Approaches to Studying Arf GAPs in Cells: In Vitro Assay with Isolated Focal Adhesions

Pei‐Wen Chen1, Xiaoying Jian1, Ruibai Luo1, Paul A. Randazzo1

1 Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 17.13
DOI:  10.1002/0471143030.cb1713s55
Online Posting Date:  June, 2012
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Abstract

The Arf GAPs are a family of proteins with a common catalytic function of hydrolyzing GTP bound to ADP‐ribosylation factors (Arf) with proposed cellular functions that are diverse (Inoue and Randazzo, 2007; Kahn et al., 2008). Understanding the biochemistry of the Arf GAPs is valuable for designing and interpreting experiments using standard cell biology techniques described elsewhere. The following briefly reviews some common approaches for in vivo studies of Arf GAPs and discusses the use of isolated cellular organelles to complement in vivo experiments. Detailed protocols for examining the activity of Arf GAPs in whole cell lysates and in association with isolated focal adhesions are provided. Curr. Protoc. Cell Biol. 55:17.13.1‐17.13.20. © 2012 by John Wiley & Sons, Inc.

Keywords: ADP‐ribosylation factor; GTPase‐activating protein; guanine nucleotide binding protein; Arf1; Arf6

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Cell Lysates for GAP Assay
  • Basic Protocol 2: Preparation of FA Fraction
  • Basic Protocol 3: Expression of Human Myristoylated Arf1, Arf5, and Arf6 in E. coli
  • Basic Protocol 4: Isolation of MyrArf1 or MyrArf5 Protein
  • Basic Protocol 5: Isolation of MyrArf6‐GDP
  • Basic Protocol 6: Loading Arf with [α32P]GTP to form Substrate for Arf GAPs
  • Basic Protocol 7: Measuring GAP Activity in Cell Lysates or Isolated FAs
  • Support Protocol 1: Determining Binding Stoichiometry of myrArf Protein for GTP
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Cell Lysates for GAP Assay

  Materials
  • HeLa cells maintained at 37°C in Dulbecco's modified Eagle's medium supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% fetal bovine serum (FBS)
  • Plasmids expressing Arf GAP or mutants of Arf GAP (e.g., ASAP1 plasmids, Randazzo et al., )
  • PBS (Quality Biological)
  • Lysis buffer for HeLa cells (see recipe)
  • BioRad protein assay reagent
  • Dilution buffer (lysis buffer without 1% Triton X‐100)
  • 1% Triton X‐100 ( appendix 2A)
  • 6‐well tissue culture plate
  • 37°C, 5% CO 2 incubator
  • Cell scraper (SARSTEDT)
  • 1.5‐ml microcentrifuge tubes

Basic Protocol 2: Preparation of FA Fraction

  Materials
  • Fibronectin (from bovine plasma; Sigma)
  • Phosphate buffered saline (PBS; Quality Biological Inc.)
  • HeLa cells maintained at 37°C in Dulbecco's modified Eagle's medium supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% fetal bovine serum (FBS)
  • Plasmids expressing Arf GAP or mutants of Arf GAP
  • Hypotonic lysis buffer, ice cold (see recipe)
  • 12‐well tissue culture plates (Corning) or microscope coverslips (no. 1.5, 12‐mm circle; Fisher)
  • 37°C, 5% CO 2 incubator
  • Repetitive pipettor (Eppendorf)

Basic Protocol 3: Expression of Human Myristoylated Arf1, Arf5, and Arf6 in E. coli

  Materials
  • Bacteria co‐transformed with expression plasmids for Arf and yeast N‐myristoyltransferase glycerol stocks
  • LB agar plate containing 100 µg/ml ampicillin and 50 µg/ml kanamycin
  • LB broth containing 100 µg/ml ampicillin and 50 µg/ml kanamycin
  • 50 µM sodium myristate (mol. wt. 250.4, 12.52 mg/1 liter)
  • 100 mM isopropyl β‐D‐1‐thiogalactopyranoside (IPTG)
  • 37°C incubator and bacteria shaker
  • Spectrophotometer
  • Refrigerated centrifuge
  • Room temperature bacteria shaker

