The dual specificity mammalian enzyme PIKfyve phosphorylates in vitro position D-5 in phosphatidylinositol (PtdIns) and PtdIns 3-P, itself or exogenous protein substrates. Here we have addressed the crucial questions for the identity of the lipid products and the role of PIKfyve enzymatic activity in mammalian cells. CHO, HEK293, and COS cells expressing PIKfyve WT at high levels and >90% efficiencies increased selectively the intracellular PtdIns 3,5-P 2 production by 30 -55%. In these cell types Recent studies in Saccharomyces cerevisiae suggest a distinct function of PtdIns 3,5-P 2 intracellular levels in yeast membrane trafficking (9 -11). The major phenotypic characteristics resulting from inactivation of yeast fab1, whose gene product is responsible for the intracellular PtdIns 3,5-P 2 production, include severe growth defect and extremely enlarged vacuoles that occupy the majority of the cell (9, 12). Despite these severe defects, however, all transport pathways to the vacuole in Fab1p-deficient cells appear intact (11). Emr and collaborators (11) therefore suggest that Fab1p kinase and PtdIns 3,5-P 2 function to maintain vacuolar size and membrane homeostasis by regulating recycling/turnover of membranes from the yeast vacuolar surface to earlier compartments.PIKfyve (phosphoinositide kinase for five position containing a fyve finger) appears to be the mammalian ortholog of Fab1p lipid kinase that rescues the vacuolar defects in the ⌬fab1 yeast strain (13,14). In vitro, PIKfyve lipid kinase synthesizes PtdIns 5-P and PtdIns 3,5-P 2 in a wortmannin-resistant fashion (ID 50 , 600 nM) (15). Recent studies indicate that PIKfyve is also a protein kinase that likely acts in intact cells to modulate PIKfyve lipid kinase activity and/or specificity by autophosphorylation (16). PIKfyve partitions between the soluble and membrane-bound intracellular pools: the membrane-bound populations being visualized as distinct vesicles largely positive for late endosomal markers, but not for protein residents of earlier compartments in the endocytic pathway (17). This characteristic intracellular localization is most likely conferred by PIKfyve's FYVE finger, a PtdIns 3-P-binding protein module found in other mammalian proteins as a major localization determinant for the endosomal membranes, enriched in PtdIns 3-P (7, 18). However, despite this intensive characterization, the functions of PIKfyve enzymatic activity as well as the identity of its lipid or protein products in the context of live mammalian cells

One or more free hydroxyls of the phosphatidylinositol (PtdIns) head group undergo enzymatic phosphorylation, yielding phosphoinositides (PIs) with key functions in eukaryotic cellular regulation. Two such species, PtdIns 5-P and PtdIns 3,5-P 2 , have now been identified in mammalian cells, but their biosynthesis remains unclear. We have isolated a novel mammalian PI kinase, p235, whose exact substrate specificity remained to be determined (Shisheva, A., Sbrissa, D., and Ikonomov, O. (1999) Mol. Cell. Biol. 19, 623-634). Here we report that recombinant p235 expressed in COS cells, like the authentic p235 in adipocytes, displays striking specificity for PtdIns over PI substrates and generates two products identified as PtdIns 5-P and PtdIns 3,5-P 2 by HPLC analyses. Synthetic PtdIns 3-P substrates were also converted to PtdIns 3,5-P 2 but to a substantially lesser extent than PtdIns isolated from natural sources. Important properties of the p235 PI 5-kinase include high sensitivity to nonionic detergents and relative resistance to wortmannin and adenosine. By analyzing deletion mutants in a heterologous cell system, we determined that in addition to the predicted catalytic domain other regions of the molecule are critical for the p235 enzymatic activity. HPLC resolution of monophosphoinositide products, generated by p235 immune complexes derived from lysates of 3T3-L1 adipocytes acutely stimulated with insulin, revealed essentially the same PtdIns 5-P levels as the corresponding p235 immune complexes of resting cells. However, the acute insulin action resulted in an increase of a wortmannin-sensitive PtdIns 3-P peak, suggestive of a plausible recruitment of wortmannin-sensitive PI 3-kinase(s) to p235. In conclusion, mouse p235 (renamed here PIKfyve) displays a strong in vitro activity for PtdIns 5-P and PtdIns 3,5-P 2 generation, implying PIKfyve has a key role in their biosynthesis.Phosphorylated species of phosphatidylinositol (PtdIns) 1 are an attribute of eukaryotes, where they regulate diverse cellular processes such as membrane ruffling, secretion, vesicular trafficking, insulin-mediated membrane translocation of GLUT4 glucose transporters, cell adhesion, chemotaxis, DNA synthesis, and cell cycle (for recent reviews see Refs. 1-8). Although the inositol head group of PtdIns contains five candidate phosphorylation positions, the hydroxyl groups only at positions D-3, D-4, and D-5 are found to be phosphorylated intracellularly, separately, or in all possible combinations, resulting in 7 phosphoinositide (PI) species, i.e. PtdIns 3-P, PtdIns 4-P, PtdIns 5-P, PtdIns 3,4-P 2 , PtdIns 4,5-P 2 , PtdIns 3,5-P 2 , and PtdIns 3,4,5-P 3 (3, 8). A wide spectrum of phosphoinositide kinases with broad or more restricted substrate specificity are responsible for their biosynthesis. These enzymes are typically grouped in three general families on the basis of their specificity for a particular position: PI 3-kinases, PI 4-kinases, and PI 5-kinases (2-4, 7-8). PI 3-kinases, usually subdivided into three classes, catalyze ...

These data demonstrate a coupling between the machinery for PtdIns(3,5)P 2 synthesis and turnover achieved through a physical assembly of PIKfyve, ArPIKfyve, and Sac3. We suggest that the tight regulation in PtdIns(3,5)P 2 homeostasis is mechanistically linked to early endosome dynamics in the course of cargo transport.

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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.