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Proc Natl Acad Sci U S A. 2015 Mar 31;112(13):E1594-603. doi: 10.1073/pnas.1503286112. Epub 2015 Mar 17.

Combinatorial proteomic analysis of intercellular signaling applied to the CD28 T-cell costimulatory receptor.

Author information

1
Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada M5G 1X5; Department of Chemistry and Shenzhen Key Laboratory of Cell Microenvironment, South University of Science and Technology of China, Shenzhen 518055, China; aweiss@medicine.ucsf.edu tian.rj@sustc.edu.cn.
2
Division of Rheumatology, Department of Medicine, Rosalind Russell and Ephraim P. Engleman Rheumatology Research Center, and.
3
Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada M5G 1X5;
4
Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037;
5
Department of Electrical Engineering and Computer Sciences, University of California, Berkeley, CA 94720; and.
6
Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada M5G 1X5; Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada M5S 1A8.
7
Division of Rheumatology, Department of Medicine, Rosalind Russell and Ephraim P. Engleman Rheumatology Research Center, and Howard Hughes Medical Institute, University of California, San Francisco, CA 94143; aweiss@medicine.ucsf.edu tian.rj@sustc.edu.cn.

Abstract

Systematic characterization of intercellular signaling approximating the physiological conditions of stimulation that involve direct cell-cell contact is challenging. We describe a proteomic strategy to analyze physiological signaling mediated by the T-cell costimulatory receptor CD28. We identified signaling pathways activated by CD28 during direct cell-cell contact by global analysis of protein phosphorylation. To define immediate CD28 targets, we used phosphorylated forms of the CD28 cytoplasmic region to obtain the CD28 interactome. The interaction profiles of selected CD28-interacting proteins were further characterized in vivo for amplifying the CD28 interactome. The combination of the global phosphorylation and interactome analyses revealed broad regulation of CD28 and its interactome by phosphorylation. Among the cellular phosphoproteins influenced by CD28 signaling, CapZ-interacting protein (CapZIP), a regulator of the actin cytoskeleton, was implicated by functional studies. The combinatorial approach applied herein is widely applicable for characterizing signaling networks associated with membrane receptors with short cytoplasmic tails.

KEYWORDS:

T cells; intercellular signaling; phosphorylation; proteomics; signal transduction

PMID:
25829543
PMCID:
PMC4386406
DOI:
10.1073/pnas.1503286112
[Indexed for MEDLINE]
Free PMC Article

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