PIKfyve Regulation of Endosome-Linked Pathways

Traffic. 2009 Jul;10(7):883-93. doi: 10.1111/j.1600-0854.2009.00915.x.

Abstract

The phosphoinositide 5-kinase (PIKfyve) is a critical enzyme for the synthesis of PtdIns(3,5)P2, that has been implicated in various trafficking events associated with the endocytic pathway. We have now directly compared the effects of siRNA-mediated knockdown of PIKfyve in HeLa cells with a specific pharmacological inhibitor of enzyme activity. Both approaches induce changes in the distribution of CI-M6PR and trans-Golgi network (TGN)-46 proteins, which cycles between endosomes and TGN, leading to their accumulation in dispersed punctae, whilst the TGN marker golgin-245 retains a perinuclear disposition. Trafficking of CD8-CI-M6PR (retromer-dependent) and CD8-Furin (retromer-independent) chimeras from the cell surface to the TGN is delayed following drug administration, as is the transport of the Shiga toxin B-subunit. siRNA knockdown of PIKfyve produced no defect in epidermal growth factor receptor (EGFR) degradation, unless combined with knockdown of its activator molecule Vac14, suggesting that a low threshold of PtdIns(3,5)P2 is necessary and sufficient for this pathway. Accordingly pharmacological inhibition of PIKfyve results in a profound block to the lysosomal degradation of activated epidermal growth factor (EGF) and Met receptors. Immunofluorescence revealed EGF receptors to be trapped in the interior of a swollen endosomal compartment. In cells starved of amino acids, PIKfyve inhibition leads to the accumulation of the lipidated form of GFP-LC3, a marker of autophagosomal structures, which can be visualized as fluorescent punctae. We suggest that PIKfyve inhibition may render the late endosome/lysosome compartment refractory to fusion with both autophagosomes and with EGFR-containing multivesicular bodies.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Androstadienes / metabolism
  • Animals
  • Autophagy / physiology
  • CD8 Antigens / metabolism
  • Endosomes / metabolism*
  • ErbB Receptors / metabolism
  • Furin / metabolism
  • HeLa Cells
  • Humans
  • Membrane Glycoproteins / metabolism
  • Microtubule-Associated Proteins / genetics
  • Microtubule-Associated Proteins / metabolism
  • Molecular Structure
  • Phosphatidylinositol 3-Kinases / genetics
  • Phosphatidylinositol 3-Kinases / metabolism*
  • Phosphoinositide-3 Kinase Inhibitors
  • Protein Kinase Inhibitors / metabolism
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / metabolism
  • Receptor Protein-Tyrosine Kinases / metabolism
  • Receptor, IGF Type 2
  • Receptors, Cytoplasmic and Nuclear / metabolism
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Shiga Toxin 2 / metabolism
  • Vacuoles / metabolism
  • Wortmannin
  • trans-Golgi Network / metabolism*

Substances

  • Androstadienes
  • CD8 Antigens
  • Membrane Glycoproteins
  • Microtubule-Associated Proteins
  • Phosphoinositide-3 Kinase Inhibitors
  • Protein Kinase Inhibitors
  • RNA, Small Interfering
  • Receptor, IGF Type 2
  • Receptors, Cytoplasmic and Nuclear
  • Recombinant Fusion Proteins
  • Shiga Toxin 2
  • TGOLN2 protein, human
  • cation-dependent mannose-6-phosphate receptor
  • light chain 3, human
  • shiga toxin 2 B subunit
  • PIKFYVE protein, human
  • ErbB Receptors
  • Receptor Protein-Tyrosine Kinases
  • Furin
  • Wortmannin