Assia Shisheva

PIKfyve, a mammalian ortholog of yeast Fab1p lipid kinase, synthesizes 5-phosphoinositides. Effect of insulin.

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-P2, 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-P2 by HPLC analyses. Synthetic PtdIns 3-P substrates were also converted to PtdIns 3,5-P2 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-P2 generation, implying PIKfyve has a key role in their biosynthesis.

PIKfyve: Partners, significance, debates and paradoxes.

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.

Complementation Analysis in PtdInsPKinase-deficient Yeast Mutants Demonstrates ThatSchizosaccharomyces pombe and Murine Fab1p Homologues Are Phosphatidylinositol 3-Phosphate 5-Kinases

Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P 2) is widespread in eukaryotic cells. In Saccharomyces cerevisiae,PtdIns(3,5)P 2 synthesis is catalyzed by the PtdIns3P 5-kinase Fab1p, and loss of this activity results in vacuolar morphological defects, indicating that PtdIns(3,5)P 2 is essential for vacuole homeostasis. We have therefore suggested that all Fab1p homologues may be PtdIns3P 5-kinases involved in membrane trafficking. It is unclear which phosphatidylinositol phosphate kinases (PIPkins) are responsible for PtdIns(3,5)P 2synthesis in higher eukaryotes. To clarify how PtdIns(3,5)P 2 is synthesized in mammalian and other cells, we determined whether yeast and mammalian Fab1p homologues or mammalian Type I PIPkins (PtdIns4P 5-kinases) make PtdIns(3,5)P 2 in vivo. The recently cloned murine (p235) and Schizosaccharomyces pombe FAB1homologues both restored basal PtdIns(3,5)P 2synthesis in Δfab1 cells and made PtdIns(3,5)P 2 in vitro. Only p235 corrected the growth and vacuolar defects of fab1 S. cerevisiae. A mammalian Type I PIPkin supported no PtdIns(3,5)P 2 synthesis. Thus, FAB1and its homologues constitute a distinct class of Type III PIPkins dedicated to PtdIns(3,5)P 2 synthesis. The differential abilities of p235 and of SpFab1p to complement the phenotypic defects of Δfab1 cells suggests that interaction(s) with other protein factors may be important for spatial and/or temporal regulation of PtdIns(3,5)P 2synthesis. These results also suggest that p235 may regulate a step in membrane trafficking in mammalian cells that is analogous to its function in yeast.

Cloning, Characterization, and Expression of a Novel Zn2+-Binding FYVE Finger-Containing Phosphoinositide Kinase in Insulin-Sensitive Cells

ABSTRACT Signaling by phosphorylated species of phosphatidylinositol (PI) appears to regulate diverse responses in eukaryotic cells. A differential display screen for fat- and muscle-specific transcripts led to identification and cloning of the full-length cDNA of a novel mammalian 2,052-amino-acid protein (p235) from a mouse adipocyte cDNA library. Analysis of the deduced amino acid sequence revealed that p235 contains an N-terminal zinc-binding FYVE finger, a chaperonin-like region in the middle of the molecule, and a consensus for phosphoinositide 5-kinases at the C terminus. p235 mRNA appears as a 9-kb transcript, enriched in insulin-sensitive cells and tissues, likely transcribed from a single-copy gene in at least two close-in-size splice variants. Specific antibodies against mouse p235 were raised, and both the endogenously and heterologously expressed proteins were biochemically detected in 3T3-L1 adipocytes and transfected COS cells, respectively. Immunofluorescence microscopy analysis of endogenous p235 localization in 3T3-L1 adipocytes with affinity-purified anti-p235 antibodies documented a punctate peripheral pattern. In COS cells, the expressed p235 N-terminal but not the C-terminal region displayed a vesicular pattern similar to that in 3T3-L1 adipocytes that became diffuse upon Zn2+ chelation or FYVE finger truncation. A recombinant protein comprising the N-terminal but not the C-terminal region of the molecule was found to bind 2.2 mole equivalents of Zn2+. Determination of the lipid kinase activity in the p235 immunoprecipitates derived from 3T3-L1 adipocytes or from COS cells transiently expressing p235 revealed that p235 displayed unique preferences for PI substrate over already phosphorylated PI. In conclusion, the mouse p235 protein determines an important novel class of phosphoinositide kinases that seems to be targeted to specific intracellular loci by a Zn-dependent mechanism.

PIKfyve: The road to PtdIns 5-P and PtdIns 3,5-P2

Phosphorylated derivatives of phosphatidylinositol (PdtIns), collectively called phosphoinositides (PIs), are an attribute of eukaryotic cell membranes. Besides structural functions, PIs are involved in complex intracellular regulation. They all stem from the same precursor, PtdIns, and differ by the degree and position of phosphorylation of the inositol head group (reviewed in Fruman et al., 1998). Out of the five candidate phosphorylation positions, only the hydroxyls at positions D-3, D-4 and D-5 are found to be phosphorylated intracellularly, separately or in all possible combinations, resulting in seven phosphoinositide species, i.e., PtdIns 3-P, PtdIns 4-P, PtdIns 5-P, PtdIns 3,4-P2, PtdIns 4,5-P2, PtdIns 3,5-P2 and PtdIns 3,4,5-P3. In mammalian cells, the most abundant are PtdIns 4,5-P2 and PtdIns 4-P. PtdIns 3-P and PtdIns 5-P, which are found in comparable quantities and represent only 0.25% of cellular PI (Rameh et al., 1997; Sbrissa et al., 2000; Ikonomov et al., 2001). The levels of the higher 3 -phosphorylated species are typically low or absent, and are subject to regulation by extracellular stimuli. Although PIs represent only a small percentage of total cellular phospholipids, they play a crucial role in regulating diverse cellular processes in eukaryotes. Not only do they provide a source of second messengers, such as DAG and IP3, but are now recognized as effector molecules in their own right. Several hallmarks make PIs ideally suited for this mission. First, PI levels can be acutely modulated by regulating the enzymatic activities responsible for their biosynthesis (kinases) or turnover (phosphatases). Next, being membrane-bound, PIs

