PIKfyve-ArPIKfyve-Sac3 Core Complex

2009/10/20 by Ognian C. Ikonomov, Diego Sbrissa, Homer Fenner +1 · 1 citation
Biochemistry, Genetics and Molecular Biology · Medicine · #Adenosine and Purinergic Signaling #Cellular transport and secretion #Pancreatic function and diabetes

paper · pdf · doi:10.1074/jbc.m109.037515

openalex publication_date 2009/10/20 · openalex created_date 2016/06/24 · openalex updated_date 2026/08/04

Abstract

The phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P(2)) metabolizing enzymes, the kinase PIKfyve and the phosphatase Sac3, constitute a single multiprotein complex organized by the PIKfyve regulator ArPIKfyve and its ability to homodimerize. We previously established that PIKfyve is activated within the triple PIKfyve-ArPIKfyve-Sac3 (PAS) core. These data assign an atypical function for the phosphatase in PtdIns(3,5)P(2) biosynthesis, thus raising the question of whether Sac3 retains its PtdIns(3,5)P(2) hydrolyzing activity within the PAS complex. Herein, we address the issue of Sac3 functionality by a combination of biochemical and morphological assays in triple-transfected COS cells using a battery of truncated or point mutants of the three proteins. We identified the Cpn60_TCP1 domain of PIKfyve as a major determinant for associating the ArPIKfyve-Sac3 subcomplex. Neither Sac3 nor PIKfyve enzymatic activities affected the PAS complex formation or stability. Using the well established formation of aberrant cell vacuoles as a sensitive functional measure of localized PtdIns(3,5)P(2) reduction, we observed a mitigated vacuolar phenotype by kinase-deficient PIKfyve(K1831E) if its ArPIKfyve-Sac3 binding region was deleted, suggesting reduced Sac3 access to, and turnover of PtdIns(3,5)P(2). In contrast, PIKfyve(K1831E), which displays intact ArPIKfyve-Sac3 binding, triggered a more severe vacuolar phenotype if coexpressed with ArPIKfyve(WT)-Sac3(WT) but minimal defects when coexpressed with ArPIKfyve(WT) and phosphatase-deficient Sac3(D488A). These data indicate that Sac3 assembled in the PAS regulatory core complex is an active PtdIns(3,5)P(2) phosphatase. Based on these and other data, presented herein, we propose a model of domain interactions within the PAS core and their role in regulating the enzymatic activities.

Citations

• PIKfyve: Partners, significance, debates and paradoxes
• ArPIKfyve–PIKfyve interaction and role in insulin-regulated GLUT4 translocation and glucose transport in 3T3-L1 adipocytes
• Kinesin Adapter JLP Links PIKfyve to Microtubule-based Endosome-to-Trans-Golgi Network Traffic of Furin
• Mammalian Cell Morphology and Endocytic Membrane Homeostasis Require Enzymatically Active Phosphoinositide 5-Kinase PIKfyve
• Phosphatidylinositol 3-Phosphate-interacting Domains in PIKfyve
• Формирование коммуникативной компетентности детей старшего дошкольного возраста в процессе обучения английскому языку
• Localized PtdIns 3,5-P2 synthesis to regulate early endosome dynamics and fusion
• A selective PIKfyve inhibitor blocks PtdIns(3,5)P2 production and disrupts endomembrane transport and retroviral budding
• The mammalian phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) regulates endosome-to-TGN retrograde transport
• VAC14 nucleates a protein complex essential for the acute interconversion of PI3P and PI(3,5)P2 in yeast and mouse
• ArPIKfyve Homomeric and Heteromeric Interactions Scaffold PIKfyve and Sac3 in a Complex to Promote PIKfyve Activity and Functionality

Cited by

• Phosphatidylinositol 3,5-bisphosphate [wikipedia]