Insulin-stimulated phosphorylation of calmodulin by rat liver insulin receptor preparations

Insulin stimulates autophosphorylation of the beta subunit of its receptor and activates the associated tyrosine kinase. This kinase, in turn, phosphorylates a number of specific protein substrates; however, the functional and structural identity of these substrates is largely unknown. In this study, we demonstrate that insulin also stimulates the phosphorylation of calmodulin by rat hepatocyte insulin receptors partially purified by wheat germ agglutinin affinity chromatography. Phosphorylation occurred predominantly on tyrosine residues and had an absolute requirement for insulin receptors, divalent cations, and certain basic proteins. Maximal 32P incorporation was observed at an insulin concentration of 5 X 10(-9) M, and the K0.5 for insulin was approximately 4 X 10(-10) M. Phosphorylation of calmodulin was dependent upon ATP, saturating at 100 microM ATP with a K0.5 of 30 microM. Insulin-stimulated phosphorylation of calmodulin was also dependent upon Mg2+ or Mn2+, but was approximately 12-fold greater in the presence of Mg2+. Maximal phosphorylation was observed in the absence of Ca2+ and was inhibited at Ca2+:EGTA ratios greater than 0.8 (0.16 microM free Ca2+). Certain basic proteins, such as polylysine, histone Hf2b, and protamine sulfate, were necessary to observe insulin-stimulated phosphorylation of calmodulin. The relative amount of insulin-stimulated phosphorylation of calmodulin observed in the presence of each of these proteins differed. Maximal insulin-stimulated phosphorylation was observed in the presence of polylysine. These data suggest that both Ca2+ and calmodulin may participate in the early post-receptor events in the cellular mechanism of insulin action in hepatocytes.     

Insulin-stimulated tyrosine protein kinase. Characterization and relation to the insulin receptor

Utilizing histone phosphorylation as the basis for a quantitative assay, the insulin-stimulated protein kinase in human placenta has been characterized. The kinase copurifies through wheat germ agglutinin-Sepharose and DEAE-cellulose in constant ratio to the insulin binding function. Both activities are bound to the same extent on insulin-Sepharose, and the immobilized kinase, after extensive washing, exhibits activity versus histone, which closely approaches that of the insulin-stimulated, solubilized kinase. In addition, the bound kinase retains the ability to phosphorylate the Mr = 95,000 subunit of the bead-bound receptor. Elution of the beads with sodium dodecyl sulfate yields on electrophoresis two major peptides of Mr = 130,000 and 95,000. Thus, insulin binding and insulin-stimulated histone kinase copurify in a constant stoichiometric ratio in close physical relation and are likely functional expressions of the same molecule. After the DEAE step, the insulin-stimulated kinase phosphorylates histone subfraction 2b exclusively on tyrosine residues. Insulin increases the Vmax for H2b by 3-5-fold and increases the rate of the histone phosphorylation in direct correspondence to the steady state level of specifically bound insulin. ATP is the preferred phosphate donor. The reaction is supported by either Mn2+ or Mg2+. At [ATP] less than 0.5 mM, insulin-stimulated kinase is substantially higher with Mn2+ as the sole divalent cation, as compared to Mg2+. At [ATP] greater than or equal to 0.5 mM, the rates observed with Mn2+ have plateaued, whereas the rates in the presence of Mg2+ show a continued increase such that maximal activity is seen with Mg2+ and 2-3 mM ATP. Under these conditions, the estimated turnover number of the kinase ranges between 30 and 100 pmol of 32P transferred per min/pmol of insulin bound. Thus, the tyrosine kinase activity of the insulin receptor is quantitatively comparable to that estimated for several serine protein kinases and is unlikely to reflect the side reaction of another enzymatic function.     

Calcium-dependence of insulin receptor phosphorylation.

