Molecular interaction between fukutin and POMGnT1 in the glycosylation pathway of alpha-dystroglycan

The recent identification of mutations in genes encoding demonstrated or putative glycosyltransferases has revealed a novel mechanism for congenital muscular dystrophy. Hypoglycosylated alpha-dystroglycan (alpha-DG) is commonly seen in Fukuyama-type congenital muscular dystrophy (FCMD), muscle-eye-brain disease (MEB), Walker-Warburg syndrome (WWS), and Large(myd) mice. POMGnT1 and POMTs, the gene products responsible for MEB and WWS, respectively, synthesize unique O-mannose sugar chains on alpha-DG. The function of fukutin, the gene product responsible for FCMD, remains undetermined. Here we show that fukutin co-localizes with POMGnT1 in the Golgi apparatus. Direct interaction between fukutin and POMGnT1 was confirmed by co-immunoprecipitation and two-hybrid analyses. The transmembrane region of fukutin mediates its localization to the Golgi and participates in the interaction with POMGnT1. Y371C, a missense mutation found in FCMD, retains fukutin in the ER and also redirects POMGnT1 to the ER. Finally, we demonstrate reduced POMGnT1 enzymatic activity in transgenic knock-in mice carrying the retrotransposal insertion in the fukutin gene, the prevalent mutation in FCMD. From these findings, we propose that fukutin forms a complex with POMGnT1 and may modulate its enzymatic activity.     

Ubiquitin ligase gp78 increases solubility and facilitates degradation of the Z variant of alpha-1-antitrypsin

Deficiency of circulating alpha-1-antitrypsin (AAT) is the most widely recognized abnormality of a proteinase inhibitor that causes lung disease. AAT-deficiency is caused by mutations of the AAT gene that lead to AAT protein retention in the endoplasmic reticulum (ER). Moreover, the mutant AAT accumulated in the ER predisposes the homozygote to severe liver injuries, such as neonatal hepatitis, juvenile cirrhosis, and hepatocellular carcinoma. Despite the fact that mutant AAT protein is subject to ER-associated degradation (ERAD), yeast genetic studies have determined that the ubiquitination machinery, Hrd1/Der3p-cue1p-Ubc7/6p, which plays a prominent role in ERAD, is not involved in degradation of mutant AAT. Here we report that gp78, a ubiquitin ligase (E3) pairing with mammalian Ubc7 for ERAD, ubiquitinates and facilitates degradation of ATZ, the classic deficiency variant of AAT having a Z mutation (Glu 342 Lys). Unexpectedly, gp78 over-expression also significantly increases ATZ solubility. p97/VCP, an AAA ATPase essential for retrotranslocation of misfolded proteins from the ER during ERAD, is involved in gp78-mediated degradation of ATZ. Surprisingly, unlike other ERAD substrates that cause ER stress leading to apoptosis when accumulated in the ER, ATZ, in fact, increases cell proliferation when over-expressed in cells. This effect can be partially inhibited by gp78 over-expression. These data indicate that gp78 assumes multiple unique quality control roles over ATZ, including the facilitation of degradation and inhibition of aggregation of ATZ.     

The heterodimer of alpha4 and PP2Ac is associated with S6 kinase1 in B cells

Alpha4 is a signal transduction molecule that is required for B cell activation. Alpha4 associates with the catalytic subunit of protein phosphatase 2A (PP2Ac) and regulates its enzymatic activity. We examined the interaction of alpha4/PP2Ac with S6 kinase1 (S6K1) as a potential downstream signal transduction molecule because both alpha4/PP2Ac association and S6K1 activity were rapamycin-sensitive. Stimulation of spleen B cells with lipopolysaccharide induced the interaction of alpha4/PP2Ac and S6K1. Pull-down assay demonstrated that alpha4 interacts with S6K1 through PP2Ac. S6K1 and alpha4 bind to the different regions of PP2Ac as S6K1 to the region from amino acid 88th to 309th of PP2Ac and alpha4 to the two separated regions of the amino-terminal (from amino acid 19th to 22nd) and the middle (from 150th to 164th) portions of PP2Ac. These results suggest that alpha4 regulates S6K1 activity through PP2Ac in B cell activation.     

