N-terminal sequence and distal histidine residues are responsible for pH-regulated cytoplasmic membrane binding of human AMP deaminase isoform E

Mammalian AMP deaminase 3 (AMPD3) enzymes reportedly bind to intracellular membranes, plasma lipid vesicles, and artificial lipid bilayers with associated alterations in enzyme conformation and function. However, proteolytic sensitivity of AMPD polypeptides makes it likely that prior studies were performed with N-truncated enzymes. This study uses erythrocyte ghosts to characterize the reversible cytoplasmic membrane association of human full-sized recombinant isoform E (AMPD3). Membrane-bound isoform E exhibits diminished catalytic activity whereas low micromolar concentrations of the cationic antibiotic, neomycin, disrupt this protein-lipid interaction and relieve catalytic inhibition. The cytoplasmic membrane association of isoform E also displays an inverse correlation with pH in the physiological range. Diethyl pyrocarbonate (DEPC) modification of isoform E nearly abolishes its cytoplasmic membrane binding capacity, and this effect can be reversed by hydroxylamine. Difference spectra reveal that 18 of 29 histidine residues in each isoform E subunit are N-carbethoxylated by DEPC. These combined data demonstrate that protonated imidazole rings of histidine residues mediate a pH-responsive association of isoform E with anionic charges on the surface of the cytoplasmic membrane, possibly phosphatidylinositol 4,5-bisphosphate, a pure noncompetitive inhibitor of the enzyme. Finally, AMPD1 and a series of N-truncated AMPD3 enzymes are used to show that these behaviors are specific to isoform E and require up to 48 N-terminal amino acids, even though this stretch of sequence contains no histidine residues. The pH-responsive cytosol-membrane partitioning of isoform E may be an important mechanism for branch point regulation of adenylate catabolism.     

Engineering the phosphoinositide-binding profile of a class I pleckstrin homology domain

Pleckstrin homology (PH) domains are protein modules that bind with varying degrees of affinity and specificity membrane phosphoinositides. Previously we have shown that although the PH domains of the Ras GTPase-activating proteins GAP1m and GAP1IP4BP are 63% identical at the amino acid level they possess distinct phosphoinositide-binding profiles. The GAP1m PH domain binds phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), whereas the domain from GAP1IP4BP binds PtdIns(3,4,5)P3 and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) equally well. These phosphoinositide specificities are translated into distinct subcellular localizations. GAP1m is cytosolic and undergoes a rapid PtdIns(3,4,5)P3-dependent association with the plasma membrane following growth factor stimulation. In contrast, GAP1IP4BP is constitutively associated, in a PtdIns(4,5)P2-dependent manner, with the plasma membrane (Cozier, G. E., Lockyer, P. J., Reynolds, J. S., Kupzig, S., Bottomley, J. R., Millard, T., Banting, G., and Cullen, P. J. (2000) J. Biol. Chem. 275, 28261-28268). In the present study, we have used molecular modeling to identify residues in the GAP1IP4BP PH domain predicted to be required for high affinity binding to PtdIns(4,5)P2. This has allowed the isolation of a mutant, GAP1IP4BP-(K591T), which while retaining high affinity for PtdIns(3,4,5)P3 has a 6-fold reduction in its affinity for PtdIns(4,5)P2. Importantly, GAP1IP4BP-(K591T) is predominantly localized to the cytosol and undergoes a PtdIns(3,4,5)P3-dependent association with the plasma membrane following growth factor stimulation. We have therefore engineered the phosphoinositide-binding profile of the GAP1IP4BP PH domain, thereby emphasizing that subtle changes in PH domain structure can have a pronounced effect on phosphoinositide binding and the subcellular localization of GAP1IP4BP.     

