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G J Sale - One of the best experts on this subject based on the ideXlab platform.

  • Studies on an Insulin-stimulated Insulin Receptor serine kinase activity: separation of the kinase activity from the Insulin Receptor and its reconstitution back to the Insulin Receptor.
    The Biochemical journal, 1995
    Co-Authors: K A Asamoah, P G Atkinson, W G Carter, G J Sale
    Abstract:

    In cells Insulin stimulates autophosphorylation of the Insulin Receptor on tyrosine and its phosphorylation on serine and threonine by poorly characterized kinases. Recently we have achieved co-purification of the Insulin Receptor with Insulin-stimulated Insulin Receptor serine kinase activity. We now show that the co-purified serine kinase activity can be removed by NaCl washing and reconstituted by adding back the NaCl eluate. Reconstitution enabled higher serine phosphorylation than achieved with the co-purified preparation. Myelin basic protein was discovered to be a potent substrate for Insulin-stimulated serine phosphorylation by the co-purified preparation, with the activity responsible having similar properties to the serine kinase activity towards the Receptor. Myelin basic protein was also phosphorylated on serine by the NaCl eluate. Myelin basic protein phosphorylated by the co-purified preparation or the NaCl eluate gave the same set of phosphoserine peptides. The major myelin basic protein serine kinase activity in the NaCl eluate co-purified exactly on Mono Q with the activity that restored Insulin-stimulated Insulin Receptor serine phosphorylation. These results provide strong evidence for the true separation of the serine kinase from the Insulin Receptor and the distinctiveness of the serine kinase activity from the Insulin Receptor tyrosine kinase and mitogen-activated protein kinases. The procedures developed for the isolation of the serine kinase and the establishment of an effective in vitro substrate should allow purification of the kinase. The protocols also provide flexible systems for identifying the functions of the Insulin-stimulated serine phosphorylations and the respective kinase(s).

Alexandra C Newton - One of the best experts on this subject based on the ideXlab platform.

  • Diacylglycerol directly stimulates the Insulin Receptor tyrosine kinase
    FEBS Letters, 1996
    Co-Authors: Rebecca S. Arnold, Alexandra C Newton
    Abstract:

    Abstract Studies with detergent: lipid mixed micelles reveal that diacylglycerol directly stimulates the intrinsic tyrosine kinase activity of the Insulin Receptor. Kinetic analyses indicate that diacylglycerol activates the kinase by causing a marked increase in the affinity of the Receptor for Insulin. In contrast, diacylglycerol has no effect on the Insulin Receptor's catalytic activity or its affinity for ATP. Stimulation of the Insulin Receptor is not a result of protein kinase C activation. First, phorbol myristate acetate, a potent activator of protein kinase C, has no effect on Insulin Receptor activity. Second, the activation by diacylglycerol is not stereospecific, in marked contrast to the specificity for 1,2-diacyl-sn-glycerol in the activation of protein kinase C. Because circulating levels of Insulin are below the Kd of the Insulin Receptor forcor∗ Insulin, the ability of diacylglycerol to modulate the affinity of the Receptor for ligand suggests that increases in cellular levels of diacylglycerol directly sensitize the Receptor to Insulin.

  • Inhibition of the Insulin Receptor tyrosine kinase by sphingosine.
    Biochemistry, 1991
    Co-Authors: Rebecca S. Arnold, Alexandra C Newton
    Abstract:

    Sphingosine inhibits autophosphorylation of the Insulin Receptor tyrosine kinase in vitro and in situ. This lysosphingolipid has been shown previously to inhibit the Ca2+/lipid-dependent protein kinase C. Here we show that Insulin-dependent autophosphorylation of partially purified Insulin Receptor is half-maximally inhibited by 145 microM sphingosine (9 mol %) in Triton X-100 micelles. Half-maximal inhibition of protein kinase C autophosphorylation occurs with 60 microM sphingosine (3.4 mol %) in Triton X-100 mixed micelles containing phosphatidylserine and diacylglycerol. Sphingomyelin does not inhibit significantly the Insulin Receptor, suggesting that, as with protein kinase C, the free amino group may be essential for inhibition. Similar to the effects observed for protein kinase C, inhibition of the Insulin Receptor kinase by sphingosine is reduced in the presence of other lipids. However, the reduction displays a marked dependence on the lipid species: phosphatidylserine, but not a mixture of lipids compositionally similar to the cell membrane, markedly reduces the potency of sphingosine inhibition. The inhibition occurs at the level of the protein/membrane interaction: a soluble form of the Insulin Receptor comprising the cytoplasmic kinase domain is resistant to sphingosine inhibition. Lastly, sphingosine inhibits the Insulin-stimulated rate of tyrosine phosphorylation of the Insulin Receptor in NIH 3T3 cells expressing the human Insulin Receptor. These results suggest that sphingosine alters membrane function independently of protein kinase C.