Structures of the N-linked carbohydrate of ascorbic acid oxidase from zucchini

N-glycan moiety of ascorbic acid oxidase from zucchini (Cucurbita pepo) has been described to be a core-pentasaccharide with a xylose [D'Andrea et al. (1988) Glycoconjugate J 5:151-7]. Ascorbic acid oxidase is sometimes used to characterize antibodies directed against carbohydrate determinants on plant glycoproteins. To prevent misinterpretations of immunological data, the structure of the N-glycan of ascorbic acid oxidase has been reinvestigated. The oligosaccharides were released by almond N-glycosidase and analysed as their pyridylamino derivatives by 2D-HPLC and exoglycosidase digestions. The main structure resembled the typical complex plant N-glycan consisting of a core-pentasaccharide decorated with xylose and 3-linked fucose. The other abundant species lacked the fucose residue. Small amounts of these glycans carried a GlcNAc residue on the 6-arm. Therefore, ascorbic acid oxidase will not only react with antibodies directed against the xylosylated region but also with those binding to N-glycans with 3-linked fucose.     

Phosphorylation on the Ser 824 residue of TRPV4 prefers to bind with F-actin than with microtubules to expand the cell surface area

Previously, we demonstrated that the transient receptor potential vanilloid 4 (TRPV4) cation channel, a member of the TRP vanilloid subfamily, is one of the serum glucocorticoid-induced protein kinase1 (SGK1) authentic substrate proteins, and that the Ser 824 residue of TRPV4 is phosphorylated by SGK1 [1]. In this study, we demonstrated that phosphorylation on the Ser 824 residue of TRPV4 is required for its interaction with F-actin, using TRPV4 mutants (S824D; a phospho-mimicking TRPV4 mutant and S824A; a non-phosphorylatable TRPV4 mutant) and its proper subcellular localization. Additionally, we noted that the phosphorylation of the Ser824 residue promotes its single channel activity, Ca^2 + influx, protein stability, and cell surface area (expansion of plasma membrane).     

A new approach to understanding kinetic cooperativity when the hill coefficients are less than 2

Dihydrofolate reductase from chicken liver has a single sulfhydryl group which reacts stoichiometrically and specifically with a wide variety of organic mercury compounds to yield an enzyme derivative which exhibits up to 10-fold the activity of the unmodified form when measured at pH 6.5, the optimum for the modified enzyme. The sulfhydryl group is apparently not at the active site since a 25-fold excess of either major cosubstrate, dihydrofolate or TPNH, affects neither the rate nor extent of the modification reaction. The reaction is essentially instantaneous and yields an enzyme with altered kinetic properties for all the substrate pairs examined (TPNH/dihydrofolate, TPNH/ folate, and DPNH/dihydrofolate) when tested near their pH optima. V values increased 3- to 10-fold when TPNH was cofactor; K_m values increased 10- to 15-fold for the TPNH/dihydrofolate pair. The mercurial-activated enzyme, unlike the native form, exhibits a markedly increased sensitivity to heat, proteolysis, and the ionic environment, losing approximately 50% of its activity under conditions where there is no loss of activity in the native form. However, substrates can afford protection, the order of effectiveness being identical with the relative affinities of the substrates for the native enzyme (Subramanian, S., and Kaufman, B. T. (1978) Proc. Nat. Acad. Sci. USA75, 3201). Thus, dihydrofolate, with the largest binding constant is the most efficient, protecting completely against trypsin digestion when present at a 1:1 ratio with enzyme. Heating the mercury enzyme in the absence of substrates gives rise to a stable but altered conformation characterized by a time course which shows marked hysteresis. The striking similarity of the properties of the mercurial-activated dihydrofolate reductase to the reductase activated by 4 m urea, a reagent known to affect the tertiary structure of proteins, suggests that covalent binding of organic mercurials to the sulfhydryl group results in a similar conformational change characterized by a marked facilitation of the dihydrofolate reductase reaction.     

