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Biomedical subjects

A R Saltiel

Publications and source records attributed to A R Saltiel.

At least 127 records · Page 7Linked to original sources
PubMed1992

Stimulation by growth hormone of MAP kinase activity in 3T3-F442A fibroblasts.

We have previously demonstrated that growth hormone (GH) promotes an increase in tyrosine kinase activity associated with the GH receptor. To gain insight into the role of GH-dependent tyrosine kinase activity in signaling by GH, we investigated the possibility that GH might stimulate MAP kinase, a serine/threonine/tyrosine kinase thought to be a common element in tyrosine kinase-initiated response cascades. Treatment of 3T3-F442A fibroblasts with 100 ng/ml GH results in a 3-6-fold increase in the ability of cell-free extracts to phosphorylate MAP-2 and myelin basic protein. GH-stimulated kinase activity is unaffected by heparin, H7, or cAMP-dependent protein kinase inhibitor peptide, partially reduced by staurosporin and inhibited by fluoride and calcium ions, indicating that the kinase is not protein kinase C or A, casein kinase, or a calcium/calmodulin-dependent protein kinase. Based on gel permeation chromatography, the molecular mass of the GH-stimulated MAP kinase is approximately kDa. Furthermore, anti-phosphotyrosine antibodies revealed the GH-dependent appearance of two phosphotyrosine-containing proteins in cell-free lysates of GH-treated cells that co-migrate with proteins recognized by anti-MAP kinase antibodies. The GH-dependent increase in MAP kinase activity displays a biphasic time course and is dependent on the concentration of GH applied to the cells. GH-dependent MAP kinase activity, partially purified by Mono-Q chromatography, is inactivated by treatment with alkaline phosphatase. Addition of H7 to the cells prior to the addition of GH has no effect, whereas addition of H8 increases MAP kinase activity in control cells with no effect in GH-treated cells, indicating that protein kinase C is unlikely to be an intermediary in the GH-dependent stimulation of MAP kinase activity. These findings indicate that signaling by GH in 3T3-F443A cells may, at least in part, utilize a kinase cascade similar to those that have been proposed for other membrane receptors with associated tyrosine kinase activity.

3T3 Cells↗
PubMed1992

Activation of phosphatidylinositol-3 kinase by nerve growth factor involves indirect coupling of the trk proto-oncogene with src homology 2 domains.

Growth factor receptor tyrosine kinases can form stable associations with intracellular proteins that contain src homology (SH) 2 domains, including the p85 regulatory subunit of phosphatidylinositol (PI)-3 kinase. The activation of this enzyme by growth factors is evaluated in PC12 pheochromocytoma cells and NIH 3T3 fibroblasts expressing the pp140c-trk nerve growth factor (NGF) receptor (3T3-c-trk). NGF causes the rapid stimulation of PI-3 kinase activity detected in anti-phosphotyrosine, but not in anti-trk, immunoprecipitates. This effect coincides with the tyrosine phosphorylation of two proteins, with molecular masses of of 100 kd and 110 kd, that coimmunoprecipitate with p85. Similar phosphorylation patterns are induced when an immobilized fusion protein containing the amino-terminal SH2 domain of p85 is used to precipitate tyrosine-phosphorylated proteins. Thus, although NGF produces the rapid activation of PI-3 kinase through a mechanism that involves tyrosine phosphorylation, there is no evidence for tyrosine phosphorylation of p85, or for its ligand-dependent association with the NGF receptor. Perhaps another phosphoprotein may link the NGF receptor to this enzyme.

3T3 Cells↗
PubMed1992

Diversity in cellular signaling for nerve growth factor and insulin: variations on a common theme.

Numerous similarities exist in the cellular signaling events observed for insulin and nerve growth factor. Because the two hormones share many functional properties, and exhibit similar effects on neurons, the possibility of common early signaling events has been explored. Many studies have focused on the important role of protein phosphorylation. Two distinct but related mechanisms are discussed that may mediate, in part, the ability of these two hormones to regulate the activities of protein kinases and phosphatases.

Amino Acid Sequence↗
PubMed1991

Nerve growth factor binds to the 140 kd trk proto-oncogene product and stimulates its association with the src homology domain of phospholipase C gamma 1.

