ReCLIP (Reversible Cross-Link Immuno-Precipitation): An Efficient Method for Interrogation of Labile Protein Complexes

Smith, Andrew L.; Friedman, David; Yu, Huapeng; Carnahan, Robert H.; Reynolds, Albert B.

doi:10.1371/journal.pone.0016206

Cited by 67 publications

(72 citation statements)

References 44 publications

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“…Easily rationalized proteins that were well tagged by EcadBL that are not identified in the 'cadherin adhesome' include members of the pleckstrin homology domain containing family A members 5 and 6, EH-domain binding protein 1-like, the coxsackie and adenovirus receptor, vang-like protein 1 and 4F2 cell surface antigen heavy chain; this last protein was previously identified as interacting with catenin delta-1 (Smith et al, 2011). Proteins that might play a role but have yet undescribed functions include sickle tail homolog and uncharacterized protein LOC100688057, which contains an actin binding/calponin homology domain.…”

Section: Discussionmentioning

confidence: 99%

“…In terms of relative abundance, the top five proteins identified are all adherens junction proteins (Fig. 2B); one of these, catenin a-2 (a-Ncatenin) is generally thought to be restricted to the nervous system (Abe et al, 2004), although it was identified in a proteomic screen in A431 cells (Smith et al, 2011). A third acatenin isoform, catenin a-3 (a-T-catenin), was also identified by EcadBL-dependent biotinylation (rank 10 in abundance); this catenin is enriched in heart and testes and has not been previously described in MDCK cells (Janssens et al, 2001).…”

Section: Ecadbl Fusion Protein Localizes To Cell-cell Contacts In MDCmentioning

confidence: 99%

“…The interactions with actin are indirect and occur through extensively studied catenin proteins that bind to the intracellular domain of E-cadherin, namely b-catenin and a-catenin, and also through several cateninassociated actin-binding proteins, including vinculin, formin-1 and VASP (reviewed by Meng and Takeichi, 2009). Many proteins have been localized to adherens junctions by biochemical and microscopic techniques (Smith et al, 2011;Zaidel-Bar, 2013), but there have been relatively few attempts at global proteomic analysis.…”

Section: Introductionmentioning

confidence: 99%

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Biotin ligase tagging identifies proteins proximal to E-cadherin, including lipoma preferred partner, a regulator of epithelial cell-cell and cell-substrate adhesion

Itallie

Tietgens

Aponte

et al. 2013

Journal of Cell Science

116

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Known proteins associated with the cell-adhesion protein Ecadherin include catenins and proteins involved in signaling, trafficking and actin organization. However, the list of identified adherens junction proteins is likely to be incomplete, limiting investigation into this essential cell structure. To expand the inventory of potentially relevant proteins, we expressed Ecadherin fused to biotin ligase in MDCK epithelial cells, and identified by mass spectrometry neighboring proteins that were biotinylated. The most abundant of the 303 proteins identified were catenins and nearly 40 others that had been previously reported to influence cadherin function. Many others could be rationalized as novel candidates for regulating the adherens junction, cytoskeleton, trafficking or signaling. We further characterized lipoma preferred partner (LPP), which is present at both cell contacts and focal adhesions. Knockdown of LPP demonstrated its requirement for Ecadherin-dependent adhesion and suggested that it plays a role in coordination of the cell-cell and cell-substrate cytoskeletal interactions. The analysis of LPP function demonstrates proof of principle that the proteomic analysis of E-cadherin proximal proteins expands the inventory of components and tools for understanding the function of E-cadherin.

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Section: Discussionmentioning

confidence: 99%

Section: Ecadbl Fusion Protein Localizes To Cell-cell Contacts In MDCmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Biotin ligase tagging identifies proteins proximal to E-cadherin, including lipoma preferred partner, a regulator of epithelial cell-cell and cell-substrate adhesion

Itallie

Tietgens

Aponte

et al. 2013

Journal of Cell Science

116

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“…37°C) standard external solution (SES; 140 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 0.8 mM MgCl2, 10 mM glucose, and 10 mM HEPES, pH 7.4) and then incubated for 30 min at 37°C with 0.5 mM dithiobis[succinimidyl propionate] (DSP; Thermo Fisher Scientific), a membrane-permeable and thiol-cleavable NHS-ester cross-linker, or DMSO in SES in the presence or the absence of 40 M Y-27632. The DSP concentration used here (i.e., 0.5 mM) was in the same range used in previous IP experiments of cell adhesionrelated proteins (28,41,56). Cross-linking reactions were quenched with 1% glycine in cold SES for 15 min on ice.…”

Section: Methodsmentioning

confidence: 99%

Force-dependent vinculin binding to talin in live cells: a crucial step in anchoring the actin cytoskeleton to focal adhesions

