Changes in Gab2 phosphorylation and interaction partners in response to interleukin (IL)-2 stimulation in T-lymphocytes

Sci Rep. 2016 Mar 30:6:23530. doi: 10.1038/srep23530.

Abstract

Interleukin-2 (IL-2) stimulation results in T-cell growth as a consequence of activation of highly sophisticated and fine-tuned signaling pathways. Despite lacking intrinsic enzymatic activity, scaffold proteins such as Gab2, play a pivotal role in IL-2-triggered signal transduction integrating, diversifying and amplifying the signal by serving as a platform for the assembly of effectors proteins. Traditionally, Gab2-mediated protein recruitment was believed to solely depend on cytokine-induced phosphotyrosine moieties. At present, phosphorylation on serine/threonine residues is also emerging as a key mediator of Gab2-dependent signal regulation. Despite its relevance, IL-2-triggered regulation on Gab2 phosphorylation is yet poorly understood. Combining antibody- and TiO2-based enrichment of the scaffold protein with SILAC quantitative mass spectrometry we disclose the prominent regulation IL-2 exerts on Gab2 serine/threonine phosphorylation by showing that at least 18 serines and 1 threonine, including previously non-reported ones, become phosphorylated in response to cytokine stimulation. Additionally, we decipher the interactome of the docking protein in resting and cytokine-treated T-lymphocytes and besides well-known Gab2 interactors we discover three novel cytokine-inducible Gab2-binding proteins. Thus, our data provide novel insights and a wealth of candidates for future studies that will shed light into the role of Gab2 in IL-2-initiated signal transduction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / genetics
  • Adaptor Proteins, Signal Transducing / metabolism*
  • Amino Acid Sequence
  • Blotting, Western
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism*
  • Cell Line, Tumor
  • Class Ia Phosphatidylinositol 3-Kinase
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Gene Expression / drug effects
  • HeLa Cells
  • Humans
  • Interleukin-2 / pharmacology*
  • Phosphatidylinositol 3-Kinases / genetics
  • Phosphatidylinositol 3-Kinases / metabolism
  • Phosphorylation / drug effects
  • Protein Binding / drug effects
  • Protein Tyrosine Phosphatase, Non-Receptor Type 11 / genetics
  • Protein Tyrosine Phosphatase, Non-Receptor Type 11 / metabolism
  • Proteomics / methods
  • Reverse Transcriptase Polymerase Chain Reaction
  • Signal Transduction / drug effects
  • Signal Transduction / genetics
  • T-Lymphocytes / drug effects*
  • T-Lymphocytes / metabolism
  • Tandem Mass Spectrometry

Substances

  • ARID3B protein, human
  • Adaptor Proteins, Signal Transducing
  • Carrier Proteins
  • DNA-Binding Proteins
  • GAB2 protein, human
  • Gab3 protein, human
  • Interleukin-2
  • PIK3R1 protein, human
  • Class Ia Phosphatidylinositol 3-Kinase
  • PTPN11 protein, human
  • Protein Tyrosine Phosphatase, Non-Receptor Type 11