An Autoinhibitory Role for the Pleckstrin Homology Domain of Interleukin-2-Inducible Tyrosine Kinase and Its Interplay with Canonical Phospholipid Recognition

Biochemistry. 2017 Jun 13;56(23):2938-2949. doi: 10.1021/acs.biochem.6b01182. Epub 2017 May 25.

Abstract

Pleckstrin homology (PH) domains are well-known as phospholipid binding modules, yet evidence that PH domain function extends beyond lipid recognition is mounting. In this work, we characterize a protein binding function for the PH domain of interleukin-2-inducible tyrosine kinase (ITK), an immune cell specific signaling protein that belongs to the TEC family of nonreceptor tyrosine kinases. Its N-terminal PH domain is a well-characterized lipid binding module that localizes ITK to the membrane via phosphatidylinositol 3,4,5-trisphosphate (PIP3) binding. Using a combination of nuclear magnetic resonance spectroscopy and mutagenesis, we have mapped an autoregulatory protein interaction site on the ITK PH domain that makes direct contact with the catalytic kinase domain of ITK, inhibiting the phospho-transfer reaction. Moreover, we have elucidated an important interplay between lipid binding by the ITK PH domain and the stability of the autoinhibitory complex formed by full length ITK. The ITK activation loop in the kinase domain becomes accessible to phosphorylation to the exogenous kinase LCK upon binding of the ITK PH domain to PIP3. By clarifying the allosteric role of the ITK PH domain in controlling ITK function, we have expanded the functional repertoire of the PH domain generally and opened the door to alternative strategies to target this specific kinase in the context of immune cell signaling.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Allosteric Regulation
  • Amino Acid Substitution
  • Animals
  • Binding Sites
  • Catalytic Domain
  • Enzyme Stability
  • Lipid Bilayers / chemistry
  • Lipid Bilayers / metabolism*
  • Mice
  • Models, Molecular*
  • Mutagenesis, Site-Directed
  • Mutation
  • Nuclear Magnetic Resonance, Biomolecular
  • Peptide Fragments / antagonists & inhibitors
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Phosphatidylinositol Phosphates / chemistry
  • Phosphatidylinositol Phosphates / metabolism*
  • Phosphorylation
  • Pleckstrin Homology Domains
  • Protein Conformation
  • Protein Interaction Domains and Motifs
  • Protein Processing, Post-Translational
  • Protein Transport
  • Protein-Tyrosine Kinases / antagonists & inhibitors
  • Protein-Tyrosine Kinases / chemistry
  • Protein-Tyrosine Kinases / genetics
  • Protein-Tyrosine Kinases / metabolism*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism

Substances

  • Lipid Bilayers
  • Peptide Fragments
  • Phosphatidylinositol Phosphates
  • Recombinant Fusion Proteins
  • phosphatidylinositol 3,4,5-triphosphate
  • Protein-Tyrosine Kinases
  • emt protein-tyrosine kinase