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Biomedical subjects

A R Saltiel

Publications and source records attributed to A R Saltiel.

At least 55 records · Page 3Linked to original sources
PubMed1997

The regulation of glycogen synthase by protein phosphatase 1 in 3T3-L1 adipocytes. Evidence for a potential role for DARPP-32 in insulin action.

The stimulation of glycogen-targeted protein phosphatase 1 (PP1), glycogen synthase, and glycogen synthesis by insulin was examined during the differentiation of 3T3-L1 fibroblasts into adipocytes. Insulin treatment barely changed the low levels of glycogen synthesis measured in fibroblasts. Following differentiation into adipocytes, insulin increased glycogen synthesis up to 40-fold. After further culturing of the adipocytes for a week, insulin stimulated glycogen accumulation 700-fold. Differentiation of 3T3-L1 cells also resulted in the increased expression of glycogen synthase and in increases in both total glycogen synthase activity and -fold stimulation by insulin. While the levels of PP1 protein were unchanged by differentiation, PP1 specific activity decreased over 60%, although sensitivity to insulin treatment was augmented. Concurrently, levels of the PP1 inhibitor protein DARPP-32 were dramatically induced upon 3T3-L1 adipogenesis. DARPP-32 in both 3T3-L1 and primary rat adipocytes was exclusively localized to the particulate fractions, including the glycogen-enriched pellet. PP1 activity from 3T3-L1 adipocytes exhibited a kinetic lag in vitro, which was not present in fibroblast extracts. Insulin pretreatment of the adipocyte cells overcame the in vitro lag in PP1 activity, resulting in up to 5-fold stimulation of PP1 activity being measured at early assay time points. These results suggest that in 3T3-L1 adipocytes, DARPP-32 may maintain glycogen-targeted PP1 activity in a low basal state, priming the phosphatase for stimulation by insulin.

3T3 Cells↗
PubMed1997

Mitogen-activated protein kinase kinase inhibition decreases growth hormone stimulated transcription mediated by STAT5.

We have investigated the possible involvement of the MAPK pathway in the growth hormone(GH)-induced activation of one of the members of signal transducers and activators of transcription, STAT5, by using the MAPK kinase (MEK) inhibitor PD98059. PD98059 treatment of Chinese hamster ovarian cells, stably transfected with the GH receptor (CHOA cells), abolished the GH-induced MAPK activity. PD98059 decreased the amount of GH-induced STAT5 in nuclear extract with DNA-binding capacity. Furthermore, GH dependent transcription of a STAT5 regulated reporter gene was inhibited by PD98059. The MEK inhibitor did not reduce GH-stimulated nuclear translocation of STAT5. We also investigated if PD98059 differentially influences the activation of the two STAT5 homologs, STAT5a and STAT5b, which differ mainly at the C-terminal end, one of the differences being the presence of a possible MAPK phosphorylation site in STAT5a. Expression plasmids for these transcription factors were transfected into CHOA cells together with a reporter gene. GH-stimulated fold induction of transcription was reduced by PD98059 in STAT5a but not in STAT5b overexpressing cells. A MAPK phosphorylation site-mutated version of STAT5a was also transfected into CHOA cells. GH-stimulated fold induction of cotransfected reporter gene was not reduced by PD98059 in cells overexpressing mutant STAT5a. The above data show that the MAPK pathway is required for the full activation of one of the STAT5 isoforms (STAT5a).

Amino Acid Sequence↗
PubMed1997

Insulin-like growth factor-I-mediated neurite outgrowth in vitro requires mitogen-activated protein kinase activation.

