Phosphorylation of glycolytic and gluconeogenic enzymes by the insulin receptor kinase

Various glycolytic and gluconeogenic enzymes were tested as substrates for the insulin receptor kinase. Phosphofructokinase and phosphoglycerate mutase were found to be the best substrates. Phosphorylation of these enzymes was rapid, stimulated 2- to 6-fold by 10(-7) M insulin and occurred exclusively on tyrosine residues. Enolase, fructose 1,6-bisphosphatase, lactate dehydrogenases in decreasing order, were also subject to insulin-stimulated phosphorylation but to a smaller extent than that for phosphofructokinase or phosphoglycerate mutase. The phosphorylation of phosphofructokinase was studied most extensively since phosphofructokinase is known to catalyze a rate-limiting step in glycolysis. The apparent Km of the insulin receptor for phosphofructokinase was 0.1 microM, which is within the physiologic range of concentration of this enzyme in most cells. Tyrosine phosphorylation of phosphofructokinase paralleled autophosphorylation of the beta-subunit of the insulin receptor with respect to time course, insulin dose response (half maximal effect between 10(-9) and 10(-8) M insulin), and cation requirement (Mn2+ greater than Mg2+ much greater than Ca2+). Further study will be required to determine whether the tyrosine phosphorylation of phosphofructokinase plays a role in insulin-stimulated increases in glycolytic flux.     

Phosphorylation of the insulin receptor by casein kinase I

Insulin receptor was examined as a substrate for the multipotential protein kinase casein kinase I. Casein kinase I phosphorylated partially purified insulin receptor from human placenta as shown by immunoprecipitation of the complex with antiserum to the insulin receptor. Analysis of the phosphorylated complex by polyacrylamide gel electrophoresis under nonreducing conditions showed a major phosphorylated band at the position of the alpha 2 beta 2 complex. When the phosphorylated receptor was analyzed on polyacrylamide gels under reducing conditions, two phosphorylated bands, Mr 95,000 and Mr 135,000, were observed which corresponded to the alpha and beta subunits. The majority of the phosphate was associated with the beta subunit with minor phosphorylation of the alpha subunit. Phosphoamino acid analysis revealed that casein kinase I phosphorylated only seryl residues. The autophosphorylated alpha 2 beta 2 receptor purified by affinity chromatography on immobilized O-phosphotyrosyl binding antibody was also a substrate for casein kinase I. Reduction of the phosphorylated alpha 2 beta 2 receptor indicated that casein kinase I incorporated phosphate into seryl residues only in the beta subunit.     

Evidence the pp60src, the product of the Rous sarcoma virus src gene, undergoes autophosphorylation.

F/St mice are unique in producing high levels of both ecotropic and xenotropic murine leukemia virus. The high ecotropic virus phenotype is determined by three or more V (virus-inducing) loci. A single locus for inducibility of xenotropic murine leukemia virus was mapped to chromosome 1 close to, but possibly not allelic to, Bxv-1. Although the high ecotropic virus phenotype is phenotypically dominant, the high xenotropic virus phenotype was recessive in all crosses tested. Suppression of xenotropic murine leukemia virus is governed by a single gene which is not linked to the xenotropic V locus.     

Phosphorylation and activation of protein tyrosine phosphatase (PTP) 1B by insulin receptor