Harai

Tatsumi

Lim

et al. 2014

American Journal of Physiology-Cell Physiology

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(FAs) where the actin cytoskeleton is anchored to the extracellular matrix through integrin and a variety of linker proteins including talin and vinculin. The localization of vinculin at FAs depends on mechanical forces. While in vitro studies have demonstrated the force-induced increase in vinculin binding to talin, it remains unclear whether such a mechanism exists at FAs in vivo. In this study, using fibroblasts cultured on elastic silicone substrata, we have examined the role of forces in modulating talin-vinculin binding at FAs. Stretching the substrata caused vinculin accumulation at talin-containing FAs, and this accumulation was abrogated by expressing the talin-binding domain of vinculin (domain D1, which inhibits endogenous vinculin from binding to talin). These results indicate that mechanical forces loaded to FAs facilitate vinculin binding to talin at FAs. In cellprotruding regions, the actin network moved backward over talincontaining FAs in domain D1-expressing cells while it was anchored to FAs in control cells, suggesting that the force-dependent vinculin binding to talin is crucial for anchoring the actin cytoskeleton to FAs in living cells. talin; vinculin; focal adhesion; mechanotransduction; molecular clutch CELL ADHESION TO EXTRACELLULAR matrices (ECMs) is crucial for cellular morphogenesis, migration, proliferation, and differentiation. Cell-to-ECM adhesion is primarily mediated by the transmembrane ECM receptors integrins. Integrin molecules are clustered at focal adhesions (FAs), where the actin cytoskeleton is anchored to the ECM through integrin clusters and plaques of a variety of linker proteins (16). There is bidirectional transmission of forces at FAs between ECM and the actin cytoskeleton (30). Thus FAs sustain tensile stress generated in the actin cytoskeleton. When integrin-actin cytoskeleton linkage is dissected, actin stress fibers are retracted and FAs are disassembled (37,43,53,54), indicating that the linkage is crucial for maintaining the integrity of FAs.Molecular processes of formation of integrin-cytoskeleton linkages have been extensively studied. Talin has both ␤-integrin-and actin-binding sites (9) and initially forms a molecular bond between ECM-bound integrin and the actin cytoskeleton in fibroblasts (17,34,65). The talin-mediated link between the actin cytoskeleton and clustered integrin is broken repeatedly by a small force of ϳ2 pN generated by the retrograde flow of actin filaments (34). On the other hand, the integrin-actin cytoskeleton linkage is strengthened when a mechanical force is loaded to it (7, 61). The strengthened linkage can sustain much larger forces (ϳ20 pN), which prevents the slippage between the actin cytoskeleton and integrin clusters (7).Vinculin also plays an important role in mediating the integrin-actin linkage because vinculin-deficient cells exhibit weaker linkage (1, 11). Vinculin binds to talin via its NH 2 -terminal domain D1, while its COOH-terminal tail domain has an actin-binding site (19,32,66). Talin has up to 11 vinculinbind...

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“…In-cell crosslinking The in-cell crosslinking of INS-1E cells was performed using 0.5 mmol/l dithiobis(succinimidyl) propionate in PBS for 3 h on ice [33]. After removal of the cross-linker, the cells were incubated 15 min with 20 mmol/l T r i s -H C l , p H 7 .…”

Section: Methodsmentioning

confidence: 99%

Protein kinase Cδ regulates nuclear export of FOXO1 through phosphorylation of the chaperone 14-3-3ζ

et al. 2015

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Aims/hypothesis Forkhead box protein O1 (FOXO1) is a transcription factor essential for beta cell fate. Protein kinase B-dependent phosphorylation of FOXO1 at S256 (P-FOXO1) enables its binding to 14-3-3 dimers and nuclear export. Dephosphorylated FOXO1 enters nuclei and activates pro-apoptotic genes. Since our previous observations suggest that protein kinase C delta (PKCδ) induces nuclear accumulation of FOXO1, the underlying mechanism was examined. Methods In human islets, genetically modified mice and INS-1E cells apoptosis was assessed by TUNEL staining. Subcellular translocation of proteins was examined by confocal microscopy and signalling pathways were analysed by western blotting and overlay assay. Results In PKCδ-overexpressing (PKCδ-tg) mouse islet cells and INS-1E cells FOXO1 accumulated in nuclei, surprisingly, as P-FOXO1. PKCδ-tg decelerated IGF-1-dependent stimulation of nuclear export, indicating that changes in export caused nuclear retention of P-FOXO1. Nuclear accumulation of P-FOXO1 was accompanied by increased phosphorylation of 14-3-3ζ at S58 and reduced dimerisation of 14-3-3ζ. Palmitic acid further augmented phosphorylation of 14-3-3ζ and triggered nuclear accumulation of FOXO1 in both INS-1E and human islet cells. Furthermore, the overexpression of a phosphomimicking mutant of 14-3-3ζ (S58D) enhanced nuclear FOXO1. In accordance with the nuclear accumulation of P-FOXO1, PKCδ overexpression alone did not increase apoptotic cell death. Additionally, insulin secretion and glucose homeostasis in PKCδ-overexpressing mice remained unaffected. Conclusions/interpretation These results suggest that PKCδ-mediated phosphorylation of 14-3-3ζ contributes to the nuclear retention of FOXO1, even when FOXO1 is phosphorylated as under non-stress conditions. P-FOXO1 does not induce pro-apoptotic genes, but may rather exert beneficial effects on beta cells.

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ReCLIP (Reversible Cross-Link Immuno-Precipitation): An Efficient Method for Interrogation of Labile Protein Complexes

Cited by 67 publications

References 44 publications

Biotin ligase tagging identifies proteins proximal to E-cadherin, including lipoma preferred partner, a regulator of epithelial cell-cell and cell-substrate adhesion

Biotin ligase tagging identifies proteins proximal to E-cadherin, including lipoma preferred partner, a regulator of epithelial cell-cell and cell-substrate adhesion

Force-dependent vinculin binding to talin in live cells: a crucial step in anchoring the actin cytoskeleton to focal adhesions

Protein kinase Cδ regulates nuclear export of FOXO1 through phosphorylation of the chaperone 14-3-3ζ

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