High-speed Pellet Fraction Research Articles

Direct dynamin–actin interactions regulate the actin cytoskeleton

The large GTPase dynamin assembles into higher order structures that are thought to promote endocytosis. Dynamin also regulates the actin cytoskeleton through an unknown, GTPase-dependent mechanism. Here, we identify a highly conserved site in dynamin that binds directly to actin filaments and aligns them into bundles. Point mutations in the actin-binding domain cause aberrant membrane ruffling and defective actin stress fibre formation in cells. Short actin filaments promote dynamin assembly into higher order structures, which in turn efficiently release the actin-capping protein (CP) gelsolin from barbed actin ends in vitro, allowing for elongation of actin filaments. Together, our results support a model in which assembled dynamin, generated through interactions with short actin filaments, promotes actin polymerization via displacement of actin-CPs.

Turnover of the Active Fraction of IRS1 Involves Raptor-mTOR- and S6K1-Dependent Serine Phosphorylation in Cell Culture Models of Tuberous Sclerosis

The TSC1-TSC2/Rheb/Raptor-mTOR/S6K1 cell growth cassette has recently been shown to regulate cell autonomous insulin and insulin-like growth factor I (IGF-I) sensitivity by transducing a negative feedback signal that targets insulin receptor substrates 1 and 2 (IRS1 and -2). Using two cell culture models of the familial hamartoma syndrome, tuberous sclerosis, we show here that Raptor-mTOR and S6K1 are required for phosphorylation of IRS1 at a subset of serine residues frequently associated with insulin resistance, including S307, S312, S527, S616, and S636 (of human IRS1). Using loss- and gain-of-function S6K1 constructs, we demonstrate a requirement for the catalytic activity of S6K1 in both direct and indirect regulation of IRS1 serine phosphorylation. S6K1 phosphorylates IRS1 in vitro on multiple residues showing strong preference for RXRXXS/T over S/T,P sites. IRS1 is preferentially depleted from the high-speed pellet fraction in TSC1/2-deficient mouse embryo fibroblasts or in HEK293/293T cells overexpressing Rheb. These studies suggest that, through serine phosphorylation, Raptor-mTOR and S6K1 cell autonomously promote the depletion of IRS1 from specific intracellular pools in pathological states of insulin and IGF-I resistance and thus potentially in lesions associated with tuberous sclerosis.

MAP kinase phosphorylation of plant profilin

Profilin is a small actin-binding protein and is expressed at high levels in mature pollen where it is thought to regulate actin filament dynamics upon pollen germination and tube growth. The majority of identified plant profilins contain a MAP kinase phosphorylation motif, P–X–T–P, and a MAP kinase interaction motif (KIM). In in vitro kinase assays, the tobacco MAP kinases p45 Ntf4 and SIPK, when activated by the tobacco MAP kinase kinase NtMEK2, can phosphorylate the tobacco profilin NtProf2. Mutagenesis of the threonine residue in this motif identified it as the site of MAP kinase phosphorylation. Fractionation of tobacco pollen extracts showed that p45 Ntf4 is found exclusively in the high-speed pellet fraction while SIPK and profilin are predominantly cytosolic. These data identify one of the first substrates to be directly phosphorylated by MAP kinases in plants.

Active PIKfyve Associates with and Promotes the Membrane Attachment of the Late Endosome-to-trans-Golgi Network Transport Factor Rab9 Effector p40

PIKfyve, a kinase that displays specificity for phosphatidylinositol (PtdIns), PtdIns 3-phosphate (3-P), and proteins, is important in multivesicular body/late endocytic function. Enzymatically inactive PIKfyve mutants elicit enormous dilation of late endocytic structures, suggesting a role for PIKfyve in endosome-to-trans-Golgi network (TGN) membrane retrieval. Here we report that p40, a Rab9 effector reported previously to bind Rab9-GTP and stimulate endosome-to-TGN transport, interacts with PIKfyve as determined by yeast two-hybrid assays, glutathione S-transferase (GST) pull-down assays, and co-immunoprecipitation in doubly transfected HEK293 cells. The interaction engages the PIKfyve chaperonin domain and four out of the six C-terminally positioned kelch repeats in p40. Differential centrifugation in a HEK293 cell line, stably expressing PIKfyveWT, showed the membrane-associated immunoreactive p40 co-sedimenting with PIKfyve in the high speed pellet (HSP) fraction. Remarkably, similar analysis in a HEK293 cell line stably expressing dominant-negative kinase-deficient PIKfyveK1831E demonstrated a marked depletion of p40 from the HSP fraction. GST-p40 failed to specifically associate with the PIKfyve lipid products PtdIns 5-P and PtdIns 3,5-P2 in a liposome binding assay but was found to be an in vitro substrate of the PIKfyve serine kinase activity. A band with the p40 electrophoretic mobility was found to react with a phosphoserine-specific antibody mainly in the PIKfyveWT-containing fractions obtained by density gradient sedimentation of total membranes from PIKfyveWT-expressing HEK293 cells. Together these results identify the Rab9 effector p40 as a PIKfyve partner and suggest that p40-PIKfyve interaction and the subsequent PIKfyve-catalyzed p40 phosphorylation anchor p40 to discrete membranes facilitating late endosome-to-TGN transport.