In primary cultures of rat hepatocytes, epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and foetal-calf serum (FCS) prevented the stimulation of amino acid transport by glucagon (cyclic AMP-dependent) and by catecholamines (cyclic AMP-independent), but not by insulin. The insulin effect, as well as the effect of other hormones, were totally inhibited by thrombin through a mechanism independent of its proteolytic activity. The inhibitory effect of growth factors, not found in freshly isolated hepatocytes, was expressed very early in culture (4h). Induction of tyrosine aminotransferase by glucagon or dexamethasone, which, like stimulation of transport, represents a late hormonal effect, was not affected by EGF, PDGF or FCS, but was inhibited by thrombin. In contrast, none of the rapid changes in protein phosphorylation caused by hormones was altered by growth factors. Thus the inhibition by growth factors of hormonal stimulation of transport presumably involves late step(s) in the cascade of events implicated in this hormonal effect.     

G-protein-coupled receptor rhodopsin regulates the phosphorylation of retinal insulin receptor

We have shown previously that phosphoinositide 3-kinase in the retina is activated in vivo through light-induced tyrosine phosphorylation of the insulin receptor (IR). The light effect is localized to photoreceptor neurons and is independent of insulin secretion (Rajala, R. V., McClellan, M. E., Ash, J. D., and Anderson, R. E. (2002) J. Biol. Chem. 277, 43319-43326). These results suggest that there exists a cross-talk between phototransduction and other signal transduction pathways. In this study, we examined the stage of phototransduction that is coupled to the activation of the IR. We studied IR phosphorylation in mice lacking the rod-specific alpha-subunit of transducin to determine if phototransduction events are required for IR activation. To confirm that light-induced tyrosine phosphorylation of the IR is signaled through bleachable rhodopsin, we examined IR activation in retinas from RPE65(-/-) mice that are deficient in opsin chromophore. We observed that IR phosphorylation requires the photobleaching of rhodopsin but not transducin signaling. To determine whether the light-dependent activation of IR is mediated through the rod or cone transduction pathway, we studied the IR activation in mice lacking opsin, a mouse model of pure cone function. No light-dependent activation of the IR was found in the retinas of these mice. We provide evidence for the existence of a light-mediated IR pathway in the retina that is different from the known insulin-mediated pathway in nonneuronal tissues. These results suggest that IR phosphorylation in rod photoreceptors is signaled through the G-protein-coupled receptor rhodopsin. This is the first study demonstrating that rhodopsin can initiate signaling pathway(s) in addition to its classical phototransduction.     

Insulin-stimulated serine/threonine phosphorylation of the insulin receptor: paucity of threonine 1348 phosphorylation in vitro indicates the involvement of more than one serine/threonine kinase in vivo.

Immunoaffinity-purified insulin receptors were used to analyse and compare the serine/threonine sites phosphorylated on the insulin receptor in vitro (isolated receptor) with the insulin-stimulated phosphorylation in vivo (intact cells in culture). In vivo, insulin-stimulation resulted in the appearance of three phosphoserine-containing phosphopeptides and a distinct phosphothreonine peptide (threonine 1348). In vitro, similar phosphoserine peptides were observed but the phosphothreonine peptide was absent. These results indicate that multiple serine sites are phosphorylated in vivo and in vitro and that an additional protein kinase mediates insulin-stimulated insulin receptor threonine phosphorylation in vivo.     

Inhibition of insulin receptor phosphorylation by indomethacin

Insulin stimulated phosphorylation of tyrosine residues by the insulin receptor kinase may be part of a signalling mechanism associated with insulin's action. We report that indomethacin inhibited the phosphorylation of the -subunit of the solubilized adipocyte insulin receptor. Indomethacin also inhibited several insulin-sensitive processes in intact rat adipocytes. Indomethacin (1 mM) inhibited basal phosphorylation of the -subunit of the solubilized insulin receptor by 6007o and insulin-stimulated phosphorylation by 30%. In adipocytes, indomethacin inhibited basal 3-0-[methyl-¹⁴C]-methyl-D glucose transport by 50070 (P < 0.01), D-[6-¹⁴C]-glucose oxidation by 5007o (P < 0.01), D-[6-¹⁴C]-glucose conversion to lipid by 30010 (P < 0.01), and D-[1-¹⁴C]-glucose conversion to lipid by 6007o (P<0.01). Similarly, indomethacin inhibited insulin-stimulated 3-0-[methyl-¹⁴C]-methyl-D-glucose transport by 75070 (P<0.01), D-[6-¹⁴C]-glucose oxidation by 20% (P<0.05), D-[1-¹⁴C]-glucose oxidation by 35070 (P<0.01), D-[6-¹⁴C] glucose conversion to lipid by 25010 (P<0.01), and D-[1-¹⁴C] glucose conversion to lipid by 4501o (P<0.01). In contrast, insulin binding to its receptor, basal D-[1-¹⁴C]-glucose oxidation and both basal and insulin-stimulated activation of glycogen synthase were unaffected by indomethacin. Thus, indomethacin partially inhibited autophosphorylation of the solubilized insulin receptor on tyrosine and partially inhibited some but not all of insulin's actions. This supports the hypothesis that insulin's metabolic effects are linked to activation of the insulin receptor protein kinase and indicates that there may be heterogeneity in the mechanisms of intracellular metabolic control by insulin.     