Graham J. Sale - One of the best experts on this subject based on the ideXlab platform.

  • Purification and characterization of an Insulin-stimulated Insulin Receptor serine kinase.
    Biochemistry, 1996
    Co-Authors: Wayne G. Carter, Alexandra C. Sullivan, Kojo A. Asamoah, Graham J. Sale
    Abstract:

    In cells, Insulin stimulates autophosphorylation of the Insulin Receptor on tyrosine and its phosphorylation on serine and threonine by poorly characterized kinases. Here we describe methods for the purification of an Insulin-stimulated Insulin Receptor serine kinase from human placenta and rat liver by sequential chromatography of solubilized membranes on wheat germ agglutinin−agarose, Mono Q, phenyl-Superose, and Superose 12. On silver-stained SDS−polyacrylamide gels, the resulting kinase was homogeneous (human) or near-homogeneous (rat) and had an apparent Mr of 40 000. The apparent Mr determined by gel filtration was also 40 000, suggesting that the kinase exists as a monomer. The kinase could be reconstituted back to the Insulin Receptor stripped of the kinase to yield a high stoichiometry of serine phosphorylation of the Insulin Receptor in the presence of Insulin (0.75 ± 0.15 mol/mol of β-subunit, mean ± SEM, n = 3). The activity of the reconstituted kinase toward the Insulin Receptor was Insulin-r...

Robert E. Lewis - One of the best experts on this subject based on the ideXlab platform.

  • Expression of the Insulin Receptor with a recombinant vaccinia virus. Biochemical evidence that the Insulin Receptor has intrinsic serine kinase activity.
    The Journal of biological chemistry, 1996
    Co-Authors: Thomas J. Tauer, Deanna J. Volle, Solon L. Rhode, Robert E. Lewis
    Abstract:

    Abstract We have previously reported the tight association of a serine kinase activity with the human Insulin Receptor (Lewis, R. E., Wu, G. P., MacDonald, R. G., and Czech, M. P.(1990) J. Biol. Chem. 265, 947-954). We tested the possibility that the associated serine kinase activity was intrinsic to the Receptor catalytic domain. The ratio of phosphoserine to phosphotyrosine on Insulin Receptors phosphorylated in vitro was used as an index of the associated serine kinase activity. Phosphorylation and phosphoamino acid analysis of Insulin proReceptors revealed associated serine kinase activity early in Receptor synthesis. Insulin Receptors were expressed in HeLa cells using a recombinant vaccinia virus. The ratio of phosphoserine to phosphotyrosine on Insulin Receptors expressed by the recombinant vaccinia virus was determined relative to endogenous Insulin Receptors in cells treated with α-amanitin to block host cell mRNA synthesis. α-Amanitin treatment had no effect on the ratio of phosphoserine to phosphotyrosine on Insulin Receptors expressed from the recombinant virus even though they were present in a 4000-fold excess above endogenous Receptors. We conclude that the serine kinase activity associated with the Insulin Receptor is intrinsic to the Receptor catalytic domain. Receptor-catalyzed autophosphorylation of serine may play an important role in modulating Insulin Receptor signaling.

Chang-kiu Moon - One of the best experts on this subject based on the ideXlab platform.

  • Brazilin inhibits activities of protein kinase C and Insulin Receptor serine kinase in rat liver
    Archives of Pharmacal Research, 1998
    Co-Authors: Seong-gon Kim, You-me Kim, Lee-yong Khil, Sun-duck Jeon, Chang-hyun Moon, Chang-kiu Moon
    Abstract:

    Hypoglycemic action of brazilin was found to be based on the improvement of peripheral glucose utility, and this action might be correlated with the Insulin action pathway. In the present study we investigated the effect of brazilin on the Insulin Receptor autophosphorylation, protein kinase C (PKC), protein phosphatase and Insulin Receptor serine kinase in order to confirm whether the hypoglycemic mechanism is concerned with Insulin action pathway. Brazilin was found to inhibit PKC and Insulin Receptor serine kinase, which are involved in the regulation of Insulin signal pathway. But any significant effect was not shown on Insulin Receptor tyrosine kinase activity, autophosphorylation and phosphatase activity. These findings suggest that brazilin might enhance Insulin Receptor function by decreasing serine phosphorylation, which might mediate hypoglycemic effect of brazilin