14-3-3 facilitates insulin-stimulated intracellular trafficking of insulin receptor substrate 1.

The appearance of a complex between tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1) and PI3K in a high-speed pellet fraction (HSP) is thought to be a key event in insulin action. Conversely, the disappearance of the IRS-1/PI3K complex from this fraction has been linked to insulin desensitization. The present study examines the role of 14-3-3, a specific phospho-serine binding protein, in mediating the disappearance of IRS-1 from the HSP after insulin treatment. An in vitro pull-down assay using recombinant 14-3-3 revealed that insulin enhances the association of 14-3-3 with IRS-1 in cultured adipocytes and that this is completely inhibited by wortmannin. An association of IRS-1 and 14-3-3 was also observed and was maximal after stimulation by insulin, when endogenous proteins were immunoprecipitated. Epidermal growth factor (EGF), 12-O-tetradecanoylphorbol-13-acetate, and okadaic acid, other agents that cause serine/threonine phosphorylation of IRS-1, also stimulated IRS binding to 14-3-3. The enhancement of IRS-1 binding to 14-3-3 by insulin was accompanied by movement of IRS-1 and the p85 subunit of PI3K from the HSP to the cytosol. In keeping with a key role of 14-3-3 in mediating this redistribution of IRS-1, the complexes of IRS-1 and 14-3-3 were found in the cytosol but not in the HSP of insulin-treated cells. In addition, colocalization of IRS-1 and 14-3-3 was observed in the cytoplasm after insulin treatment by confocal microscopy. Finally, the addition of a phosphorylated 14-3-3 binding peptide to an adipocyte homogenate (to remove 14-3-3 from IRS-1) increased the abundance of IRS-1/PI3K complexes in the HSP and decreased their abundance in the cytosol. These findings strongly suggest that 14-3-3 participates in the intracellular trafficking of IRS-1 by promoting the displacement of serine-phosphorylated IRS-1 from particular structures. They also suggest that 14-3-3 proteins could play an integral role in the process of insulin desensitization.

Insulin-regulatable phosphoproteins in 3T3-L1 adipocytes form detergent-insoluble complexes not associated with caveolin.

Whole body glucose homeostasis is dependent on the action of insulin. In muscle and adipose tissues, insulin stimulates glucose uptake by inducing the translocation of vesicles containing the glucose transporter GLUT4 to the cell surface. While the mechanisms of insulin-regulated GLUT4 translocation are not fully understood, some signaling intermediates have been implicated in this process. Interestingly, some of these intermediates, including IRS-1 and PI3K, have been localised to the same intracellular membrane fraction as the GLUT4 storage pool, designated here as the high-speed pellet (HSP) fraction. This raises the possibility that many of the downstream insulin signaling intermediates may be located within close proximity to intracellular GLUT4. The goal of this study was to test this hypothesis in 3T3-L1 adipocytes. A large proportion of adipocyte phosphoproteins co-fractionated in the HSP fraction. In an attempt to resolve insulin-regulatable phosphoproteins, we subjected 32P-labeled subcellular fractions to two-dimensional gel electrophoresis (2-DE). Insulin reproducibly stimulated the phosphorylation of 12 spots in the HSP fraction. Most of the HSP phosphoproteins were insoluble in the nonionic detergent Triton X-100, whereas integral membrane proteins such as GLUT4 and intracellular caveolin were soluble under the same conditions. These results suggest that insulin-regulatable phosphoproteins in adipocytes may be organized in microdomains within the cell and that this assembly may act as an efficient conductor of the signaling proteins to rapidly facilitate downstream biological responses. Further study is required to establish the molecular basis for these detergent-insoluble signaling complexes.

