Mark G. Waugh

Phosphatidylinositol 4-kinase is required for endosomal trafficking and degradation of the EGF receptor

The type II alpha isoform of phosphatidylinositol 4-kinase has recently been shown to function in the recruitment of adaptor protein-1 complexes to the trans-Golgi network. Here we show that phosphatidylinositol 4-kinase IIα is also a component of highly dynamic membranes of the endosomal system where it colocalises with protein markers of the late endosome and with endocytosed epidermal growth factor. When phosphatidylinositol 4-kinase IIα activity was inhibited in vivo using the monoclonal antibody 4C5G or by depression of endogenous phosphatidylinositol 4-kinase IIα protein levels using RNA interference, ligand-bound epidermal growth factor receptor failed to traffic to late endosomes and instead accumulated in vesicles in a sub-plasma membrane compartment. Furthermore, lysosomal degradation of activated epidermal growth factor receptor was dramatically impaired in small inhibitory RNA-treated cells. We demonstrate that phosphatidylinositol 4-kinase IIα is necessary for the correct endocytic traffic and downregulation of activated epidermal growth factor receptor.

Localization of a highly active pool of type II phosphatidylinositol 4-kinase in a p97/valosin-containing-protein-rich fraction of the endoplasmic reticulum

Different phosphoinositides are synthesized in cell membranes in order to perform a variety of functions. One of the most abundant of these lipids is phosphatidylinositol (PI) 4-phosphate (PI4P), which is formed in human eukaryotes by type II and type III phosphatidylinositol 4-kinase (PI4K II and III) activities. PI4K II activity occurs in many different subcellular membranes, although no detailed analysis of the distribution of this activity has been reported. Using density gradient ultracentrifugation, we have previously found that in A431 cells the predominant PI4K activity arises from a type II alpha enzyme that is localized to a buoyant membrane fraction of unknown origin [Waugh, Lawson, Tan and Hsuan (1998) J. Biol. Chem. 273, 17115-17121]. We show here that these buoyant membranes contain an activated form of PI4K II alpha that can be separated from the bulk of the PI4K II alpha protein in A431 and COS-7 cells. Proteomic analysis revealed that the buoyant membrane fraction contains numerous endoplasmic reticulum (ER)-marker proteins, although it was separated from the bulk of the ER, ER-Golgi intermediate compartment, transitional ER, Golgi and other major subcellular membranes. Furthermore, the majority of the cytoplasmic valosin-containing protein (VCP), an AAA+ATPase implicated in various ER, transitional ER, Golgi and nuclear functions, was almost completely localized to the same buoyant membrane fraction. Co-localization of VCP and PI4K activity was confirmed by co-immunoprecipitation. These results suggest the previously unsuspected existence of an ER-related domain in which the bulk of the cellular PI4P synthesis and VCP are localized.

Loss of phosphatidylinositol 4-kinase 2α activity causes late onset degeneration of spinal cord axons

Phosphoinositide (PI) lipids are intracellular membrane signaling intermediates and effectors produced by localized PI kinase and phosphatase activities. Although many signaling roles of PI kinases have been identified in cultured cell lines, transgenic animal studies have produced unexpected insight into the in vivo functions of specific PI 3- and 5-kinases, but no mammalian PI 4-kinase (PI4K) knockout has previously been reported. Prior studies using cultured cells implicated the PI4K2α isozyme in diverse functions, including receptor signaling, ion channel regulation, endosomal trafficking, and regulated secretion. We now show that despite these important functions, mice lacking PI4K2α kinase activity initially appear normal. However, adult Pi4k2aGT/GT animals develop a progressive neurological disease characterized by tremor, limb weakness, urinary incontinence, and premature mortality. Histological analysis of aged Pi4k2aGT/GT animals revealed lipofuscin-like deposition and gliosis in the cerebellum, and loss of Purkinje cells. Peripheral nerves are essentially normal, but massive axonal degeneration was found in the spinal cord in both ascending and descending tracts. These results reveal a previously undescribed role for aberrant PI signaling in neurological disease that resembles autosomal recessive hereditary spastic paraplegia.