Unique presence of 2-methylthio-ribosylzeatin in the transfer ribonucleic acid of the bacterium Pseudomonas aeruginosa

Analysis of ³⁵S labled nucleosides prepared from tRNA of Pseudomonas aeruginosa by phosphocellulose column chromatography, thin layer chromatography and Sephadex LH-20 column chromatography revealed the presence of 2-methylthioribosylzeatin in it.     

Absorbance changes due to the charge-accumulating species in System 2 of photosynthesis

Absorbance changes are reported associated with Photosystem II and showing a periodicity of two and four as a function of flash number.

The absorbance changes showing a periodicity of two were found to occur in the presence of artificial electron donors as well and are presumably caused by the secondary electron acceptor R of Photosystem II. The absorbance difference spectra suggest that R is a plastoquinone molecule, which is reduced to its semiquinone anion after an uneven number of flashes. After an even number of flashes, the semiquinone is reoxidized. The absorbance changes showing a periodicity of four are tentatively ascribed to the charge accumulating donor complex of Photosystem II.     

Determination of 6,4′-bis-(2-imidazolinylhydrazone)-2-phenylimidazo[1,2-a]pyridine in plasma and whole blood by high-performance liquid chromatography

A selective and sensitive HPLC assay for the quantitative determination of a new antifilarial drug, 6,4′-bis-(2-imidazolinylhydrazone)-2-phenylimidazo[1,2-a]pyridine (CDR 101) is described. After extraction from plasma and blood, CDR 101 was analysed using a C₁₈ Nucleosil ODS column (250×4.6 mm, 5 μm particle size) and mobile phase of acetonitrile-0.05 M ammonium acetate adjusted to pH 3.0, with UV detection at 318 nm. The mean recoveries of CDR 101 in plasma and blood over a concentration range of 25–500 ng/ml were 95.5±2.01% and 83.3±1.87%, respectively. The within-day and day-to-day coefficient of variations for plasma were 3.23-6.21% and 2.59-9.90%, respectively, those for blood were 2.59-5.92% and 2.89-6.82%, respectively. The minimum detectable concentration for CDR 101 was 1 ng/ml in plasma and 2.5 ng/ml in whole blood. This method was found to be suitable for clinical pharmacokinetic studies.     

Molecular dynamics simulations of a cyclic-beta-(1-->2) glucan containing an alpha-(1-->6) linkage as a 'molecular alleviator' for the macrocyclic conformational strain

The conformational preferences of a cyclic osmoregulated periplasmic glucan of Ralstonia solanacearum (OPGR), which is composed of 13 glucose units and linked entirely via beta-(1-->2) linkages excluding one alpha-(1-->6) linkage, were characterized by molecular dynamics simulations. Of the three force fields modified for carbohydrates that were applied to select a suitable one for the cyclic glucan, the carbohydrate solution force field (CSFF) was found to most accurately simulate the cyclic molecule. To determine the conformational characteristics of OPGR, we investigated the glycosidic dihedral angle distribution, fluctuation, and the potential energy of the glucan and constructed hypothetical cyclic (CYS13) and linear (LINEAR) glucans. All beta-(1-->2)-glycosidic linkages of OPGR adopted stable conformations, and the dihedral angles fluctuated in this energy region with some flexibility. However, despite the inherent flexibility of the alpha-(1-->6) linkage, the dihedral angles have no transition and are more rigid than that in a linear glucan. CYS13, which consists of only beta-(1-->2) linkages, is somewhat less flexible than other glycans, and one of its linkages adopts a higher energy conformation. In addition, the root-mean-square fluctuation of this linkage is lower than that of other linkages. Furthermore, the potential energy of glucans increases in the order of LINEAR, OPGR, and CYS13. These results provide evidence of the existence of conformational constraints in the cyclic glucan. The alpha-(1-->6)-glycosidic linkage can relieve this constraint more efficiently than the beta-(1-->2) linkage. The conformation of OPGR can reconcile the tendency for individual glycosidic bonds to adopt energetically favorable conformations with the requirement for closure of the macrocyclic ring by losing the inherent flexibility of the alpha-(1-->6)-glycosidic linkage.     

