In vitro, insulin receptor catalyses phosphorylation of clathrin heavy chain and a plasma membrane 180,000 molecular weight protein

Insulin receptor mutation studies that the receptor tyrosine kinase activity is necessary for receptor endocytosis, and several insulin receptor-containing tissues have a plasma membrane-associated protein (M_r 180,000, p180) whose tyrosine phosphorylation is receptor catalysed. Since clathrin heavy chain (M_r 180,000 in dodecyl sulphate gel electrophoresis) is a major component of coated vesicles, the latter functioning in receptor endocytosis, we investigated whether insulin receptors can catalyse clathrin phosphorylation and whether p180 is clathrin. Bovine brain triskelion or coated vesicles and ³²P-ATP were added to prephosphorylated insulin receptor preparations (wheat ferm agglutinin-purified human placenta membrane proteins). Antiphosphotyrosine immunoprecipitated a phosphorylated 180,000 molecular weight protein. Insulin (10⁻⁷M) increased the rate of phosphorylation. Monoclonal anti-clathrin antibody immunoprecipitated the phosphorylated 180,000 molecular weight protein, whereas monoclonal anti-insulin receptor antibodies (-IR1, MA10) immunoprecipitated both insulin receptors and the phosphorylated 180,000 molecular weight protein. In the absence of added clathrin, anticlathrin immunoprecipitated no proteins, and -IR1 imunoprecipitated only the insulin receptor. Density gradient (glycerol 7.5–30%, w/v) centrifugation separated human placenta microsomal membrane proteins into endosomal, plasma membrane, cytoplasmic and coated vesicle fractions. Antiphosphotyrosine immunoprecipitated phosphorylated-microsomal proteins that centrifugated into endosomal and plasma membrane fractions. Addition of glycerol gradient fractions to a prephosphorylated insulin receptor preparation, however, gave a tyrosine-phosphorylated 180,000 molecular weight protein when cytoplasmic and coated vesicle fractions were added. Taken together these results suggest: (1) that, in vitro, human placenta insulin receptors can phosphorylate bovine brain and human placenta clathrin heavy chain; (2) that both assembled and unassembled clathrin can be phosphorylated; and (3) that p180, the plasma membrane-associated insulin receptor substrate, is not clathrin heavy chain.     

Substrates and signalling complexes: the tortured path to insulin action.

In the last few years several potential substrates of the insulin receptor tyrosine kinase have been identified, purified, and their cDNAs isolated. These putative substrates include: 1) pp15, a fatty acid-binding protein; 2) pp120, a plasma membrane ecto-ATPase; 3) pp42, a MAP serine/threonine kinase; 4) pp85, a subunit of the Type 1 phosphatidylinositol kinase; and 5) pp185, a phosphatidylinositol kinase binding protein. Although the tyrosine phosphorylation of several of these substrates correlates with the signalling capabilities of various mutant receptors, the role of these substrates in mediating any one of insulin's many biological responses is still unknown. In addition, recent data indicate that the tyrosine phosphorylation of pp42 may in fact be due to autophosphorylation, thereby removing it from the list of putative substrates of the insulin receptor kinase. Finally, the present review discusses the question of whether signalling occurs as a result of the tyrosine phosphorylation of substrates or via the formation of signalling complexes.     

The 180000 molecular weight plasma membrane insulin receptor substrate is a protein tyrosine phosphatase and is elevated in diabetic plasma membranes

Wheat germ agglutinin-purified non-diabetic and diabetic human placenta membranes were or were not depleted of EGF receptor with monoclonal anti-EGF receptor antibody B1D8, and subsequently phosphorylated. Phosphorylated insulin receptor beta-subunit was lower and pp180 was higher in diabetic placenta membranes than in non-diabetic membranes. Phosphorylated-beta-subunit was also lower in diabetic (streptozotocin-induced) rat liver whereas the amount of pp180 was dependent on membrane protein concentration. When rat liver tyrosine-phosphorylated proteins were incubated 30 min, 4 degrees C with EDTA-terminated 32P-phosphorylation reaction mixtures of wheat germ agglutinin-purified rat liver proteins, less phosphorylated proteins were immunoprecipitated with antiphosphotyrosine. The decrease in tyrosine-phosphorylated products suggested that pp180 was a protein tyrosine phosphatase. Taken together, the results suggest that diabetic plasma membranes contain more tyrosine phosphatase than non-diabetic membranes.     