Relationship between phosphatidylinositol 4-phosphate synthesis, membrane organization, and lateral diffusion of PI4KIIα at the trans-Golgi network

Type II phosphatidylinositol 4-kinase IIα (PI4KIIα) is the dominant phosphatidylinositol kinase activity measured in mammalian cells and has important functions in intracellular vesicular trafficking. Recently PI4KIIα has been shown to have important roles in neuronal survival and tumorigenesis. This study focuses on the relationship between membrane cholesterol levels, phosphatidylinositol 4-phosphate (PI4P) synthesis, and PI4KIIα mobility. Enzyme kinetic measurements, sterol substitution studies, and membrane fragmentation analyses all revealed that cholesterol regulates PI4KIIα activity indirectly through effects on membrane structure. In particular, we found that cholesterol levels determined the distribution of PI4KIIα to biophysically distinct membrane domains. Imaging studies on cells expressing enhanced green fluorescent protein (eGFP)-tagged PI4KIIα demonstrated that cholesterol depletion resulted in morphological changes to the juxtanuclear membrane pool of the enzyme. Lateral membrane diffusion of eGFP-PI4KIIα was assessed by fluorescence recovery after photobleaching (FRAP) experiments, which revealed the existence of both mobile and immobile pools of the enzyme. Sterol depletion decreased the size of the mobile pool of PI4KIIα. Further measurements revealed that the reduction in the mobile fraction of PI4KIIα correlated with a loss of trans-Golgi network (TGN) membrane connectivity. We conclude that cholesterol modulates PI4P synthesis through effects on membrane organization and enzyme diffusion.

Lipid and Peptide Control of Phosphatidylinositol 4-Kinase IIα Activity on Golgi-endosomal Rafts

The most abundant and widely expressed mammalian phosphoinositide kinase activity is contributed by phosphatidylinositol 4-kinase IIα (PI4KIIα). In this study we demonstrate that PI4KIIα is a novel GTP-independent target of the wasp venom tetradecapeptide mastoparan and that different mechanisms of activation occur in different subcellular membranes. Following cell membrane fractionation mastoparan specifically stimulated a high activity Golgi/endosomal pool of PI4KIIα independently of exogenous guanine nucleotides. Conversely, GTPγS stimulated a low activity pool of PI4KIIα in a separable dense membrane fraction and this response was further enhanced by mastoparan. Overexpression of PI4KIIα increased the basal phosphatidylinositol 4-kinase activity of each membrane pool, as well as the mastoparan-dependent activities, thereby demonstrating that mastoparan specifically activates this isozyme. Both mastoparan and M7, at concentrations known to invoke secretion, stimulated PI4KIIα with similar efficacies, resulting in an increase in the apparent Vmax and decrease in Km for exogenously added PI. Mastoparan also stimulated PI4KIIα immunoprecipitated from the raft fraction, indicating that PI4KIIα is a direct target of mastoparan. Finally we reveal a striking dependence of both basal and mastoparan-stimulated PI4KIIα activity on endogenous cholesterol concentration and therefore conclude that changes in membrane environment can regulate PI4KIIα activity.

EGF receptors as transcription factors: ridiculous or sublime?

The notion that a transmembrane receptor at the cell surface can somehow reappear as a transcription factor in the nucleus is bound to be controversial. However, there are two reported examples of this. If this hypothesis can withstand the inevitable and necessary battery of additional empirical tests then our understanding of signal transduction needs to move in a new direction.

Mammalian phosphatidylinositol 4-kinases as modulators of membrane trafficking and lipid signaling networks.

Abstract The four mammalian phosphatidylinositol 4-kinases modulate inter-organelle lipid trafficking, phosphoinositide signalling and intracellular vesicle trafficking. In addition to catalytic domains required for the synthesis of PI4P, the phosphatidylinositol 4-kinases also contain isoform-specific structural motifs that mediate interactions with proteins such as AP-3 and the E3 ubiquitin ligase Itch, and such structural differences determine isoform-specific roles in membrane trafficking. Moreover, different permutations of phosphatidylinositol 4-kinase isozymes may be required for a single cellular function such as occurs during distinct stages of GPCR signalling and in Golgi to lysosome trafficking. Phosphatidylinositol 4-kinases have recently been implicated in human disease. Emerging paradigms include increased phosphatidylinositol 4-kinase expression in some cancers, impaired functioning associated with neurological pathologies, the subversion of PI4P trafficking functions in bacterial infection and the activation of lipid kinase activity in viral disease. We discuss how the diverse and sometimes overlapping functions of the phosphatidylinositol 4-kinases present challenges for the design of isoform-specific inhibitors in a therapeutic context.

