Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes

Trinkle-Mulcahy, Laura; Boulon, Séverine; Lam, Yun Wah; Urcia, Roby; Boisvert, François-Michel; Vandermoere, Franck; Morrice, Nick; Swift, Sam; Rothbauer, Ulrich; Leonhardt, Heinrich; Lamond, Angus I.

doi:10.1083/jcb.200805092

Cited by 412 publications

(450 citation statements)

References 43 publications

Supporting

Mentioning

440

Contrasting

Unclassified

Order By: Relevance

“…2). The list was refined to 209 by curation against a database of polypeptides that nonselectively bind to anti-FLAG beads from untransfected DSP cross-linked cell extracts (26)(27)(28). A total of 93 of these proteins were enriched more than twofold and 73 were enriched more than fivefold in the Ano1-expressing cells ( Fig.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Anoctamin 1 (Tmem16A) Ca ² ⁺ -activated chloride channel stoichiometrically interacts with an ezrin–radixin–moesin network

Pérez‐Cornejo

Gokhale

Duran

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

114

139

View full text Add to dashboard Cite

The newly discovered Ca 2+ -activated Cl − channel (CaCC), Anoctamin 1 (Ano1 or TMEM16A), has been implicated in vital physiological functions including epithelial fluid secretion, gut motility, and smooth muscle tone. Overexpression of Ano1 in HEK cells or Xenopus oocytes is sufficient to generate Ca 2+ -activated Cl − currents, but the details of channel composition and the regulatory factors that control channel biology are incompletely understood. We used a highly sensitive quantitative SILAC proteomics approach to obtain insights into stoichiometric protein networks associated with the Ano1 channel. These studies provide a comprehensive footprint of putative Ano1 regulatory networks. We find that Ano1 associates with the signaling/scaffolding proteins ezrin, radixin, moesin, and RhoA, which link the plasma membrane to the cytoskeleton with very high stoichiometry. Ano1, ezrin, and moesin/radixin colocalize apically in salivary gland epithelial cells, and overexpression of moesin and Ano1 in HEK cells alters the subcellular localization of both proteins. Moreover, interfering RNA for moesin modifies Ano1 current without affecting its surface expression level. Another network associated with Ano1 includes the SNARE and SM proteins VAMP3, syntaxins 2 and -4, and syntaxin-binding proteins munc18b and munc18c, which are integral to translocation of vesicles to the plasma membrane. A number of other regulatory proteins, including GTPases, Ca 2+ -binding proteins, kinases, and lipid-interacting proteins are enriched in the Ano1 complex. These data provide stoichiometrically prioritized information about mechanisms regulating Ano1 function and trafficking to polarized domains of the plasma membrane.calcium | interactome | cross-linker | apical targeting

show abstract

Section: Resultsmentioning

confidence: 99%

“…2 and Dataset S1). A twofold SILAC enrichment is a stringent cutoff criterion to reliably detect differences between two samples (26,28,29).…”

Section: Resultsmentioning

confidence: 99%

Anoctamin 1 (Tmem16A) Ca ² ⁺ -activated chloride channel stoichiometrically interacts with an ezrin–radixin–moesin network

Pérez‐Cornejo

Gokhale

Duran

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

114

139

View full text Add to dashboard Cite

show abstract

“…16,45,77,78 SMN is thought to dissociate from snRNPs soon after their import, as the protein does not co-purify with mature snRNP mono-particles. 79,80 Once released from the SMN complex, the newly assembled snRNP is then free to diffuse throughout the interchromatin space. The fate of the SMN complex following snRNP release in the Cajal body is mostly unknown.…”

Section: Nuclear Functions Of the Smn Complexmentioning

confidence: 99%

SMN - A chaperone for nuclear RNP social occasions?

