Proteomic Analysis of Rat Liver Phosphoproteins after Treatment with Protein Kinase Inhibitor H89 (N-(2-[p-Bromocinnamylamino-]ethyl)-5-isoquinolinesulfonamide)
| Autor: | Douglas Hinerfeld, Myrtle Davis, Sun W. Tam, Jennifer Rutherford-Bethard, Naijia H. Huang, Sajan Joseph, Yu-Hua Hui, John D. Leszyk |
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| Rok vydání: | 2006 |
| Předmět: |
Male
Proteomics medicine.drug_class Biology MAP2K7 Rats Sprague-Dawley In vivo Databases Genetic Image Processing Computer-Assisted medicine Animals Electrophoresis Gel Two-Dimensional Protein phosphorylation Enzyme Inhibitors Phosphorylation Protein Kinase Inhibitors Pharmacology Sulfonamides Kinase Protein kinase inhibitor Isoquinolines Phosphoproteins Cyclic AMP-Dependent Protein Kinases Molecular biology Rats Liver Biochemistry Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Phosphoprotein Molecular Medicine |
| Zdroj: | Journal of Pharmacology and Experimental Therapeutics. 318:589-595 |
| ISSN: | 1521-0103 0022-3565 |
| DOI: | 10.1124/jpet.105.100032 |
| Popis: | 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 (N-(2-[p-bromocinnamylamino]-ethyl)-5-isoquinolinesulfonamide) as a tool to determine whether differential profiling of tissue phosphoproteins can be used to detect treatment-related effects of a protein kinase A (PKA) inhibitor in vivo. With a combination of phosphoprotein column enrichment, high-throughput two-dimensional gel electrophoresis, differential gel staining with Pro-Q Diamond/SYPRO Ruby, statistical analysis, and matrix-assisted laser desorption ionization/time of flight mass spectrometry 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. |
| Databáze: | OpenAIRE |
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