PIKfyve Kinase and SKD1 AAA ATPase define distinct endocytic compartments. Only PIKfyve expression inhibits the cell-vacoulating activity of Helicobacter pylori VacA toxin.
The mammalian phosphatidylinositol (PtdIns)- 5-P/PtdIns-3,5-P(2)-producing kinase PIKfyve and AAA ATPase SKD1, as their yeast counterparts, are implicated in the formation and function of multivesicular bodies/late endosomes. Point mutations inhibiting the enzyme activities convert PIKfyve and SKD1 into dominant-negative mutants (PIKfyve(K1831E) and SKD1(E235Q)), whose expression in cells of kidney origin induces a vacuolation phenotype. This phenotype closely resembles the changes in late endosomal-lysosomal morphology that occur following cell exposure to the vacuolating cytotoxin (VacA) from Helicobacter pylori. Here we have examined the possible functional relationship between PIKfyve and SKD1 as well as the role of these enzymes in the molecular mechanism of VacA-induced intracellular vacuolation. When co-expressed in COS cells, PIKfyve(WT) reduced SKD1(E235Q)dependent vacuole formation, whereas SKD1(WT) did not alter the vacuolation induced by PIKfyve(K1831E). In addition, SKD1(E235Q) disrupted the normal distribution of PIKfyve(WT). Expression of PIKfyve(WT) in COS and HEK293 cells inhibited vacuolation induced by subsequent intoxication with VacA, and microinjection of the PIKfyve lipid product PtdIns-3,5-P(2) produced a similar inhibitory effect. In contrast, in COS cells expressing SKD1(WT), VacA induced the formation of characteristic vacuoles with an efficiency similar to that in the control cells. These observations demonstrate that, although PIKfyve and SKD1 are functionally related, only PIKfyve regulates VacA action, and suggest that the inhibition of PIKfyve PtdIns-3,5-P(2)-producing activity is a key molecular event in VacA-induced cellular vacuolation.
Ikonomov
Ognian C
OC
Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Sbrissa
Diego
D
Yoshimori
Tamotsu
T
Cover
Timothy L
TL
Shisheva
Assia
A
D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000633:toxicity; D002451:Cell Compartmentation; D002460:Cell Line; D004705:Endocytosis; D056827:Endosomal Sorting Complexes Required for Transport; D006801:Humans; D008845:Microinjections; D008856:Microscopy, Fluorescence; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism*; D025262:Vacuolar Proton-Translocating ATPases; D033921:Vesicular Transport Proteins
D001426:Bacterial Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D012097:Repressor Proteins; C074689:VacA protein, Helicobacter pylori; D033921:Vesicular Transport Proteins; D019869:Phosphatidylinositol 3-Kinases; C492139:PIKFYVE protein, human; D000251:Adenosine Triphosphatases; D025262:Vacuolar Proton-Translocating ATPases; D000074183:ATPases Associated with Diverse Cellular Activities; C490305:VPS4A protein, human