Insulin-like growth factor-I (IGF-I) induces neuronal differentiation in vitro. In the present study, we examined the signaling pathway underlying IGF-I-mediated neurite outgrowth. In SH-SY5Y human neuroblastoma cells, treatment with IGF-I induced concentration- and time-dependent tyrosine phosphorylation of the type I IGF receptor (IGF-IR) and extracellular signal-regulated protein kinases (ERK) 1 and 2. These effects of IGF-I were blocked by a neutralizing antibody against IGF-IR. Whereas IGF-IR phosphorylation was observed within 1 min, maximal phosphorylation of ERKs was not reached for 30 min. Both IGF-IR and ERK phosphorylation were maintained for at least 24 h. Also, the concentration dependence of IGF-I-stimulated IGF-IR and ERK tyrosine phosphorylation paralleled that of IGF-I-mediated neurite outgrowth. We further examined the role of mitogen-activated protein kinase activation in IGF-I-stimulated neuronal differentiation using the mitogen-activated protein kinase/ERK kinase inhibitor PD98059. Whereas PD98059 had no effect on IGF-IR phosphorylation, PD98059 reduced IGF-I-mediated ERK tyrosine phosphorylation and ERK phosphorylation of the substrate Elk-1. PD98059 also produced a parallel reduction of IGF-I-stimulated neurite outgrowth. Finally, consistent with its ability to block neuronal differentiation, PD98059 inhibited IGF-I-dependent changes of GAP-43 and c-myc gene expression. Together these results suggest that activation of ERKs is essential for IGF-I-stimulated neuronal differentiation.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1997

Insulin-stimulated tyrosine phosphorylation of caveolin is specific for the differentiated adipocyte phenotype in 3T3-L1 cells.

Previous work from this laboratory has shown that insulin stimulates the tyrosine phosphorylation of caveolin in 3T3-L1 adipocytes (Mastick, C. C., Brady, M. J., and Saltiel, A. R. (1995) J. Cell Biol. 129, 1523-1531). This phosphorylation is specific for insulin and involves the activation of a tyrosine kinase downstream of the insulin receptor. We report here that tyrosine phosphorylation of caveolin is detected only in fully differentiated adipocytes, not in fibroblasts (preadipocytes), despite the fact that both cell types express caveolin-1 and active insulin receptor. Caveolin copurifies with caveolin tyrosine kinase activity in both preadipocytes and adipocytes. Accumulating evidence indicates that this kinase is the Src family kinase Fyn. Fyn is expressed in the preadipocytes and the adipocytes and is colocalized with caveolin in low density Triton-insoluble complexes in both cell types. In adipocytes, overexpression of wild type Fyn leads to increased basal phosphorylation of caveolin and hyperphosphorylation of caveolin in response to insulin. In vitro kinase assays suggest that Fyn may be activated in response to insulin through the binding of a tyrosine-phosphorylated insulin receptor substrate protein. Previous work suggested that this protein may be c-Cbl (Ribon, V., and Saltiel, A. R. (1997) Biochem. J. 324, 839-846). In 3T3-L1 adipocytes, Cbl binds to Fyn in an insulin-dependent manner, and Cbl phosphorylation is adipocyte-specific. Here we show that phosphorylated Cbl is translocated into caveolin-enriched Triton-insoluble complexes after insulin stimulation. This may lead to the cell type-specific, compartmentalized activation of Fyn and the specific phosphorylation of proteins in the caveolae in response to insulin in adipocytes.

3T3 Cells↗
PubMed1997

Role of protein targeting to glycogen (PTG) in the regulation of protein phosphatase-1 activity.

We have recently cloned from 3T3-L1 adipocytes a novel glycogen-targeting subunit of protein phosphatase-1, termed PTG (Printen, J. A., Brady, M. J., and Saltiel, A. R. (1997) Science 275, 1475-1478). Differentiation of 3T3-L1 fibroblasts into highly insulin-responsive adipocytes resulted in a marked increase in PTG expression. Immobilized glutathione S-transferase (GST)-PTG fusion protein specifically bound either PP1 or phosphorylase a. Addition of soluble GST-PTG to 3T3-L1 lysates increased PP1 activity against 32P-labeled phosphorylase a by decreasing the Km of PP1 for phosphorylase 5-fold, while having no effect on the Vmax of the dephosphorylation reaction. Alternatively, PTG did not affect PP1 activity against hormone-sensitive lipase. PTG was not a direct target of intracellular signaling, as insulin or forskolin treatment of cells did not activate a kinase capable of phosphorylating PTG in vivo or in vitro. Finally, PTG decreased the ability of DARPP-32 to inhibit PP1 activity from 3T3-L1 adipocyte lysates. These data cumulatively suggest that PTG increases PP1 activity against specific proteins by several distinct mechanisms.

3T3 Cells↗
PubMed1997

Insulin stimulates tyrosine phosphorylation of the proto-oncogene product of c-Cbl in 3T3-L1 adipocytes.

