeled `Y' and numbered; identified Src and Abl targets are indicated. (B) LEM-domain amino acid sequences of all known human LEM-domain proteins (Wagner and Krohne, 2007). Conserved residues are light gray. All tyrosines are dark gray and numbered; black dots indicate known phosphorylation sites (Fig. 1A). (C) Surface representation of the NMR co-structure of BAF (gray) and the emerin LEM domain (white) (Cai et al., 2007); emerin residues Y4, Y19, Y34 and Y41 are shaded black. (D,E) Effects of Y-to-F mutations on emerin binding to BAF in vitro. Recombinant GST or GST-emerin (wild-type residues 1-176 or Y-to-F mutants) was incubated with HeLa cell lysates and pelleted using glutathione beads and western blotted for GST and BAF. Blots shown are representative of n>3 experiments and quantified in E as the BAF-to-His signal expressed as a percentage and normalized to wild-type GST-emerin. Error bars show s.e.m.; ^*P<0.05, ^***P<0.005 (n=3). (F,G) Effects of Y-to-F mutations on emerin binding to endogenous BAF in HeLa cells. HeLa cells that transiently expressed GFP or GFP-emerin (wild type, or each Y-to-F missense mutation) for 24 hours were immunoprecipitated with GFP antibodies (GFP IP); pellets were resolved by SDS-PAGE and western blotted for GFP and BAF. As loading controls (Input), 1% of each input lysate was directly probed with antibodies against GFP, BAF or actin. Blots shown in F are representative of n>3 experiments and quantified in G as the BAF-to-GFP signal expressed as a percentage and normalized to wild-type GFP-emerin. Error bars represent the s.e.m.; ^*P<0.05, ^**P<0.01 (n>3). (H,I) FFF mutation reduces binding of endogenous BAF from HeLa cell lysates to recombinant His-emerin residues 1-176. Blots shown in H are representative of n=4 experiments and were quantified in I as the BAF-to-His signal expressed as a percentage and normalized to wild-type His-emerin. Error bars indicate s.e.m.; ^***P<0.005 (n=4). (J,K) HeLa cells that transiently expressed GFP or GFP-emerin for 24 hours were treated with PBS or 1 μM PV for 30 minutes, then lysed, immunoprecipitated with GFP antibodies, resolved by 16% Tris-Tricine SDS-PAGE and western blotted for GFP, BAF or Tyr-P. Results from J were quantified in K as the BAF-to-GFP signal expressed as a percentage and normalized to PBS-treated cells. Error bars show s.e.m.; ^***P<0.005 (n=3).