Regulation of insulin receptor kinase by multisite phosphorylation

The regulation of the insulin receptor kinase by phosphorylation and dephosphorylation has been examined. Under in vitro conditions, the tyrosine kinase activity of the insulin receptor toward histone is markedly activated when the receptor either undergoes autophosphorylation or is phosphorylated by a purified preparation of src tyrosine kinase on tyrosine residues of its beta subunit. The elevated kinase activity of the phosphorylated insulin receptor is readily reversed when the receptor is dephosphorylated with alkaline phosphatase. Analysis of tryptic digests of phosphorylated insulin receptor using reverse-phase high pressure liquid chromatography suggests that phosphorylation of a specific tyrosine site on the receptor beta subunit may be involved in the mechanism of the receptor kinase activation. Further studies indicate that tyrosine phosphorylation-mediated increase in insulin receptor activity also occurs in intact cells. Thus, when the histone kinase activities of insulin receptor from control and insulin-treated H-35 hepatoma cells are assayed in vitro following the purification of the receptors under conditions which preserve the phosphorylation state of the receptors, the insulin receptors extracted from insulin-treated cells exhibit histone kinase activities 100% higher than those from control cells. The elevated receptor kinase activity from insulin-treated cells appears to result from the increase in phosphotyrosine content of the receptor. Taken together, these results indicate that tyrosine phosphorylation of the insulin receptor beta subunit exerts a major stimulatory effect on the kinase activity of the receptor. Insulin receptor partially purified by specific immunoprecipitation from detergent extracts of control and isoproterenol-treated cells have similar basal but diminished insulin-stimulated beta subunit autophosphorylation activities when incubated with [gamma-32 P]ATP. Similarly, the ability of insulin to stimulate the receptor beta subunit phosphorylation in intact isoproterenol-treated adipocytes is greatly attenuated, whereas, the basal phosphorylation of the insulin receptor is slightly increased by the beta-catecholamine. These data indicate that in rat adipocytes, a cyclic AMP-mediated mechanism, possibly through serine and threonine phosphorylation of the receptor or its regulatory components, may uncouple the receptor tyrosine kinase activity from activation by insulin. Treatment of 32P-labeled H-35 hepatoma cells with phorbol myristate acetate (PMA) results in a marked increase in serine phosphorylation of the insulin receptor beta subunit.(ABSTRACT TRUNCATED AT 400 WORDS)     

The insulin receptor catalyzes the tyrosine phosphorylation of caveolin-1

Our previous studies revealed that insulin stimulates the tyrosine phosphorylation of caveolin in 3T3L1 adipocytes. To explore the mechanisms involved in this event, we evaluated the association of the insulin receptor with caveolin. The receptor was detected in a Triton-insoluble low density fraction, co-sedimenting with caveolin and flotillin on sucrose density gradients. We also detected the receptor in caveolin-enriched rosette structures by immunohistochemical analysis of plasma membrane sheets from 3T3L1 adipocytes. Insulin stimulated the phosphorylation of caveolin-1 on Tyr(14). This effect of the hormone was not blocked by overexpression of mutant forms of the Cbl-associated protein that block the translocation of phospho-Cbl to the caveolin-enriched, lipid raft microdomains. Moreover, this phosphorylation event was also unaffected by inhibitors of the MAPK and phosphatidylinositol 3-kinase pathways. Although previous studies demonstrated that the Src family kinase Fyn was highly enriched in caveolae, an inhibitor of this kinase had no effect on insulin-stimulated caveolin phosphorylation. Interestingly, overexpression of a mutant form of caveolin that failed to interact with the insulin receptor did not undergo phosphorylation. Taken together, these data indicate that the insulin receptor directly catalyzes the tyrosine phosphorylation of caveolin.     