Nuclear translocation of phospholipase C-delta1 is linked to the cell cycle and nuclear phosphatidylinositol 4,5-bisphosphate

Nuclear phosphoinositides, especially phosphatidylinositol 4,5-bisphosphate, fluctuate throughout the cell cycle and are linked to proliferation and differentiation. Here we report that phospholipase C-delta(1) accumulates in the nucleus at the G(1)/S boundary and in G(0) phases of the cell cycle. Furthermore, as wild-type protein accumulated in the nucleus, nuclear phosphatidylinositol 4,5-bisphosphate levels were elevated 3-5-fold, whereas total levels were decreased compared with asynchronous cultures. To test whether phosphatidylinositol 4,5-bisphosphate binding is important during this process, we introduced a R40D point mutation within the pleckstrin homology domain of phospholipase C-delta(1), which disables high affinity phosphatidylinositol 4,5-bisphosphate binding, and found that nuclear translocation was significantly reduced at G(1)/S and in G(0). These results demonstrate a cell cycle-dependent compartmentalization of phospholipase C-delta(1) and support the idea that relative levels of phosphoinositides modulate the portioning of phosphoinositide-binding proteins between the nucleus and other compartments.     

The p40phox and p47phox PX domains of NADPH oxidase target cell membranes via direct and indirect recruitment by phosphoinositides.

The Phox homology (PX) domain has recently been reported to bind to phosphoinositides, and some PX domains can localize to endosomes in vivo. Here we show data to support the conclusion that the p40(phox) PX domain binds to phosphatidylinositol 3-phosphate specifically in vitro and localizes to endosomes in intact cells. In addition, its Y59A/L65Q mutant, which has decreased affinity for phosphatidylinositol 3-phosphate in vitro, fails to target EGFP-p40-PX to endosomes. However, unlike published results, we find that the p47(phox) PX domain weakly binds to many phosphoinositides in vitro showing slightly higher affinity for phosphatidylinositol 3,4,5-trisphosphate. Moreover, we show for the first time that upon insulin-like growth factor-1 stimulation of COS cells, the p47(phox) PX domain is localized to the plasma membrane, and this subcellular localization is dependent on PI 3-kinase activity. Unexpectedly, its R42Q mutant that loses in vitro phosphoinositide-binding ability can still target EGFP-p47-PX to the plasma membrane. Our data suggest that the translocation of p47(phox) PX domain to the plasma membrane does involve 3'-phosphoinositide(s) in the process, but the phosphoinositide-binding of p47(phox) PX domain is not sufficient to recruit it to the plasma membrane. Therefore, the p40(phox) and p47(phox) PX domains can target subcellular membranes via direct or indirect recruitment by phosphoinositides, while both are under the control of phosphatidylinositol 3-kinase activity.     

Pharmacological inhibition of AMP-deaminase in rat cardiac myocytes

Because mutation of AMP deaminase 1 gene leading to reduced AMP deaminase activity may result in protection of cardiac function in patients with heart disease, inhibitors of AMP deaminase (AMPD) may have therapeutic applications. This study evaluated the effect of a specific inhibitor of AMP deaminase 3-[2-(3-carboxy-4-bromo-5,6,7,8-tetrahydronaphthyl)ethyl]-3,6,7,8-tetrahydroimidazo [4,5-d][1,3]diazepin-8-ol (AMPDI) on the isolated human enzyme and on nucleotide catabolism in rat cardiomyocytes. AMPDI effectively inhibited isolated human AMPD with an IC(50) = 0.5 micro M. AMPDI was much less effective with isolated cardiomyocytes (IC(50) = 0.5 mM). AMPDI is a very effective inhibitor of AMPD that despite lower efficiency in the cell system examined could be useful for in vivo studies.     

The pleckstrin homology domain of the Arf6-specific exchange factor EFA6 localizes to the plasma membrane by interacting with phosphatidylinositol 4,5-bisphosphate and F-actin