We have previously reported a direct in vivo interaction between the activated insulin receptor and protein-tyrosine phosphatase-1B (PTP1B), which leads to an increase in PTP1B tyrosine phosphorylation. In order to determine if PTP1B is a substrate for the insulin receptor tyrosine kinase, the phosphorylation of the Cys 215 Ser, catalytically inactive mutant PTP1B (CS-PTP1B) was measured in the presence of partially purified and activated insulin receptor. In vitro, the insulin receptor tyrosine kinase catalyzed the tyrosine phosphorylation of PTP1B. 53% of the total cellular PTP1B became tyrosine phosphorylated in response to insulin in vivo. Tyrosine phosphorylation of PTP1B by the insulin receptor was absolutely dependent upon insulin-stimulated receptor autophosphorylation and required an intact kinase domain, containing insulin receptor tyrosines 1146, 1150 and 1151. Tyrosine phosphorylation of wild type PTP1B by the insulin receptor kinase increased phosphatase activity of the protein. Intermolecular transdephosphorylation was demonstrated both in vitro and in vivo, by dephosphorylation of phosphorylated CS-PTP1B by the active wild type enzyme either in a cell-free system or via expression of the wild type PTP1B into Hirc-M cell line, which constitutively overexpress the human insulin receptor and CS-PTP1B. These results suggest that PTP1B is a target protein for the insulin receptor tyrosine kinase and PTP1B can regulate its own phosphatase activity by maintaining the balance between its phosphorylated (the active form) and dephosphorylated (the inactive form) state.     

Highly specific antibody to Rous sarcoma virus src gene product recognizes a novel population of pp60v-src and pp60c-src molecules

Antiserum to the Rous sarcoma virus (RSV)-transforming protein, pp60v-src, was produced in rabbits immunized with p60 expressed in Escherichia coli. alpha p60 serum immunoprecipitated quantitatively more pp60v-src than did tumor-bearing rabbit (TBR) sera. When RSV-transformed cell lysates were preadsorbed with TBR serum, the remaining lysate contained additional pp60v-src, which was recognized only by reimmunoprecipitation with alpha p60 serum and not by TBR serum. In subcellular fractions of RSV-infected chicken embryo fibroblasts (RSV-CEFs) and field vole cells probed with TBR serum, the majority of the pp60v-src was associated with the plasma membrane-enriched P100 fraction. However, alpha p60 serum revealed equal distribution of pp60v-src and its kinase activity between the P1 (nuclear) and P100 fractions. The same results were obtained for pp60c-src in uninfected CEFs. On discontinuous sucrose gradients nearly 50% of the P1-pp60v-src sedimented with nuclei, in fractions where no plasma membrane was detected. Indirect immunofluorescence microscopy of RSV-CEFs with alpha p60 serum revealed a distinct pattern of perinuclear fluorescence, in addition to staining at the cell periphery. Thus the use of a highly specific antibody reveals that enzymatically active pp60v-src and pp60c-src molecules are present in other intracellular structures, probably juxtareticular nuclear membranes, in addition to the plasma membrane in normal, uninfected, and wild-type RSV-infected cells.     

The P300 acetyltransferase inhibitor C646 promotes membrane translocation of insulin receptor protein substrate and interaction with the insulin receptor

Inhibition of P300 acetyltransferase activity by specific inhibitor C646 has been shown to improve insulin signaling. However, the underlying molecular mechanism of this improvement remains unclear. In this study, we analyzed P300 levels of obese patients and found that they were significantly increased in liver hepatocytes. In addition, large amounts of P300 appeared in the cytoplasm. Inhibition of P300 acetyltransferase activity by C646 drastically increased tyrosine phosphorylation of the insulin receptor protein substrates (IRS1/2) without affecting the tyrosine phosphorylation of the beta subunit of the insulin receptor (IRβ) in hepatocytes in the absence of insulin. Since IRS1/2 requires membrane translocation and binding to inositol compounds for normal functions, we also examined the role of acetylation on binding to phosphatidylinositol(4,5)P2 and found that IRS1/2 acetylation by P300 reduced this binding. In contrast, we show that inhibition of IRS1/2 acetylation by C646 facilitates IRS1/2 membrane translocation. Intriguingly, we demonstrate that C646 activates IRβ′s tyrosine kinase activity and directly promotes IRβ interaction with IRS1/2, leading to the tyrosine phosphorylation of IRS1/2 and subsequent activation of insulin signaling even in the absence of insulin. In conclusion, these data reveal the unique effects of C646 in activating insulin signaling in patients with obesity and diabetes.     

Amino acid alterations within a highly conserved region of the Rous sarcoma virus src gene product pp60src inactivate tyrosine protein kinase activity.