Rates and properties of endogenous cyclic photophosphorylation of isolated intact chloroplasts measured by CO2 fixation in the presence of dihydroxyacetone phosphate

1. Dihydroxyacetone phosphate in concentrations ? 2.5 mM completely inhibits CO₂-dependent O₂ evolution in isolated intact spinach chloroplasts. This inhibition is reversed by the addition of equimolar concentrations of P_i, but not by addition of 3-phosphoglycerate. In the absence of P_i, 3-phosphoglycerate and dihydroxyacetone phosphate, only about 20% of the ¹⁴C-labelled intermediates are found in the supernatant, whereas in the presence of each of these substances the percentage of labelled intermediates in the supernatant is increased up to 70–95%. Based on these results the mechanism of the inhibition of O₂ evolution by dihydroxyacetone phosphate is discussed with respect to the function of the known phosphate translocator in the envelope of intact chloroplasts.2. Although O₂ evolution is completely suppressed by dihydroxyacetone phosphate, CO₂ fixation takes place in air with rates of up to 65μ mol · mg^?1 chlorophyll · h^?1. As non-cyclic electron transport apparently does not occur under these conditions, these rates must be due to endogenous pseudocyclic and/or cyclic photophosphorylation.3. Under anaerobic conditions, the rates of CO₂ fixation in presence of dihydroxyacetone phosphate are low (2.5–7 μmol · mg^?1 chlorophyll · h^?1), but they are strongly stimulated by addition of dichlorophenyl-dimethylurea (e.g. 2 · 10^?7 M) reaching values of up to 60 μmol · mg^?1 chlorophyll · h^?1. As under these conditions the ATP necessary for CO₂ fixation can be formed by an endogenous cyclic photophosphorylation, the capacity of this process seems to be relatively high, so it might contribute significantly to the energy supply of the chloroplast. As dichlorophenyl-dimethylurea stimulates CO₂ fixation in presence of dihydroxyacetone phosphate under anaerobic but not under aerobic conditions, it is concluded that only under anaerobic conditions an “overreduction” of the cyclic electron transport system takes place, which is removed by dichlorophenyl-dimethylurea in suitable concentrations. At concentrations above 5 · 10^?7 M dichlorophenyl-dimethylurea inhibits dihydroxyacetone phosphate-dependent CO₂ fixation under anaerobic as well as under aerobic conditions in a similar way as normal CO₂ fixation. Therefore, we assume that a properly poised redox state of the electron transport chain is necessary for an optimal occurrence of endogenous cyclic photophosphorylation.4. The inhibition of dichlorophenyl-dimethylurea-stimulated CO₂ fixation in presence of dihydroxyacetone phosphate by dibromothymoquinone under anaerobic conditions indicates that plastoquinone is an indispensible component of the endogenous cyclic electron pathway.     

Diethyldithiocarbamate Potentiates the Neurotoxicity of In Vivo l-Methyl-4-Phenyl-1, 2, 3, 6-Tetrahydropyridine and of In Vitro 1-Methyl-4-Phenylpyridinium