Characterization of an endogenous substrate of the insulin receptor in cultured cells

Using antiphosphotyrosine antibodies, we have characterized the tyrosine phosphorylation of an endogenous substrate of the insulin receptor in Fao hepatoma cells and in Chinese hamster ovary cells transfected with a eukaryotic expression vector containing the human insulin receptor cDNA. In Fao cells, besides the beta-subunit of the insulin receptor, a protein with a molecular mass between 170 and 210 kDa designated pp185, undergoes tyrosine phosphorylation immediately after insulin stimulation reaching a maximum level within 30 s. After 4 h of continuous insulin stimulation, the labeling of pp185 decreased to less than half of its original intensity, whereas the insulin receptor was unchanged. After 24 h of insulin stimulation, the phosphotyrosine-containing insulin receptor decreased by 75% owing to down-regulation, whereas the pp185 was completely undetectable. By several biochemical and physiological criteria, the pp185 is distinct from the insulin receptor. The pp185 and the beta-subunit of the insulin receptor were strongly labeled with [32P]orthophosphate, but in contrast to the insulin receptor, the pp185 was not labeled by cross-linking with 125I-insulin or surface 125I iodination. Unlike the insulin receptor, the pp185 was extracted from Fao cells without detergent, and tryptic phosphopeptide mapping of the pp185 and the insulin receptor yielded distinct patterns. Thus, the pp185 is not located at the external face of the plasma membrane and does not bind insulin. Treatment of Fao cells with the phorbol ester, phorbol 12-myristate 13-acetate, stimulated the phosphorylation of two proteins with molecular weights of 170 and 210 kDa which were immunoprecipitated with the anti-phosphotyrosine antibody. Subsequent insulin stimulation increased the phosphorylation of the 210 kDa protein, but the pp185 was not detected. Increasing the concentration of the human insulin receptor in the Chinese hamster ovary cells by transfection with a plasmid containing the human insulin receptor cDNA caused a higher level of tyrosine phosphorylation of the beta-subunit and the pp185. These data support the notion that the insulin signal may be transmitted to a cellular substrate (pp185) which may initiate insulin action at intracellular sites.     

Knock-down of LAR protein tyrosine phosphatase induces insulin resistance

To test the role of the leukocyte common antigen-related protein tyrosine phosphatase (LAR) as a regulator of insulin receptor (IR) signalling, an siRNA probe against LAR was developed. Knock-down of LAR induced post-receptor insulin resistance with the insulin-induced activation of PKB/Akt and MAP kinases markedly inhibited. The phosphorylation and dephosphorylation of the IR and insulin receptor substrate (IRS) proteins were unaffected by LAR knock-down. These results identify LAR as a crucial regulator of the sensitivity of two key insulin signalling pathways to insulin. Moreover, the siRNA probe provides a molecular tool of general applicability for further dissecting the precise targets and roles of LAR.     

An Mr 180,000 protein is an endogenous substrate for the insulin-receptor-associated tyrosine kinase in human placenta.