The cellular actions of nerve growth factor (NGF) involve regulation of protein phosphorylation. In PC-12 pheochromocytoma cells, exposure of [125I]NGF followed by crosslinking indicates that the ligand binds to two discreet receptors, the previously described 75 kd protein, as well as the trk proto-oncogene product pp140c-trk. Competition experiments reveal that of the two, pp 140c-trk binds to NGF with higher affinity. Following exposure to NGF, pp140c-trk undergoes a rapid autophosphorylation on tyrosine residues, and concomitantly phosphorylates and associates with phospholipase C gamma 1 (PLC gamma 1), through interaction with its src homology domains. The binding of NGF to pp140c-trk with high affinity, the NGF-dependent homology domains. The binding of NGF to pp140c-trk with high affinity, the NGF-dependent activation of its tyrosine kinase activity and the specific association with the effector molecule, PLC gamma 1, suggests that this is the biologically relevant signaling receptor for NGF.

Adrenal Gland Neoplasms↗
PubMed1991

Phospholipase C-gamma 1 directly associates with the p70 trk oncogene product through its src homology domains.

Tyrosine phosphorylation of proteins was examined in NIH3T3 cells transformed by an oncogenic form of the trk protein. Proteins of 148, 140, 70, and 55 kDa were phosphorylated on tyrosine residues in trk-transformed cells but not control NIH3T3 cells. The 70-kDa protein may represent the trk oncogene protein itself which was shown to be tyrosine-phosphorylated in vivo using trk-specific antiserum. Phospholipase C-gamma 1 (PLC-gamma 1) was also found to be constitutively tyrosine-phosphorylated in trk-transformed cells and the trk protein co-immunoprecipitated with PLC-gamma. The GTPase-activating protein of ras (GAP) and the 62-kDa GAP-associated protein were tyrosine-phosphorylated in trk-transformed cells, and a lesser amount of trk co-immunoprecipitated with GAP relative to with PLC-gamma. The trk oncogene product bound specifically to a bacterially expressed fusion protein containing the src homology domains of PLC-gamma. The data suggest a significant role for PLC-gamma in intracellular signaling by the trk oncogene.

Blotting, Western↗
PubMed1991

Synthesis of phosphatidylinositol in rat liver microsomes is accompanied by the rapid formation of lysophosphatidylinositol.

In mammalian cells, newly synthesized phosphatidylinositol (PI) has a fatty acid composition similar to its precursors, phosphatidic acid and CDP-diacylglycerol (DAG). It is then remodelled by deacylation/reacylation cycles to the predominant form, 1-stearoyl, 2-arachidonoyl PI. Incubation of dipalmitoyl CDP-DAG, [3H]inositol and Mg2+ with rat liver microsomes results in the rapid synthesis of PI, along with the simultaneous formation of multiple species of lysoPI. Analysis of the kinetics of formation of PI and lysoPI reveals no lag in the formation of lysoPI from PI. Moreover, evaluation of the concentration dependencies indicate nearly identical apparent Km values for PI synthesis compared with lysoPI synthesis for the substrates inositol (180 microM) and CDP-DAG (100 microM). The dependence on pH and the requirement for Mg2+ or Mn2+ are nearly identical for PI and lysoPI formation and the labelling of both lipids is similarly inhibited by submicromolar concentrations of calcium and by NEM. These results suggest that the formation of lysoPI is dependent on the initial, rate-limiting synthesis of PI. Pulse-chase analysis of the labelling of these lipids indicates that PI and lysoPI rapidly equilibrate after the initial slow synthesis of PI. In addition, it appears that only newly synthesized PI is involved in lysoPI formation. The extent of lysoPI formation depends upon the fatty acid composition of the added CDP-DAG. A number of experimental approaches demonstrate that lysoPI is not formed when pre-existing microsomal PI is labelled by head group exchange, perhaps because this PI has already undergone remodelling to polyenoic forms. These data suggest that the rapid deacylation of newly synthesized PI may represent the first step in PI remodeling.

Acylation↗
PubMed1991

Fatty acid remodelling of phosphatidylinositol under conditions of de novo synthesis in rat liver microsomes.