2016

View full text Add to dashboard Cite

Survival Motor Neuron (SMN) protein localizes to both the nucleus and the cytoplasm. Cytoplasmic SMN is diffusely localized in large oligomeric complexes with core member proteins, called Gemins. Biochemical and cell biological studies have demonstrated that the SMN complex is required for the cytoplasmic assembly and nuclear transport of Sm-class ribonucleoproteins (RNPs). Nuclear SMN accumulates with spliceosomal small nuclear (sn)RNPs in Cajal bodies, sub-domains involved in multiple facets of snRNP maturation. Thus, the SMN complex forms stable associations with both nuclear and cytoplasmic snRNPs, and plays a critical role in their biogenesis. In this review, we focus on potential functions of the nuclear SMN complex, with particular emphasis on its role within the Cajal body.

show abstract

“…Therefore, strategies commonly used to identify binding partners based on the overexpression of the protein bait may not always identify functional binders because functional interactions may on occasion occur through modifications or conformations not known a priori and not necessarily present in overexpressed proteins. We designed an AP-MS strategy based on quantitative LC-MS, which is emerging as a powerful method for the identification of protein-protein interactions (26,(38)(39)(40) to identify functional binding partners of endogenous class IA PI3K. The reasoning was that regulatory proteins would be present at different levels in PI3K IPs extracted from stimulated and resting cells.…”

Section: Discussionmentioning

confidence: 99%

Calpain interacts with class IA phosphoinositide 3-kinases regulating their stability and signaling activity

Beltran¹,

Chaussade

Vanhaesebroeck

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Class IA phosphoinositide 3-kinases (PI3Ks) are signaling enzymes with key roles in the regulation of essential cellular functions and disease, including cancer. Accordingly, their activity is tightly controlled in cells to maintain homeostasis. The formation of multiprotein complexes is a ubiquitous mechanism to regulate enzyme activity but the contribution of protein-protein interactions to the regulation of PI3K signaling is not fully understood. We designed an affinity purification quantitative mass spectrometry strategy to identify proteins interacting dynamically with PI3K in response to pathway activation, with the view that such binding partners may have a functional role in pathway regulation. Our study reveals that calpain small subunit 1 interacts with PI3K and that the association between these proteins is lower in cells stimulated with serum compared to starved cells. Calpain and PI3K activity assays confirmed these results, thus demonstrating that active calpain heterodimers associate dynamically with PI3K. In addition, calpains were found to cleave PI3K proteins in vitro (resulting in a reduction of PI3K lipid kinase activity) and to regulate endogenous PI3K protein levels in vivo. Further investigations revealed that calpains have a role in the negative regulation of PI3K/Akt pathway activity (as measured by Akt and ribosomal S6 phosphorylation) and that their inhibition promotes cell survival during serum starvation. These results indicate that the interaction between calpain and PI3K is a novel mechanism for the regulation of class IA PI3K stability and activity.lipid signaling | proteomics | quantitative analysis

show abstract

Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes

Abstract: Abbreviations used in this paper: FP, fl uorescent protein; SILAC, stable isotope labeling with amino acids in cell culture.The online version of this article contains supplemental material.

Cited by 412 publications

References 43 publications

Anoctamin 1 (Tmem16A) Ca ² ⁺ -activated chloride channel stoichiometrically interacts with an ezrin–radixin–moesin network

Anoctamin 1 (Tmem16A) Ca ² ⁺ -activated chloride channel stoichiometrically interacts with an ezrin–radixin–moesin network

SMN - A chaperone for nuclear RNP social occasions?

Calpain interacts with class IA phosphoinositide 3-kinases regulating their stability and signaling activity

Contact Info

Product

Resources

About

Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes

Abstract: Abbreviations used in this paper: FP, fl uorescent protein; SILAC, stable isotope labeling with amino acids in cell culture.The online version of this article contains supplemental material.

Cited by 412 publications

References 43 publications

Anoctamin 1 (Tmem16A) Ca 2 + -activated chloride channel stoichiometrically interacts with an ezrin–radixin–moesin network

Anoctamin 1 (Tmem16A) Ca 2 + -activated chloride channel stoichiometrically interacts with an ezrin–radixin–moesin network

SMN - A chaperone for nuclear RNP social occasions?

Calpain interacts with class IA phosphoinositide 3-kinases regulating their stability and signaling activity

Contact Info

Product

Resources

About

Anoctamin 1 (Tmem16A) Ca ² ⁺ -activated chloride channel stoichiometrically interacts with an ezrin–radixin–moesin network

Anoctamin 1 (Tmem16A) Ca ² ⁺ -activated chloride channel stoichiometrically interacts with an ezrin–radixin–moesin network