Identification of the ligands of protein interaction domains through a functional approach

Ginevra Caratù, Danilo Allegra, Marida Bimonte, Gabriele Giacomo Schiattarella, Chiara D'Ambrosio, Andrea Scaloni, Maria Napolitano, Tommaso Russo, Nicola Zambrano

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

The identification of protein-protein interaction networks has often given important information about the functions of specific proteins and on the cross-talk among metabolic and regulatory pathways. The availability of entire genome sequences has rendered feasible the systematic screening of collections of proteins, often of unknown function, aimed to find the cognate ligands. Once identified by genetic and/or biochemical approaches, the interaction between two proteins should be validated in the physiologic environment. Herein we describe an experimental strategy to screen collections of protein-protein interaction domains to find and validate candidate interactors. The approach is based on the assumption that the overexpression in cultured cells of protein-protein interaction domains, isolated from the context of the whole protein, could titrate the endogenous ligand and, in turn, exert a dominant negative effect. The identification of the ligand could provide us with a tool to check the relevance of the interaction because the contemporary overexpression of the isolated domain and of its ligand could rescue the dominant negative phenotype. We explored this approach by analyzing the possible dominant negative effects on the cell cycle progression of a collection of phosphotyrosine binding (FTB) domains of human proteins. Of 47 PTB domains, we found that the over-expression of 10 of them significantly interfered with the cell cycle progression of NIH3T3 cells. Four of them were used as baits to identify the cognate interactors. Among these proteins, CARM1, interacting with the PTB domain of Rab-GAP1, and EF1α, interacting with RGS12, were able to rescue the block of the cell cycle induced by the isolated PTB domain of the partner protein, thus confirming in vivo the relevance of the interaction. These results suggest that the described approach can be used for the systematic screening of the ligands of various protein-protein interaction domains also by using different biological assays.

Original languageEnglish
Pages (from-to)333-345
Number of pages13
JournalMolecular and Cellular Proteomics
Volume6
Issue number2
DOIs
Publication statusPublished - Feb 2007

Fingerprint

Protein Interaction Domains and Motifs
Ligands
Cell Cycle
Proteins
Cells
Protein Interaction Maps
Phosphotyrosine
Screening
Metabolic Networks and Pathways
Biological Assay
Molecular Biology
Cultured Cells
Genome
Phenotype
Assays
Genes
Availability

ASJC Scopus subject areas

  • Biochemistry

Cite this

Caratù, G., Allegra, D., Bimonte, M., Schiattarella, G. G., D'Ambrosio, C., Scaloni, A., ... Zambrano, N. (2007). Identification of the ligands of protein interaction domains through a functional approach. Molecular and Cellular Proteomics, 6(2), 333-345. https://doi.org/10.1074/mcp.M600289-MCP200

Identification of the ligands of protein interaction domains through a functional approach. / Caratù, Ginevra; Allegra, Danilo; Bimonte, Marida; Schiattarella, Gabriele Giacomo; D'Ambrosio, Chiara; Scaloni, Andrea; Napolitano, Maria; Russo, Tommaso; Zambrano, Nicola.

In: Molecular and Cellular Proteomics, Vol. 6, No. 2, 02.2007, p. 333-345.

Research output: Contribution to journalArticle

Caratù, G, Allegra, D, Bimonte, M, Schiattarella, GG, D'Ambrosio, C, Scaloni, A, Napolitano, M, Russo, T & Zambrano, N 2007, 'Identification of the ligands of protein interaction domains through a functional approach', Molecular and Cellular Proteomics, vol. 6, no. 2, pp. 333-345. https://doi.org/10.1074/mcp.M600289-MCP200
Caratù G, Allegra D, Bimonte M, Schiattarella GG, D'Ambrosio C, Scaloni A et al. Identification of the ligands of protein interaction domains through a functional approach. Molecular and Cellular Proteomics. 2007 Feb;6(2):333-345. https://doi.org/10.1074/mcp.M600289-MCP200
Caratù, Ginevra ; Allegra, Danilo ; Bimonte, Marida ; Schiattarella, Gabriele Giacomo ; D'Ambrosio, Chiara ; Scaloni, Andrea ; Napolitano, Maria ; Russo, Tommaso ; Zambrano, Nicola. / Identification of the ligands of protein interaction domains through a functional approach. In: Molecular and Cellular Proteomics. 2007 ; Vol. 6, No. 2. pp. 333-345.
@article{a6d1a7845b164639841b350a3b1c2b50,
title = "Identification of the ligands of protein interaction domains through a functional approach",
abstract = "The identification of protein-protein interaction networks has often given important information about the functions of specific proteins and on the cross-talk among metabolic and regulatory pathways. The availability of entire genome sequences has rendered feasible the systematic screening of collections of proteins, often of unknown function, aimed to find the cognate ligands. Once identified by genetic and/or biochemical approaches, the interaction between two proteins should be validated in the physiologic environment. Herein we describe an experimental strategy to screen collections of protein-protein interaction domains to find and validate candidate interactors. The approach is based on the assumption that the overexpression in cultured cells of protein-protein interaction domains, isolated from the context of the whole protein, could titrate the endogenous ligand and, in turn, exert a dominant negative effect. The identification of the ligand could provide us with a tool to check the relevance of the interaction because the contemporary overexpression of the isolated domain and of its ligand could rescue the dominant negative phenotype. We explored this approach by analyzing the possible dominant negative effects on the cell cycle progression of a collection of phosphotyrosine binding (FTB) domains of human proteins. Of 47 PTB domains, we found that the over-expression of 10 of them significantly interfered with the cell cycle progression of NIH3T3 cells. Four of them were used as baits to identify the cognate interactors. Among these proteins, CARM1, interacting with the PTB domain of Rab-GAP1, and EF1α, interacting with RGS12, were able to rescue the block of the cell cycle induced by the isolated PTB domain of the partner protein, thus confirming in vivo the relevance of the interaction. These results suggest that the described approach can be used for the systematic screening of the ligands of various protein-protein interaction domains also by using different biological assays.",
author = "Ginevra Carat{\`u} and Danilo Allegra and Marida Bimonte and Schiattarella, {Gabriele Giacomo} and Chiara D'Ambrosio and Andrea Scaloni and Maria Napolitano and Tommaso Russo and Nicola Zambrano",
year = "2007",
month = "2",
doi = "10.1074/mcp.M600289-MCP200",
language = "English",
volume = "6",
pages = "333--345",
journal = "Molecular and Cellular Proteomics",
issn = "1535-9476",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "2",

