Dissecting Kinase Effector Signaling Using the RapRTAP Methodology

Methods Mol Biol. 2017:1636:21-33. doi: 10.1007/978-1-4939-7154-1_2.

Abstract

Kinases are involved in a broad spectrum of cell behaviors. A single kinase can interact with different ligands each eliciting a specific cellular response. Dissecting downstream signaling pathways of kinases is a key step to understanding physiological and pathological cell process. However, directing kinase activity to specific substrates remains challenging. Here, we present a new tool to selectively activate a kinase in a specific protein complex in living cells. This technology uses a rapamycin-inducible kinase activation coupled to interaction with FKBP12-binding domain (FRB) tagged protein. Here, we demonstrate application of this method by targeting Src to either p130Cas or FAK and discriminating cell mophodynamic changes downstream each of these signaling complexes.

Keywords: FKBP12; Kinase; Phosphorylation; Rapamycin; Targeted signaling pathway.

Publication types

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

MeSH terms

  • Animals
  • Carrier Proteins
  • Focal Adhesion Kinase 1 / metabolism
  • Gene Expression Regulation / drug effects*
  • Humans
  • Molecular Imaging
  • Phosphorylation
  • Protein Binding
  • Protein Interaction Domains and Motifs*
  • Protein Kinases / metabolism*
  • Signal Transduction / drug effects*
  • Sirolimus / pharmacology*
  • Tacrolimus Binding Protein 1A / chemistry*
  • Tacrolimus Binding Protein 1A / metabolism*
  • src-Family Kinases / metabolism

Substances

  • Carrier Proteins
  • Protein Kinases
  • Focal Adhesion Kinase 1
  • src-Family Kinases
  • Tacrolimus Binding Protein 1A
  • Sirolimus