Diethyldithiocarbamic acid (DDC) potentiates in vivo neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and in vitro neurotoxicity of 1-methyl-4-phenylpyridinium (MPP+). Male C57B1/6 mice were given two or five injections of MPTP (30 mg/kg i.p.) preceded 0.5 h by DDC (400 mg/kg i.p.). The mice were tested for catalepsy, akinesia, or motor activity during and after the period of dosing. Striatal and hippocampal tissues were obtained at 2 and 7 days following the last injection and evaluated for dopamine and norepinephrine levels, respectively. These same tissues were also analyzed for the levels of glial fibrillary acidic protein (GFAP), an astrocyte-localized protein known to increase in response to neural injury. Pretreatment with DDC potentiated the effect of MPTP in striatum and resulted in substantially greater dopamine depletion, as well as a more pronounced elevation in GFAP. In hippocampus, the levels of norepinephrine and GFAP were not different from controls in mice receiving only MPTP, but pretreatment with DDC resulted in a sustained depletion of norepinephrine and an elevation of GFAP, suggesting that damage was extended to this brain area by the combined treatment. Mice receiving MPTP preceded by DDC also demonstrated a more profound, but reversible, catalepsy and akinesia compared to those receiving MPTP alone. Systemically administered MPP+ decreased heart norepinephrine, but did not alter the striatal levels of dopamine or GFAP, and pretreatment with DDC did not alter these effects, but did increase lethality. DDC is known to increase brain levels of MPP+ after MPTP, but our data indicate that this is not due to a movement of peripherally generated MPP+ into CNS.(ABSTRACT TRUNCATED AT 250 WORDS)     

New synthesis of 6[3-(1-adamantyl)-4-methoxyphenyl]-2-naphthoic acid and evaluation of the influence of adamantyl group on the DNA binding of a naphthoic retinoid

6[3-(1-Adamantyl)-4-methoxyphenyl]-2-naphthoic acid (Adapalene®), a synthetic aromatic retinoid specific for RARβ and RARγ receptors, has been prepared utilizing a Pd/C-mediated Suzuki coupling between 6-bromo-2-naphthoic acid and 4-methoxyphenyl boronic acid, followed by introduction of an adamantyl group in the position 3 of the formed 6-(4-methoxyphenyl)-2-naphthoic acid. The interaction of 6-(4-methoxyphenyl)-2-naphthoic acid/ethyl ester and the 3-adamantyl analogs with DNA was studied in aqueous solution at physiological conditions by UV–vis spectroscopy. The calculated binding constants K_ligand–DNA ranged between 1.1 × 10⁴ M⁻¹ and 1.1 × 10⁵ M⁻¹, the higher values corresponding to those of the adamantylated compounds. Molecular modeling studies have emphasized that the intercalative binding of adapalene and its derivatives to DNA is mainly stabilized by hydrophobic interactions related to the presence of the adamantyl group.     

Clofibrate treatment promotes branched-chain amino acid catabolism and decreases the phosphorylation state of mTOR, eIF4E-BP1, and S6K1 in rat liver

Leucine stimulates protein synthesis by modulating the mammalian target of rapamycin (mTOR) signaling pathway. We hypothesized that promotion of the branched-chain amino acid (BCAA) catabolism might influence the leucine-induced protein synthesis. Clofibric acid (an active metabolite of clofibrate) is known to promote the BCAA catabolism by activation of branched-chain alpha-keto acid dehydrogenase complex (BCKDC), the rate-limiting enzyme of the BCAA catabolism. In the present study, we examined the phosphorylation state of mTOR, eukaryotic initiation factor 4E-binding protein-1 (4E-BP1), and ribosomal protein S6 kinase 1 (S6K1) in liver of rats with or without activation of the BCKDC by clofibrate treatment. Clofibrate-treated rats were prepared by oral administration of clofibrate 5 h before sacrifice. In order to stimulate phosphorylation of components in the mTOR signaling pathway, rats were orally administered with leucine 1 h before sacrifice. Clofibrate treatment almost fully activated hepatic BCKDC and significantly decreased the plasma leucine concentration in rats without leucine administration, resulting in decreased mTOR and 4E-BP1 phosphorylation. Similarly, in rats administered with leucine, clofibrate treatment attenuated the predicted increase in plasma leucine concentration as well as the phosphorylation of mTOR, 4E-BP1, and S6K1. These results suggest that BCAA catabolism enhanced by clofibrate treatment has significant influences on the leucine-induced activation of translation initiation processes.     