The beta-subunit of the insulin receptor contains a tyrosine-specific protein kinase. Insulin binding activates this kinase and causes phosphorylation of the beta-subunit of the insulin receptor. It is believed that phosphorylation of other proteins might transmit the insulin signal from the receptor to the cell. In the present study we used a polyclonal anti-phosphotyrosine antibody to detect other proteins that become tyrosine phosphorylated upon insulin stimulation. Glycoproteins from human placenta membranes were enriched by wheat germ agglutinin chromatography and phosphorylation was studied with [gamma-32P]ATP and insulin in vitro. Phosphorylated proteins were immunoprecipitated by antibodies against the insulin receptor and by serum containing the anti-phosphotyrosine antibody. Beside the insulin-stimulated phosphorylation of the 95 kDa beta-subunit of the insulin receptor, an insulin-stimulated phosphorylation of a 180 kDa protein was found. The phosphorylation of both proteins occurred only on tyrosine residues. Insulin increased 32P incorporation into the 180 kDa band 2.7-fold (S.E.M. +/- 0.3, n = 5). The 180 kDa protein was not precipitated by antibodies against the insulin receptor. H.p.l.c. chromatograms of tryptic fragments of the phosphorylated 180 kDa protein and of the beta-subunit of the insulin receptor revealed different patterns for both proteins. Insulin-stimulated phosphorylation of the 180 kDa protein was also detectable in unfractionated detergent-solubilized membranes. The phosphorylation of the 180 kDa protein was stimulated by insulin with the same dose-response curve as the phosphorylation of the beta-subunit, suggesting that this protein might be another endogenous substrate of the insulin receptor kinase.     

Reverse phase chromatography of trypsin digests of a plasma membrane and a cytoplasmic insulin receptor substrate.

The 180,000 molecular weight protein from [32P]phosphorylated wheat germ agglutinin-purified rat liver plasma membranes was digested with trypsin. NIH 3T3 HIR 3.5 cells were [32P]phosphate-labelled in the presence of 10(-7) M insulin, and the 185,000 molecular weight cytoplasmic protein was digested with trypsin. Digests were applied to a C18-mu Bondapak column, eluted with acetonitrile gradients, and radioactivity in the eluate was monitored. The chromatogram for the cytoplasmic protein was similar but not identical to chromatograms of trypsin digests of insulin receptor substrates from other cultured cells. Thirteen and seven phosphopeptides were obtained from the plasma membrane and cytoplasmic substrate, respectively. One phosphopeptide from the two digests eluted at the same acetonitrile concentration; however, dissimilarity in elution profiles and dissimilarity in relative yields of individual phosphopeptides, suggest that the primary structures of tyrosine phosphorylation sites in the two insulin receptor substrates are different.     

Insulin receptor substrates 1 and 2 but not Shc can activate the insulin receptor independent of insulin and induce proliferation in CHO-IR cells

Ligand-activated insulin receptor (IR) attracts and phosphorylates various substrates such as insulin receptor substrates 1-4 (IRS) and Shc. To investigate how binding affinity for substrate affects signalling we generated chimeric receptors with the beta-chain of the insulin receptor containing NPXY motives with different affinities for receptor substrates. We found that the extent of receptor tyrosine phosphorylation positively correlates with binding affinity towards IRS1/2 but not towards Shc. Moreover, overexpression of IRS1 or IRS2 but not of Shc increased IR tyrosine phosphorylation in a dose-dependent manner, also independent of insulin. Molecular truncations of IRS1 revealed that neither the isolated PH and PTB domains nor the C-terminus with the tyrosine phosphorylation sites alone are sufficient for substrate-dependent receptor activation. Overexpression of IRS1 and IRS2 impaired insulin-induced internalization of the IR in a dose-dependent manner suggesting that IRS proteins prevent endosome-associated receptor dephosphorylation/inactivation. IRS1 and IRS2 could therefore target the activated IR to different cellular compartments. Overexpression of IRS1 and IRS2 inhibited insulin-stimulated activation of the MAP kinases Erk1/2 while it increased/induced activation of Akt/PKB. Finally, overexpression of IRS1 and IRS2 but not of Shc induced DNA synthesis in starved CHO-IR cells independent of exogenous growth factors. Our results demonstrate that variations in cellular IRS1 and IRS2 concentration affect insulin signalling both upstream and downstream and that IRS proteins could play instructive rather than just permissive roles in signal transmission.     

Insulin-sensitive myelin basic protein phosphorylation on tyrosine residues.