Bisulfite mutagenesis techniques have been used to introduce single-point mutations within a region of the Rous sarcoma virus src gene defined by a BglI restriction endonuclease cleavage site. The mutants of Rous sarcoma virus that are produced by these techniques encode src proteins which contain single amino acid changes within a highly conserved amino acid sequence encompassing residues 430 to 433. DNA from the mutants CHpm26 ( Ala430 to Val), CHpm9 ( Pro431 to Ser), CHpm6 ( Glu432 to Lys), and CHpm65 ( Ala433 to Thr) each failed to transform chicken cells upon transfection, whereas DNA from CHpm59 (a third base alteration in the codon for Glu432 ) readily transformed chicken cells. Analysis of immune complexes containing the altered src proteins indicates that these proteins have decreased tyrosine protein kinase activity in vitro. In vivo labeling of cells infected with the mutant virus revealed diminished levels of the tyrosine-phosphorylated 34,000-molecular-weight protein. These data indicate that mutations within the sequence Ala430 - Pro431 - Glu432 - Ala433 lead to alterations in pp60src-specific tyrosine protein kinase activity and a concomitant loss of transforming potential of the mutant virus.     

Ethanol inhibits L1 cell adhesion molecule tyrosine phosphorylation and dephosphorylation and activation of pp60src

Fetal alcohol syndrome is a leading cause of mental retardation. The neuropathology found in patients with fetal alcohol syndrome overlaps with those with mutations in the gene for cell adhesion molecule (L1). We have previously shown that L1-mediated neurite outgrowth and L1 activation of extracellular receptor kinases 1/2 are inhibited at low concentrations of ethanol. One possible mechanism for this effect is through disruption of a tyrosine-based sorting signal, Y(1176)RSLE, on the cytoplasmic domain of L1. Our goal was to determine if ethanol inhibited the sorting signal or its phosphorylation state. Using cerebellar granule neurons and dorsal root ganglion neurons, we found that ethanol had no effect on L1 distribution to the growth cone or its ability to be expressed on the cell surface as determined by confocal microscopy. In cerebellar granule neurons, clustering of L1 resulted in increased dephosphorylation of Y(1176), increased L1 tyrosine phosphorylation, and an increase in the activation of pp60^src as measured by immunoblot. All changes were inhibited by 25 mM ethanol. Using PP2 to inhibit pp60^src activation resulted in inhibition of increases in L1 tyrosine and extracellular receptor kinases 1/2 phosphorylation, and Y(1176) dephosphorylation. We conclude that ethanol disrupts L1 trafficking/signaling following its expression on the surface of the growth cone, and prior to its activation of pp60^src.     

Interaction of the insulin receptor kinase with serine/threonine kinases in vitro

Insulin causes rapid phosphorylation of the beta subunit (Mr = 95,000) of its receptor in broken cell preparations. This occurs on tyrosine residues and is due to activation of a protein kinase which is contained in the receptor itself. In the intact cell, insulin also stimulates the phosphorylation of the receptor and other cellular proteins on serine and threonine residues. In an attempt to find a protein that might link the receptor tyrosine kinase to these serine/threonine phosphorylation reactions, we have studied the interaction of a partially purified preparation of insulin receptor with purified preparations of serine/threonine kinases known to phosphorylate glycogen synthase. No insulin-dependent phosphorylation was observed when casein kinases I and II, phosphorylase kinase, or glycogen synthase kinase 3 was incubated in vitro with the insulin receptor. These kinases also failed to phosphorylate the receptor. By contrast, the insulin receptor kinase catalyzed the phosphorylation of the calmodulin-dependent kinase and addition of insulin in vitro resulted in a 40% increase in this phosphorylation. In the presence of calmodulin-dependent kinase and the insulin receptor kinase, insulin also stimulated the phosphorylation of calmodulin. Phosphoamino acid analysis showed an increase of phosphotyrosine content in both calmodulin and calmodulin-dependent protein kinase. These data suggest that the insulin receptor kinase may interact directly and specifically with the calmodulin-dependent kinase and calmodulin. Further studies will be required to determine if these phosphorylations modify the action of these regulatory proteins.     