Reactions of hybrid organotellurium ligands 1-(4-methoxyphenyl telluro)-2-[3-(6-methyl-2-pyridyl)propoxy]ethane (L) and 1-ethylthio-2-[2-thienyltelluro]ethane (L) with mercury (II) bromide: formation of complexes and their decomposition

Two tellurium ligands 1-(4-methoxyphenyltelluro)-2-[3-(6-methyl-2-pyridyl)propoxy]ethane (L¹) and 1-ethylthio-2-[2-thienyltelluro]ethane (L²) have been synthesized by reacting nucleophiles [4-MeO-C₆H₄Te⁻] and [C₄H₃S-2-Te⁻] with 2-[3-(6-methyl-2-pyridyl)propoxy]ethylchloride and chloroethyl ethyl sulfide, respectively. Both the ligands react with HgBr₂ resulting in complexes of stoichiometry [HgBr₂ · L¹/L²] (1/4), which show characteristic NMR (¹H and ¹³C{¹H}). On crystallization of 1 from acetone-hexane (2:1) mixture, the cleavage of L¹ occurs resulting in 4-MeOC₆H₄HgBr (2) and [RTe⁺→HgBr₂]Br⁻ (3) (where R = -CH₂CH₂OCH₂CH₂CH₂-(2-(6-CH₃-C₅H₃N))). The 2 is characterized by X-ray diffraction on its single crystal. It is a linear molecule and is the first such system which is fully characterized structurally. The Hg-C and Hg-Br bond lengths are 2.085(6) and2.4700(7) Å. The distance of four bromine atoms (3.4041(7)-3.546(7) Å) around Hg (cis to C) is greater than the sum of van der Waal’s radii 3.30 Å. This mercury promoted cleavage is observed for an acyclic ligand of RArTe type for the first time and is unique, as there appears to be no strong intramolecular interaction to stabilize the cleavage products. The 4 on crystallization shows the cleavage of organotellurium ligand L² and formation of a unique complex [(EtS(CH₂)₂SEt)HgBr(μ-Br)Hg(Br)(μ-Br)₂Hg(Br)(μ-Br)BrHg(EtS(CH₂)₂SEt)] · 2HgBr₂ (5), which has been characterized by single crystal structure determination and ¹H and ¹³C{¹H} NMR spectra. The elemental tellurium and [C₄H₃SCH₂]₂ are the other products of dissociation as identified by NMR (proton and carbon-13). The cleavage appears to be without any transmetalation and probably first of its kind. The centrosymmetric structure of 5 is unique as it has [HgBr₃]⁻ unit, one Hg in distorted tetrahedral geometry and one in pseudo-trigonal bipyramidal one. The molecule of 5 may also be described as having [(EtSCH₂CH₂SEt)HgBr]⁺ [HgBr₃]⁻ units, which dimerize and co-crystallize with two HgBr₂ moieties. There are very weak Hg?Br interactions between co-crystallized HgBr₂ units and rest of the molecule. [Hg(3)-Br(1)/Hg(3)-Br(4) = 3.148(1)/3.216(1) Å]. The bridging Hg?Br distances, Hg(2)-Br(4)′, Hg(2)′-Br(4) and Hg(1)-Br(2), are from 2.914(1) to 3.008(1) Å.     

Identification of the role of a cysteine-rich region of PC6B by determining the enzymatic characteristics of its mutants

In the proprotein convertases family, mouse PC6B (mPC6B) has a very large cysteine-rich region (CRR), consisting of 22 tandem cysteine-rich (Cys-rich) repeated segments. The role of this region remains elusive. In this report, to get insight on the possible role of the CRR, we constructed four truncated mPC6B mutant genes with 0, 5, 11, and 22 Cys-rich repeated segments remaining; using the baculovirus-expression system and a simple purification method, we obtained four enzyme mutants of mPC6B. By determining their optimal pH and calcium ion concentration for enzymatic activity and their thermal stability, we found that CRR did not affect pH optimum and Ca²⁺ optimum compared with the p-domain. However, CRR acted as a stabilizing domain in addition to the p-domain. By kinetic analyses of four mutants, we found that the long Cys-rich repeats in the native form of mPC6B reduced its V _max. These facts suggest that CRR acts as an important part of functional domain.     