Detergent-free isolation and characterization of cholesterol-rich membrane domains from trans-Golgi network vesicles.

Cholesterol is an abundant lipid of the trans-Golgi network (TGN) and of certain endosomal membranes where cholesterol-rich microdomains are important in the organization and compartmentalization of vesicular trafficking. Here we describe the development of a rapid method to isolate a cholesterol-rich endomembrane fraction. We show that widely used subcellular fractionation techniques incompletely separate cholesterol-rich membranes, such as the TGN, from organelles, such as late endosomes and lysosomes. To address this issue, we devised a new subcellular fractionation scheme involving two rounds of velocity centrifugation, membrane sonication, and discontinuous sucrose density gradient centrifugation. This strategy resulted in the isolation of a cholesterol and GM1 glycosphingolipid-enriched membrane fraction that was completely cleared of plasma membrane, endoplasmic reticulum, and mitochondria. This buoyant fraction was enriched for the TGN and recycling endosome proteins Rab11 and syntaxin-6, and it was well resolved from cis-Golgi and early and late endosomal membranes. We demonstrate that this technique can give useful insights into the compartmentation of phosphoinositide synthesis, and it facilitates the isolation of cholesterol-rich membranes from a population of TGN-trafficking vesicles.

The Phosphatidylinositol 4-Kinases: Don’t Call it a Comeback

Phosphatidylinositol 4-phosphate (PtdIns4P) is a quantitatively minor membrane phospholipid which is the precursor of PtdIns(4,5)P (2) in the classical agonist-regulated phospholipase C signalling pathway. However, PtdIns4P also governs the recruitment and function of numerous trafficking molecules, principally in the Golgi complex. The majority of phosphoinositides (PIs) phosphorylated at the D4 position of the inositol headgroup are derived from PtdIns4P and play roles in a diverse array of fundamental cellular processes including secretion, cell migration, apoptosis and mitogenesis; therefore, PtdIns4P biosynthesis can be regarded as key point of regulation in many PI-dependent processes.Two structurally distinct sequence families, the type II and type III PtdIns 4-kinases, are responsible for PtdIns4P synthesis in eukaryotic organisms. These important proteins are differentially expressed, localised and regulated by distinct mechanisms, indicating that the enzymes perform non-redundant roles in trafficking and signalling. In recent years, major advances have been made in our understanding of PtdIns4K biology and here we summarise current knowledge of PtdIns4K structure, function and regulation.

Identification and characterization of differentially active pools of type IIalpha phosphatidylinositol 4-kinase activity in unstimulated A431 cells.

The seven known polyphosphoinositides have been implicated in a wide range of regulated and constitutive cell functions, including cell-surface signalling, vesicle trafficking and cytoskeletal reorganization. In order to understand the spatial and temporal control of these diverse cell functions it is necessary to characterize the subcellular distribution of a wide variety of polyphosphoinositide synthesis and signalling events. The predominant phosphatidylinositol kinase activity in many mammalian cell types involves the synthesis of the signalling precursor, phosphatidylinositol 4-phosphate, in a reaction catalysed by the recently cloned PI4KIIalpha (type IIalpha phosphatidylinositol 4-kinase). However the regulation of this enzyme and the cellular distribution of its product in different organelles are very poorly understood. This report identifies the existence, in unstimulated cells, of two major subcellular membrane fractions, which contain PI4KIIalpha possessing different levels of intrinsic activity. Separation of these membranes from each other and from contaminating activities was achieved by density gradient ultracentrifugation at pH 11 in a specific detergent mixture in which both membrane fractions, but not other membranes, were insoluble. Kinetic comparison of the purified membrane fractions revealed a 4-fold difference in K (m) for phosphatidylinositol and a 3.5-fold difference in V (max), thereby indicating a different mechanism of regulation to that described previously for agonist-stimulated cells. These marked differences in basal activity and the occurrence of this isozyme in multiple organelles emphasize the need to investigate cell signalling via PI4KIIalpha at the level of individual organelles rather than whole-cell lysates.