Mammalian Cell Morphology and Endocytic Membrane Homeostasis Require Enzymatically Active Phosphoinositide 5-Kinase PIKfyve

Abstract

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

Core Protein Machinery for Mammalian Phosphatidylinositol 3,5-Bisphosphate Synthesis and Turnover That Regulates the Progression of Endosomal Transport

“…Intriguingly, Myc-Sac3/eGFPPIKfyve-or Myc-Sac3/eGFP-ArPIKfyve-positive vesicles appeared significantly enlarged as compared with the fine puncta seen typically upon expression of the eGFP-PIKfyve protein alone (Fig. 7B) (10,19,21). These data are consistent with the notion that Sac3 localizes onto ArPIKfyve/PIKfyve sites and induces vesicle enlargement because of increased rate of PtdIns(3,5)P 2 turnover.…”

“…Proteins were produced in Escherichia coli BL21(DE3) strain and purified as described (47). Construction of pCMV5-HA-hArPIKfyve, pEGFP-HA-hArPIKfyve, or pEGFP-HA-mPIKfyve WT was detailed elsewhere (10,38,43).…”

“…By contrast, ECV/MVB formation/detachment, but not the inward invagination of the MVE limiting membrane, is reportedly dependent on annexin2 (37). Whether the enlarged size of MVEs observed in cell models with loss-of-function or expressing dominant-negative mutants of PIKfyve (10,16,19,20) is associated with arrested ECV/MVB formation/detachment (or maturation) because of perturbed PtdIns(3,5)P 2 endosome membrane remodeling has never been examined.…”

“…The ability to undergo acute and reversible phosphorylation by kinases and phosphatases makes the PIs indispensable and versatile membrane-anchored signals that, by recruiting distinct effector proteins, govern diverse and essential cellular processes, such as intracellular membrane trafficking and signaling (3)(4)(5)(6)(7)(8)(9). PtdIns(3,5)P 2 , one of the seven PIs, is widespread in eukaryotes but is present only in minute quantity, comprising as little as 0.8% of total PIs (10,11). Identified first in smooth muscle cells ϳ17 years ago (12), steady-state PtdIns(3,5)P 2 levels are now detected in all mammalian cell types examined as well as in yeast and plants (13).…”

Assembly of a Fab1 Phosphoinositide Kinase Signaling Complex Requires the Fig4 Phosphoinositide Phosphatase

“…Although it is likely that Fab1 CCR(C/A) , and especially Fab1 CCT(T/I) , are hypomorphs rather than representing full impairment of the respective domains, our data still support an important role for the GroL-related region. This is also consistent with inactivation of PIKfyve upon deletion of the CCT domain Ikonomov et al, 2001).…”