Purification and characterisation of an insulin-simulated insulin receptor serine kinase

Sullivan, Alexandra Catherine (1997) Purification and characterisation of an insulin-simulated insulin receptor serine kinase. University of Southampton, Doctoral Thesis.

Abstract

This research project is concerned with clarifying the biochemical processes involved in the insulin signalling pathway. This may provide an understanding of abnormalities that can arise in non insulin dependent diabetes mellitus (NIDDM) and insulin resistance. Research is focused on the pathways that link the binding of insulin to its receptor and the intracellular activities that ensue.

In cells insulin stimulates autophosphorylation of the insulin receptor on tyrosine and its phosphorylation on serine and threonine residues by poorly characterised kinases. This project is concerned with studying a kinase responsible for the serine phosphorylation, namely, the insulin-stimulated insulin receptor serine kinase (IRSK). The IRSK is closely associated with the insulin receptor and it is important to determine its roles in propagating the insulin signal and in the control of insulin receptor function.

Here we describe methods for the purification of the IRSK from human placenta to apparent homogeneity by affinity chromatography, ion-exchange chromatography and gel filtration. The kinase had an apparent molecular weight of 38,000 on silver stained gels and also by gel filtration, suggesting it exists as a monomer. Phosphorylation of the insulin receptor by the IRSK did not affect insulin-stimulated exogenous protein tyrosine kinase activity of the receptor. The IRSK was found to be activated by autophosphorylation on serine.

The IRSK was characterised to distinguish it from previously identified insulin-stimulated kinases, and was found to be a novel entity. Partial amino acid sequence information for the protein was obtained.

An anti-phosphopeptide antibody raised against a major known site of serine phosphorylation of the insulin receptor (serine 1078) was characterised to facilitate studying phosphorylation at this site.

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