We report here that the product of the c-Cbl proto-oncogene is prominently tyrosine phosphorylated in response to insulin in 3T3-L1 adipocytes. The tyrosine phosphorylation of c-Cbl reaches a maximum within 1-2 min after stimulation by insulin and gradually declines thereafter. The tyrosine phosphorylation of c-Cbl was also observed after treatment of 3T3-L1 adipocytes with epidermal growth factor, whereas platelet-derived growth factor had no effect. After insulin-dependent tyrosine phosphorylation, c-Cbl specifically associates with fusion proteins containing the Src homology 2 (SH2) domains of Crk and the Fyn tyrosine kinase, but not with fusion proteins containing the SH2 domains of either the p85 subunit of phosphatidylinositol 3'-kinase or the tyrosine phosphatase SHPTP2/Syp. Furthermore insulin stimulates the association of c-Cbl with endogenous c-Crk and Fyn in intact 3T3-L1 adipocytes. The tyrosine phosphorylation of c-Cbl is regulated during adipocyte differentiation. Neither insulin-like growth factor 1 nor insulin stimulated the tyrosine phosphorylation of c-Cbl in 3T3-L1 fibroblasts. Moreover, c-Cbl is not tyrosine phosphorylated in response to insulin in cells expressing high levels of the human insulin receptor, or in hepatocytes, despite comparable levels of c-Cbl expression. These results suggest that c-Cbl might have a novel function in the regulation of insulin receptor intracellular signalling in 3T3-L1 adipocytes.

3T3 Cells↗
PubMed1997

PTG, a protein phosphatase 1-binding protein with a role in glycogen metabolism.

Protein dephosphorylation by phosphatase PP1 plays a central role in mediating the effects of insulin on glucose and lipid metabolism. A PP1C-targeting protein expressed in 3T3-L1 adipocytes (called PTG, for protein targeting to glycogen) was cloned and characterized. PTG was expressed predominantly in insulin-sensitive tissues. In addition to binding and localizing PP1C to glycogen, PTG formed complexes with phosphorylase kinase, phosphorylase a, and glycogen synthase, the primary enzymes involved in the hormonal regulation of glycogen metabolism. Overexpression of PTG markedly increased basal and insulin-stimulated glycogen synthesis in Chinese hamster ovary cells overexpressing the insulin receptor, which do not express endogenous PTG. These results suggest that PTG is critical for glycogen metabolism, possibly functioning as a molecular scaffold.

3T3 Cells↗
PubMed1997

Negative modulation of membrane localization of the Raf-1 protein kinase by hyperphosphorylation.

The serine/threonine-specific protein kinase Raf-1 plays a key role in mitogenic signal transduction by coupling Ras to the mitogen-activated protein (MAP) kinase cascade. Ras-mediated translocation to the plasma membrane represents a crucial step in the process of serum-stimulated Raf-1 kinase activation. The exact role of the multisite phosphorylation in Raf regulation, however, is not clear. We have previously reported that the mobility shift-associated hyperphosphorylation of Raf correlates with a reduction of serum-stimulated Raf kinase activity (Wartmann, M., and Davis, R. J. (1994) J. Biol. Chem. 269, 6695-6701). Here we show that incubation of serum-starved CHO cells with D609, a purported inhibitor of phosphatidylcholine-specific phospholipase C, also results in a mobility shift of Raf-1 that is due to hyperphosphorylation on sites identical to those observed following mitogen stimulation. Subcellular fractionation analyses revealed that D609-induced mobility shift-associated hyperphosphorylation was paralleled by a decreased membrane association of Raf-1. Similar results were obtained in an in vitro reconstitution system. Furthermore, PD98059, a specific inhibitor of activation of the MAP kinase kinase MEK, prevented D609-induced Raf hyperphosphorylation and restored the amount of membrane-bound Raf to control levels. Taken together, these data suggest that mobility shift-associated hyperphosphorylation of Raf-1, by virtue of reducing the amount of plasma membrane-bound Raf-1, represents a negative feedback mechanism contributing to the desensitization of the MAP kinase signaling cascade.

