Effect of cyclic AMP-dependent protein kinases on gene expression

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Mechanisms for regulating activation of cyclic AMP-dependent protein kinase were investigated and a possible biological role of this enzyme was elucidated. Autophosphorylation of cyclic AMP-dependent protein kinase was shown to occur via an intramolecular mechanism: the regulatory subunit undergoes phosphorylation only within the holoenzyme. The phosphoform of the catalytic subunit has the capacity to phosphorylate the regulatory subunit. The phosphotransferase reaction and the reaction of autophosphorylation were found to proceed with the involvement of the same active site. Autophosphorylation was demonstrated to lead to almost complete dissociation of the holoenzyme under the influence of cyclic AMP. Circular dichroism spectra of the phosphorylated and nonphosphorylated forms of protein kinase were studied. The relative content of the secondary structure elements in proteins was estimated and conformational changes were detected in the enzyme upon its interaction with cyclic AMP. By the method of circular dichroism it was also shown that cyclic AMP and its 8-Br-substituted analog were bound in the cyclic AMP-binding site of the protein kinase in anti-conformation. The data obtained suggest the change of conformation of the 8-Br-cyclic AMP molecule during activation of protein kinase: while having a preferential syn-conformation in the free state in solution, the molecule of 8-Br-cyclic AMP acquires anti-conformation in the fixed complex with the regulatory subunit. A study was made of nuclear translocation of cyclic AMP-dependent protein kinase and its subunits, as well as of the binding of these proteins to metaphase chromosomes. The CHO cell cultures were treated with 3H-labeled protein kinase and its subunits. The results indicate that cyclic AMP-dependent protein kinase becomes translocated into the nucleus in a dissociated state and that the subunits have specific binding sites on chromatin. Transformation of normal mouse fibroblasts by virus SV 40 interferes with the nuclear translocation of the regulatory subunit. The process is restored when the level of cyclic AMP in the system is increased. Binding of the regulatory subunit to metaphase chromosomes of cells transformed by virus SV 40 does not change. In the case of spontaneous cancer (KB cells) translocation of the regulatory subunit remains unaffected, whereas acceptance of the protein by the metaphase chromosomes is impeded. The results of this work suggest that compartmentalization of cyclic AMP-dependent protein kinase, and particularly of its regulatory subunit, in the cell is highly significant for cellular processes. Disorders arising as a result of neoplastic transformation involve changes in nuclear translocation of the regulatory subunit and in its binding to the structural elements of the genome. The effect of the regulatory subunit of cyclic AMP-dependent protein kinase on the process of transcription in vitro was shown. The regulatory subunit was found to increase the number of initiation sites on chromatin for RNA polymerase. In this connection, an attempt was made to detect more directly expression of the genome by the regulatory subunit and to identify the final protein product formed. The regulatory subunit induced a specific synthesis of new proteins, the direct and first response of 3T3 cells to the introduction of the regulatory subunit being the synthesis of protein P-15. The molecular weight of the protein was 15,000, the isoelectric point 6.3. The electrophoretic analysis of the cytosol of SV 40-3T3 cells revealed a general derepression of the genome of the virus-transformed cells. A protein identical with P-15 was detected to be present in SV 40-3T3 cells. The treatment of these cells with the regulatory subunit as well as with cyclic AMP separately did not affect the synthesis of P-15, whereas introduction of the cyclic AMP-regulatory subunit complex caused a significant expression of protein P-15. The data obtained indicate that the protein synthesis is dependent on nuclear translocation of the regulatory subunit.