The Arf6-specific exchange factor EFA6 coordinates membrane trafficking with actin cytoskeleton remodeling. It localizes to the plasma membrane where it catalyzes Arf6 activation and induces the formation of actin-based membrane ruffles. We have shown previously that the pleckstrin homology (PH) domain of EFA6 was responsible for its membrane localization. In this study we looked for the partners of the PH domain at the plasma membrane. Mutations of the conserved basic residues suspected to be involved in the binding to phosphoinositides redistribute EFA6-PH to the cytosol. In addition, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) breakdown also leads to the solubilization of EFA6-PH. Direct binding measured by surface plasmon resonance gives an apparent affinity of approximately 0.5 microm EFA6-PH for PI(4,5)P2. Moreover, we observed in vitro that the catalytic activity of EFA6 is strongly increased by PI(4,5)P2. These results indicate that the plasma membrane localization of EFA6-PH is based on its interaction with PI(4,5)P2, and this interaction is necessary for an optimal catalytic activity of EFA6. Furthermore, we demonstrated by fluorescence recovery after photobleaching and Triton X-100 detergent solubility experiments that in addition to the phophoinositides, EFA6-PH is linked to the actin cytoskeleton. We observed both in vivo and in vitro that EFA6-PH interacts directly with F-actin. Finally, we demonstrated that EFA6 could bind simultaneously filamentous actin and phospholipids vesicles. Our results explain how the exchange factor EFA6 via its PH domain could coordinate at the plasma membrane actin cytoskeleton organization with membrane trafficking.     

Pharmacological Inhibition of AMP-Deaminase in Rat Cardiac Myocytes

Because mutation of AMP deaminase 1 gene leading to reduced AMP deaminase activity may result in protection of cardiac function in patients with heart disease, inhibitors of AMP deaminase (AMPD) may have therapeutic applications. This study evaluated the effect of a specific inhibitor of AMP deaminase 3-[2-(3-carboxy-4-bromo-5,6,7,8-tetrahydronaphthyl)ethyl]-3,6,7,8-tetrahydroimidazo [4,5-d][1,3]diazepin-8-ol (AMPDI) on the isolated human enzyme and on nucleotide catabolism in rat cardiomyocytes. AMPDI effectively inhibited isolated human AMPD with an IC ₅₀ = 0.5 μ M. AMPDI was much less effective with isolated cardiomyocytes (IC ₅₀ = 0.5 mM). AMPDI is a very effective inhibitor of AMPD that despite lower efficiency in the cell system examined could be useful for in vivo studies.     

Calcium activates erythrocyte AMP deaminase [isoform E (AMPD3)] through a protein-protein interaction between calmodulin and the N-terminal domain of the AMPD3 polypeptide

Erythrocyte AMP deaminase [isoform E (AMPD3)] is activated in response to increased intracellular calcium levels in Tarui's disease, following exposure of ionophore-treated cells to extracellular calcium, and by the addition of calcium to freshly prepared hemolysates. However, the assumption that Ca(2+) is a positive effector of isoform E is inconsistent with the loss of sensitivity to this divalent cation following dilution of erythrocyte lysates or enzyme purification. Ca(2+) regulation of isoform E was studied by examining in vitro effects of calmodulin (CaM) on this enzyme and by monitoring the influence of CaM antagonists on purine catabolic flow in freshly prepared erythrocytes under various conditions of energy imbalance. Erythrocyte and recombinant isoform E both adsorb to immobilized Ca(2+)-CaM, and relative adsorption across a series of N-truncated recombinant enzymes localizes CaM binding determinants to within residues 65-89 of the AMPD3 polypeptide. Ca(2+)-CaM directly stimulates isoform E catalytic activity through a K(mapp) effect and also antagonizes the protein-lipid interaction between this enzyme and intracellular membranes that inhibits catalytic activity. AMP is the predominant purine catabolite in erythrocytes deprived of glucose or exposed to A23187 ionophore alone, whereas IMP accumulates when Ca(2+) is included under the latter conditions and also during autoincubation at 37 degrees C. Preincubation with a CaM antagonist significantly slows the accumulation of erythrocyte IMP under both conditions. The combined results reveal a protein-protein interaction between Ca(2+)-CaM and isoform E and identify a mechanism that advances our understanding of erythrocyte purine metabolism. Ca(2+)-CaM overcomes potent isoform E inhibitory mechanisms that function to maintain the total adenine nucleotide pool in mature erythrocytes, which are unable to synthesize AMP from IMP because of a developmental loss of adenylosuccinate synthetase. This may also explain why Tarui's disease erythrocytes exhibit accelerated adenine nucleotide depletion in response to an increase in intracellular Ca(2+) concentration. This regulatory mechanism could also play an important role in purine metabolism in other human tissues and cells where the AMPD3 gene is expressed.     

