The overall objective of this research proposal is to investigate the structure, function and regulation of a novel insulin- stimulated cytosolic serine kinase that we have discovered in rat adipocytes. In his proposed study, attempts will be made to purify this kinase to homogeneity by sequential liquid chromatographies through DEAE anion exchange, S-200 gel filtration, hydrophobic interaction and ATP citrate lyase-agarose affinity columns.
The aim i s to obtain purified cytosolic kinase for partial sequencing and for generation of polyclonal and monoclonal antibodies. These partial kinase sequences will e scrutinized and their relationship to other known protein kinases will be evaluated. The long term goal is to utilize this partial sequence information for the molecular cloning to this kinase. The complete primary structure of the cytosolic kinase can then be deduced from its cDNA. The kinase antibody preparations will be used for cell biology studies of this enzyme and to improve upon the original purification protocol. We will investigate the y=hypothesis that this enzyme is activated by hormone-induced phosphorylation. In these studies, we will examine whether a direct correlation exists between the activity of the cytosolic kinase and its phosphate content. If true, the nature of the insulin-stimulated phosphorylation will be analyzed in order to determine whether the cytosolic kinase is activated by serine/threonine or tyrosine phosphorylation. These results will be instrumental in deducing insulin-sensitive changes preceding the activation of the cytosolic kinase. To evaluate the role of the cytosolic kinase in mediating insulin action on cellular metabolism, antibodies inhibitory to the kinase will be selected and microinjected into insulin responsive cells by osmotic lysis of pinocytic vesicles. If some of these insulin sensitive metabolic parameters are indeed regulated by the cytosolic kinase, their insulin responsiveness should be inhibited in cells injected with the antikinase antibodies. Furthermore, attempts will be made to identify the physiological substrate proteins for the insulin-stimulated cytosolic kinase. Several typical insulin sensitive phosphoproteins such as the insulin receptor, acetyl CoA carboxylase and ATP citrate lyase will be evaluated as potential substrates for the kinase. If true, the effects of kinase catalyzed serine phosphorylation on their functions will be investigated. In addition, a cytosolic kinase- agarose column will be constructed and utilized to extract other potential substrate proteins for adipocytes.
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|Klarlund, J K; Bradford, A P; Milla, M G et al. (1990) Purification of a novel insulin-stimulated protein kinase from rat liver. J Biol Chem 265:227-34|