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

被引:29
作者
Caratu, Ginevral
Allegra, Danilo
Bimonte, Marida
Schiattarella, Gabriele Giacomo
D'Ambrosio, Chiara
Scaloni, Andrea
Napolitano, Maria
Russo, Tommaso
Zambrano, Nicola
机构
[1] Univ Naples Federico II, Dipartimento Biochim & Biotecnol Med, CEINGE Biotecnol Avanzate, I-80145 Naples, Italy
[2] Fdn Pascales, Ist Nazl Tumori, I-80131 Naples, Italy
[3] CNR, ISPAAM, Proteom & Mass Spectrometry Lab, I-80147 Naples, Italy
关键词
D O I
10.1074/mcp.M600289-MCP200
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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 (PTB) domains of human proteins. Of 47 PTB domains, we found that the overexpression 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 EF1 alpha, 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.
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页码:333 / 345
页数:13
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