Modulation of oncogenic DBL activity by phosphoinositol phosphate binding to pleckstrin homology domain

The Dbl family guanine nucleotide exchange factors (GEFs) contain a region of sequence similarity consisting of a catalytic Dbl homology (DH) domain in tandem with a pleckstrin homology (PH) domain. PH domains are involved in the regulated targeting of signaling molecules to plasma membranes by protein-protein and/or protein-lipid interactions. Here we show that Dbl PH domain binding to phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-triphosphate results in the inhibition of Dbl GEF activity on Rho family GTPase Cdc42. Phosphatidylinositol 4,5-bisphosphate binding to the PH domain significantly inhibits the Cdc42 interactive activity of the DH domain suggesting that the DH domain is subjected to the PH domain modulation under the influence of phosphoinositides (PIPs). We generated Dbl mutants unable to interact with PIPs. These mutants retained GEF activity on Cdc42 in the presence of PIPs and showed a markedly enhanced activating potential for both Cdc42 and RhoA in vivo while displaying decreased cellular transforming activity. Immunofluorescence analysis of NIH3T3 transfectants revealed that whereas the PH domain localizes to actin stress fibers and plasma membrane, the PH mutants are no longer detectable on the plasma membrane. These results suggest that modulation of PIPs in both the GEF catalytic activity and the targeting to plasma membrane determines the outcome of the biologic activity of Dbl.     

Activation of plant phospholipase Dbeta by phosphatidylinositol 4,5-bisphosphate: characterization of binding site and mode of action

Hydrolysis of phospholipids by plant phospholipase Dbeta (PLDbeta) requires phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Here we show that PLDbeta is stimulated by different polyphosphoinositides, among which PI(4,5)P2 is most effective. On the basis of amino acid sequence analysis, PI(4,5)P2 binding assay, and protein engineering studies, we have identified in the catalytic region of PLDbeta a new PI(4,5)P2 binding region (PBR1), which is conserved in eukaryotic PLDs. PBR1 is a second domain besides the previously characterized N-terminal C2 domain of PLDbeta which also binds PI(4,5)P2. Submillimolar levels of calcium ions, while inhibiting PI(4,5)P2 binding by the C2 domain, enhanced the affinity of PBR1 for that phosphoinositide. Substrate binding by PLDbeta was promoted by PI(4,5)P2-bound PBR1. Isolated, recombinant PBR1 bound PI(4,5)P2 specifically and in a saturable manner. Deletion of PBR1 from PLDbeta or mutation of the conserved basic amino acid residues in PBR1 (K437G/K440G) abolished the enzymatic activity. Circular dichroism spectroscopy revealed a conformational change caused by PI(4,5)P2 binding to the catalytic region of PLD. The conformational change apparently helps in the recruitment of the substrate to the active site of the enzyme. The results taken together allow us to describe an anchorage-scooting model for the synergistic activation of PLDbeta by PI(4,5)P2 and Ca2+.     

Enzymes of adenylate metabolism and their role in hibernation of the white-tailed prairie dog, Cynomys leucurus

AMP deaminase (AMPD) and adenylate kinase (AK) were purified from skeletal muscle of the white-tailed prairie dog, Cynomus leucurus, and enzyme properties were assayed at temperatures characteristic of euthermia (37 degrees C) and hibernation (5 degrees C) to analyze their role in adenylate metabolism during hibernation. Total adenylates decreased in muscle of torpid individuals from 6.97 +/- 0. 31 to 4.66 +/- 0.58 micromol/g of wet weight due to a significant drop in ATP but ADP, AMP, IMP, and energy charge were unchanged. The affinity of prairie dog AMPD for AMP was not affected by temperature and did not differ from that of rabbit muscle AMPD, used for comparison. However, both prairie dog and rabbit AMPD showed much stronger inhibition by ions and GTP at 5 degrees C, versus 37 degrees C, and inhibition by inorganic phosphate, NH(4)Cl, and (NH(4))(2)SO(4) was much stronger at 5 degrees C for the prairie dog enzyme. Furthermore, ATP and ADP, which activated AMPD at 37 degrees C, were strong inhibitors of prairie dog AMPD at 5 degrees C, with I(50) values of 1 and 14 microM, respectively. ATP also inhibited rabbit AMPD at 5 degrees C (I(50) = 103 microM). Strong inhibition of AMPD at 5 degrees C by several effectors suggests that enzyme function is specifically suppressed in muscle of hibernating animals. By contrast, AK showed properties that would maintain or even enhance its function at low temperature. K(m) values for substrates (ATP, ADP, AMP) decreased with decreasing temperature, the change in K(m) ATP paralleling the decrease in muscle ATP concentration. AK inhibition by ions was also reduced at 5 degrees C. The data suggest that adenylate degradation via AMPD is blocked during hibernation but that AK maintains its function in stabilizing energy charge.     