Monoclonal antibody against the carboxy terminal peptide of pp60src of Rous sarcoma virus reacts with native pp60src.

A monoclonal mouse antibody has been prepared against a synthetic peptide corresponding to the six carboxy-terminal amino acids (C' peptide) of the src gene product pp60v -src of Rous sarcoma virus (RSV). The antibody was able to precipitate pp60v -src and to bind pp60v -src kinase activity in a competition test, indicating that this peptide can serve as an antibody-binding site (epitope). Furthermore, the finding that three out of 28 pp60src-specific tumor-bearing rabbit (TBR) sera contained antibody against the C' peptide argues for an in vivo role for the carboxy terminus of pp60src. C' peptide-specific IgG was purified from one TBR serum using affinity chromatography, and was shown to precipitate significant amounts of pp60src, and bind most of the pp60src kinase activity from SRA, PrA, and B77-C strains of avian sarcoma virus (ASV), but not endogenous pp60c -src, a cellular homologue to the viral pp60v -src. Similar results were obtained with IgG isolated from a C' peptide immune rabbit serum. None of the three C' peptide-specific IgGs could serve as a phosphate acceptor in an immune complex protein kinase reaction.     

Binding of SH2 containing proteins to the insulin receptor: A new way for modulating insulin signalling

Prior studies have established a role in insulin action for the tyrosine phosphorylation of substrates and their subsequent complexing with SH2 containing proteins. More recently, SH2 proteins have been identified which can tightly bind to the tyrosine phosphorylated insulin receptor. The major protein identified so far (called Grb-IR or Grb10) of this type appears to be present in at least 3 isoforms, varying in the presence of a pleckstrin homology domain and in the sequence of its amino terminus. The binding of this protein to the insulin receptor appears to inhibit signalling by the receptor. The present review will discuss the current knowledge of the structure and function of this protein.     

Cytoskeletal association of pp60src. The transforming protein of the Rous sarcoma virus

Immunoferritin labelling methods have been employed to examine the distribution of the Rous Sarcoma virus (RSV)-transforming protein pp60src in the detergent-resistant cytoskeleton of transformed cells. pp60src was found to be localized on actin microfilaments present in adhesion plaques, at adherens junctions between cells and also in microfilament bundles. This localization is consistent with the hypothesis that some of the morphological effects of transformation result from the interaction in situ of pp60src with microfilament-bound target proteins.     

Monoclonal antibodies to Rous sarcoma virus pp60src react with enzymatically active cellular pp60src of avian and mammalian origin

The derivation and characterization of 22 hybridoma clones producing monoclonal antibodies (Mabs) specific for the transforming protein of Rous sarcoma virus, pp60src, are described. All Mabs reacted with pp60v-src encoded by Prague, Schmidt-Ruppin, and Bratislava 77 strains of Rous sarcoma virus. Of these Mabs, 10 efficiently immunoprecipitated pp60c-src from chicken embryo cells. Of these 10 Mabs, 2 (GD11 and EB8) readily detected pp60c-src from a variety of rodent and human cultured cells and from rat brain tissue in an in vitro immune complex kinase assay. Mapping experiments have tentatively localized the determinant(s) recognized by GD11 and EB8 to a region of the src protein bounded by amino acid residues 82 to 169, whereas the remaining Mabs appeared to recognize determinants residing within residues 1 to 82 or 169 to 173. Most of the Mabs complexed denatured pp60v-src in a Western immunoblot, and several were used to localize pp60v-src in Rous sarcoma virus-transformed chicken embryo cells by indirect immunofluorescence microscopy.     

