Serine residues 994 and 1023/25 are important for insulin receptor kinase inhibition by protein kinase C isoforms beta2 and theta

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 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. 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 beta2 and theta on insulin receptor autophosphorylation.

A 350 ka record of climate change from Lake El'gygytgyn, Far East Russian Arctic: refining the pattern of climate modes by means of cluster analysis

Abstract. Rock magnetic, biochemical and inorganic records of the sediment cores PG1351 and Lz1024 from Lake El’gygytgyn, Chukotka peninsula, Far East Russian Arctic, were subject to a hierarchical agglomerative cluster analysis in order to refine and extend the pattern of climate modes as defined by Melles et al. (2007). Cluster analysis of the data obtained from both cores yielded similar results, differentiating clearly between the four climate modes warm, peak warm, cold and dry, and cold and moist. In addition, two transitional phases were identified, representing the early stages of a cold phase and slightly colder conditions during a warm phase. The statistical approach can thus be used to resolve gradual changes in the sedimentary units as an indicator of available oxygen in the hypolimnion in greater detail. Based upon cluster analyses on core Lz1024, the published succession of climate modes in core PG1351, covering the last 250 ka, was modified and extended back to 350 ka. Comparison to the marine oxygen isotope (�18O) stack LR04 (Lisiecki and Raymo, 2005) and the summer insolation at 67.5� N, with the extended Lake El’gygytgyn parameter records of magnetic susceptibility (�LF), total organic carbon content (TOC) and the chemical index of alteration (CIA; Minyuk et al., 2007), revealed that all stages back to marine isotope stage (MIS) 10 and most of the substages are clearly reflected in the pattern derived from the cluster analysis.