Autophosphorylation of type I phosphatidylinositol phosphate kinase regulates its lipid kinase activity

Phosphatidylinositol phosphate kinases (PIPKs) have important roles in the production of various phosphoinositides. For type I PIP5Ks (PIP5KI), a broad substrate specificity is known. They phosphorylate phosphatidylinositol 4-phosphate most effectively but also phosphorylate phosphatidylinositol (PI), phosphatidylinositol 3-phosphate, and phosphatidylinositol (3,4)-bisphosphate (PI(3, 4)P(2)), resulting in the production of phosphatidylinositol (4, 5)-bisphosphate (PI(4,5)P(2)), phosphatidylinositol 3-phosphate, phosphatidylinositol (3,4)-bisphosphate (PI(3,4)P(2)), phosphatidylinositol (3,5)-bisphosphate (PI(3,5)P(2)), and phosphatidylinositol (3,4,5)-trisphosphate. We show here that PIP5KIs have also protein kinase activities. When each isozyme of PIP5KI (PIP5KIalpha, -beta, and -gamma) was subjected to in vitro kinase assay, autophosphorylation occurred. The lipid kinase-negative mutant of PIP5KIalpha (K138A) lost the protein kinase activity, suggesting the same catalytic mechanism for the lipid and the protein kinase activities. PIP5KIbeta expressed in Escherichia coli also retains this protein kinase activity, thus confirming that no co-immunoprecipitated protein kinase is involved. In addition, the autophosphorylation of PIP5KI is markedly enhanced by the addition of PI. No other phosphoinositides such as phosphatidylinositol phosphate, phosphatidylinositol bisphosphate, or phosphatidylinositol trisphosphate have such an effect. We also found that the PI-dependent autophosphorylation strongly suppresses the lipid kinase activity of PIP5KI. The lipid kinase activity of PIP5KI was decreased to one-tenth upon PI-dependent autophosphorylation. All these results indicate that the lipid kinase activity of PIP5KI that acts predominantly for PI(4,5)P(2) synthesis is regulated by PI-dependent autophosphorylation in vivo.     

Counteracting Roles of AMP Deaminase and AMP Kinase in the Development of Fatty Liver

Fatty liver (hepatic steatosis) is associated with nucleotide turnover, loss of ATP and generation of adenosine monophosphate (AMP). It is well known that in fatty liver, activity of the AMP-activated kinase (AMPK) is reduced and that its stimulation can prevent hepatic steatosis by both enhancing fat oxidation and reducing lipogenesis. Here we show that another AMP dependent enzyme, AMPD2, has opposing effects on fatty acid oxidation when compared to AMPK. In human hepatocytres, AMPD2 activation –either by overexpression or by lowering intracellular phosphate levels with fructose- is associated with a significant reduction in AMPK activity. Likewise, silencing of AMPK spontaneously increases AMPD activity, demonstrating that these enzymes counter-regulate each other. Furthermore, we show that a downstream product of AMP metabolism through AMPD2, uric acid, can inhibit AMPK activity in human hepatocytes. Finally, we show that fructose-induced fat accumulation in hepatocytes is due to a dominant stimulation of AMPD2 despite stimulating AMPK. In this regard, AMPD2-deficient hepatocytes demonstrate a further activation of AMPK after fructose exposure in association with increased fatty acid oxidation, and conversely silencing AMPK enhances AMPD-dependent fat accumulation. In vivo, we show that sucrose fed rats also develop fatty liver that is blocked by metformin in association with both a reduction in AMPD activity and an increase in AMPK activity. In summary, AMPD and AMPK are both important in hepatic fat accumulation and counter-regulate each other. We present the novel finding that uric acid inhibits AMPK kinase activity in fructose-fed hepatocytes thus providing new insights into the pathogenesis of fatty liver.     

