Robert H. Michell

Phosphoinositide signalling links O -GlcNAc transferase to insulin resistance

Glucose flux through the hexosamine biosynthetic pathway leads to the post-translational modification of cytoplasmic and nuclear proteins by O-linked β-N-acetylglucosamine (O-GlcNAc). This tandem system serves as a nutrient sensor to couple systemic metabolic status to cellular regulation of signal transduction, transcription, and protein degradation. Here we show that O-GlcNAc transferase (OGT) harbours a previously unrecognized type of phosphoinositide-binding domain. After induction with insulin, phosphatidylinositol 3,4,5-trisphosphate recruits OGT from the nucleus to the plasma membrane, where the enzyme catalyses dynamic modification of the insulin signalling pathway by O-GlcNAc. This results in the alteration in phosphorylation of key signalling molecules and the attenuation of insulin signal transduction. Hepatic overexpression of OGT impairs the expression of insulin-responsive genes and causes insulin resistance and dyslipidaemia. These findings identify a molecular mechanism by which nutritional cues regulate insulin signalling through O-GlcNAc, and underscore the contribution of this modification to the aetiology of insulin resistance and type 2 diabetes.

Osmotic stress activates phosphatidylinositol-3,5-bisphosphate synthesis.

Inositol phospholipids play multiple roles in cell signalling systems. Two widespread eukaryotic phosphoinositide-based signal transduction mechanisms, phosphoinositidase C-catalysed phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) hydrolysis and 3-OH kinase-catalysed PtdIns(4,5)P2 phosphorylation, make the second messengers inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) sn-1,2-diacylglycerol and PtdIns(3,4,5)P3 (refs 1,2,3,4,5,6,7). In addition, PtdIns(4,5)P2 and PtdIns3P have been implicated in exocytosis and membrane trafficking. We now show that when the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe are hyperosmotically stressed, they rapidly synthesize phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P2) by a process that involves activation of a PtdIns3P 5-OH kinase. This PtdIns(3,5)P2 accumulation only occurs in yeasts that have an active vps34-encoded PtdIns 3-OH kinase, showing that this latter kinase makes the PtdIns3P needed for PtdIns(3,5)P2 synthesis and indicating that PtdIns(3,5)P2 may have a role in sorting vesicular proteins. PtdIns(3,5)P2 is also present in mammalian and plant cells: in monkey Cos-7 cells, its labelling is inversely related to the external osmotic pressure. The stimulation of a PtdIns3P 5-OH kinase-catalysed synthesis of PtdIns(3,5)P2, a molecule that might be a new type of phosphoinositide ‘second messenger’, thus appears to be central to a widespread and previously uncharacterized regulatory pathway.

Diacylglycerols and phosphatidates: which molecular species are intracellular messengers?

In eukaryotes, many receptor agonists use phospholipase-generated lipids as intracellular messengers. Receptor occupation stimulates the production of polyunsaturated 1,2-diacylglycerols by phosphatidylinositol-4,5-bisphosphate specific phospholipases C and/or of mono-unsaturated and saturated phosphatidates by phospholipase-D-catalysed phosphatidylcholine breakdown. The primary phospholipase products are rapidly metabolized: polyunsaturated 1,2-diacylglycerols are converted to polyunsaturated phosphatidates by diacylglycerol kinase; mono-unsaturated and saturated phosphatidates are dephosphorylated to give mono-unsaturated and saturated 1,2-diacylglycerols by phosphatidate phosphohydrolase. The phospholipase-generated polyunsaturated 1,2-diacylglycerols and mono-unsaturated and saturated phosphatidates appear to be intracellular messengers, whereas their immediate metabolites probably do not have signalling functions.

Identification of ARAP3, a novel PI3K effector regulating both Arf and Rho GTPases, by selective capture on phosphoinositide affinity matrices

We show that matrices carrying the tethered homologs of natural phosphoinositides can be used to capture and display multiple phosphoinositide binding proteins in cell and tissue extracts. We present the mass spectrometric identification of over 20 proteins isolated by this method, mostly from leukocyte extracts: they include known and novel proteins with established phosphoinositide binding domains and also known proteins with surprising and unusual phosphoinositide binding properties. One of the novel PtdIns(3,4,5)P3 binding proteins, ARAP3, has an unusual domain structure, including five predicted PH domains. We show that it is a specific PtdIns(3,4,5)P3/PtdIns(3,4)P2-stimulated Arf6 GAP both in vitro and in vivo, and both its Arf GAP and Rho GAP domains cooperate in mediating PI3K-dependent rearrangements in the cell cytoskeleton and cell shape.