Rat brain plasma membranes were solubilized in detergent and a glycoprotein-enriched fraction was obtained by lectin affinity chromatography. This glycoprotein fraction contained insulin receptors, as well as protein kinases capable of phosphorylating some exogenously added substrates such as MAP2 (microtubule associated protein 2) and MBP (myelin basic protein), but not ribosomal protein S6. Phosphoamino acid analysis of MAP2 and MBP showed that phosphotyrosine residues, as well as phosphoserine/phosphotheronine residues, were present in both proteins under basal conditions. Whereas the addition of insulin to the rat brain membrane glycoprotein fraction in vitro had no effect on MAP2 phosphorylation, MBP phosphorylation was stimulated 2.7-fold in response to insulin. This phenomenon was dose-dependent, with half-maximal stimulation of MBP phosphorylation observed with 2 nM insulin. Phosphoamino acid analysis of MBP indicated that insulin stimulated the phosphorylation of tyrosine residues nearly three-fold, whereas the phosphorylation of serine or threonine residues was not increased. These results identify MBP as a substrate for the rat brain insulin receptor tyrosine-specific protein kinase in vitro.     

An altered IGF-I receptor is present in human leukemic cells.

We have characterized and analyzed IGF-I- and insulin-stimulated cell growth, receptor binding, and autophosphorylation in the human leukemic cell line HL-60. IGF-I-stimulated cell growth occurred at low (5 ng/ml) and insulin stimulated only at high (500 ng/ml) concentrations. Binding of 125I-IGF-I to partially purified plasma membrane proteins followed the characteristics of IGF-I receptor binding. 125I-IGF-I binding, as determined by chemical cross-linking, occurred to a 145-kDa protein. IGF-I, as well as insulin, stimulated the autophosphorylation of a 105-kDa band (pp105), but we could not detect a 95-kDa band corresponding to the known molecular mass of the IGF-I and insulin receptor beta-subunits. Phosphorylation of pp105 followed the dose-response characteristics of the IGF-I receptor. The phosphorylation of pp105 occurred at tyrosine and threonine, and the pattern of HPLC tryptic peptide maps showed marked differences when compared with that of a phosphorylated insulin receptor beta-subunit. Enzymatic deglycosylation of pp105 resulted only in a slight reduction of the molecular weight. These data suggest that pp105 is the beta-subunit of an IGF-I receptor variant with a higher molecular weight, similar to that found in fetal tissue. The HL-60 cell may acquire, at least in part, malignant growth characteristics through reexpression of the fetal version of the IGF-I receptor.     

Insulin and insulin-like growth factor-I stimulate a common endogenous phosphoprotein substrate (pp185) in intact neuroblastoma cells

Mouse neuroblastoma N18 cells contain specific high affinity insulin and insulin-like growth factor-I (IGF-I) receptors. Insulin and IGF-I induce phosphorylation, in intact cells, of their respective receptor beta subunits. The insulin receptor beta subunit is represented by a 95-kDa phosphoprotein that is recognized by a specific antiserum (B10). The IGF-I receptor beta subunit is represented by two phosphoproteins of molecular mass 95 and 105 kDa. The hormone-induced phosphorylation was rapid and dose-dependent occurring on both phosphoserine and phosphotyrosine residues. In addition, both insulin and IGF-I induced phosphorylation of an endogenous protein of molecular mass 185 kDa (pp185). The rapidity and dose dependency of the phosphorylation of pp185 suggested that it may represent a common endogenous substrate for the insulin and IGF-I receptors in these neural-derived cells. Phosphorylation was primarily on phosphoserine and phosphotyrosine residues. pp185 did not absorb to wheat germ agglutinin-agarose and was not stimulated by either epidermal growth factor or platelet-derived growth factor. The finding of pp185 in these neural-related cells as well as in non-neural tissues suggests that it may represent a ubiquitous endogenous substrate for both the insulin and IGF-I receptor kinases.     