Phosphatidylinositol (PI) is initially synthesized in mammalian cells with a fatty acid composition similar to that of its precursor, primarily monounsaturated forms of cytidine diphosphodiglyceride (CDP-DAG). However, at the steady state, over 80% of PI exists in the 1-stearoyl, 2-arachidonoyl form. The fatty acid remodelling of PI is due to a number of deacylation/reacylation mechanisms. In the preceding paper we demonstrated that de novo synthesized PI is rapidly deacylated and subsequently reacylated. In this report we present further evidence that cycles of deacylation and reacylation are involved in the remodelling of PI. Incubation of microsomes with CDP-DAG of different fatty acid composition results in quantitative and qualitative differences in lysoPI formation. Additionally, analyses of the resulting lysoPI and PI species reveal that multiple species of fatty acids are incorporated into the 1-position of both PI and lysoPI. Addition of acylation cofactors (fatty acyl CoAs or ATP plus CoA) potentiate reacylation in this system. The addition of stearoyl or myristoyl CoA during de novo synthesis of PI results in the incorporation of these added fatty acids into the I-positive of PI. In addition, some evidence is presented that multiple mechanisms for remodelling of the 1-position of PI may be active in the microsomes, including ATP- and CoA-dependent acylation, ATP-independent, CoA-dependent acylation and CoA-independent mechanisms. Finally, the disappearance of only a subset of lysoPI species upon the addition of acylation cofactors suggests that the reacylation step exhibits some substrate specificity.

Acyl Coenzyme A↗
PubMed1991

Coenzyme A-dependent, ATP-independent acylation of 2-acyl lysophosphatidylinositol in rat liver microsomes.

Phosphatidylinositol (PI) is synthesized from cytidine-diphosphodiacylglycerol (CDP-DAG) and inositol by the enzyme PI synthase. CDP-DAG is itself synthesized from phosphatidic acid and CTP. The observation that PI differs in fatty acid composition from its precursors CDP-DAG and phosphatidic acid led to the proposal that following its synthesis the fatty acids of PI are removed and replaced by others in a process called fatty acid remodelling. Previously, we used rat liver microsomes to study the molecular mechanisms of PI remodelling. Following its synthesis, PI is rapidly deacylated to form lysoPI which is reacylated to form new PI species. PI remodelling occurs predominantly at the 1-position. We demonstrate here that lysoPI can be acylated in the 1-position in an ATP-independent manner. The acylation of 2-acyl lysoPI by the coenzyme A-dependent, ATP-independent mechanism was examined. The acylation exhibits a pH optimum of 7.5, does not require a divalent cation, and is not inhibited by Ca2+ or Mg2+, although Zn2+ is a potent inhibitor. The apparent Km values for coenzyme A and 2-acyl lysoPI are 14 microM and 30 microM, respectively. The acylation of 2-acyl lysoPI incorporates primarily stearic acid into the 1-position of PI, as would be expected based on the fatty acid composition of steady-state PI in rat hepatocytes.

Acylation↗
PubMed1991

Amylin increases cyclic AMP formation in L6 myocytes through calcitonin gene-related peptide receptors.

The cellular function of amylin is investigated in L6 myocytes, a rat skeletal muscle cell line. Both rat amylin and human amylin-amide acutely cause a dose-dependent increase in cyclic AMP formation in L6 myocytes. 100 nM amylin stimulates intracellular cyclic AMP concentrations 12-fold, whereas human amylin-amide at this concentration causes only a 2-fold increase. Up to 10 mM human amylin has no effect on cyclic AMP levels. Rat calcitonin gene-related peptide (CGRP) is more potent than amylin, causing a 60-fold increase over basal at 1 nM, with an EC50 value of 0.2 nM. The CGRP receptor antagonist, human CGRP8-37 (hCGRP8-37), completely blocks the stimulatory effect of both rat amylin and human amylin-amide on cyclic AMP production. [125I]CGRP binds specifically to a membrane fraction prepared from L6 [125I]CGRP with a Ki of 0.9 nM, while rat amylin also displaces [125I]CGRP with a Ki of 91 nM. Specific binding of [125I]CGRP to plasma membranes of rat liver and brain is also displaced by rat amylin with Ki values of 35 nM and 37 nM, respectively. In contrast, specific binding of [125I]amylin to numerous cells and tissues, under similar conditions, can not be demonstrated. These results suggest that the cellular effects and physiological actions of amylin may be mediated through receptors for CGRP.

Amyloid↗
PubMed1991

Nerve growth factor stimulates protein tyrosine phosphorylation in PC-12 pheochromocytoma cells.

