Studies on protein phosphorylation in response to insulin in isolated cellular fractions reconstituted with insulin receptors
Creators: name: Lew, Gregory John
Publisher: name: University of British Columbia
Year: 2010

description: The mechanism by which insulin and other polypeptide growth factors alter cellular metabolism is not fully understood. In the case of insulin, it is thought that phosphorylation/dephosphorylation mechanisms may play a central role in the signalling pathway. This is based on evidence which includes demonstration that the receptor for insulin is a tyrosine-specific protein kinase which is activated in response to insulin binding. Ultimately, insulin binding to its receptor on the surface of intact fat cells leads to altered levels of serine phosphorylation of several soluble proteins, including the phosphorylation of ATP-citrate lyase and acetyi-CoA carboxylase. Recently, studies involving site-specific mutagenesis have shown that the tyrosine kinase function of the insulin receptor is essential for insulin signalling. The studies described in this thesis have addressed the problem of how activation of the insulin receptor/tyrosine kinase results in the altered serine phosphorylation observed in intact cells in response to insulin. To gain further understanding of the cellular components required for insulin signalling, reconstitution experiments have been carried out mixing isolated cellular fractions with preparations of insulin receptors. The effects of insulin on altering protein-serine and protein-tyrosine phosphorylation have been determined in this reconstituted system. Results show that in a high-speed (100,000 x g) supernatant fraction prepared from rat adipose tissue endogenous protein-serine kinases are sensitive to conditions which are commonly employed for assaying insulin receptor/kinase activity. This includes inhibition by micromolar concentrations of MnCI₂, by 40 mM NaF, and by low reaction temperature (0°C). When the insulin receptor, present in a WGA-Sepharose-purified preparation of detergent-solublized rat liver membranes, was assayed in the complete absence of both MnCI₂ and NaF, receptor/tyrosine kinase activity was only slightly reduced with little or no decrease in the responsiveness to insulin. Furthermore, when the WGA-Sepharose-purified membrane fraction was incubated at 37°C in the presence of [ɣ -³²P]ATP several endogenous proteins were observed to be phosphorylated in addition to the β-subunit of the insulin receptor. These membrane proteins appear to be phosphorylated on tyrosine as indicated by their resistance to alkali hydrolysis. Upon reconstitution of the adipose tissue high-speed supernatant fraction with the WGA-Sepharose-purified preparation of insulin receptors the most striking effects observed were the phosphorylation of a 40 kd protein subunit (pp40) and the dephosphorylation of a 25 kd protein subunit (pp25) present in adipose tissue. The phosphorylation of pp40 occurs on tyrosine and is insulin-responsive, whereas the dephosphorylation of pp25 occurs following reconstitution with either untreated control, or insulin-activated insulin receptors. To assess the effect that reconstituted insulin receptors may have on the phosphorylation of endogenous ATP-citrate lyase in adipose tissue high-speed supernatant, it was found that a more pure preparation of insulin receptors was required. Further purification of the insulin receptor to homogeneity was therefore attempted using insulin-agarose affinity chromatography. However, difficulties including low yield and instability of the receptor through purification have prevented progress with these studies at present. In a separate study, highly purified acetyl-CoA carboxylase was reconstituted with a crude fraction consisting of total Triton-solublized membrane proteins. In this reconstituted system phosphorylation of acetyl-CoA carboxylase was enhanced to an extent greater than 6-fold after incubation with [ɣ -³²P]ATP. Following chromatography of the crude Triton-solublized extract over WGA-Sepharose this acetyl-CoA carboxylase kinase activity was found to be present in the flow-through void fraction and not in the N-acetylglucosamine eluted fraction. The acetyl-CoA carboxylase kinase, at present, does not appear to be insulin-responsive, but further studies are needed to confirm this observation.