ArPIKfyve Homomeric and Heteromeric Interactions Scaffold PIKfyve and Sac3 in a Complex to Promote PIKfyve Activity and Functionality

Abstract

PtdIns(3,5)P 2 is vital in differentiation and development of multicellular organisms because the knockout of the PtdIns(3,5)P 2 -synthesizing enzyme PIKfyve or its associated regulator, ArPIKfyve, is lethal. In previous work with endogenous proteins we identified that Sac3, a phosphatase that turns over PtdIns(3,5)P 2 , associates with the PIKfyve-ArPIKfyve biosynthetic complex. However, whether the three proteins suffice for the organization/maintenance of this complex (referred to as PAS complex), how they interact with each other and what the functional relevance of this ternary association would be, remained unresolved. Using coimmunoprecipitation analyses in transfected mammalian cells with increased or decreased levels of the three proteins, singly or in double vs. triple combinations, we report here that the triad is sufficient to form and maintain the PAS complex. ArPIKfyve is the principal organizer interacting with both Sac3 and PIKfyve, whereas Sac3 is permissive for maximal PIKfyve-ArPIKfyve association in the PAS complex. We further identified that ArPIKfyve scaffolds the PAS complex through homomeric interactions, mediated via its conserved C-terminal domain. Introduction of the C-terminal peptide fragment of the ArPIKfyveArPIKfyve contact sites effectively disassembled the PAS complex and reduced the in vitro PIKfyve lipid kinase activity. Exploring insulin-regulated GLUT4 translocation in 3T3L1 adipocytes as a functional readout, a process that is positively regulated by PIKfyve activity and ArPIKfyve levels, we determined that ectopic expression of the ArPIKfyve C-terminal peptide inhibits GLUT4 surface accumulation. Our data indicate that the PAS complex is organized to provide optimal PIKfyve functionality and is maintained via ArPIKfyve homomeric and heteromeric interactions.

PIKfyve-ArPIKfyve-Sac3 Core Complex

“…In line with the requirement for PtdIns(3,5)P 2 in maintaining proper balance between membrane removal (fission) and membrane insertion (fusion), disrupted function of PIKfyve, the sole enzyme for PtdIns(3,5)P 2 synthesis, is phenotypically manifested by endosome vesicle swelling and endomembrane vacuolation seen in a number of mammalian cell types (7). As unraveled recently, PIKfyve is engaged in an unusual physical interaction with the phosphatase Sac3 that turns over PtdIns(3,5)P 2 , forming a common endogenous complex (the PAS core complex) organized by the PIKfyve activator ArPIKfyve (9,14). The ternary association, scaffolded by ArPIKfyve homomeric interactions, activates the PIKfyve kinase as evidenced by recent data for reduced PIKfyve activity upon disintegration of the PAS core (14).…”

The Phosphoinositide Kinase PIKfyve Is Vital in Early Embryonic Development

“…WT/KO -We have recently revealed that PIKfyve associates with the ArPIKfyve scaffold and Sac3 phosphatase in a regulatory ternary assembly, called the PAS complex, that relays two opposing enzymatic activities, synthesis and breakdown of PtdIns(3,5)P 2 (17,18). Considering a potential regulatory mechanism at the level of the Sac3 phosphatase as a means to counterbalance the loss of PtdIns(3,5)P 2 (and probably PtdIns5P) in the PIKfyve WT/KO genotype, we next examined the total protein levels of Sac3 and ArPIKfyve as well as their relative amounts engaged in the association with PIKfyve.…”

“…the PtdIns(3,5)P 2 -specific phosphatase Sac3, which turns over PtdIns(3,5)P 2 to PtdIns3P (13,14). This interaction is indirect and is promoted by ArPIKfyve, an adapter protein that associates with and stabilizes Sac3 in a heterooligomeric complex (15)(16)(17). PIKfyve binds efficiently only to the ArPIKfyve-Sac3 complex to form a triple heterooligomer in which Sac3 fulfills a dual function (17,18).…”

“…This interaction is indirect and is promoted by ArPIKfyve, an adapter protein that associates with and stabilizes Sac3 in a heterooligomeric complex (15)(16)(17). PIKfyve binds efficiently only to the ArPIKfyve-Sac3 complex to form a triple heterooligomer in which Sac3 fulfills a dual function (17,18). Not only does it turn over PtdIns (3,5)P 2 at the site of production, but together with ArPIKfyve, it secures robust PtdIns(3,5)P 2 synthesis through activating PIKfyve (17,18).…”

“…PIKfyve binds efficiently only to the ArPIKfyve-Sac3 complex to form a triple heterooligomer in which Sac3 fulfills a dual function (17,18). Not only does it turn over PtdIns (3,5)P 2 at the site of production, but together with ArPIKfyve, it secures robust PtdIns(3,5)P 2 synthesis through activating PIKfyve (17,18). Clearly, the PIKfyve molecule displays a unique combination of features and binding partners, reflecting the critical requirement for its rapid recruitment at the proper endosomes and a dynamic balance between the substrate and the product of its enzymatic activity.…”