A genetic library screen for signaling proteins that interact with phosphorylated T cell costimulatory receptors.
In the past decade, the fundamental importance and therapeutic potential of costimulatory signals for lymphocyte activation have spurred a large amount of work in immunology, infection, cancer, autoimmune diseases, etc. However, the mechanisms behind T cell costimulation remain unclear, partly due to the lack of suitable techniques. There is an urgent need for functional genomic research to develop comprehensive approaches to direct identification of protein-protein interactions that are dependent on the posttranslational modification of one component of the complex, particularly in the field of T cell immunology. Using inducible costimulator (ICOS) as a model, we failed to find any proteins that associated with the cytoplasmic tail of ICOS by the yeast two-hybrid approach. Therefore, we have developed a new yeast three-hybrid system that facilitates the rapid screening of cDNA libraries to find signaling molecules that interact with phosphorylated T cell costimulatory receptors. We demonstrate the utility of this technique to detect the interaction between ICOS and the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K). The p85 unit of PI3K is the only signaling molecule identified so far that interacts with ICOS. This system may be of great help in dissecting the mechanisms of T cell costimulation and could be applied to other receptors.
Zang
Xingxing
X
Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
Loke
P'ng
P
Tropical Disease Research Unit, University of California at San Francisco, San Francisco, CA 94143, USA.
Kim
Jayon
J
Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
Wojnoonski
Kathleen
K
Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
Kusdra
Leonard
L
Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720, USA.
Allison
James P
JP
Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA. Electronic address: allisonj@mskcc.org.
D000818:Animals; D000945:Antigens, Differentiation, T-Lymphocyte / Q000737:chemistry / Q000378:metabolism*; D015723:Gene Library; D060889:Inducible T-Cell Co-Stimulator Protein; D008213:Lymphocyte Activation; D051379:Mice; D008822:Mice, Transgenic; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation; D011948:Receptors, Antigen, T-Cell / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000276:immunology; D020798:Two-Hybrid System Techniques
D000945:Antigens, Differentiation, T-Lymphocyte; D060889:Inducible T-Cell Co-Stimulator Protein; D019869:Phosphatidylinositol 3-Kinases; D011948:Receptors, Antigen, T-Cell; C556694:Icos protein, mouse