Calcium compartments and fluxes are affected by the src gene product of Rat-1 cells transformed by temperature-sensitive Rous sarcoma virus

Total cellular calcium content (determined by atomic absorption spectrometry) of Rat-1 cells transformed by temperature-sensitive Rous sarcoma virus decreases with cell density, but is found not significantly different at permissive and at non-permissive temperature. Kinetic analysis of 45Ca efflux from preloaded cells exhibits three separable pools of exchangeable calcium. The ratio of pool size of the fast-exchanging Ca-compartment (bound to cell surface) to pool size of the intermediate Ca-compartment (cytoplasmic) was found to decrease from 2.5 to 1.3 upon shift from non-permissive to permissive temperature. The slowly exchanging Ca-pool (presumably mitochondrial) did not change significantly upon temperature shift. These and further data demonstrate a close correlation between distribution of cellular Ca among different cellular compartments and characteristics of cellular proliferation, both attributable to the function(s) of a single oncogene.     

Identification of an autoinhibitory domain in the insulin receptor tyrosine kinase

We have tested the hypothesis that activation of the insulin receptor tyrosine kinase is due to autophosphorylation of tyrosines 1146, 1150 and 1151 within a putative autoinhibitory domain. A synthetic peptide corresponding to residues 1134–1162, with tyrosines substituted by alanine or phenylalanine, of the insulin receptor subunit was tested for its inhibitory potency and specificity towards the tyrosine kinase activity. This synthetic peptide gave inhibition of the insulin receptor tyrosine kinase autophosphorylation and phosphorylation of the exogenous substrate poly(Glu, Tyr) with an approximate IC₅₀ of 100 M. Inhibition appeared to be independent of the concentrations of insulin or the substrate poly(Glu, Tyr) but was decreased by increasing concentrations of ATP. This same peptide also inhibited the EGF receptor tyrosine kinase but not a serine/threonine protein kinase. These results are consistent with the hypothesis that this autophosphorylation domain contains an autoinhibitory sequence. (Mol Cell Biochem120: 103–110, 1993)Abbreviations IR Insulin Receptor - SDS/PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis - CaM Calmodulin - HEPES 4-(2-Hydroxyethyl)-Piperazineethane-Sulfonic Acid - DMEM Dulbecco's Modified Eagle' Medium - PMSF Phenylmethyl-Sulfonyl Fluoride - HPLC High Performance Liquid Chromatography - PKC Protein Kinase C - PKI Inhibitory Peptide for cAMP-Kinase - CaMK II Ca²⁺/Calmodulin-Dependent Protein Kinase II - CaN A A Subunit of Calcineurin     

In vivo activation of insulin receptor tyrosine kinase by melatonin in the rat hypothalamus

Melatonin is the pineal hormone that acts via a pertussis toxin-sensitive G-protein to inhibit adenylate cyclase. However, the intracellular signalling effects of melatonin are not completely understood. Melatonin receptors are mainly present in the suprachiasmatic nucleus (SCN) and pars tuberalis of both humans and rats. The SCN directly controls, amongst other mechanisms, the circadian rhythm of plasma glucose concentration. In this study, using immunoprecipitation and immunoblotting, we show that melatonin induces rapid tyrosine phosphorylation and activation of the insulin receptor beta-subunit tyrosine kinase (IR) in the rat hypothalamic suprachiasmatic region. Upon IR activation, tyrosine phosphorylation of IRS-1 was detected. In addition, melatonin induced IRS-1/PI3-kinase and IRS-1/SHP-2 associations and downstream AKT serine phosphorylation and MAPK (mitogen-activated protein kinase) phosphorylation, respectively. These results not only indicate a new signal transduction pathway for melatonin, but also a potential cross-talk between melatonin and insulin.     

Association of Rous sarcoma virus DNA with Xenopus laevis spermatozoa and its transfer to ova through fertilization