Validation of a cell-based screen for insulin receptor modulators by quantification of insulin receptor phosphorylation

Stimulation of a cell with insulin initiates a signal transduction cascade that results in cellular activities that include phosphorylation of the receptor itself. Measurement of the degree of phosphorylation can serve as a marker for receptor activation. Receptor phosphorylation has been measured using Western blot analysis, which is very low throughput and not easily quantifiable. The goal of this project was to develop a cell-based assay to measure receptor phosphorylation in high throughput. This report describes a cell-based assay for insulin receptor phosphorylation that is robust and amenable to high-volume screening in a microwell format.     

Insulin-stimulated insulin secretion in single pancreatic beta cells

Functional insulin receptors are known to occur in pancreatic beta cells; however, except for a positive feedback on insulin synthesis, their physiological effects are unknown. Amperometric measurements at single, primary pancreatic beta cells reveal that application of exogenous insulin in the presence or absence of nonstimulatory concentrations of glucose evokes exocytosis mediated by the beta cell insulin receptor. Insulin also elicits increases in intracellular Ca2+ concentration in beta cells but has minimal effects on membrane potential. Conditions where the insulin receptor is blocked or cell surface concentration of free insulin is reduced during exocytosis diminishes secretion induced by other secretagogues, providing evidence for direct autocrine action of insulin upon secretion from the same cell. These results indicate that the beta cell insulin receptor can mediate positive feedback for insulin secretion. The presence of a positive feedback mechanism for insulin secretion mediated by the insulin receptor provides a potential link between impaired insulin secretion and insulin resistance.     

Diacylglycerols modulate phosphorylation of the insulin receptor from human mononuclear cells

It has been found that 1,2- but not 1,3-diacylglycerols stimulated phosphorylation of the insulin receptor of cultured human monocyte-like (U-937) and lymphoblastoid (IM-9) cells both in the intact- and broken-cell systems. The stimulation of the receptor's beta-subunit phosphorylation was dose-dependent, with optimal effect at 100 micrograms/ml of diacylglycerol. The effects of insulin and 1,2-diacylglycerols on the phosphorylation of partially purified insulin receptors were additive. Phosphoamino acid analysis showed a major effect of diacylglycerols on phosphorylation of tyrosine residues. The diacylglycerols also stimulated tyrosine kinase activity of the partially purified U-937 and IM-9 insulin receptors 2.5-3.5-fold when measured by phosphorylation of an exogenous substrate, poly(Glu80Tyr20) in the absence of any added insulin, calcium or phospholipid. Since this diacylglycerol effect could not be reproduced under conditions optimal for protein kinase C activation and the purified protein kinase C did not stimulate phosphorylation of the beta-subunit of the insulin receptor in this system, it is unlikely that the diacylglycerol effect was mediated by protein kinase C. Since these exogenous 1,2-diacylglycerols at the same high concentration also inhibited 125I-insulin binding to the insulin receptor of the intact U-937 and IM-9 cells, diacylglycerols could modulate the function of the insulin receptor and insulin action in human mononuclear cells.     

The in vitro synthesized and processed human insulin receptor precursor binds insulin.

