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.

Key components of membrane trafficking and signaling machinery in eukaryotic cells are proteins that bind or synthesize phosphoinositides. PIKfyve, a product of an evolutionarily conserved single-copy gene has both these features. It binds to membrane phosphatidylinositol (PtdIns)3P and synthesizes PtdIns(3,5)P2 and PtdIns5P. Molecular functions of PIKfyve are elusive but recent advances are consistent with a key role in the course of endosomal transport. PIKfyve dysfunction induces endosome enlargement and profound cytoplasmic vacuolation, likely as a result of impaired normal endosome processing and membrane exit out of endosomes. Multicellular organisms with genetically impaired function of PIKfyve or that of the PIKfyve protein partners regulating PtdIns(3,5)P2 homeostasis display severe disorders, including embryonic/perinatal death. This review describes recent advances on PIKfyve functionality in higher eukaryotes, with particular reference to biochemical and genetic insights in PIKfyve protein partners.

Myotubularin and related proteins constitute a large and highly conserved family possessing phosphoinositide 3-phosphatase activity, although not all members possess this activity. This family contains a conserved region called the GRAM domain that is found in a variety of proteins associated with membrane-coupled processes and signal transduction. Mutations of myotubularin are found in X-linked myotubular myopathy, a severe muscle disease. Mutations in the GRAM domain are responsible for this condition, suggesting crucial roles for this region. Here, we show that the GRAM domain of myotubularin binds to phosphoinositide with the highest affinity to phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P 2 ). In patients with myotubular myopathy, mutations in the myotubularin GRAM domain eliminate this binding, indicating that the PtdIns(3,5)P 2 binding ability of the GRAM (glucosyltransferases, Rablike GTPase activators and myotubularin) domain is crucial for the functions of myotubularin in vivo. Stimulation of epidermal growth factor recruits myotubularin to the late endosomal compartment in a manner dependent on the phosphoinositide binding. Overexpression of myotubularin inhibits epidermal growth factor receptor trafficking from late endosome to lysosome and induces the large endosomal vacuoles. Thus, our data suggest that myotubularin phosphatase physiologically functions in late endosomal trafficking and vacuolar morphology through interaction with PtdIns(3,5)P 2 .In eukaryotic cells, D3-phosphorylated phosphoinositides such as phosphatidylinositol 3-phosphate (PtdIns3P) 1 play key roles in the vesicular trafficking through direct interaction with phosphoinositide-binding domains such as the PH domain, FYVE finger domain, and PX (Phox) domain found in effector proteins that control vesicular trafficking (1-3). Previous studies have revealed that PtdIns3P binding is essential for the recruitment/activation of these effector proteins at unique membrane sites (4, 5). PtdIns(3,5)P 2 was one of the phosphoinositide species identified recently in both yeast and mammalian cells (6). PtdIns(3,5)P 2 is thought to be involved in osmotic stress responsiveness and essential for the maintenance of vacuole size and homeostasis in yeast (7). Recently, it was reported that PtdIns(3,5)P 2 is necessary for late endosomal trafficking in yeast (8). However, the mechanisms for cellular PtdIns(3,5)P 2 regulation are unknown. Intracellular levels of these phosphoinositide species are strictly regulated by enzymes that dephosphorylate at the D3-position of the inositol ring. Myotubularin and its related proteins (myotubularinrelated proteins; MTMRs) constitute a large and highly conserved subfamily of dual specific phosphatases that were recently revealed to be phosphoinositide 3-phosphatases (9 -11). Among those proteins, myotubularin is encoded by the MTM1 gene, which is mutated in X-linked myotubular myopathy (12), whereas MTMR2 is associated with neurodegenerative disorder Charcot-Marie-tooth disease type 4B (13). Myopathy patie...