A significant soluble keratin fraction in ‘simple’ epithelial cells Lack of an apparent phosphorylation and glycosylation role in keratin solubility

We studied the solubility of keratin polypeptides 8 and 18 (K8/18), which are the predominant intermediate filaments in the human colonic epithelial cell line HT29. We find that asynchronously growing cells (G0/G1 stage of the cell cycle) have a substantial pool of soluble keratin that constitutes approx. 5% of total cellular keratin. This soluble keratin pool was observed after immunoprecipitation of K8/18 from the cytosolic fraction of cells disrupted using three detergent-free methods. Several other cell lines showed a similar significant soluble cytosolic K8/18 pool. Arrest of HT29 cells in G2/M stage of the cell cycle was associated with a concurrent increase in keratin solubility. Comparison of K8/18 obtained from the soluble cytosolic fraction and the insoluble high-speed pellet fraction showed similar levels of phosphorylation and glycosylation and similar tryptic radiolabeled phospho- and glycopeptide patterns. Soluble K8/18 can form characteristic 10 nm filaments in vitro as determined by electron microscopy. Cross-linking of soluble K8/18 followed by immunoprecipitation resulted in dimeric and tetrameric forms, based on migration in SDS-polyacrylamide gels. In addition, cross-linked and native soluble K8/18 showed similar migration on nondenaturing gels and similar sedimentation after sucrose density gradient centrifugation. Our results indicate that simple epithelial keratins are appreciably more soluble than previously recognized. The soluble keratin form is assembly competent and appears to be primarily tetrameric. Although K8/18 solubility was found to increase during mitotic arrest, glycosylation and phosphorylation did not play an obvious role in generating the soluble fraction, suggesting an alternate mechanism for keratin solubility.

The pattern of accumulation of cauliflower mosaic virus-specific products in infected turnips

The concentrations of cauliflower mosaic virus (CaMV) DNA and protein products in the developing leaves of a host, turnip, have been measured and the results have been correlated with symptom production. Virus-specific products were limited to the symptomatic leaves. CaMV DNA was detected in the youngest foliar tissues showing full systemic symptoms and continued to accumulate as the leaf expanded, indicating that virus multiplication was not restricted to meristematic tissues of the host plant and that virus concentration was not a primary determinant for symptom production. Using specific antisera for Western blot analysis, the distribution of CaMV-specific proteins (P1-P6) in a range of subcellular fractions of infected tissue was determined. The protein products (P2-P6) of genes II–VI were all detected in fractions enriched for virus inclusion bodies, although P5 was present only at low levels. A high-speed pellet fraction enriched for virus replication complexes revealed P5 in higher concentrations, and also contained P4 and small amounts of P6 in proportions which indicated that replication complexes had been released from inclusion bodies. In the different leaves of the host, P2, 3, 4, 5, and 6 all increased in concentration in parallel with viral DNA, although there appeared to be a bias toward protein rather than DNA synthesis in the very young leaves. P1 showed a different pattern of accumulation; it was most concentrated in the very young and the oldest infected tissues, and showed a different spectrum of products between leaves. The experiments described provide a more complete picture of the relationship between CaMV multiplication and expression, and leaf development, and an increased understanding of how the disease syndrome is established.

Translocation of glucose transporters in response to anoxia in heart.

We tested whether translocation of glucose transporters between subcellular membrane fractions is involved in the stimulation of glucose transport by anoxia by perfusing rat hearts in the presence or absence of oxygen. The hearts were then fractionated by a modification of the procedures of Watanabe, et al. (Watanabe, T., Smith, M. M., Robinson, F. W., and Kono, T. (1984) J. Biol. Chem. 259, 13117-13122), who previously demonstrated translocation in response to insulin in heart, to give plasma membrane and high-speed pellet fractions. The contents of glucose transporters in the two fractions were determined by reconstitution of transport activity, D-glucose-reversible binding of cytochalasin B, and labeling with antibodies against the erythrocyte transporter. The heart transporter was also recognized by antibodies against the COOH-terminal peptide of the glucose transporter. All three types of assays revealed a decrease (20-30%) in the high-speed pellet fraction and an increase (20-70%) in the plasma membranes in response to anoxia. Treatment of hearts with insulin produced a similar extent of translocation and a similar stimulation (about 2-fold) of glucose uptake, indicating that translocation plays a role of similar importance in the stimulation of transport by both of these effectors.

