Analysing c-kit internalization using a functional c-kit-EGFP chimera containing the fluorochrome within the extracellular domain

Oncogene. 2002 Jul 4;21(29):4508-20. doi: 10.1038/sj.onc.1205559.


In order to investigate activation and internalization of c-kit we created a functional c-kit-EGFP chimera by inserting EYFP (enhanced yellow fluorescent protein) within the extracellular domain of c-kit immediately downstream of the signal sequence, SS-EYFP-kit. This location was chosen because the C-terminal fusion of EGFP to c-kit unexpectedly caused constitutive activation of the c-kit tyrosine kinase. As analysed in fixed cells and by real time imaging in vivo, SCF induced activation led to internalization of the fusion construct and translocation to punctate structures resembling vesicles. Analysis of the internalization process by time lapse imaging revealed high mobility and discontinuous movement of these vesicles and their predominantly radial tracks. Two subsets of vesicles were observed: Traffic of the majority of vesicles was directed from the periphery to the center of the cell and most likely represents the internalization of activated receptor molecules via the endosomal pathway. However, some vesicular structures were observed to move towards the periphery of the cell and probably contain newly synthesized protein to replace internalized receptor molecules. The calculated velocity of moving vesicles ranged from 0.05 to 0.2 microm per se. Vesicle formation upon SCF induced dimerization of the receptor was strictly dependent on kinase activity of c-kit. Treatment of cells with phenylarsine oxide, an agent blocking receptor internalization, prior to SCF stimulation resulted in abrogation of the translocation of the chimera to vesicles whereas accumulation of vesicles was observed when cells were treated with proteasome inhibitors. Cholesterol depletion of the cell membrane by methyl-beta-cyclodextrin resulted in dose dependent reduction of receptor internalization indicating that c-kit may be present in lipid rafts or that intact lipid rafts are required for efficient internalization of the receptor. Using the induction of vesicular structures as a sign of efficient internalization of the receptor analysis of mutant c-kit constructs deficient either in activation of PI3-Kinase or Src revealed that internalization of c-kit is dependent on recruitment of Src but not PI3-Kinase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Arsenicals / pharmacology
  • Blotting, Western
  • COS Cells
  • Cell Line
  • Cholesterol / metabolism
  • Cloning, Molecular
  • Cyclodextrins / pharmacology
  • Cysteine Endopeptidases / metabolism
  • Dimerization
  • Endocytosis* / drug effects
  • Endosomes / drug effects
  • Endosomes / metabolism
  • Enzyme Activation
  • Flow Cytometry
  • Green Fluorescent Proteins
  • Luminescent Proteins / chemistry
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism*
  • Membrane Microdomains / drug effects
  • Membrane Microdomains / metabolism
  • Microscopy, Fluorescence
  • Multienzyme Complexes / antagonists & inhibitors
  • Multienzyme Complexes / metabolism
  • Proteasome Endopeptidase Complex
  • Protein Structure, Tertiary
  • Protein Transport / drug effects
  • Proto-Oncogene Proteins c-kit / chemistry
  • Proto-Oncogene Proteins c-kit / genetics
  • Proto-Oncogene Proteins c-kit / metabolism*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Time Factors
  • Tyrosine / genetics
  • Tyrosine / metabolism
  • beta-Cyclodextrins*


  • Arsenicals
  • Cyclodextrins
  • Luminescent Proteins
  • Multienzyme Complexes
  • Recombinant Fusion Proteins
  • beta-Cyclodextrins
  • methyl-beta-cyclodextrin
  • oxophenylarsine
  • Green Fluorescent Proteins
  • Tyrosine
  • Cholesterol
  • Proto-Oncogene Proteins c-kit
  • Cysteine Endopeptidases
  • Proteasome Endopeptidase Complex