Tyrosine phosphorylation of pp185 by insulin receptor kinase in a cell-free system

Insulin treatment of rat H-35 hepatoma cells causes rapid tyrosine phosphorylation of a high molecular weight protein termed pp185 besides autophosphorylation of the beta-subunit of the insulin receptor (IR) in an intact cell system. To elucidate the molecular basis for tyrosine phosphorylation of pp185, cell-free phosphorylation of pp185 was performed using phosphotyrosine-containing proteins (PYPs) purified from detergent-solubilized cell lysates by immunoprecipitation with anti-phosphotyrosine antibody. After insulin treatment of cells, marked increases of tyrosine phosphorylation of pp185 and IR were observed compared to noninsulin-treated cells. Site-specific antibodies that specifically inactivate IR kinase inhibited tyrosine phosphorylation of pp185 as well as the beta-subunit of IR. PYPs purified from detergent-free cell extracts contained pp185 but little IR; tyrosine phosphorylation of pp185 did not occur. Addition of IR kinase purified from human placenta to these PYPs restored insulin-dependent tyrosine phosphorylation of pp185. These results suggest that tyrosine phosphorylation of pp185 is catalyzed directly by IR kinase in this cell-free system.     

Improved glucose homeostasis and enhanced insulin signalling in Grb14-deficient mice

Gene targeting was used to characterize the physiological role of growth factor receptor-bound (Grb)14, an adapter-type signalling protein that associates with the insulin receptor (IR). Adult male Grb14(-/-) mice displayed improved glucose tolerance, lower circulating insulin levels, and increased incorporation of glucose into glycogen in the liver and skeletal muscle. In ex vivo studies, insulin-induced 2-deoxyglucose uptake was enhanced in soleus muscle, but not in epididymal adipose tissue. These metabolic effects correlated with tissue-specific alterations in insulin signalling. In the liver, despite lower IR autophosphorylation, enhanced insulin-induced tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and activation of protein kinase B (PKB) was observed. In skeletal muscle, IR tyrosine phosphorylation was normal, but signalling via IRS-1 and PKB was increased. Finally, no effect of Grb14 ablation was observed on insulin signalling in white adipose tissue. These findings demonstrate that Grb14 functions in vivo as a tissue-specific modulator of insulin action, most likely via repression of IR-mediated IRS-1 tyrosine phosphorylation, and highlight this protein as a potential target for therapeutic intervention.     

Phosphorylation of the solubilized insulin receptor by the gene product of the Rous sarcoma virus, pp60src

Both the insulin receptor and the gene product of the Rous sarcoma virus, pp60src, are protein kinases which phosphorylate themselves and other proteins on tyrosine residues. Addition of the solubilized insulin receptor to purified pp60src increased the phosphorylation of the beta-subunit of the insulin receptor. Phosphorylation of the insulin receptor by pp60src occurred both in the absence and presence of insulin but did not alter the insulin dose response for autophosphorylation of the receptor. Increasing concentrations of pp60src increased the phosphorylation of the receptor and at high concentrations equaled the maximal effect produced by insulin. Our observations suggest a possible mechanism by which the metabolically regulated insulin receptor tyrosine kinase could be altered by other tyrosine kinases such as that associated with pp60src. Further studies will be required to determine if the insulin receptor is phosphorylated by pp60src in Rous sarcoma virus-infected cells.     

Insulin-like growth factor I rapidly stimulates tyrosine phosphorylation of a Mr 185,000 protein in intact cells

