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

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


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / metabolism
  • CD28 Antigens / metabolism*
  • Cell Communication*
  • Cell Line, Tumor
  • Cytoskeleton / metabolism
  • Enzyme-Linked Immunosorbent Assay
  • Flow Cytometry
  • Gene Expression Regulation*
  • Humans
  • Intracellular Signaling Peptides and Proteins / metabolism
  • Jurkat Cells
  • Mass Spectrometry
  • Phosphoproteins / metabolism
  • Phosphorylation
  • Protein Binding
  • Protein Structure, Tertiary
  • Proteomics
  • Receptors, Peptide / metabolism*
  • Signal Transduction


  • Actins
  • CD28 Antigens
  • Intracellular Signaling Peptides and Proteins
  • Phosphoproteins
  • Receptors, Peptide