Shamshad Cockcroft

Polyphosphoinositide phosphodiesterase: regulation by a novel guanine nucleotide binding protein, Gp

Abstract A novel guanine nucleotide binding protein, G p , may be invoveld in coupling receptor activation to the breakdown of phosphatidylinositol 4,5-bisphosphate by a phosphodiesterase. This generates two second messengers, inositol 1,4,5,-triphosphate and diacylglycerol.

Stimulated neutrophils from patients with autosomal recessive chronic granulomatous disease fail to phosphorylate a Mr-44,000 protein.

Phagocytosing neutrophils, monocytes, macrophages and eosino-phils produce a burst of non-mitochondrial respiration that is important for the killing and digestion of microbes. Much of the information about the oxidase system involved comes from studies on patients with chronic granulomatous disease (CGD), a syndrome in which an undue predisposition to infection results from complete absence of this burst of stimulated respiratory activity1. The basis of the oxidase activity is an electron transport chain, the only established component of which is a very unusual b-type cyto-chrome (b−245) (ref. 2). The molecular defect in the X-linked subgroup of CGD is the absence of this cytochrome b−245, which, however, appears to be normal in those subjects with the autosomal recessive mode of inheritance3. In an attempt to identify an abnormality of activation, or an absence or malfunction of a proximal component of the electron transport chain in this latter group, we examined protein phosphorylation in neutrophils after activation of the oxidase with phorbol myristate acetate. All four of the patients studied demonstrated a selective lack of the enhanced phosphorylation of a protein of relative molecular mass (Mr) 44,000 (44K) that was observed in normal subjects and in two CGD patients with an X-linked inheritance. This molecule, therefore, could be an important functional component of the oxidase.

Characterization of p150, an Adaptor Protein for the Human Phosphatidylinositol (PtdIns) 3-Kinase SUBSTRATE PRESENTATION BY PHOSPHATIDYLINOSITOL TRANSFER PROTEIN TO THE p150·;PtdIns 3-KINASE COMPLEX

Genetic and biochemical studies have shown that the phosphatidylinositol (PtdIns) 3-kinase encoded by the yeast VPS34 gene is required for the efficient sorting and delivery of proteins to the vacuole. A human homologue of the yeast VPS34 gene product has recently been characterized as part of a complex with a cellular protein of 150 kDa (Volinia, S., Dhand, R., Vanhaesebroeck, B., MacDougall, L. K., Stein, R., Zvelebil, M. J., Domin, J., Panaretou, C., and Waterfield, M. D. (1995) EMBO J. 14, 3339-3348). Here, cDNA cloning is used to show that the amino acid sequence of this protein, termed p150, is 29.6% identical and 53% similar to the yeast Vps15p protein, an established regulator of Vps34p. Northern blot analysis showed a ubiquitous tissue distribution for p150 similar to that previously observed with PtdIns 3-kinase. Recombinant p150 associated with PtdIns 3-kinase in vitro in a stable manner, resulting in a 2-fold increase in lipid kinase activity. Addition of phosphatidylinositol transfer protein (PI-TP) further stimulated the lipid kinase activity of the p150·;PtdIns 3-kinase complex 3-fold. A PtdIns 3-kinase activity could also be co-immunoprecipitated from human cell lysates using anti-PI-TP antisera. This observation demonstrates that an interaction between a PtdIns 3-kinase and PI-TP occurs in vivo, which further implicates lipid transfer proteins in the regulation of PtdIns 3-kinase activity. These results suggest that the Vps15p·;Vps34p complex has been conserved from yeast to man and in both species is involved in protein trafficking.

PHOSPHOLIPASE D AND MEMBRANE TRAFFIC. POTENTIAL ROLES IN REGULATED EXOCYTOSIS, MEMBRANE DELIVERY AND VESICLE BUDDING

It is now well-established that phospholipase D is transiently stimulated upon activation by G-protein-coupled and receptor tyrosine kinase cell surface receptors in mammalian cells. Over the last 5 years, a tremendous effort has gone to identify the major intracellular regulators of mammalian phospholipase D and to the cloning of two mammalian phospholipase D enzymes (phospholipase D1 and D2). In this chapter, we review the physiological function of mammalian phospholipase D1 that is synergistically stimulated by ADP ribosylation factor, Rho and protein kinase Calpha. We discuss the function of this enzyme in membrane traffic, emphasising the possible integrated relationships between consumption of vesicles in regulated exocytosis, membrane delivery and constitutive membrane traffic.