Subcellular distribution of lysophospholipase of rat intestinal mucosa

We have studied the subcellular localization of rat intestinal lysophospholipase activity and some of the biochemical properties of this enzyme. After subcellular fractionation, an enriched activity was found in the high-speed pellet fraction containing the microsomes and the brush border membranes. Subsequently, these organelles were isolated. Using the classical calcium-precipitation method to isolate brush border membranes, we failed to demonstrate any significant recovery of lysophospholipase activity associated with this fraction. The microsomal fraction was further isolated after density gradient centrifugation, and most of the lysophospholipase activity was recovered with this fraction. Because further purification of the enzyme was unsuccessful, some of the biochemical properties of the enzyme were determined on the partially purified microsomal fraction. The optimum pH of the activity was centered at 7.0, and the enzyme did not require bivalent cations. By using double reciprocal plots, we determined the Kapp(m) to be 0.4 mM; the Vapp(max), 23 mumol.h-1.mg protein-1. The enzyme was strongly inhibited by detergents having a low critical micellar concentration and less inhibited by those having a higher critical micellar concentration.

Apparent translocation of glucose transport activity in rat epididymal adipocytes by insulin-like effects of high pH or hyperosmolarity.

The basal and plus insulin states of glucose transport activity in adipocytes are known to show different responses to changes in the pH or osmolarity of the incubation mixture. When the pH was raised from 7 to 8, the basal glucose transport activity (assessed from the rate of 3-O-methyl-D-glucose uptake) was increased approximately 3-fold while the plus insulin activity remained virtually unaffected. Likewise, when cells were exposed to 300 mM sorbitol, the basal glucose transport activity, but not the plus insulin activity, was considerably increased. In both cases, the change in the transport activity was ATP-dependent and was completed in approximately 60 min. The increase in the cellular glucose transport activity was accompanied, in both cases, by an increase in the glucose transport activity in the plasma membrane fraction and a decrease in the activity in the high-speed pellet fraction. The transport activity in the subcellular fractions was determined after reconstitution into egg lecithin liposomes. Both isotonic buffer at pH 8.0 and hypertonic buffer at pH 7.4 significantly stimulated membrane-bound cAMP phosphodiesterase in adipocytes. It is concluded that the above two experimental conditions may induce insulin-like effects in fat cells and may facilitate translocation of the glucose transport activity from an intracellular site to the plasma membrane.

Activation of the respiratory burst enzyme from human neutrophils in a cell-free system. Evidence for a soluble cofactor.

Activation of the respiratory burst in phagocytic cells, an important host defense process, is not yet well understood. We now report the development of a cell-free system for activation of NADPH oxidase, the respiratory burst enzyme, in human neutrophils. Activation was achieved by the addition of arachidonic acid to a postnuclear supernatant (500 g) from disrupted unstimulated cells (no arachidonate, 0.2; with arachidonate, 3.4 nmol superoxide anion/min per mg) and was dependent on both the concentration of arachidonate and on the amount of cellular material present. Activity stimulated by arachidonate appeared to be NADPH oxidase based on a Michaelis constant for NADPH of 32 microM and a pH optimum of 7.0-7.5. Separation of the 500-g supernatant by high speed centrifugation revealed a requirement for both soluble and particulate cofactors. Activation of NADPH oxidase by arachidonate did not occur in the high speed pellet fraction from unstimulated cells but could be restored by the addition of the high speed supernatant. In addition, priming of intact neutrophils with low concentrations of the chemoattractant N-formyl-methionyl-leucyl-phenylalanine or the tumor promoter phorbol myristate acetate replaced the soluble factor requirement for NADPH oxidase activation by arachidonate in the high speed pellet. This cell-free system can now be used to provide further insight into the biochemical basis of priming and the terminal mechanisms involved in the activation of NADPH oxidase.