The cellular actions of nerve growth factor (NGF) and epidermal growth factor (EGF) may be mediated by changes in protein phosphorylation. The tyrosine phosphorylation of two predominant proteins of molecular mass 40 and 42 kDa is seen in PC-12 cells treated with NGF or EGF, correlating with activation of a previously identified serine/threonine protein kinase that phosphorylates microtubule-associated protein (MAP). Stimulation of phosphoprotein (pp) 40 and 42 phosphorylation and MAP kinase activity by NGF but not EGF is selectively attenuated by staurosporine and K-252A. Moreover, the time courses of pp40/42 phosphorylation and MAP kinase activation produced by NGF or EGF are identical. Chromatography of lysates from growth factor-treated cells on ion-exchange or hydrophobic-interaction HPLC resolves MAP kinase into two peaks, neither of which precisely coelutes with pp40 or pp42. One of these peaks (II) exhibits no detectable phosphotyrosine. The other peak (I) has some overlap with pp40. However, the activity residing in both peaks is almost completely inhibited after treatment with alkaline phosphatase, suggesting that, at least, serine/threonine phosphorylation is required for the activity of these enzymes. These data indicate that while tyrosine phosphorylation appears to be a critical early event in NGF action, the role of this modification in activation of MAP kinases remains unclear.

Adrenal Gland Neoplasms↗
PubMed1991

The specific protein phosphatase inhibitor okadaic acid differentially modulates insulin action.

The pleiotropic nature of insulin action suggests diverse mechanisms of signal transduction for the hormone. The specific protein phosphatase inhibitor, okadaic acid, is utilized to differentiate metabolic pathways that may be regulated by phosphorylation or dephosphorylation of key enzymes. In H-35 hepatoma cells, okadaic acid inhibits insulin-stimulated glycogen synthesis with an IC50 of 400 nM. In contrast, activation of lipogenesis by insulin is inhibited with an IC50 of 50 nM okadaic acid. The toxin also inhibits stimulation of lipogenesis in these cells by the insulin-sensitive inositol glycan enzyme modulator. In isolated rat adipocytes, insulin-stimulated lipogenesis is also inhibited by okadaic acid with an IC50 of approximately 1,700 nM. The antilipolytic effect of insulin in these cells is more sensitive to okadaic acid, exhibiting an IC50 of 150 nM. Maximal activation of lipogenesis by insulin is dramatically reduced by okadaic acid with no effect on the concentration required for half-maximal activation, whereas the sensitivity of insulin-induced antilipolysis is attenuated by okadaic acid, with no apparent reduction in the maximal effect of the hormone. Taken together, these data suggest that specific phosphatases may be differentially involved in some of the metabolic pathways regulated by insulin.

Adipose Tissue↗
PubMed1991

Short-term action of insulin on Aplysia neurons: generation of a possible novel modulator of ion channels.

In mollusks as in other animals, peptides can act as hormones, growth factors, and neurotransmitters. The presence of insulin in vertebrate brain as well as its actions on nerve cells led us to examine the electrophysiological effects of the mammalian hormone on Aplysia neurons. Application of insulin extracellularly causes hyperpolarization of L14 and L10, identified neurons of the abdominal ganglion. This hyperpolarization is associated with a decreased membrane conductance that reverses at -35 mV. We also injected inositol phosphate glycan (IPG) into the identified neurons. This complex sugar, which was purified from rat liver and which is a putative second messenger for insulin in nonneural vertebrate cells (Saltiel and Cuatrecasas, 1986; Saltiel, Osterman, and Darnell, 1988), causes hyperpolarization with decreased membrane conductance in L14 and L10 similar to the effects of insulin. Furthermore, exposure of isolated ganglia to insulin results in the generation of IPG with a compensating decrease in its glycosyl-phosphatidylinositol precursor. We suggest that, in addition to its other roles, insulin may function as a neuropeptide transmitter using IPG as a second messenger.

Animals↗
PubMed1991

The role of glycosyl-phosphoinositides in hormone action.