Purification and characterization of a phosphoinositide-specific phospholipase C from guinea pig uterus. Phosphorylation by protein kinase C in vivo

Two peaks of phosphoinositide-specific phospholipase C (PI-PLC) activity were resolved when guinea pig uterus cytosolic proteins were chromatographed on a DEAE-Sepharose column. The first peak of enzyme activity eluting from the DEAE-Sepharose column (PI-PLC I) was further purified to homogeneity, whereas the second peak of enzyme activity was enriched 300-fold. PI-PLC I migrated as a 62-kDa protein on sodium dodecyl sulfate-polyacrylamide gels. Antibodies prepared against PI-PLC I failed to react with PI-PLC II. PI-PLC I hydrolyzed all three phosphoinositides, exhibiting a greater Vmax for phosphatidylinositol 4,5-bisphosphate greater than phosphatidylinositol 4-phosphate greater than phosphatidylinositol. Hydrolysis of phosphatidylinositol was calcium-dependent, whereas significant hydrolysis of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate occurred in the presence of 2.5 mM EGTA. At physiological concentrations of calcium, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate were the preferred substrates. Antibodies specific for PI-PLC I reacted with a 62-kDa protein in both the cytosol and membrane fractions from guinea pig uterus. Quantitation of the immunoblots revealed that 25% of the 62-kDa protein was membrane-associated, whereas only 5% of the total enzyme activity was membrane-associated. Approximately 20% of the membrane-bound phospholipase C activity and immunoreactive material were loosely bound, whereas the remainder required detergent extraction for complete solubilization. The 62-kDa protein associated with the membrane fractions did not bind lectin affinity columns, suggesting that it was not glycosylated. PI-PLC I was identified as a phosphoprotein in [32P]orthophosphate-labeled rat basophilic leukemia (RBL-1) cells by two-dimensional gel electrophoresis followed by immunoblotting. In untreated cells, 32P-labeled PI-PLC I was found in the cytosolic fraction. Treatment of RBL-1 cells with those phorbol esters which are known to activate the Ca2+/phospholipid-dependent enzyme protein kinase C, resulted in a time-dependent increase in the phosphorylation of both membrane-bound and cytosolic PI-PLC I. Thus, in RBL-1 cells, protein kinase C may play an important role in the regulation of phospholipase C through protein phosphorylation.     

Identification of nuclear phosphatidylinositol 4,5-bisphosphate-interacting proteins by neomycin extraction

Considerable insight into phosphoinositide-regulated cytoplasmic functions has been gained by identifying phosphoinositide-effector proteins. Phosphoinositide-regulated nuclear functions however are fewer and less clear. To address this, we established a proteomic method based on neomycin extraction of intact nuclei to enrich for nuclear phosphoinositide-effector proteins. We identified 168 proteins harboring phosphoinositide-binding domains. Although the vast majority of these contained lysine/arginine-rich patches with the following motif, K/R-(X(n= 3-7)-K-X-K/R-K/R, we also identified a smaller subset of known phosphoinositide-binding proteins containing pleckstrin homology or plant homeodomain modules. Proteins with no prior history of phosphoinositide interaction were identified, some of which have functional roles in RNA splicing and processing and chromatin assembly. The remaining proteins represent potentially other novel nuclear phosphoinositide-effector proteins and as such strengthen our appreciation of phosphoinositide-regulated nuclear functions. DNA topology was exemplar among these: Biochemical assays validated our proteomic data supporting a direct interaction between phosphatidylinositol 4,5-bisphosphate and DNA Topoisomerase IIα. In addition, a subset of neomycin extracted proteins were further validated as phosphatidyl 4,5-bisphosphate-interacting proteins by quantitative lipid pull downs. In summary, data sets such as this serve as a resource for a global view of phosphoinositide-regulated nuclear functions.     