An essential role for phosphatidylinositol transfer protein in phospholipase C-mediated inositol lipid signaling

Transmembrane signaling by the phospholipase C-beta (PLC-beta) pathway is known to require at least three components: the receptor, the G protein, and the PLC. Recent studies have indicated that if the cytosol is allowed to leak out of HL60 cells, then G protein-stimulated PLC activity is greatly diminished, indicating an essential role for a cytosolic component(s). We now report the complete purification of one component based on its ability to reconstitute GTP gamma S-mediated PLC activity and identify it as the phosphatidylinositol transfer protein (PI-TP). Based on the in vitro effects of PI-TP, we surmise that it is involved in transporting PI from intracellular compartments for conversion to PI bisphosphate (PIP2) prior to hydrolysis by PLC-beta 2/PLC-beta 3, the endogenous PLC isoforms present in these cells.

ARF and PITP restore GTPγS-stimulated protein secretion from cytosol-depleted HL60 cells by promoting PIP2 synthesis

BACKGROUND In many cell types, including neutrophils and HL60 cells, there is an absolute requirement for a GTP-dependent step to elicit Ca(2+)-regulated secretion. Neutrophils and HL60 cells secrete lysosomal enzymes from azurophilic granules; this secretion is inhibited by 1% ethanol, indicating that phosphatidate (PA) produced by phospholipase D (PLD) activity may be involved. PLD can use primary alcohols in preference to water during the hydrolytic step, generating the corresponding phosphatidylalcohol instead of PA, its normal product. As ARF (ADP-ribosylation factor) proteins regulate PLD activity and are implicated in constitutive vesicular traffic, we have investigated whether ARF is also required for GTP-dependent secretion in HL60 cells. RESULTS We have used a cell-permeabilization protocol that allows HL60 cells to become refractory to stimulation with GTP gamma S plus 10 microM Ca2+ with regard to secretion and PLD activity. Permeabilization with streptolysin O for 10 minutes permitted the loss of freely diffusable cytosolic proteins, including ARF proteins. Fractions derived from brain cytosol, enriched in ARF proteins, restored secretory function and PLD activity. The major contaminating protein present in these ARF-enriched fractions was identified as phosphatidylinositol transfer protein (PITP). Unexpectedly, PITP was also found to restore GTP gamma S-dependent secretion. Restoration of secretory function was characterized using recombinant proteins, rARF1 and rPITP alpha and rPITP beta. The rARF1 protein restored both secretory function and PLD activity, whereas PITP only restored secretory function. However, both ARF and PITP were capable of stimulating phosphatidylinositol bis phosphate (PIP2) synthesis. CONCLUSIONS ARF and PITP restore secretory function in cytosol-depleted cells when stimulated with GTP gamma S plus Ca2+. We have previously shown that PITP participates in the synthesis of PIP2. In comparison, ARF1 activates PLD, producing PA, which is a known activator of phosphatidylinositol-4-phosphate 5 kinase, the enzyme responsible for PIP2 synthesis. We propose that ARF and PITP both restore exocytosis by a common mechanism-promoting PIP2 synthesis.

Membrane targeting and activation of the Lowe syndrome protein OCRL1 by rab GTPases

The X‐linked disorder oculocerebrorenal syndrome of Lowe is caused by mutation of the OCRL1 protein, an inositol polyphosphate 5‐phosphatase. OCRL1 is localised to the Golgi apparatus and early endosomes, and can translocate to lamellipodia upon growth factor stimulation. We show here that OCRL1 interacts with several members of the rab family of small GTPases. Strongest interaction is seen with Golgi‐associated rab1 and rab6 and endosomal rab5. Point mutants defective in rab binding fail to target to the Golgi apparatus and endosomes, strongly suggesting rab interaction is required for targeting of OCRL1 to these compartments. Membrane recruitment via rab binding is required for changes in Golgi and endosomal dynamics induced by overexpression of catalytically inactive OCRL1. In vitro experiments demonstrate that rab5 and rab6 directly stimulate the 5‐phosphatase activity of OCRL1. We conclude that rabs play a dual role in regulation of OCRL1, firstly targeting it to the Golgi apparatus and endosomes, and secondly, directly stimulating the 5‐phosphatase activity of OCRL1 after membrane recruitment.

