Received for publication December 21, 2005.
Revised May 8, 2006.
Accepted for publication May 8, 2006.

Proteomic analysis of rat liver phosphoproteins following treatment with protein kinase inhibitor H89 (N-(2-[p- bromocinnamylamino-]ethyl)-5-isoquinoline-sulfonamide)

Abstract

Phosphorylation of kinase targets are the essential measure of kinase inhibitor activity. Therapeutic strategies focused on kinase inhibition rely heavily on surrogate measures of kinase inhibition obtained from in vitro assay systems. There is a need to develop methodology that will facilitate measurement of kinase inhibitor activity or specificity in tissue samples from whole animals treated with these compounds. Many of the current methods are limited by the use of antibodies; many of which do not cross react between several species. The proteomics approach described herein has the potential to reveal novel tissue substrates, potential new pathway interconnections and inhibitor specificity by monitoring differences in protein phosphorylation. We used the protein kinase inhibitor H89 as a tool to ask whether differential profiling of tissue phosphoproteins can be used to detect treatment related effects of a PKA inhibitor in vivo. With a combination of phosphoprotein column enrichment, high-throughput 2D gel electrophoresis, differential gel staining with ProQ Diamond/Sypro Ruby, statistical analysis and MALDI-TOF MS analysis, we were able to show clear differences between the phosphoprotein profiles of rat liver protein extract from control and treated animals. Moreover, several proteins that show a potential change in phosphorylation were previously identified as PKA substrates or have putative PKA phosphorylation sites. The data presented support the use of differential proteomic methods to measure effects of kinase inhibitor treatment on protein phosphorylation in vivo.

Key words: Protein Kinase A, kinase inhibitor, kinases, proteomics, screening, toxicology

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