SAC1-like domains of yeast SAC1, INP52, and INP53 and of human synaptojanin encode polyphosphoinositide phosphatases

The SAC1 gene product has been implicated in the regulation of actin cytoskeleton, secretion from the Golgi, and microsomal ATP transport; yet its function is unknown. Within SAC1 is an evolutionarily conserved 300-amino acid region, designated a SAC1-like domain, that is also present at the amino termini of the inositol polyphosphate 5-phosphatases, mammalian synaptojanin, and certain yeast INP5 gene products. Here we report that SAC1-like domains have intrinsic enzymatic activity that defines a new class of polyphosphoinositide phosphatase (PPIPase). Purified recombinant SAC1-like domains convert yeast lipids phosphatidylinositol (PI) 3-phosphate, PI 4-phosphate, and PI 3,5-bisphosphate to PI, whereas PI 4,5-bisphosphate is not a substrate. Yeast lacking Sac1p exhibit 10-, 2.5-, and 2-fold increases in the cellular levels of PI 4-phosphate, PI 3,5-bisphosphate, and PI 3-phosphate, respectively. The 5-phosphatase domains of synaptojanin, Inp52p, and Inp53p are also catalytic, thus representing the first examples of an inositol signaling protein with two distinct lipid phosphatase active sites within a single polypeptide chain. Together, our data provide a long sought mechanism as to how defects in Sac1p overcome certain actin mutants and bypass the requirement for yeast phosphatidylinositol/phosphatidylcholine transfer protein, Sec14p. We demonstrate that PPIPase activity is a key regulator of membrane trafficking and actin cytoskeleton organization and suggest signaling roles for phosphoinositides other than PI 4,5-bisphosphate in these processes. Additionally, the tethering of PPIPase and 5-phosphatase activities indicate a novel mechanism by which concerted phosphoinositide hydrolysis participates in membrane trafficking.     

Differential roles of phosphatidylserine, PtdIns(4,5)P2, and PtdIns(3,4,5)P3 in plasma membrane targeting of C2 domains. Molecular dynamics simulation, membrane binding, and cell translocation studies of the PKCalpha C2 domain

Many cytosolic proteins are recruited to the plasma membrane (PM) during cell signaling and other cellular processes. Recent reports have indicated that phosphatidylserine (PS), phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)), and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) that are present in the PM play important roles for their specific PM recruitment. To systematically analyze how these lipids mediate PM targeting of cellular proteins, we performed biophysical, computational, and cell studies of the Ca(2+)-dependent C2 domain of protein kinase Calpha (PKCalpha) that is known to bind PS and phosphoinositides. In vitro membrane binding measurements by surface plasmon resonance analysis show that PKCalpha-C2 nonspecifically binds phosphoinositides, including PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3), but that PS and Ca(2+) binding is prerequisite for productive phosphoinositide binding. PtdIns(4,5)P(2) or PtdIns(3,4,5)P(3) augments the Ca(2+)- and PS-dependent membrane binding of PKCalpha-C2 by slowing its membrane dissociation. Molecular dynamics simulations also support that Ca(2+)-dependent PS binding is essential for membrane interactions of PKCalpha-C2. PtdIns(4,5)P(2) alone cannot drive the membrane attachment of the domain but further stabilizes the Ca(2+)- and PS-dependent membrane binding. When the fluorescence protein-tagged PKCalpha-C2 was expressed in NIH-3T3 cells, mutations of phosphoinositide-binding residues or depletion of PtdIns(4,5)P(2) and/or PtdIns(3,4,5)P(3) from PM did not significantly affect the PM association of the domain but accelerated its dissociation from PM. Also, local synthesis of PtdIns(4,5)P(2) or PtdIns(3,4,5)P(3) at the PM slowed membrane dissociation of PKCalpha-C2. Collectively, these studies show that PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3) augment the Ca(2+)- and PS-dependent membrane binding of PKCalpha-C2 by elongating the membrane residence of the domain but cannot drive the PM recruitment of PKCalpha-C2. These studies also suggest that effective PM recruitment of many cellular proteins may require synergistic actions of PS and phosphoinositides.     

