caa a22 4500 475772377 CHVBK 20180406123621.0 cr unu---uuuuu 170329e20000401xx s 000 0 eng 10.1007/s001250051327 doi (NATIONALLICENCE)springer-10.1007/s001250051327 Serine residues 994 and 1023/25 are important for insulin receptor kinase inhibition by protein kinase C isoforms β 2 and θ [Elektronische Daten] [V. Strack, A. M. Hennige, J. Krützfeldt, B. Bossenmaier, H. -H. Klein, M. Kellerer, R. Lammers, H. -U. Häring] Aims/hypothesis. Inhibition of the signalling function of the human insulin receptor (HIR) is one of the principle mechanisms which induce cellular insulin resistance. It is speculated that serine residues in the insulin receptor β-subunit are involved in receptor inhibition either as inhibitory phosphorylation sites or as part of receptor domains which bind inhibitory proteins or tyrosine phosphatases. As reported earlier we prepared 16 serine to alanine point mutations of the HIR and found that serine to alanine mutants HIR-994 and HIR-1023/25 showed increased tyrosine autophosphorylation when expressed in human embryonic kidney (HEK) 293 cells. In this study we examined whether these mutant receptors have a different susceptibility to inhibition by serine kinases or an altered tyrosine kinase activity.¶Methods. Tyrosine kinase assay and transfection studies.¶Results. In an in vitro kinase assay using IRS-1 as a substrate we could detect a higher intrinsic tyrosine kinase activity of both receptor constructs. Additionally, a higher capacity to phosphorylate the adapter protein Shc in intact cells was seen. To test the inhibition by serine kinases, the receptor constructs were expressed in HEK 293 cells together with IRS-1 and protein kinase C isoforms β2 and θ. Phorbol ester stimulation of these cells reduced wild-type receptor autophosphorylation to 58 % or 55 % of the insulin simulated state, respectively. This inhibitory effect was not observed with HIR-994 and HIR-1023/25, although all other tested HIR mutants showed similar inhibition induced by protein kinase C.¶Conclusion/interpretation. The data suggest that the HIR-domain which contains the serine residues 994 and 1023/25 is important for the inhibitory effect of protein kinase C isoforms β2 and θ on insulin receptor autophosphorylation. [Diabetologia (2000) 43: 443-449] Springer-Verlag Berlin Heidelberg, 2000 Keywords Insulin receptor inhibition, tyrosine kinase activity, serine phosphorylation, protein kinase C nationallicence Strack V. Medical Clinic Department IV, Eberhard-Karls-University of Tübingen, Germany, DE aut Hennige A. M. Medical Clinic Department IV, Eberhard-Karls-University of Tübingen, Germany, DE aut Krützfeldt J. Medical Clinic Department IV, Eberhard-Karls-University of Tübingen, Germany, DE aut Bossenmaier B. Roche Diagnostics, Penzberg, Germany, DE aut Klein H. -H Medical Clinic I, Medical University of Lübeck, Germany, DE aut Kellerer M. Medical Clinic Department IV, Eberhard-Karls-University of Tübingen, Germany, DE aut Lammers R. Medical Clinic Department IV, Eberhard-Karls-University of Tübingen, Germany, DE aut Häring H. -U Medical Clinic Department IV, Eberhard-Karls-University of Tübingen, Germany, DE aut https://doi.org/10.1007/s001250051327 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s001250051327 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Strack V. Medical Clinic Department IV, Eberhard-Karls-University of Tübingen, Germany, DE aut NATIONALLICENCE 700 1- Hennige A. M. Medical Clinic Department IV, Eberhard-Karls-University of Tübingen, Germany, DE aut NATIONALLICENCE 700 1- Krützfeldt J. Medical Clinic Department IV, Eberhard-Karls-University of Tübingen, Germany, DE aut NATIONALLICENCE 700 1- Bossenmaier B. Roche Diagnostics, Penzberg, Germany, DE aut NATIONALLICENCE 700 1- Klein H. -H Medical Clinic I, Medical University of Lübeck, Germany, DE aut NATIONALLICENCE 700 1- Kellerer M. Medical Clinic Department IV, Eberhard-Karls-University of Tübingen, Germany, DE aut NATIONALLICENCE 700 1- Lammers R. Medical Clinic Department IV, Eberhard-Karls-University of Tübingen, Germany, DE aut NATIONALLICENCE 700 1- Häring H. -U Medical Clinic Department IV, Eberhard-Karls-University of Tübingen, Germany, DE aut Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer