Allosteric Activation of Kinases: Design and Application of RapR Kinases

Andrei V. Karginov1, Klaus M. Hahn1

1 Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 14.13
DOI:  10.1002/0471143030.cb1413s53
Online Posting Date:  December, 2011
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Abstract

Here we describe a method for the engineered regulation of protein kinases in living cells, the design and application of RapR (rapamycin regulated) kinases. The RapR kinase method enables activation of kinases with high specificity and precise temporal control. Insertion of an engineered allosteric switch, the iFKBP domain, at a structurally conserved position within the kinase catalytic domain makes the modified kinase inactive. Treatment with rapamycin or its non‐immunosuppressive analogs triggers interaction with a small FKBP‐rapamycin‐binding domain (FRB), restoring the activity of the kinase. The reagents used in this method are genetically encoded or membrane permeable, enabling ready application in many systems. Based on the structural similarity of kinase catalytic domains, this method is likely applicable to a wide variety of kinases. Successful regulation has already been demonstrated for three kinases representing both tyrosine and serine/threonine kinase families (p38, FAK, Src). Procedures for designing and testing RapR kinases are discussed. Curr. Protoc. Cell Biol. 53:14.13.1‐14.13.16. © 2011 by John Wiley & Sons, Inc.

Keywords: kinase; allosteric; activation; phosphorylation

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Generation of Kinase Constructs with iFKBP Insertion
  • Basic Protocol 2: Analysis of RapR‐Kinase Activity
  • Basic Protocol 3: Imaging Changes in Cell Phenotype Initiated by Activation of RapR‐Kinases in Living Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Generation of Kinase Constructs with iFKBP Insertion

  Materials
  • DNA primers for iFKBP “megaprimer” synthesis (Fig. )
  • pmyc‐iFKBP‐FAK expression plasmid (Addgene)
  • PfuTurbo DNA polymerase and 10× reaction buffer (Agilent Technologies)
  • Qiagen DNA gel purification kit or equivalent
  • pmyc‐FAK expression plasmid (developed in Dr. Steven Hanks laboratory)
  • 10 mM dNTP mix (New England Biolabs)
  • Transformation‐competent DH5α bacterial cells or equivalent (available from various molecular biology providers)
  • LB medium and agar plates ( appendix 2A) with appropriate antibiotic for selection (50 µg/ml carbenicillin for pmyc‐FAk‐based constructs)
  • Apex 2.0× Taq RED Master Mix Kit (Genesee Scientific, https://www.geneseesci.com/) or equivalent reagents
  • DNA primers for colony PCR screen (Fig. ; also see Sandhu et al., )
  • PCR tubes
  • PCR thermal cycler
  • Additional reagents and equipment for PCR (Kramer and Coen, ) and PCR screening of obtained colonies (Sandhu et al., )

Basic Protocol 2: Analysis of RapR‐Kinase Activity

  Materials
  • HEK293T cells (ATCC has only derivatives of this cell line; the closest derivative is HEK293T/17, ATCC cat. no. CRL‐11268, which was selected for high transfection efficiency and should generate same results as HEK293T cells)
  • DMEM medium containing 10% FBS ( appendix 2A) and 1× GlutaMAX supplement (Invitrogen)
  • pEGFP‐FRB plasmid (Addgene)
  • pmyc‐RapR‐FAK plasmid (Addgene)
  • pmyc‐FAK plasmid (generated in Dr. Steven Hanks laboratory)
  • pmyc‐iFKBP‐FAK plasmid (Addgene)
  • pmyc‐RapR‐FAK‐D546R plasmid (Addgene)
  • FuGene6 transfection reagent (Roche)
  • Protein G‐coupled agarose beads (Millipore or other manufacturer)
  • Lysis buffer (see recipe)
  • Bovine serum albumin (BSA)
  • 1 mg/ml 4A6 anti‐myc antibody (Millipore)
  • 1 mM rapamycin (LC Laboratories) stock solution in ethanol
  • Ethanol
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Kinase buffer (see recipe)
  • Paxillin/ATP mix (see recipe)
  • 2× Laemmli SDS‐PAGE protein sample buffer ( appendix 2A)
  • JL8 anti‐GFP antibody (Clontech)
  • Anti‐phospho‐Tyr31 paxillin antibody (Invitrogen)
  • 6‐well tissue culture plates
  • Cell scraper
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1) and immunoblotting (unit 6.2)

Basic Protocol 3: Imaging Changes in Cell Phenotype Initiated by Activation of RapR‐Kinases in Living Cells

  Materials
  • HeLa cells (ATCC, cat. no. CCL‐2)
  • DMEM medium containing 10% FBS and 1× GlutaMAX supplement (Invitrogen)
  • pGFP‐RapR‐FAK plasmid (Addgene)
  • pmCherry‐FRB plasmid (Addgene)
  • FuGene6 transfection reagent (Roche)
  • 5 mg/ml fibronectin in phosphate‐buffered saline (PBS; appendix 2A)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Mineral oil, sterile filtered, suitable for mouse embryo cell culture (Sigma‐Aldrich)
  • L15 Leibovitz medium (Invitrogen) containing 5% FBS
  • 1 mM rapamycin (LC Laboratories) stock solution in ethanol
  • 35‐mm tissue culture plates
  • 25‐mm round glass coverslips, 0.17 mm thick (Fisher Scientific)
  • Attofluor cell chamber (Invitrogen)
  • Imaging system: Olympus IX‐81 microscope equipped with an oil‐immersion 40× Olympus UIS2 DIC objective lens, a Photometrix CoolSnap ES2 CCD camera controlled by Metamorph software, and an open heated chamber (Warner Instruments)
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Figures

Videos

Literature Cited

Literature Cited
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