Yeast Sec14p Deficient in Phosphatidylinositol Transfer Activity Is Functional In Vivo

Yeast phosphatidylinositol transfer protein (Sec14p) is essential for Golgi secretory function. It is widely accepted, though unproven, that phosphatidylinositol transfer between membranes represents the physiological activity of phosphatidylinositol transfer proteins (PITPs). We report that Sec14pK66,239A is inactivated for phosphatidylinositol, but not phosphatidylcholine (PC), transfer activity. As expected, Sec14pK66,239A fails to meet established criteria for a PITP in vitro and fails to stimulate phosphoinositide production in vivo. However, its expression efficiently rescues the lethality and Golgi secretory defects associated with sec14-1ts and sec14 null mutations. This complementation requires neither phospholipase D activation nor the involvement of a novel class of minor yeast PITPs. These findings indicate that PI binding/transfer is remarkably dispensable for Sec14p function in vivo.

Ca2+-dependent conversion of phosphatidylinositol to phosphatidate in neutrophils stimulated with fMet-Leu-Phe or ionophore A23187

Human and rabbit neutrophils stimulated with formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe) and A23187 show a loss of phosphatidylinositol and an increase in phosphatidate. In cells prelabelled with 32Pi it would be expected that the newly synthesised phosphatidate would have the same specific activity as cellular ATP, provided that the loss of phosphatidylinositol is by phospholipase C attack and the resultant diacyglycerol is phosphorylated by ATP. Instead, it is demonstrated that the specific activity of newly-formed phosphatidate is less than a tenth of the specific activity of ATP initially followed by a gradual increase. The time-course of mass and of [3H]glycerol-labelled phosphatidate formation (from cells pulse-labelled with [3H]glycerol) is similar to enzyme release but differs from the generation of 32P-labelled phosphatidate (from cells prelabelled with 32Pi). The source of the newly formed phosphatidate is most likely from phosphatidylinositol because: (a) The increase in phosphatidate is always accompanied by a loss of phosphatidylinositol with no changes in the other lipids. (b) Cells pulse-labelled with [3H]glycerol lose label from phosphatidylinositol only and this is accompanied by an increase in label in phosphatidate. (c) The specific activity of the newly synthesised phosphatidate is closest to the specific activity of phosphatidylinositol. One plausible explanation for these results is that phosphatidylinositol is directly converted to phosphatidate by phospholipase D action and the resulting phosphatidate accumulates radioactivity by exchange of its phosphate group with ATP. It is also shown that enzyme secretion and conversion of phosphatidylinositol to phosphatidate can depend on both intra- as well as extracellular Ca2+. Depletion of the intracellular pool of Ca2+ is essential to inhibit totally the enzyme secretion and the conversion of phosphatidylinositol to phosphatidate in agreement with our previous results on rabbit neutrophils (Cockcroft, S., et al. (1981) Biochem. J. 200, 501-508).

Rat mast cells permeabilized with ATP secrete histamine in response to calcium ions buffered in the micromolar range.

1. Rat mast cells with ATP (0.5‐4 micro M) in the absence of divalent cations (so that almost all the ATP is present as ATP4‐) became permeable to normally impermeant aqueous solutes, added extracellularly. These include the stable complexes Co HEDTA and Ca EDTA, and 6‐carboxyfluorescein but not inulin. At 4 micro M‐ATP4‐ the space accessible to Ca EDTA is 89% of that occupied by 3H2O. 2. The kinetics of solute entry are regulated by the concentration of ATP4‐. 3. Ca2+, buffered in the range 1‐10 micro M with HEDTA, causes histamine secretion from mast cells that have been rendered permeable with ATP4‐. The extent of secretion increases as the concentration of ATP4‐ is raised from 3 to 5 micro M. With extracellular Ca2+ present as physiological (millimolar) concentrations, the effect of increasing ATP4‐ through this range is to inhibit secretion. 4. The rates of histamine secretion and of 32P‐metabolite leakage from 32P‐prelabelled cells in the presence of micromolar concentrations of Ca2+, were compared. The kinetics of both processes are regulated by the concentration of ATP4‐, but not Ca2+.