Animals↗
PubMed1997

Insulin-like growth factor 1 inhibits apoptosis using the phosphatidylinositol 3'-kinase and mitogen-activated protein kinase pathways.

The role of insulin-like growth factor 1 (IGF-1) in preventing apoptosis was examined in differentiated PC12 cells. Induction of differentiation was achieved using nerve growth factor, and apoptosis was provoked by serum withdrawal. After 4-6 h of serum deprivation, apoptosis was initiated, concomitant with a 30% decrease in cell number and a 75% decrease in MTT activity. IGF-1 was capable of preventing apoptosis at concentrations as low as 10(-9) M and as early as 4 h. The phosphatidylinositol 3' (PI3')-kinase inhibitors wortmannin (at concentrations of 10(-8) M) and LY294002 (10(-6) M) blocked the effect of IGF-1. The pp70 S6 kinase (pp70S6K) inhibitor rapamycin (10(-8) M) was, however, less effective in blocking IGF-1 action. Moreover, stable transfection of a dominant-negative p85 (subunit of PI3'-kinase) construct in PC12 cells enhanced apoptosis provoked by serum deprivation. Interestingly, in the cells overexpressing the dominant-negative p85 protein, IGF-1 was still capable of inhibiting apoptosis, suggesting the existence of a second pathway involved in the IGF-1 effect. Blocking the mitogen-activated protein kinase pathway with the specific mitogen-activated protein kinase/extracellular-response kinase kinase inhibitor PD098059 (10(-5) M) inhibited the IGF-1 effect. When wortmannin and PD098059 were given together, the effect was synergistic. The results presented here suggest that IGF-1 is capable of preventing apoptosis by activation of multiple signal transduction pathways.

Androstadienes↗
PubMed1997

Metabolic effects of troglitazone in the Goto-Kakizaki rat, a non-obese and normolipidemic rodent model of non-insulin-dependent diabetes mellitus.

Troglitazone (TRG) is an orally active antidiabetic agent that increases insulin sensitivity in models of non-insulin-dependent diabetes mellitus (NIDDM), subsequently reducing hyperinsulinemia and hyperglycemia. We examined the effects of TRG on the development and severity of diabetes in the Goto-Kakizaki (GK) rat, a spontaneous, non-obese model of NIDDM. TRG was administered at a dose of 30 mg/kg/d beginning at 4 weeks of age. TRG-treated GK rats were evaluated against Wistar and untreated GK rats at 8, 12, and 16 weeks of age. Untreated GK rats were nonketotic, normolipidemic, hyperglycemic, and had normal fasting insulin levels compared with Wistar rats. TRG treatment decreased glycosylated hemoglobin levels in the GK rat independently of its effects on plasma insulin. In untreated GK rats, intravenous glucose tolerance tests (IVGTTs) showed a hyperglycemic response to glucose loading with severely impaired glucose disposal relative to Wistar controls. TRG treatment was successful in decreasing the glucose area under the curve (AUC) (P < .03) but did not improve glucose disposal, suggesting a direct hepatic effect. Ex vivo evaluation of hepatic glucose output (HGO) further supported a direct hepatic action, with 50% reduction in HGO in TRG-treated GK rats (P < .004). A euglycemic-hyperinsulinemic clamp performed at 16 weeks of age showed severe insulin resistance in the untreated GK rat, with a glucose infusion rate (GIR) 33% lower than in Wistar rats (P < .004). TRG treatment had no effect on this insulin resistance. These results indicate that TRG selectively decreases hepatic glucose production in this unique model of NIDDM independently of its action on peripheral insulin sensitivity or hyperlipidemia.

Animals↗
PubMed1997

Proliferin induces endothelial cell chemotaxis through a G protein-coupled, mitogen-activated protein kinase-dependent pathway.