Despite significant advances in the past few years on the chemistry and biology of insulin and its receptor, the molecular events that couple the insulin-receptor interaction to the regulation of cellular metabolism remain uncertain. Progress in this area has been complicated by the pleiotropic nature of insulin's actions. These most likely involve a complex network of pathways resulting in the coordination of mechanistically distinct cellular effects. Since the well-recognized mechanisms of signal transduction (i.e., cyclic nucleotides, ion channels) appear not to be central to insulin action, investigators have searched for a novel second messenger system. A low-molecular-weight substance has been identified that mimics certain actions of insulin on metabolic enzymes. This substance has an inositol glycan structure, and is produced by the insulin-sensitive hydrolysis of a glycosyl-phosphatidylinositol in the plasma membrane. This hydrolysis reaction, which is catalyzed by a specific phospholipase C, also results in the production of a structurally distinct diacylglycerol that may selectively regulate one or more of the protein kinases C. The glycosyl-phosphatidylinositol precursor for the inositol glycan enzyme modulator is structurally analogous to the recently described glycosyl-phosphatidylinositol membrane protein anchor. Preliminary studies suggest that a subset of proteins anchored in this fashion might be released from cells by a similar insulin-sensitive, phospholipase-catalyzed reaction. Future efforts will focus on the precise role of the metabolism of glycosyl-phosphatidylinositols in insulin action.

Animals↗
PubMed1991

Nerve growth factor induces the association of a 130-Kd phosphoprotein with its receptor in PC-12 pheochromocytoma cells.

To explore the molecular mechanisms of nerve growth factor (NGF) action, we have attempted to identify proteins that immunoprecipitate with the NGF receptor. An anti-NGF receptor antibody was developed that immunoprecipitated the 75-Kd receptor in PC-12 cells. In [35S]methionine-labeled cells lysed with nonionic detergent, immunoprecipitation with this antireceptor antisera specifically brought down several associated proteins, although prior treatment of cells with NGF produced no apparent change in the distribution of these proteins. However, in vitro phosphorylation assays of the immunoprecipitated complex revealed the presence of a serine kinase that phosphorylated two predominant substrates with Mrs of 60 and 130 Kd. Prior treatment of cells produced no change in the appearance of the 60-Kd phosphoprotein, but NGF did stimulate the appearance of the 130-Kd protein. This effect was observed with as little as 0.1 nM NGF and was maximal at 5 min, but declined thereafter. Prior treatment of cells with NGF did not increase the phosphorylation of enolase added exogenously to the immunoprecipitates, suggesting that this action of NGF may have reflected the hormone-dependent association of the 130-Kd protein with the receptor, rather than activation of a receptor-associated kinase. Thus the association of the NGF 75-Kd receptor with a 130-Kd protein may be involved in signal transduction for the growth factor, although the role of this receptor in the NGF-dependent tyrosine phosphorylation remains unclear.

Amino Acids↗
PubMed1991

Phosphatidylinositol-3 kinase is activated in v-src, v-yes, and v-fps transformed chicken embryo fibroblasts.

PI-3 kinase activity has been shown to associate with p60v-src. We found that immunoprecipitates of p60v-src exhibit an activity that catalyzes the formation of PI-3-P, PI-3,4-P2 and PIP3 from PI, PI-4-P, and PI-4,5-P2, respectively. Transformation of chicken embryo fibroblasts (CEF) by p60v-src of Rous sarcoma virus (RSV) caused elevation of PI-3-P, PI-3,4-P2, and PIP3, suggesting that the PI-3 kinase may be activated in these cells. Similar elevations were seen in cells transformed with the v-yes or v-fps oncogenes, but not with v-ros or v-ras. We have established also a system that allows the binding of PI-3 kinase to purified p60v-src in vitro, reproducing the binding seen in vivo. This assay indicated that more PI-3 kinase activity binds to purified p60v-src in cell lysates from CEF transformed with v-yes or v-fps, suggesting that some modification or over-expression of PI-3 kinase takes place in these cells.

1-Phosphatidylinositol 4-Kinase↗
PubMed1990

Okadaic acid stimulates the activity of microtubule associated protein kinase in PC-12 pheochromocytoma cells.

PC-12 pheochromocytoma cells contain a growth factor-sensitive protein kinase that phosphorylates microtubule associated protein 2 (MAP-2). This MAP kinase is also activated by the protein phosphatase inhibitor okadaic acid (OA). Additionally, OA potentiates the NGF-dependent activation of MAP kinase, but causes only a modest potentiation (20%) of the maximal activation observed with EGF. Since OA is a specific serine/threonine phosphatase inhibitor, these results suggest that serine/threonine phosphorylation may be involved in the hormonal regulation of MAP kinase.

Adrenal Gland Neoplasms↗