The cell-free examination of the human insulin receptor during biogenesis may provide a greater understanding of the elements that contribute to the acquisition of receptor function. The insulin receptor precursor components were produced in a cell-free system and the insulin binding ability of the [35S]methionine-labeled translation products was determined. The processed proreceptor represented by a 190 kDa band was retained on insulin-linked biotin-streptavidin agarose or an insulin column. The insulin binding 190 kDa band migrated slower than the non-binding 190 kDa band on SDS-PAGE which suggests that covalent modifications account for these differences. The trypsin-digested product of the 190 kDa proreceptor was also retained on insulin-linked biotin-streptavidin agarose, however the alpha-subunit precursor was retained on insulin agarose to a much lesser degree. We conclude that a significant fraction of the processed, in vitro translated insulin proreceptor acquires insulin binding ability.     

Differential modulation of the tyrosine phosphorylation state of the insulin receptor by IRS (insulin receptor subunit) proteins.

In response to insulin, tyrosine kinase activity of the insulin receptor is stimulated, leading to autophosphorylation and tyrosine phosphorylation of proteins including insulin receptor subunit (IRS)-1, IRS-2, and Shc. Phosphorylation of these proteins leads to activation of downstream events that mediate insulin action. Insulin receptor kinase activity is requisite for the biological effects of insulin, and understanding regulation of insulin receptor phosphorylation and kinase activity is essential to understanding insulin action. Receptor tyrosine kinase activity may be altered by direct changes in tyrosine kinase activity, itself, or by dephosphorylation of the insulin receptor by protein-tyrosine phosphatases. After 1 min of insulin stimulation, the insulin receptor was tyrosine phosphorylated 8-fold more and Shc was phosphorylated 50% less in 32D cells containing both IRS-1 and insulin receptors (32D/IR+IRS-1) than in 32D cells containing only insulin receptors (32D/IR), insulin receptors and IRS-2 (32D/IR+IRS-2), or insulin receptors and a form of IRS-1 that cannot be phosphorylated on tyrosine residues (32D/IR+IRS-1F18). Therefore, IRS-1 and IRS-2 appeared to have different effects on insulin receptor phosphorylation and downstream signaling. Preincubation of cells with pervanadate greatly decreased protein-tyrosine phosphatase activity in all four cell lines. After pervanadate treatment, tyrosine phosphorylation of insulin receptors in insulin-treated 32D/IR, 32D/ IR+IRS-2, and 32D/IR+IRS-1F18 cells was markedly increased, but pervanadate had no effect on insulin receptor phosphorylation in 32D/IR+IRS-1 cells. The presence of tyrosine-phosphorylated IRS-1 appears to increase insulin receptor tyrosine phosphorylation and potentially tyrosine kinase activity via inhibition of protein-tyrosine phosphatase(s). This effect of IRS-1 on insulin receptor phosphorylation is unique to IRS-1, as IRS-2 had no effect on insulin receptor tyrosine phosphorylation. Therefore, IRS-1 and IRS-2 appear to function differently in their effects on signaling downstream of the insulin receptor. IRS-1 may play a major role in regulating insulin receptor phosphorylation and enhancing downstream signaling after insulin stimulation.     

The APS adapter protein couples the insulin receptor to the phosphorylation of c-Cbl and facilitates ligand-stimulated ubiquitination of the insulin receptor

The APS adapter protein is rapidly tyrosine-phosphorylated following insulin stimulation. In insulin-stimulated 3T3-L1 adipocytes, APS co-precipitated with phosphorylated c-Cbl. In CHO.T-APS cells overexpressing the insulin receptor and APS, APS co-precipitated with c-Cbl but not in CHO.T cells which do not express APS. APS-mediated recruitment of c-Cbl to the insulin receptor led to rapid ubiquitination of the insulin receptor beta-subunit in CHO. T-APS but not in parental CHO.T cells. These results suggest that the function of APS is to facilitate coupling of the insulin receptor to c-Cbl in order to catalyse the ubiquitination of the receptor and initiation of internalisation or degradation.     

src kinase catalyzes the phosphorylation and activation of the insulin receptor kinase