To investigate the mechanism of action of the placental angiogenic hormone proliferin (PLF), we analyzed the signaling components in endothelial cells that are required for PLF-induced chemotaxis. Pertussis toxin, which inactivates Gi proteins, inhibited PLF-induced chemotaxis of endothelial cells. Gi proteins can lead to activation of the mitogen-activated protein kinase (MAPK) pathway; PLF was found to stimulate MAPK activity, and this induction was blocked by both pertussis toxin and a specific inhibitor of MAPK kinase, PD 098059. Furthermore, a blockade of MAPK activation prevented endothelial cell movement in response to PLF. As PLF functionally interacts with the insulin-like growth factor II (IGF-II)/mannose 6-phosphate receptor, we also examined the effects of pertussis toxin and PD 098059 on another ligand for this receptor, a mutant form of IGF-II; both inhibitors also block the action of this factor on endothelial cells. These data suggest that chemotaxis initiated by PLF and mediated by the IGF-II/mannose 6-phosphate receptor occurs through a G protein-coupled pathway, and that MAPK activation is necessary for the chemotactic response.

Animals↗
PubMed1996

Nuclear magnetic resonance solution structure of the growth factor receptor-bound protein 2 Src homology 2 domain.

A family of NMR solution structures of the growth factor receptor-bound protein 2 (Grb2) SH2 domain has been determined by heteronuclear multidimensional NMR. Proton, nitrogen, and carbon chemical shift assignments have been made for the SH2 domain of Grb2. Assignments were made from a combination of homonuclear two-dimensional and 15N- and 13C-edited three-dimensional spectra at pH 6.2 and 298 K. Structure-induced proton and carbon secondary shifts were calculated and used to facilitate the spectral assignment process. NOE, scalar coupling, secondary chemical shift, and amide proton exchange data were used to characterize the secondary structural elements and hydrogen-bonding network in the Grb2 SH2 domain. The three-dimensional structure of the Grb2 SH2 domain was calculated using 1112 restraints obtained from NOE, coupling constant, and amide proton exchange data. The rmsd for the 24 calculated structures to the mean structure of the Grb2 SH2 domain was 0.75 A for backbone and 1.28 A for all heavy atoms. The three-dimensional fold of the Grb2 SH2 domain is similar to that observed for other SH2 domains and consists of two alpha-helical segments and eight beta-strands, six strands that make up two contiguous antiparallel beta-sheets, and two strands that form two short parallel beta-sheets. The structure of the phosphotyrosine binding pocket of Grb2 is similar to that observed for other SH2 domains. The hydrophobic binding pocket of Grb2 is similar to that observed for Src with the exception that tryptophan 121 of Grb2 occupies part of the pY+3 binding pocket. Structural implications for the Grb2 SH2 domain selectivity at the pY+2 and pY+3 sites are discussed.

Amino Acid Sequence↗
PubMed1996

Role of mitogen-activated protein kinase kinase in regulation of the epidermal growth factor receptor by protein kinase C.

The epidermal growth factor receptor (EGFR) is regulated by at least two mechanisms involving protein kinase C (PKC), inhibition of EGF binding and inhibition of EGF-stimulated tyrosine kinase activity. In this study we investigated whether mitogen-activated protein kinase (MAPK) mediates the inhibitory effects of PKC on EGFR binding or kinase activity by pretreating NIH3T3 and Chinese hamster ovary cells expressing the EGFR with PD98059, an inhibitor of MAPK/extracellular signal-regulated kinase kinase (MEK). We also determined whether substitution of cysteine for threonine at residue 669, the site of MAPK phosphorylation of the EGFR, alters the inhibition of kinase activity by PKC. The results indicate that 1) PKC down-regulates EGFR tyrosine kinase activity by an MEK-dependent mechanism presumably involving MAPK; 2) the inhibition by PKC is not a direct result of phosphorylation of the EGFR by PKC or MAPK; 3) activation of MAPK is not sufficient to regulate EGFR kinase activity; and 4) PKC-mediated down-regulation of EGF binding and EGFR kinase activity occur by different mechanisms. These data are consistent with a model for regulation of the EGFR by other receptors whereby their activation of PKC, in conjunction with MAPK, results in the phosphorylation of a protein(s) that modulates EGFR kinase activity.

3T3 Cells↗
PubMed1996

Epidermal growth factor receptor targeting prevents uncoupling of the Grb2-SOS complex.