Fragment-based design,synthesis, biological evaluation,and SAR of 1H-benzo[d]imidazol-2-yl)-1H-indazol derivatives as potent PDK1 inhibitors

In this work, we describe the use of the rule of 3 fragment-based strategies from biochemical screening data of 1100 in-house, small, low molecular weight fragments. The sequential combination of in silico fragment hopping and fragment linking based on S160/Y161/A162 hinge residues hydrogen bonding interactions leads to the identification of novel 1H-benzo[d]imidazol-2-yl)-1H-indazol class of Phosphoinositide-Dependent Kinase-1 (PDK1) inhibitors. Consequent SAR and follow-up screening data led to the discovery of two potent PDK1 inhibitors: compound 32 and 35, with an IC₅₀ of 80?nM and 94?nM, respectively. Further biological evaluation showed that, at the low nanomolar concentration, the drug had potent ability to inhibit phosphorylation of AKT and p70S6, and selectively kill the cancer cells with mutations in both PTEN and PI3K. The microarray data showed that DUSP6, DUSP4, and FOSL1 were down-regulated in the sensitive cell lines with the compound treatment. The in vivo test showed that 35 can significantly inhibit tumor growth without influencing body weight growth. Our results suggest that these compounds, especially 35, merit further pre-clinical evaluation.     

Calmodulin and the target size of the (Ca2+ + Mg2+)-ATPase of human red-cell ghosts

An average target size of 251 kDa has been obtained for the (Ca²⁺ + Mg²⁺)-ATPase of calmodulin-depleted erythrocyte ghosts by radiation inactivation with 16 MeV electrons. This is close to twice the size of the purified calcium-pump polypeptide. When calmodulin was included during the ATPase assay, a component of about 1 MDa appeared in addition to the activated dimer.     

PP2A: more than a reset switch to activate pRB proteins during the cell cycle and in response to signaling cues

In their active hypophosphorylated state, members of the retinoblastoma family of pocket proteins negatively regulate cell cycle progression at least in part by repressing expression of E2F-dependent genes. Mitogen-dependent activation of G1 and G1/S Cyclin Dependent Kinases (CDKs) results in coordinated hyperphosphorylation and inactivation of these proteins, which no longer bind and repress E2Fs. S and G2/M CDKs maintain pocket protein hyperphosphorylated through the end of mitosis. The inactivating action of inducible CDKs is opposed by the Ser/Thr protein phosphatases PP2A and PP1. Various trimeric PP2A holoenzymes have been implicated in dephosphorylation of pocket proteins in response to specific cellular signals and stresses or as part of an equilibrium with CDKs throughout the cell cycle. PP1 has specifically been implicated in dephosphorylation of pRB in late mitosis and early G1. This review is particularly focused on the emerging role of PP2A as a major hub for integration of growth suppressor signals that require rapid inactivation of pocket proteins. Of note, activation of particular PP2A holoenzymes triggers differential activation of pocket proteins in the presence of active CDKs.     

Synaptotagmin I and Ca(2+) promote half fusion more than full fusion in SNARE-mediated bilayer fusion

Synaptic membrane fusion, which is necessary for neurotransmitter release, may be mediated by SNAREs and regulated by synaptotagmin (Syt) and Ca(2+). Fusion of liposomes mediated by reconstituted SNAREs produces full fusion and hemifusion, a membrane structure in which outer leaflets are mixed but the inner leaflets remain intact. Here, using the liposome fusion assay, it is shown that Syt promoted both hemifusion and full fusion in a Ca(2+)-dependent manner. Syt.Ca(2+) increased hemifusion more than full fusion, modulating the ratio of hemifusion to full fusion. Unlike the case of neuronal SNAREs, stimulation of fusion by Syt.Ca(2+) was not seen for other SNAREs involved in trafficking in yeast, indicating that the Syt.Ca(2+) stimulation was SNARE-specific. We constructed hybrid SNAREs in which transmembrane domains were swapped between neuronal and yeast SNAREs. With these hybrid SNAREs, we demonstrated that the interaction between the SNARE motifs of neuronal proteins and Syt.Ca(2+) was required for the stimulation of fusion.