PIKfyve regulation of endosome-linked pathways.
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.
de Lartigue
Jane
J
Secretory Pathways Laboratory, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, UK.
Polson
Hannah
H
Feldman
Morri
M
Shokat
Kevan
K
Tooze
Sharon A
SA
Urbé
Sylvie
S
Clague
Michael J
MJ
D000730:Androstadienes / Q000378:metabolism; D000818:Animals; D001343:Autophagy / Q000502:physiology; D016827:CD8 Antigens / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism; D045683:Furin / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D015394:Molecular Structure; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D017527:Receptor, IGF Type 2; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D022641:Shiga Toxin 2 / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism; D000077191:Wortmannin; D021601:trans-Golgi Network / Q000378:metabolism*
D000730:Androstadienes; D016827:CD8 Antigens; D066246:ErbB Receptors; D045683:Furin; D008562:Membrane Glycoproteins; D008869:Microtubule-Associated Proteins; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; D020794:Receptor Protein-Tyrosine Kinases; D017527:Receptor, IGF Type 2; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D022641:Shiga Toxin 2; D000077191:Wortmannin; C477012:MAP1LC3A protein, human; C072237:TGOLN2 protein, human; C034304:cation-dependent mannose-6-phosphate receptor; C450841:shiga toxin 2 B subunit; C492139:PIKFYVE protein, human