Regulation of cellular events by reversible protein phosphorylation has been well established. In this proposal the biochemistry of a cyclic AMP-independent protein kinase (H4-PK) which has been isolated from lymphosarcoma cells will be investigated. Previous studies have shown that this enzyme exists in an inactivated conformation which can be converted to an active enzyme in an ATP-dependent reaction requiring a second enzyme. The active enzyme appears to be most abundant in proliferating tissues such as the lymphosarcoma cells, 3T3 cells, regenerating liver and testes. Preliminary results obtained in this laboratory and confirmed by a second laboratory have suggested that the enzyme is activated in response to insulin stimulation of cells. The overall goal of this proposal is to elucidate further the regulation and reactivity of H4-PK in order to resolve the cellular function of this enzyme.
The specific aims are as follows: (1) to investigate the mechanism by which the activator protein regulates H4-PK activity by purification of the activator protein, characterization of the activation reaction, and analysis of the physical properties of the activated and non-activated H4-PK; (2) to characterize the tissue specificity and cellular localization of the H4-PK using antibodies directed against the purified H4-PK and the H4-PK activating enzyme; (3) to analyze the phosphorylation of ribosomal protein S6 in vivo and to compare and contrast the characteristics of this reaction with those observed using purified H4-PK and 40S ribosomes in vitro. In all studies, enzymes prepared from the P1798 murine lymphosarcoma will be utilized. Standard techniques for protein purification will be utilized to purify the component enzymes in the H4-PK activation cascade. Elucidation of the activation mechanism will be pursued using the purified components. Particular emphasis will be placed on the role of ATP in the reaction. Concomitant with those studies, antibodies to H4-PK and the activating complex will be prepared. These antibodies will be used in a series of experiments designed to elucidate the selected aspects of H4-PK biochemistry in order to provide a more extensive basis for the understanding of the significance of this enzyme in cellular regulation. The long term objective of these studies is to identify the biochemical pathway which includes the H4-PK, i.e. activation reaction, substrate phosphorylation, and modification of cellular activity which occurs in response to a specific extracellular signal, and to evaluate the potential role for this enzyme in cell regulation.
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