Essential Ca(2+)-independent role of the group IVA cytosolic phospholipase A(2) C2 domain for interfacial activity

The cytosolic Group IVA phospholipase A2 (GIVAPLA2) translocates to intracellular membranes to catalyze the release of lysophospholipids and arachidonic acid. GIVAPLA2 translocation and subsequent activity is regulated by its Ca2+-dependent phospholipid binding C2 domain. Phosphatidylinositol 4,5-bisphosphate (PI-4,5-P2) also binds with high affinity and specificity to GIVAPLA2, facilitating membrane binding and activity. Herein, we demonstrate that GIVAPLA2 possessed full activity in the absence of Ca2+ when PI-4,5-P2 or phosphatidylinositol 3,4,5-trisphosphate were present. A point mutant, D43N, that is unable to bind Ca2+ also had full activity in the presence of PI-4,5-P2. However, when GIVAPLA2 was expressed without its Ca2+-binding C2 domain (DeltaC2), there was no interfacial activity. GIVAPLA2 and DeltaC2 both had activity on monomeric lysophospholipids. DeltaC2, but not the C2 domain alone, binds to phosphoinositides (PIPns) in the same manner as the full-length GIVAPLA2, confirming the location of the PIPn binding site as the GIVAPLA2 catalytic domain. Moreover, proposed PIPn-binding residues in the catalytic domain (Lys488, Lys541, Lys543, and Lys544) were confirmed to be essential for PI-4,5-P2-dependent activity increases. Exploiting the effects of PI-4,5-P2, we have discovered that the C2 domain plays a critical role in the interfacial activity of GIVAPLA2 above and beyond its Ca2+-dependent phospholipid binding.     

Point mutations in the HIV-1 matrix protein turn off the myristyl switch

During the late phase of human immunodeficiency virus type-1 (HIV-1) replication, newly synthesized retroviral Gag proteins are targeted to lipid raft regions of specific cellular membranes, where they assemble and bud to form new virus particles. Gag binds preferentially to the plasma membrane (PM) of most hematopoietic cell types, a process mediated by interactions between the cellular PM marker phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P(2)) and Gag's N-terminally myristoylated matrix (MA) domain. We recently demonstrated that PI(4,5)P(2) binds to a conserved cleft on MA and promotes myristate exposure, suggesting a role as both a direct membrane anchor and myristyl switch trigger. Here we show that PI(4,5)P(2) is also capable of binding to MA proteins containing point mutations that inhibit membrane binding in vitro, and in vivo, including V7R, L8A and L8I. However, these mutants do not exhibit PI(4,5)P(2) or concentration-dependent myristate exposure. NMR studies of V7R and L8A MA reveal minor structural changes that appear to be responsible for stabilizing the myristate-sequestered (myr(s)) species and inhibiting exposure. Unexpectedly, the myristyl group of a revertant mutant with normal PM targeting properties (V7R,L21K) is also tightly sequestered and insensitive to PI(4,5)P(2) binding. This mutant binds PI(4,5)P(2) with twofold higher affinity compared with the native protein, suggesting a potential compensatory mechanism for membrane binding.     

Direct modulation of Kir channel gating by membrane phosphatidylinositol 4,5-bisphosphate

Multiple ion channels have now been shown to be regulated by phosphatidylinositol 4,5-bisphosphate (PIP2) at the cytoplasmic face of the membrane. However, direct evidence for a specific interaction between phosphoinositides and ion channels is critically lacking. We reconstituted pure KirBac1.1 and KcsA protein into liposomes of defined composition (3:1 phosphatidylethanolamine:phosphatidylglycerol) and examined channel activity using a 86Rb+ uptake assay. We demonstrate direct modulation by PIP2 of KirBac1.1 but not KcsA activity. In marked contrast to activation of eukaryotic Kir channels by PIP2, KirBac1.1 is inhibited by PIP2 incorporated in the membrane (K(1/2) = 0.3 mol %). The dependence of inhibition on the number of phosphate groups and requirement for a lipid tail matches that for activation of eukaryotic Kir channels, suggesting a fundamentally similar interaction mechanism. The data exclude the possibility of indirect modulation via cytoskeletal or other intermediary elements and establish a direct interaction of the channel with PIP2 in the membrane.