Mature Xenopus laevis spermatozoa are capable of binding plasmid pAPrC carrying the complete Rous sarcoma virus (RSV) DNA. Each sperm cell associates, on an average, with 70–160 molecules of the plasmid DNA in a DNase resistant form, if the spermatozoa were exposed to the DNA at a concentration of 1.0–1.4 μg/10⁷ sperm cells. Fertilization with pAPrC-treated spermatozoa induced developmental malformations in 25–30% of embryos. Immunohistochemical analysis of tissue sections from defective animals revealed aberrations in myotomal structures, and increased expression of pp60^src protein in myoblasts, neuronal tube, and epidermis. The presence of characteristic v-src and RSV-long terminal repeat (LTR) sequences in X. laevis DNA was detected by PCR analysis. Embryonic RNA hybridized with a src-specific and an RSV-LTR specific probes indicating expression of the viral DNA. Plasmid DNAs without the v-src gene (pATV9) or completely free of any RSV sequences (pBR322) did not induce any changes in embryonic development. Our results provide evidence that the pBR322-cloned DNA form of the RSV genome associates with frog sperm cells in a DNase-resistant manner suggesting internalization and may be subsequently carried into eggs during the process of artificial fertilization. Correlation between the defective morphogenesis of X. laevis and increased expression of the src gene as well as an interference of RSV DNA with the developmental programs of frog embryos are discussed. © 1996 Wiley-Liss, Inc.     

The specific interaction of the Rous sarcoma virus transforming protein, pp60src, with two cellular proteins

Sera from rabbits bearing tumors induced by Rous sarcoma virus (RSV) were previously found to contain antibody to the RSV transforming protein, pp60^src. Two additional transformation-specific phosphoproteins from RSV-transformed avian cells are immunoprecipitated with these sera. These proteins, having molecular weights of 90,000 (pp90) and 50,000 (pp50), are not precipitated from uninfected or transformation-defective virus-infected cells and are not related to any RSV structural proteins. Neither pp50 nor pp90 shares any partial or complete proteolytic cleavage peptides with pp60^src, suggesting that pp90 and pp50 do not represent either a precursor or a cleavage product of pp60^src. Sedimentation analysis of RSV-transformed cell lysates on glycerol gradients revealed that the RSV pp60^src protein is present as two forms, one of which represents the majority (95%) of pp60^src and sediments as a monomer, 60,000 molecular weight protein and the other of which sediments with pp90 and pp50 as an apparent 200,000 molecular weight complex. Lysates from cells transformed by viruses containing a temperature-sensitive defect in the src gene contain a greater percentage of pp60^src associated with pp90 and pp50 under both permissive (35°C) and nonpermissive (41°C) conditions compared to wild-type virus-infected cell lysates. Phosphoserine and phosphotyrosine were found associated with pp60^src molecules that sedimented as a monomer, whereas pp60^src molecules that are complexed with pp90 and pp50 contain phosphoserine and greatly reduced amounts of phosphotyrosine. Only the monomer form of pp60^src is capable of phosphorylating IgG in the immune complex phosphotransferase reaction. Normal uninfected chicken cells contain a protein that shares identical partial proteolytic cleavage peptides with the pp90 protein immunoprecipitated from RSV-transformed cells. This pp90 protein is one of the major cytoplasmic proteins in uninfected cells. Antibody directed against pp90 also immunoprecipitates pp60^src and pp50 from lysates of RSV-transformed chicken cells.     

In vitro and in vivo prevention of HIV protease inhibitor-induced insulin resistance by a novel small molecule insulin receptor activator

Protease inhibitor (PI) therapy for the treatment of patients infected with human immunodeficiency virus is frequently associated with insulin resistance and diabetic complications. These adverse effects of PI treatment result to a large extent from their inhibition of insulin-stimulated glucose transport. Insulin receptor (IR) activators that enhance the insulin signaling pathway could be effective in treating this resistance. However, there are no agents reported that reverse inhibition of insulin action by PIs. Herein, we describe the effects of TLK19781. This compound is a non-peptide, small molecule, activator of the IR. We now report in cultured cells, made insulin resistant HIV by PI treatment, that TLK19781 both increased the content of insulin-stimulated GLUT4 at the plasma membrane, and enhanced insulin-stimulated glucose transport. In addition, oral administration of TLK19781 with the PI, indinavir improved glucose tolerance in rats made insulin resistant. These results suggest, therefore, that IR activators such as TLK19781 may be useful in treating the insulin resistance associated with PIs.