Inositol derivatives: evolution and functions

Current research on inositols mainly focuses on myo-inositol (Ins) derivatives in eukaryotic cells, and in particular on the many roles of Ins phospholipids and polyphosphorylated Ins derivatives. However, inositols and their derivatives are more versatile than this — they have acquired diverse functions over the course of evolution. Given the central involvement of primordial bacteria and archaea in the emergence of eukaryotes, what is the status of inositol derivatives in these groups of organisms, and how might inositol, inositol lipids and inositol phosphates have become ubiquitous constituents of eukaryotes? And how, later, might the multifarious functions of inositol derivatives have emerged during eukaryote diversification?

Inositol phospholipids in membrane function

Abstract Inositol lipids appear to be intimately involved in Ca 2+ -mediated control of cell functions by hormones and other ligands, in cell proliferation and in the attachment of enzymes to the plasma membrane.

Svp1p defines a family of phosphatidylinositol 3,5‐bisphosphate effectors

Phosphatidylinositol 3,5‐bisphosphate (PtdIns(3,5)P2), made by Fab1p, is essential for vesicle recycling from vacuole/lysosomal compartments and for protein sorting into multivesicular bodies. To isolate PtdIns(3,5)P2 effectors, we identified Saccharomyces cerevisiae mutants that display fab1Δ‐like vacuole enlargement, one of which lacked the SVP1/YFR021w/ATG18 gene. Expressed Svp1p displays PtdIns(3,5)P2 binding of exquisite specificity, GFP‐Svp1p localises to the vacuole membrane in a Fab1p‐dependent manner, and svp1Δ cells fail to recycle a marker protein from the vacuole to the Golgi. Cells lacking Svp1p accumulate abnormally large amounts of PtdIns(3,5)P2. These observations identify Svp1p as a PtdIns(3,5)P2 effector required for PtdIns(3,5)P2‐dependent membrane recycling from the vacuole. Other Svp1p‐related proteins, including human and Drosophila homologues, bind PtdIns(3,5)P2 similarly. Svp1p and related proteins almost certainly fold as β‐propellers, and the PtdIns(3,5)P2‐binding site is on the β‐propeller. It is likely that many of the Svp1p‐related proteins that are ubiquitous throughout the eukaryotes are PtdIns(3,5)P2 effectors. Svp1p is not involved in the contributions of FAB1/PtdIns(3,5)P2 to MVB sorting or to vacuole acidification and so additional PtdIns(3,5)P2 effectors must exist.

The stress-activated phosphatidylinositol 3-phosphate 5-kinase Fab1p is essential for vacuole function in S. cerevisiae

Polyphosphoinositides have many roles in cell signalling and vesicle trafficking [1-3]. Phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), a recently discovered PIP2 isomer, is ubiquitous in eukaryotic cells and rapidly accumulates in hyperosmotically stressed yeast. PI(3,5)P2 is synthesised from PI(3)P in both yeast and mammalian cells [4,5]. A search of the Saccharomyces cerevisiae genome database identified FAB1, a gene encoding a PIP kinase homologue and potential PI(3)P 5-kinase. Fab1p shows PI(3)P 5-kinase activity both in vivo and in vitro. A yeast strain in which FAB1 had been deleted was unable to synthesise PI(3,5)P2, either in the presence or absence of osmotic shock. A loss of PI(3,5)P2 was observed also in a temperature-sensitive FAB1 strain at the non-permissive temperature. A recombinant glutathione-S-transferase (GST)-Fab1p fusion protein was shown to have selective PI(3)P 5-kinase activity in vitro. Thus, we have demonstrated that Fab1p is a PI(3)P-specific 5-kinase and represents a third class of PIP kinase activity, which we have termed type III. Deletion of the FAB1 gene produces a loss of vacuolar morphology [6]; it is therefore concluded that PI(3,5)P2, the lipid product of Fab1p, is required for normal vacuolar function.

Phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate: Lipids in search of a function

Recent years have seen a rapid accumulation of information which relates to the idea that receptor-controlled effects on inositol lipid metabolism might somehow be involved in the control of stimulated cells (see refs 1–14 for reviews). Most of these studies have been concerned only with phosphatidylinositol (PtdIns) and have tended to ignore the polyphosphoinositides, phosphatidyl-inositol 4-phosphate (PtdIns4P), and phosphatidylinositol 4,5-bisphosphate (PtdIns4,5P2) (see Fig. 1 for structures). Although relatively few people work on these minor, but very active, lipids at present, recent observations should soon attract others into this field. It was with this in mind that we decided to collect together some thoughts on the published work on polyphosphoinositides, together with ideas and strictures that might inform future work. Detailed background information and discussion of early work can be found in reviews (1,9,15–18), and references to this work will usually be made via these reviews.

A purified plasma membrane fraction isolated from rat liver under isotonic conditions.

Abstract A fraction rich in plasma membrane has been isolated from perfused rat liver. The purity of the preparation is comparable with that of material isolated by other procedures and the yield of the purified fraction is appreciably greater. Isotonic sucrose is used in the early stages of the cell fractionation and it is therefore possible to isolate both a plasma membrane fraction and other cell components from the same homogenate.