The Phosphoinositide Kinase PIKfyve Mediates Epidermal Growth Factor Receptor Trafficking to the Nucleus

ErbB receptor tyrosine kinases can transit to nuclei in tumor cells, where they have been shown to regulate gene expression as components of transcriptional complexes. Quantitative analysis of a human bladder cancer tissue microarray identified nuclear epidermal growth factor receptor (EGFR) in tumor cells and also showed an increased frequency of this histologic feature in cancer relative to normal tissues. This observation suggests a potential role for nuclear EGFR in bladder cancer. We confirmed that EGFR could be induced to transit to nuclei in cultured human bladder cancer cells in response to the urothelial cell growth factor and EGFR ligand heparin-binding EGF-like growth factor (HB-EGF). Mass spectrometric analysis of EGFR immune complexes from a transitional carcinoma cell line (TCCSUP) identified the phosphoinositide kinase, PIKfyve, as a potential component of the EGFR trafficking mechanism. RNA silencing indicated that PIKfyve is a mediator of HB-EGF-stimulated EGFR nuclear trafficking, EGFR binding to the cyclin D1 promoter, and cell cycle progression. These results identify a novel mediator of the EGFR transcription function and further suggest that nuclear EGFR and the lipid kinase PIKfyve may play a role in bladder oncogenesis.

A Mammalian Ortholog of Saccharomyces cerevisiae Vac14 That Associates with and Up-Regulates PIKfyve Phosphoinositide 5-Kinase Activity

ABSTRACT Multivesicular body morphology and size are controlled in part by PtdIns(3,5)P2, produced in mammalian cells by PIKfyve-directed phosphorylation of PtdIns(3)P. Here we identify human Vac14 (hVac14), an evolutionarily conserved protein, present in all eukaryotes but studied principally in yeast thus far, as a novel positive regulator of PIKfyve enzymatic activity. In mammalian cells and tissues, Vac14 is a low-abundance 82-kDa protein, but its endogenous levels could be up-regulated upon ectopic expression of hVac14. PIKfyve and hVac14 largely cofractionated, populated similar intracellular locales, and physically associated. A small-interfering RNA-directed gene-silencing approach to selectively eliminate endogenous hVac14 rendered HEK293 cells susceptible to morphological alterations similar to those observed upon expression of PIKfyve mutants deficient in PtdIns(3,5)P2 production. Largely decreased in vitro PIKfyve kinase activity and unaltered PIKfyve protein levels were detected under these conditions. Conversely, ectopic expression of hVac14 increased the intrinsic PIKfyve lipid kinase activity. Concordantly, intracellular PtdIns(3)P-to-PtdIns(3,5)P2 conversion was perturbed by hVac14 depletion and was elevated upon ectopic expression of hVac14. These data demonstrate a major role of the PIKfyve-associated hVac14 protein in activating PIKfyve and thereby regulating PtdIns(3,5)P2 synthesis and endomembrane homeostasis in mammalian cells.

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

PtdIns(3,5)P(2) (with PtdIns indicating phosphatidylinositol) is vital in the differentiation and development of multicellular organisms because knockout of the PtdIns(3,5)P(2)-synthesizing enzyme PIKfyve (phosphoinositide kinase for position 5 containing a FYVE finger domain) 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 the PAS (PIKfyve-ArPIKfyve-Sac3) complex], how they interact with one another, and what the functional relevance of this ternary association would be remained unresolved. Using co-immunoprecipitation analyses in transfected mammalian cells with increased or decreased levels of the three proteins, singly or in double versus triple combinations, herein we report 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 ArPIKfyve-ArPIKfyve 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.

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.

YM201636, an inhibitor of retroviral budding and PIKfyve-catalyzed PtdIns(3,5)P2 synthesis, halts glucose entry by insulin in adipocytes.

Silencing of PIKfyve, the sole enzyme for PtdIns(3,5)P(2) biosynthesis that controls proper endosome dynamics, inhibits retroviral replication. A novel PIKfyve-specific inhibitor YM201636 disrupts retroviral budding at 800 nM, suggesting its potential use as an antiretroviral therapeutic. Because PIKfyve is also required for optimal insulin activation of GLUT4 surface translocation and glucose influx, we tested the outcome of YM201636 application on insulin responsiveness in 3T3L1 adipocytes. YM201636 almost completely inhibited basal and insulin-activated 2-deoxyglucose uptake at doses as low as 160 nM, with IC(50)=54+/-4 nM for the net insulin response. Insulin-induced GLUT4 translocation was partially inhibited at substantially higher doses, comparable to those required for inhibition of insulin-induced phosphorylation of Akt/PKB. In addition to PIKfyve, YM201636 also completely inhibited insulin-dependent activation of class IA PI 3-kinase. We suggest that apart from PIKfyve, there are at least two additional targets for YM201636 in the context of insulin signaling to GLUT4 and glucose uptake: the insulin-activated class IA PI 3-kinase and a here-unidentified high-affinity target responsible for the greater inhibition of glucose entry vs. GLUT4 translocation. The profound inhibition of the net insulin effect on glucose influx at YM201636 doses markedly lower than those required for efficient retroviral budding disruption warns of severe perturbations in glucose homeostasis associated with potential YM201636 use in antiretroviral therapy.