The type I insulin-like growth factor (IGF) receptor, like the insulin receptor, contains a ligand-stimulated protein-tyrosine kinase activity in its beta-subunit. However, in vivo, no substrates have been identified. We used anti-phosphotyrosine antibodies to identify phosphotyrosine-containing proteins which occur during IGF-I stimulation of normal rat kidney and Madin-Darby canine kidney cells labeled with ortho[32P]phosphate. Both cells provide a good system to study the function of the type I IGF receptors because they contain high concentrations of these receptors but no insulin receptors. In addition, physiological levels of IGF-I, but not insulin, stimulated DNA synthesis in growth-arrested cells. IGF-I stimulated within 1 min of tyrosine phosphorylation of two proteins. One of them, with a molecular mass between 97 and 102 kDa, was supposed to be the beta-subunit of the type I IGF receptor previously identified. The other protein had an approximate molecular mass of 185 kDa, which resembled, by several criteria, pp 185, originally identified during the initial response of Fao cells to insulin binding (White, M. F., Maron, R., and Kahn, C. R. (1985) Nature 318, 183-186). These data suggest that tyrosine phosphorylation of pp 185 may occur during activation of both the type I IGF receptor and the insulin receptor, and it could be a common substrate that transmits important metabolic signals during ligand binding.     

Lipid rafts in T cell receptor signalling

The molecular events and the protein components that are involved in signalling by the T cell receptor (TCR) for antigen have been extensively studied. Activation of signalling cascades following TCR stimulation depends on the phosphorylation of the receptor by the tyrosine kinase Lck, which localizes to the cytoplasmic face of the plasma membrane by virtue of its post-translational modification. However, the precise order of events during TCR phosphorylation at the plasma membrane, remains to be defined. A current theory that describes early signalling events incorporates the function of lipid rafts, microdomains at the plasma membrane with distinct lipid and protein composition. Lipid rafts have been implicated in diverse biological functions in mammalian cells. In T cells, molecules with a key role in TCR signalling, including Lck, localize to these domains. Importantly, mutant versions of these proteins which fail to localise to raft domains were unable to support signalling by the TCR. Biochemical studies using purified detergent-resistant membranes (DRM) and confocal microscopy have suggested that upon stimulation, the TCR and Lck-containing lipid rafts may come into proximity allowing phosphorylation of the receptor. Further, there are data suggesting that phosphorylation of the TCR could depend on a transient increase in Lck activity that takes place within lipid rafts to initiate signalling. Current results and a model of how lipid rafts may regulate TCR signalling are discussed.     

Characterization of a Novel Receptor in Toad Retina with Dual Specificity for Insulin and Insulin-Like Growth Factor I

The biochemical properties of insulin receptors from toad retinal membranes were examined in an effort to gain insight into the role this receptor plays in the retina. Competition binding assays revealed that toad retinal membranes contained binding sites that displayed an equal affinity for insulin and insulin-like growth factor I (IGF-I). Affinity labeling of toad retinal membrane proteins with 125I-insulin resulted in the specific labeling of insulin receptor alpha-subunits of approximately 105 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of partially reduced (alpha beta-heterodimer) receptors affinity-labeled with 125I-insulin indicated the presence of a disulfide-linked beta-subunit of approximately 95 kDa. Endoglycosidase F digestion of the affinity-labeled alpha-subunits increased their mobility by reducing their apparent mass to approximately 83 kDa. This receptor was not detected by immunoblot analysis with a site-specific antipeptide antibody directed against residues 657-670 of the carboxy terminal of the human insulin receptor alpha-subunit, whereas this antibody did label insulin receptor alpha-subunits from pig, cow, rabbit, and chick retinas. In in vitro autophosphorylation assays insulin stimulated the tyrosine phosphorylation of toad retina insulin receptor beta-subunits. These data indicate that toad retinal insulin receptors have a heterotetrameric structure whose alpha-subunits are smaller than other previously reported neuronal insulin receptors. They further suggest that a single receptor may account for both the insulin and IGF-I binding activities associated with toad retinal membranes.     

