Active PIKfyve associates with and promotes the membrane attachment of the late endosome-to-trans-Golgi network transport factor Rab9 effector p40.
PIKfyve, a kinase that displays specificity for phosphatidylinositol (PtdIns), PtdIns 3-phosphate (3-P), and proteins, is important in multivesicular body/late endocytic function. Enzymatically inactive PIKfyve mutants elicit enormous dilation of late endocytic structures, suggesting a role for PIKfyve in endosome-to-trans-Golgi network (TGN) membrane retrieval. Here we report that p40, a Rab9 effector reported previously to bind Rab9-GTP and stimulate endosome-to-TGN transport, interacts with PIKfyve as determined by yeast two-hybrid assays, glutathione S-transferase (GST) pull-down assays, and co-immunoprecipitation in doubly transfected HEK293 cells. The interaction engages the PIKfyve chaperonin domain and four out of the six C-terminally positioned kelch repeats in p40. Differential centrifugation in a HEK293 cell line, stably expressing PIKfyveWT, showed the membrane-associated immunoreactive p40 co-sedimenting with PIKfyve in the high speed pellet (HSP) fraction. Remarkably, similar analysis in a HEK293 cell line stably expressing dominant-negative kinase-deficient PIKfyveK1831E demonstrated a marked depletion of p40 from the HSP fraction. GST-p40 failed to specifically associate with the PIKfyve lipid products PtdIns 5-P and PtdIns 3,5-P2 in a liposome binding assay but was found to be an in vitro substrate of the PIKfyve serine kinase activity. A band with the p40 electrophoretic mobility was found to react with a phosphoserine-specific antibody mainly in the PIKfyveWT-containing fractions obtained by density gradient sedimentation of total membranes from PIKfyveWT-expressing HEK293 cells. Together these results identify the Rab9 effector p40 as a PIKfyve partner and suggest that p40-PIKfyve interaction and the subsequent PIKfyve-catalyzed p40 phosphorylation anchor p40 to discrete membranes facilitating late endosome-to-TGN transport.
Ikonomov
Ognian C
OC
Department of Physiology, Wayne State University School of Medicine, Detroit, MI 48201, USA.
Sbrissa
Diego
D
Mlak
Krzysztof
K
Deeb
Robert
R
Fligger
Jason
J
Soans
Aleric
A
Finley
Russell L
RL
Shisheva
Assia
A
D001665:Binding Sites; D002458:Cell Fractionation; D002460:Cell Line; D011992:Endosomes / Q000378:metabolism; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism* / Q000502:physiology; D010766:Phosphorylation; D021381:Protein Transport; D020798:Two-Hybrid System Techniques; D020691:rab GTP-Binding Proteins / Q000378:metabolism*; D021601:trans-Golgi Network / Q000378:metabolism
C107297:Rab9-binding protein, 40-kDa; D019869:Phosphatidylinositol 3-Kinases; C492139:PIKFYVE protein, human; D020691:rab GTP-Binding Proteins