Basic Protocol 4: Isolation of MyrArf1 or MyrArf5 Protein

  Materials
  • 2‐liter bacterial culture
  • T 20M 100M 1D 1 (see recipe)
  • Complete protease inhibitor tablets (Roche)
  • 5 M NaCl ( appendix 2A)
  • HiLoad 16/10 phenyl Sepharose HP column (GE Healthcare Life Sciences)
  • SDS‐PAGE apparatus (unit 6.1)
  • Coomassie blue dye
  • Hiload 26/60 Superdex 75 column (GE Healthcare Life Sciences)
  • Buffer for phenylsepharose column (T 50N 3000M 1D 1; see recipe)
  • French press or cell disruptor (e.g., Microfluidics M‐110P)
  • Refrigerated ultracentrifuge with Ti45 fixed‐angle rotor (Beckman)
  • 5‐ml HiTrap Q HP column (GE Healthcare Life Sciences)
  • AKTA FPLC (GE Healthcare Life Sciences) or similar protein chromatography system
  • 50‐ml Amicon stirred ultrafiltration cell attached to nitrogen gas tank or 50‐ml Amicon centrifugal filters (Ultracel 10K)
  • Amicon 43‐mm YM‐10 filters (if using the ultrafiltration cell)

Basic Protocol 5: Isolation of MyrArf6‐GDP

  Materials
  • Bacterial culture pellet
  • T 20N 25M 1D 1G 10 plus protease inhibitor (see recipe)
  • T 20N 100M 1D 1 (see recipe)
  • Ammonium sulfate
  • 0.5 M EDTA ( appendix 2A)
  • GDP, disodium salt (Sigma)
  • GST‐GEP100 sec7‐PH (protein preparation described in Sakurai et al., )
  • Glutathione Sepharose 4B beads (GE Healthcare)
  • Hiload 16/60 Superdex 75 column
  • SDS‐PAGE
  • Microfluidics M‐110P cell disruptor
  • Refrigerated centrifuge

Basic Protocol 6: Loading Arf with [α32P]GTP to form Substrate for Arf GAPs

  Materials
  • Lipids (Avanti Polar Lipids)
    • Phosphatidylcholine (PC, chicken egg)
    • Phosphatidylethanolamine (PE, bovine liver)
    • Phosphatidylserine (PS, porcine brain)
    • Phosphatidylinositol (PI, bovine liver)
    • Phosphatidylinositol 4,5‐bisphosphate (PIP2, porcine brain)
    • Cholesterol
    • Phosphatidylinositol 3,4,5‐trisphosphate (PIP3)
  • Chloroform
  • Nitrogen source
  • Lipid hydration buffer (see )
  • Ethanol/dry ice bath
  • 5× exchange buffer (see recipe)
  • 32P]GTP (Perkin Elmer)
  • 100 µM GTP
  • 2.5 mM MgCl 2 ( appendix 2A)
  • Arf
  • 12 ×75–mm siliconized glass tube
  • Lyophilizer
  • Vortexer
  • 37°C water bath
  • Lipid extruder (Avanti Polar Lipids)
  • Whatman Nuclepore Track Etched membrane with 1.0‐µm pore size

Basic Protocol 7: Measuring GAP Activity in Cell Lysates or Isolated FAs

  Materials
  • myrArf protein labeled with [α32P]GTP (see protocol 6)
  • Cell lysates
  • 5× GAP reaction buffer (see recipe)
  • LUVs
  • T 25N 100M 10D 1 wash buffer (see recipe)
  • 2 N formic acid
  • 1 N LiCl
  • Focal adhesion preparations (FAs)
  • Nitrocellulose membranes
  • Vacuum manifold
  • Polyethyleneimine‐cellulose (TLC) plate
  • Phosphorimager (e.g., StormImager, GE Healthcare Lifesciences) with screens
  • 12‐well plates

Support Protocol 1: Determining Binding Stoichiometry of myrArf Protein for GTP

  Materials
  • 5× exchange buffer (see protocol 6, except with the addition of 50 µM GTPγS)
  • LUVs
  • myrArf proteins
  • [35S]GTPγS (PerkinElmer)
  • T 25N 100M 10D 1 (see recipe)
  • Nitrocellulose filters
  • Liquid scintillation counter
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Figures

Videos

Literature Cited

Literature Cited
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