Detoxification of the phytoalexin pisatin by a fungal cytochrome P-450

The fungus Nectria haematococca, a pathogen of garden pea ( Pisum sativum), can demethylate pisatin, an antimicrobial compound synthesized by infected pea tissue. The phenolic product is less toxic than pisatin to many microorganisms. Cell extracts catalyzing pisatin demethylation were obtained from N. haematococca, and the properties of the reaction were examined. The enzyme activity was greatest in the high-speed pellet fraction, in which rates up to 20 nmol/min/mg protein were observed. The K m for pisatin was relatively low, less than 5 μ m. The reaction was dependent on NADPH, which could not be replaced by any other cofactor tested. However, in the presence of NADPH, NADH increased the rate of demethylation. Oxygen uptake by the enzyme was stimulated by addition of pisatin, the increment of oxygen utilization being approximately equimolar with pisatin added. Formaldehyde was a product of the reaction. The effects of various inhibitors were tested to determine whether this reaction is mediated by cytochrome P-450. The respiratory inhibitors KCN (1 m m) and antimycin A strongly inhibited the demethylation of pisatin by intact cells of the fungus, but not by the NADPH-supplemented enzyme. The cytochrome P-450 inhibitors SKF 525-A and 1-(2-isopropylphenyl)imidazole inhibited demethylation both in whole cells and in the enzyme preparation, though the latter compound was effective only at high concentrations. Most other cytochrome P-450 inhibitors tested had little effect. However the reaction was quite sensitive to CO, and this inhibition was readily reversed by light at wavelengths near 450 nm. It is concluded that pisatin demethylase is a cytochrome P-450 monooxygenase.

DNA polymerase activity of cultured rheumatoid synovial cells

Cultured synovial cells from 8 patients with rheumatoid arthritis (RA) and 7 subjects without joint disease were assayed and comapred for RNA-directed and DNA-directed DNA polymerase activity. No activity was found in either RA or control specimens using a synthetic RNA template that specificically detects oncornavirus RNA-directed DNA polymerase. The DNA-directed DNA polymerase activity of RA specimens was increased (P less than 0.10) in the high-speed pellet fraction of cell lysates. The possible relationship of these finding to virus infection in RA is disscussed.

Characterization of a new murine cellular DNA polymerase.

A new DNA polymerase (peak A) has been identified in the high-speed pellet fraction of two subclones of nonvirus producing Balb/3T3 cells. The activity is associated with a molecule of approximately 70,000 molecular weight and chromatographs in two systems like the mouse type-C viral reverse transcriptase and a similar enzyme from the high-speed pellet fraction of a virus producing Balb/3T3 subclone, S(2)Cl(3). Comparable quantities of peak A are present in both nonvirus infected (A31) and mouse sarcoma virus transformed nonproducer (KA31) subclones. Virus producing cells contain 10-20 times more peak A polymerase activity. A31 and KA31 peak A are comparably inhibited by anti-mouse type C virus reverse transcriptase IgG but to a lesser degree than S(2)Cl(3) peak A or authentic viral reverse transcriptase. They can also be differentiated from the latter two enzymes by template preference studies. KA31 peak A can be distinguished from three other KA31 DNA polymerases (R-DNA polymerase and DNA polymerase N and C), and thus appears to be a new species of cellular DNA polymerase.

Ribonuclease-sensitive deoxyribonucleic acid polymerase activity in uninfected rat cells and rat cells infected with Rous sarcoma virus.

Rat cells infected with the B77 strain of avian sarcoma virus [R(B77) cells] produced no virus-like particles but contained information for the production of infectious B77 virus. (3)H-labeled deoxyribonucleic acid (DNA) product of the B77 virus endogenous DNA polymerase system was used to determine the relative amounts of B77 virus-specific ribonucleic acid (RNA) in B77 virus-infected chicken and R(B77) cells. R(B77) cells were found to contain much less B77 virus RNA than did B77 virus-infected chicken cells. Ribonuclease-sensitive DNA polymerase activity was present in high-speed pellet fractions from Nonidet extracts of B77 virus-infected rat cells. Similar preparations from some uninfected rat cells contained lesser amounts of a similar ribonuclease-sensitive DNA polymerase activity. The endogenous template for the DNA polymerase activity in high-speed pellet fractions from R(B77) cells was not related to B77 virus RNA or to RNA of a rat C-type virus. The DNA product of the endogenous DNA polymerase in high-speed pellet fractions of R(B77) cells hybridized to a small extent with RNA from the same fraction and to a similar extent with RNA from uninfected rat cells.