When a partially purified insulin receptor preparation immobilized on insulin-agarose is incubated with [gamma-32P]ATP, Mn2+, and Mg2+ ions, the receptor beta subunit becomes 32P-labeled. The 32P-labeling of the insulin receptor beta subunit is increased by 2-3-fold when src kinase is included in the phosphorylation reaction. In addition, the presence of src kinase results in the phosphorylation of a Mr = 125,000 species. The Mr = 93,000 receptor beta subunit and the Mr = 125,000 32P-labeled bands are absent when an insulin receptor-deficient sample, prepared by the inclusion of excess free insulin to inhibit the adsorption of the receptor to the insulin-agarose, is phosphorylated in the presence of the src kinase. These results indicate that the insulin receptor alpha and beta subunits are phosphorylated by the src kinase. The src kinase-catalyzed phosphorylation of the insulin receptor is not due to the activation of receptor autophosphorylation because a N-ethylmaleimide-treated receptor preparation devoid of receptor kinase activity is also phosphorylated by the src kinase. Conversely, the insulin receptor kinase does not catalyze phosphorylation of the active or N-ethylmaleimide-inactivated src kinase. Subsequent to src kinase-mediated tyrosine phosphorylation, the insulin receptor, either immobilized on insulin-agarose or in detergent extracts, exhibits a 2-fold increase in associated kinase activity using histone as substrate. src kinase mediates phosphorylation of predominantly tyrosine residues on both alpha and beta subunits of the insulin receptor. Tryptic peptide mapping of the 32P-labeled receptor alpha and beta subunits by high pressure liquid chromatography reveals that the src kinase-mediated phosphorylation sites on both receptor subunits exhibit elution profiles identical with those phosphorylated by the receptor kinase. Furthermore, the HPLC elution profile of the receptor auto- or src kinase-catalyzed phosphorylation sites on the receptor alpha subunit are also identical with that on the receptor beta subunit. These results indicate that: the src kinase catalyzes tyrosine phosphorylation of the insulin receptor alpha and beta subunits; and src kinase-catalyzed phosphorylation of insulin receptor can mimic the action of autophosphorylation to activate the insulin receptor kinase in vitro, although whether this occurs in intact cells remains to be determined.     

The role of antireceptor antibodies in stimulating phosphorylation of the insulin receptor

Four polyclonal antisera directed against the insulin receptor were tested for their capability to activate the tyrosine-specific protein kinase associated with the receptor. All four antisera were shown to inhibit insulin binding to the receptor in cultured human lymphoblastoid cells and to stimulate lipogenesis in isolated rat adipocytes. Although two antisera (B-d, B-8) stimulated the activity of the tyrosine kinase of partially purified receptor preparations from rat liver, two other antisera (B-2 and B-10) failed to do so. This failure could not be explained by lack of antibody binding to receptor, by interference with the receptor as a substrate for the kinase, or by blocking of the enzyme's active site. We conclude that these two antireceptor antibodies bind to the receptor but fail to activate the kinase. The simplest interpretation of these observations is that activation of the tyrosine-specific protein kinase might not be an obligatory step in coupling insulin binding to insulin action. However, it is also possible that the mechanism by which polyclonal antireceptor antisera mimic insulin's bioactivity may differ from the mechanism of action of insulin itself.     

Insulin-like effect of trypsin on the phosphorylation of rat adipocyte insulin receptor

Trypsin treatment of a partially purified insulin receptor preparation from rat adipocytes stimulated the phosphorylation of 90,000- and 72,000-Da polypeptides immunoprecipitated by anti-insulin receptor antibody. The phosphorylation of tyrosine residues alone was observed in both polypeptides. Trypsin concentrations which stimulated insulin receptor phosphorylation were the same as those previously shown to activate rat adipocyte glycogen synthase. Trypsin treatment of the insulin receptor fraction also stimulated the phosphorylation of an exogenous substrate of tyrosine kinase similarly to insulin treatment. Trypsin treatment of a highly purified insulin receptor from human placenta also activated the phosphorylation of the receptor-derived peptides. These results suggest that the insulin-stimulated protein kinase, a component of the insulin receptor, was activated by tryptic digestion to phosphorylate polypeptides derived from the insulin receptor itself. Thus, it is suggested that stimulation by trypsin of phosphorylation of the insulin receptor may be related to the insulin-like metabolic actions of trypsin observed in rat adipocytes.     