Insulin stimulates the Ras/Raf/MEK/ERK pathway leading to feedback phosphorylation of the Ras guanylnucleotide exchange protein SOS and dissociation of Grb2 from SOS. Even though epidermal growth factor (EGF) also stimulates ERK activity and phosphorylation of SOS similar to insulin, EGF induces a dissociation of the Grb2-SOS complex from Shc. To determine the molecular basis for this difference, we examined the signaling properties of a mutant EGF receptor lacking the five major autophosphorylation sites. Although EGF stimulation of the mutant EGF receptor activates ERK and phosphorylation of both Shc and SOS, it fails to directly associate with either Shc or Grb2. However, under these conditions EGF induces a dissociation of the Grb2-SOS complex suggesting a role for receptor and/or plasma membrane targeting in the stabilization of Grb2-SOS interaction. Consistent with this hypothesis, expression of an SH2 domain Grb2 mutant which is unable to mediate plasma membrane targeting of the Grb2-SOS complex results in both insulin- and EGF-stimulated uncoupling of Grb2 from SOS. Furthermore, a plasma membrane-bound Grb2 fusion protein remains constitutively associated with SOS. Together, these data demonstrate that EGF stimulation prevents the feedback uncoupling of Grb2 from SOS by inducing a persistent plasma membrane receptor targeting of the Grb2-SOS complex.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Nerve growth factor stimulates the tyrosine phosphorylation of endogenous Crk-II and augments its association with p130Cas in PC-12 cells.

The cellular homologs of the v-Crk oncogene product consist primarily of Src homology region 2 (SH2) and 3 (SH3) domains. v-Crk overexpression causes cell transformation and elevation of tyrosine phosphorylation in fibroblasts and accelerates differentiation of PC-12 cells in response to nerve growth factor (NGF). To further explore the role of Crk in NGF-induced PC-12 cell differentiation, we found that both NGF and epidermal growth factor stimulate the tyrosine phosphorylation of endogenous Crk II. Moreover, hormone stimulation enhanced the specific association of Crk proteins with the tyrosine-phosphorylated p130Cas, the major phosphotyrosine-containing protein in cells transformed with v-Crk. This interaction is mediated by the SH2 domain of Crk and can be inhibited with a phosphopeptide containing the Crk-SH2 binding motif. Furthermore, the Crk-SH2 domain binds tyrosine-phosphorylated paxillin, a cytoskeletal protein, following treatment of PC-12 cells with NGF or epidermal growth factor. These data suggest that Crk functions in a number of signaling processes in PC-12 cells.

Adrenal Gland Neoplasms↗
PubMed1996

Regulation of both glycogen synthase and PHAS-I by insulin in rat skeletal muscle involves mitogen-activated protein kinase-independent and rapamycin-sensitive pathways.

Incubating rat diaphragm muscles with insulin increased the glycogen synthase activity ratio (minus glucose 6-phosphate/plus glucose 6-phosphate) by approximately 2-fold. Insulin increased the activities of mitogen-activated protein (MAP) kinase and the Mr = 90,000 isoform of ribosomal protein S6 kinase (Rsk) by approximately 1.5-2.0-fold. Epidermal growth factor (EGF) was more effective than insulin in increasing MAP kinase and Rsk activity, but in contrast to insulin, EGF did not affect glycogen synthase activity. The activation of both MAP kinase and Rsk by insulin was abolished by incubating muscles with the MAP kinase kinase (MEK) inhibitor, PD 098059; however, the MEK inhibitor did not significantly reduce the effect of insulin on activating glycogen synthase. Incubating muscles with concentrations of rapamycin that inhibited activation of p70S6K abolished the activation of glycogen synthase. Insulin also increased the phosphorylation of PHAS-I (phosphorylated heat- and acid-stable protein) and promoted the dissociation of the PHAS-I*eIF-4E complex. Increasing MAP kinase activity with EGF did not mimic the effect of insulin on PHAS-I phosphorylation, and the effect of insulin on increasing MAP kinase could be abolished with the MEK inhibitor without decreasing the effect of insulin on PHAS-I. The effects of insulin on PHAS-I were attenuated by rapamycin. Thus, activation of the MAP kinase/Rsk signaling pathway appears to be neither necessary nor sufficient for insulin action on glycogen synthase and PHAS-I in rat skeletal muscle. The results indicate that the effects of insulin on increasing the synthesis of glycogen and protein in skeletal muscle, two of the most important actions of the hormone, involve a rapamycin-sensitive mechanism that may include elements of the p70S6K signaling pathway.

Amino Acid Sequence↗