IP-FCM Measures Physiologic Protein-Protein Interactions Modulated by Signal Transduction and Small-Molecule Drug Inhibition

Stephen E P Smith, Anya T. Bida, Tessa R. Davis, Hugues Sicotte, Steven E. Patterson, Diana Gil, Adam G. Schrum

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Protein-protein interactions (PPI) mediate the formation of intermolecular networks that control biological signaling. For this reason, PPIs are of outstanding interest in pharmacology, as they display high specificity and may represent a vast pool of potentially druggable targets. However, the study of physiologic PPIs can be limited by conventional assays that often have large sample requirements and relatively low sensitivity. Here, we build on a novel method, immunoprecipitation detected by flow cytometry (IP-FCM), to assess PPI modulation during either signal transduction or pharmacologic inhibition by two different classes of small-molecule compounds. First, we showed that IP-FCM can detect statistically significant differences in samples possessing a defined PPI change as low as 10%. This sensitivity allowed IP-FCM to detect a PPI that increases transiently during T cell signaling, the antigen-inducible interaction between ZAP70 and the T cell antigen receptor (TCR)/CD3 complex. In contrast, IP-FCM detected no ZAP70 recruitment when T cells were stimulated with antigen in the presence of the src-family kinase inhibitor, PP2. Further, we tested whether IP-FCM possessed sufficient sensitivity to detect the effect of a second, rare class of compounds called SMIPPI (small-molecule inhibitor of PPI). We found that the first-generation non-optimized SMIPPI, Ro-26-4550, inhibited the IL-2:CD25 interaction detected by IP-FCM. This inhibition was detectable using either a recombinant CD25-Fc chimera or physiologic full-length CD25 captured from T cell lysates. Thus, we demonstrate that IP-FCM is a sensitive tool for measuring physiologic PPIs that are modulated by signal transduction and pharmacologic inhibition.

Original languageEnglish (US)
Article numbere45722
JournalPLoS One
Volume7
Issue number9
DOIs
StatePublished - Sep 21 2012

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Signal transduction
Flow cytometry
protein-protein interactions
Immunoprecipitation
flow cytometry
signal transduction
Signal Transduction
Flow Cytometry
drugs
Molecules
Pharmaceutical Preparations
T-lymphocytes
Proteins
T-cells
antigens
T-Lymphocytes
chimerism
pharmacology
T-Cell Antigen Receptor-CD3 Complex
interleukin-2

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Smith, S. E. P., Bida, A. T., Davis, T. R., Sicotte, H., Patterson, S. E., Gil, D., & Schrum, A. G. (2012). IP-FCM Measures Physiologic Protein-Protein Interactions Modulated by Signal Transduction and Small-Molecule Drug Inhibition. PLoS One, 7(9), [e45722]. https://doi.org/10.1371/journal.pone.0045722

IP-FCM Measures Physiologic Protein-Protein Interactions Modulated by Signal Transduction and Small-Molecule Drug Inhibition. / Smith, Stephen E P; Bida, Anya T.; Davis, Tessa R.; Sicotte, Hugues; Patterson, Steven E.; Gil, Diana; Schrum, Adam G.

In: PLoS One, Vol. 7, No. 9, e45722, 21.09.2012.

Research output: Contribution to journalArticle

Smith, Stephen E P ; Bida, Anya T. ; Davis, Tessa R. ; Sicotte, Hugues ; Patterson, Steven E. ; Gil, Diana ; Schrum, Adam G. / IP-FCM Measures Physiologic Protein-Protein Interactions Modulated by Signal Transduction and Small-Molecule Drug Inhibition. In: PLoS One. 2012 ; Vol. 7, No. 9.
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