The emerin-binding transcription factor Lmo7 is regulated by association with p130Cas at focal adhesions

Abstract

Loss of function mutations in the nuclear inner membrane protein, emerin, cause X-linked Emery-Dreifuss muscular dystrophy (X-EDMD). X-EDMD is characterized by contractures of major tendons, skeletal muscle weakening and wasting, and cardiac conduction system defects. The transcription factor Lmo7 regulates muscle- and heart-relevant genes and is inhibited by binding to emerin, suggesting Lmo7 misregulation contributes to EDMD disease. Lmo7 associates with cell adhesions and shuttles between the plasma membrane and nucleus, but the regulation and biological consequences of this dual localization were unknown. We report endogenous Lmo7 also associates with focal adhesions in cells, and both co-localizes and co-immunoprecipitates with p130Cas, a key signaling component of focal adhesions. Lmo7 nuclear localization and transcriptional activity increased significantly in p130Cas-null MEFs, suggesting Lmo7 is negatively regulated by p130Cas-dependent association with focal adhesions. These results support EDMD models in which Lmo7 is a downstream mediator of integrin-dependent signaling that allows tendon cells and muscles to adapt to and withstand mechanical stress.

Keywords: Emerin; Emery-Dreifuss muscular dystrophy; Focal adhesions; LEM-domain; Laminopathy; Lmo7; Nuclear envelope; Nucleoskeleton; Tendon; p130Cas.

Figure 1. Endogenous Lmo7 co-localizes with vinculin and pFAK at focal adhesions in HeLa cells.

HeLa cells plated on fibronectin-coated coverslips for two hours (A, B) or other times (30 min or six hours; C) were fixed and stained by indirect immunofluorescence for endogenous Lmo7 (green) plus endogenous vinculin or pFAK (red). Cells were imaged by epifluorescence (A) or TIRF (B, C) microscopy, and co-localization was highlighted by cross-correlation analysis. Scale bars, 10 µm. Insets show each white-boxed region at higher magnification.

Figure 2. Lmo7 association with focal adhesion protein p130Cas.

(A) Schematic of the rat Lmo7a polypeptide and GFP- or GST-fused constructs used in this study. CH, predicted Calponin Homology domain; F-BOX, predicted F-box domain; PDZ, predicted PSD95/Dlg1/Zo-1 domain; NLS, predicted nuclear localization signal; LIM, LIM-domain. Boxes above rLmo7a indicate regions sufficient for direct binding to α-actinin (Ooshio et al., 2004), afadin (Ooshio et al., 2004) or emerin (Holaska, Rais-Bahrami & Wilson, 2006). (B) Whole cell protein lysates from HeLa cells that expressed GFP or GFP-rLmo7a for two days were immunoprecipitated with GFP antibodies, resolved by SDS-PAGE and immunoblotted with antibodies specific for paxillin or p130Cas. I, input (5% loaded); P, pellet (80% loaded). (C) Purified recombinant GST-fused Lmo7 polypeptides, resolved by SDS-PAGE and stained with Coomassie. (D–E) GST-pulldowns from whole HeLa cell lysates. Each GST-fused Lmo7 polypeptide (GST-CH, GST-F-box, GST-PDZ, GST-LIM), or GST alone, was incubated with HeLa cell lysates, then bound to glutathione, washed, eluted and resolved by SDS-PAGE. Bound proteins were detected in separate gels that were either stained with Coomassie (D), or immunoblotted for p130Cas (E) or paxillin (F). The black boxes in (D) indicate GST-fused proteins that migrated as SDS-resistant dimers.

Figure 3. p130Cas associates with Lmo7 in HeLa cells and MEFs.

(A) HeLa cells that transiently expressed Myc-tagged p130Cas were immunoprecipitated with Myc antibodies and immunoblotted for endogenous Lmo7. (B, C) HeLa cells or MEFs were plated on fibronectin-coated coverslips two hours, fixed and double-stained by indirect immunofluorescence for endogenous Lmo7 (green) and endogenous p130Cas (red), then imaged by epifluorescence (B) or TIRF microscopy (C). Scale bars, 10 µm. Insets show each white-boxed region at higher magnification.

Figure 4. p130Cas regulates Lmo7 localization and Lmo7-dependent transcription.

(A) Indirect immunofluorescence images of wildtype (control) and p130Cas null (−/−) MEFs plated on fibronectin for two hours, then fixed and stained for endogenous Lmo7 (green) and pFAK (red). Scale bars, 10 µm (1 µm in insets; boxed). (B) Immunoblot of nuclear versus cytoplasmic fractions of wildtype (control) and p130Cas null (−/−) MEFs, resolved by SDS-PAGE and probed with antibodies specific for Lmo7, A-type lamins (nuclear marker), or β-tubulin (n = 3). (C) Quantification of the nuclear-to-cytoplasmic ratio of Lmo7 in wildtype (control) and p130Cas-null (−/−) MEFs (*p < 0.05, paired t-test, n = 3). (D) Quantitative real-time PCR analysis of mRNAs from genes known to be activated by Lmo7 (Mef2C, Id2, Crebbp, Pcaf, Mbnl, Mef2B, emerin) or repressed by Lmo7 (Mef2D, Rbl1), in control or p130Cas(-/-) MEFs. *p < 0.05 by the paired t-test; n = 4.