Identification of the insulin-responsive tyrosine phosphorylation sites on IRSp53

The 53-kDa insulin receptor substrate protein (IRSp53) is part of a regulatory network that organises the actin cytoskeleton in response to stimulation by small GTPases, promoting formation of actin-rich cell protrusions such as filopodia and lamellipodia. It had been established earlier that IRSp53 is tyrosine phosphorylated in response to stimulation of the insulin and insulin-related growth factor receptors, but the consequences of tyrosine phosphorylation for IRSp53 function are unknown. Here, we have used a variety of IRSp53 truncation and point mutants to identify insulin-responsive tyrosine phosphorylation sites on IRSp53. We have found that the C-terminal half of IRSp53 (residues 251-521) undergoes tyrosine phosphorylation in response to insulin stimulation of the insulin beta receptor or epidermal growth factor stimulation via the epidermal growth factor receptor, and that the key residue for insulin receptor-mediated phosphorylation is tyrosine 310, located in a region between the N-terminal IRSp53/MIM homology domain (IMD, residue 1-250) and the central SH3 domain (residues 374-438) that is predicted to be natively unstructured. Mutation of tyrosine 310 to phenylalanine or glutamic acid abrogates the phosphorylation in response to insulin stimulation, but not in response to stimulation of the epidermal growth factor receptor. The N-terminal IMD, which mediates dimerisation of IRSp53, is required for efficient tyrosine phosphorylation downstream of either the insulin or epidermal growth factor receptor stimulation, yet does not appear to include a tyrosine-phosphorylated site itself. Thus, we have identified tyrosine 310 as a primary site of tyrosine phosphorylation in response to insulin signalling and we have shown that although IRSp53 is tyrosine phosphorylated in response to epidermal growth factor receptor signalling, tyrosine 310 is not crucial. Furthermore, the tyrosine phosphorylation status does not appear to affect the cell morphology and production of filopod-like structures upon expression of IRSp53.     

Basic polycations activate the insulin receptor kinase and a tightly associated serine kinase.

The effects of cationic polyamino acids on phosphorylation of the insulin and insulin-like growth factor 1 receptor kinases were studied and the following observations were made. (a) Polylysine stimulated both tyrosine and serine phosphorylation of the insulin receptor and of additional proteins present in lectin-purified membrane preparations from rat liver. (b) Polylysine synergized with insulin to enhance phosphorylation of the insulin receptor and of additional proteins (pp40 and pp110). (c) Polylysine effects were more pronounced upon increasing the polylysine chain length. (d) The effect of polylysine was biphasic with an optimum at 100 micrograms/ml. (e) Polylysine was found ineffective in stimulating the phosphorylation of immobilized insulin receptors. Taken together, these findings support the notion that the action of polylysine involves conformational changes and presumably aggregation of soluble receptors. The same effects of polylysine were obtained with highly purified insulin receptor preparations. Under these conditions polylysine enhanced both serine and tyrosine phosphorylation of the insulin receptor, suggesting that polylysine stimulates the activity of the insulin receptor kinase, and of a serine kinase that is tightly associated with the insulin receptor.     

Palmitate modulates the early steps of insulin signalling pathway in pancreatic islets

Insulin stimulates its own secretion and synthesis by pancreatic beta-cells. Although the exact molecular mechanism involved is unknown, changes in beta-cell insulin signalling have been recognized as a potential link between insulin resistance and its impaired release, as observed in non-insulin-dependent diabetes. However, insulin resistance is also associated with elevated plasma levels of free fatty acids (FFA) that are well known modulators of insulin secretion by pancreatic islets. This information led us to investigate the effect of FFA on insulin receptor signalling in pancreatic islets. Exposure of pancreatic islets to palmitate caused up-regulation of several insulin-induced activities including tyrosine phosphorylation of insulin receptor and pp185. This is the first evidence that short exposure of these cells to 100 microM palmitate activates the early steps of insulin receptor signalling. 2-Bromopalmitate, a carnitine palmitoyl-CoA transferase-1 inhibitor, did not affect the effect of the fatty acid. Cerulenin, an acylation inhibitor, abolished the palmitate effect on protein levels and phosphorylation of insulin receptor. This result supports the proposition that protein acylation may be an important mechanism by which palmitate exerts its modulating effect on the intracellular insulin signalling pathway in rat pancreatic islets.