Tyrosine phosphorylation of phosphoinositide-dependent kinase 1 by the insulin receptor is necessary for insulin metabolic signaling

In L6 myoblasts, insulin receptors with deletion of the C-terminal 43 amino acids (IR(Delta43)) exhibited normal autophosphorylation and IRS-1/2 tyrosine phosphorylation. The L6 cells expressing IR(Delta43) (L6(IRDelta43)) also showed no insulin effect on glucose uptake and glycogen synthase, accompanied by a >80% decrease in insulin induction of 3-phosphoinositide-dependent protein kinase 1 (PDK-1) activity and tyrosine phosphorylation and of protein kinase B (PKB) phosphorylation at Thr(308). Insulin induced the phosphatidylinositol 3 kinase-dependent coprecipitation of PDK-1 with wild-type IR (IR(WT)), but not IR(Delta43). Based on overlay blotting, PDK-1 directly bound IR(WT), but not IR(Delta43). Insulin-activated IR(WT), and not IR(Delta43), phosphorylated PDK-1 at tyrosines 9, 373, and 376. The IR C-terminal 43-amino-acid peptide (C-terminal peptide) inhibited in vitro PDK-1 tyrosine phosphorylation by the IR. Tyr-->Phe substitution prevented this inhibitory action. In the L6(hIR) cells, the C-terminal peptide coprecipitated with PDK-1 in an insulin-stimulated fashion. This peptide simultaneously impaired the insulin effect on PDK-1 coprecipitation with IR(WT), on PDK-1 tyrosine phosphorylation, on PKB phosphorylation at Thr(308), and on glucose uptake. Upon insulin exposure, PDK-1 membrane persistence was significantly reduced in L6(IRDelta43) compared to control cells. In L6 cells expressing IR(WT), the C-terminal peptide also impaired insulin-dependent PDK-1 membrane persistence. Thus, PDK-1 directly binds to the insulin receptor, followed by PDK-1 activation and insulin metabolic effects.     

Identification of major tyrosine phosphorylation sites in the human insulin receptor substrate Gab-1 by insulin receptor kinase in vitro

Gab-1 (Grb2-associated binder-1), which appears to play a central role in cellular growth response, transformation, and apoptosis, is a member of the insulin receptor substrate (IRS) family. IRS proteins act downstream in the signaling pathways of different receptor tyrosine kinases, including the insulin receptor (IR). In this paper, we characterize the phosphorylation of recombinant human Gab-1 (hGab-1) by IR in vitro. Kinetic phosphorylation data revealed that hGab-1 is a high affinity substrate for the IR (K(M): 12.0 microM for native IR vs 23.3 microM for recombinant IR). To elucidate the IR-specific phosphorylation pattern of hGab-1, we used phosphopeptide mapping by two-dimensional HPLC analysis. Phosphorylated tyrosine residues were subsequently identified by sequencing the separated phosphopeptides by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) and Edman degradation. Our results demonstrate that hGab-1 was phosphorylated by IR at eight tyrosine residues (Y242, Y285, Y373, Y447, Y472, Y619, Y657, and Y689). Seventy-five percent of the identified radioactivity was incorporated into tyrosine residues Y447, Y472, and Y619 exhibiting features (NYVPM motif) of potential binding sites for the regulatory subunit (p85) of phosphatidylinositol (PI)-3 kinase. Accordingly, pull down assays with human HepG2 cell lysates showed that IR-specific phosphorylation of wild-type hGab-1 strongly enhanced PI-3 kinase binding. This is still the case when a single tyrosine residue in the NYVPM motif was mutated to phenylalanine. In contrast, phosphorylation-dependent binding of PI-3 kinase was completely abolished by changing a second tyrosine residue in a NYVPM motif independent from its location. Recently, we identified a similar cohort of tyrosine phosphorylation sites for the epidermal growth factor receptor (EGFR) with a predominant phosphorylation of tyrosine residue Y657 and binding of Syp [Lehr, S. et al. (1999) Biochemistry 38, 151-159]. These differences in the phosphorylation pattern of hGab-1 may contribute to signaling specificity by different tyrosine kinase receptors engaging distinct SH2 signaling molecules.