title: Serine residues 994 and 1023/25 are important for insulin receptor kinase inhibition by protein kinase C isoforms beta2 and theta.
abstract: OBJECTIVE
  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 beta-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 beta2 and theta. 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.
  
  
  CONCLUSIONS
  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 beta2 and theta on insulin receptor autophosphorylation.
authors.affiliation: Medical Clinic Department IV, Eberhard-Karls-University of Tübingen, Germany.
authors.forename: V
authors.initials: V
authors.lastname: Strack
authors.forename: A M
authors.initials: AM
authors.lastname: Hennige
authors.forename: J
authors.initials: J
authors.lastname: Krützfeldt
authors.forename: B
authors.initials: B
authors.lastname: Bossenmaier
authors.forename: H H
authors.initials: HH
authors.lastname: Klein
authors.forename: M
authors.initials: M
authors.lastname: Kellerer
authors.forename: R
authors.initials: R
authors.lastname: Lammers
authors.forename: H U
authors.initials: HU
authors.lastname: Häring
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000409:Alanine; D002460:Cell Line; D006801:Humans; D007333:Insulin Resistance; D007527:Isoenzymes / Q000494:pharmacology*; D010766:Phosphorylation; D017354:Point Mutation; D011493:Protein Kinase C / Q000494:pharmacology*; D011972:Receptor, Insulin / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics; D012694:Serine; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
chemical_list: D007527:Isoenzymes; D012694:Serine; D000255:Adenosine Triphosphate; D011972:Receptor, Insulin; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D000409:Alanine