}

TY - JOUR

T1 - Identification of the ligands of protein interaction domains through a functional approach

AU - Caratù, Ginevra

AU - Allegra, Danilo

AU - Bimonte, Marida

AU - Schiattarella, Gabriele Giacomo

AU - D'Ambrosio, Chiara

AU - Scaloni, Andrea

AU - Napolitano, Maria

AU - Russo, Tommaso

AU - Zambrano, Nicola

PY - 2007/2

Y1 - 2007/2

N2 - The identification of protein-protein interaction networks has often given important information about the functions of specific proteins and on the cross-talk among metabolic and regulatory pathways. The availability of entire genome sequences has rendered feasible the systematic screening of collections of proteins, often of unknown function, aimed to find the cognate ligands. Once identified by genetic and/or biochemical approaches, the interaction between two proteins should be validated in the physiologic environment. Herein we describe an experimental strategy to screen collections of protein-protein interaction domains to find and validate candidate interactors. The approach is based on the assumption that the overexpression in cultured cells of protein-protein interaction domains, isolated from the context of the whole protein, could titrate the endogenous ligand and, in turn, exert a dominant negative effect. The identification of the ligand could provide us with a tool to check the relevance of the interaction because the contemporary overexpression of the isolated domain and of its ligand could rescue the dominant negative phenotype. We explored this approach by analyzing the possible dominant negative effects on the cell cycle progression of a collection of phosphotyrosine binding (FTB) domains of human proteins. Of 47 PTB domains, we found that the over-expression of 10 of them significantly interfered with the cell cycle progression of NIH3T3 cells. Four of them were used as baits to identify the cognate interactors. Among these proteins, CARM1, interacting with the PTB domain of Rab-GAP1, and EF1α, interacting with RGS12, were able to rescue the block of the cell cycle induced by the isolated PTB domain of the partner protein, thus confirming in vivo the relevance of the interaction. These results suggest that the described approach can be used for the systematic screening of the ligands of various protein-protein interaction domains also by using different biological assays.

AB - The identification of protein-protein interaction networks has often given important information about the functions of specific proteins and on the cross-talk among metabolic and regulatory pathways. The availability of entire genome sequences has rendered feasible the systematic screening of collections of proteins, often of unknown function, aimed to find the cognate ligands. Once identified by genetic and/or biochemical approaches, the interaction between two proteins should be validated in the physiologic environment. Herein we describe an experimental strategy to screen collections of protein-protein interaction domains to find and validate candidate interactors. The approach is based on the assumption that the overexpression in cultured cells of protein-protein interaction domains, isolated from the context of the whole protein, could titrate the endogenous ligand and, in turn, exert a dominant negative effect. The identification of the ligand could provide us with a tool to check the relevance of the interaction because the contemporary overexpression of the isolated domain and of its ligand could rescue the dominant negative phenotype. We explored this approach by analyzing the possible dominant negative effects on the cell cycle progression of a collection of phosphotyrosine binding (FTB) domains of human proteins. Of 47 PTB domains, we found that the over-expression of 10 of them significantly interfered with the cell cycle progression of NIH3T3 cells. Four of them were used as baits to identify the cognate interactors. Among these proteins, CARM1, interacting with the PTB domain of Rab-GAP1, and EF1α, interacting with RGS12, were able to rescue the block of the cell cycle induced by the isolated PTB domain of the partner protein, thus confirming in vivo the relevance of the interaction. These results suggest that the described approach can be used for the systematic screening of the ligands of various protein-protein interaction domains also by using different biological assays.

UR - http://www.scopus.com/inward/record.url?scp=33847632605&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33847632605&partnerID=8YFLogxK

U2 - 10.1074/mcp.M600289-MCP200

DO - 10.1074/mcp.M600289-MCP200

M3 - Article

VL - 6

SP - 333

EP - 345

JO - Molecular and Cellular Proteomics

JF - Molecular and Cellular Proteomics

SN - 1535-9476

IS - 2

ER -