guanosine-triphosphate and bryostatin-1

Protein kinase C delta (PKC delta) from porcine spleen exhibits a marked capacity for autophosphorylation. Autophosphorylation is much more efficient in the presence of GTP than of ATP (6-fold). 15 mol phosphate/mol enzyme is incorporated with GTP as phosphate donor. The activity of PKC delta for autophosphorylation with ATP is around 4 times that of the isoenzymes alpha, beta, gamma (cPKC), and with GTP it is around 24 times that of cPKC. The catalytic subunit of protein kinase A and the tyrosine kinase src are not or only slightly autophosphorylated in the presence of GTP. The autophosphorylation of PKC delta with GTP does not differ from that with ATP regarding its activation by TPA or bryostatin, its inhibition by staurosporine, the type of phosphorylated amino acids (serine and threonine) and the mode of reaction (intrapeptide reaction). However, different sites are phosphorylated with GTP and ATP, as indicated by the amount of phosphate incorporated and by phosphopeptide mapping.

Topics: Adenosine Triphosphate; Animals; Bryostatins; Electrophoresis, Polyacrylamide Gel; Guanosine Triphosphate; Isoenzymes; Kinetics; Lactones; Macrolides; Mitogens; Peptide Mapping; Phosphopeptides; Phosphorylation; Protein Kinase C; Spleen; Substrate Specificity; Swine; Tetradecanoylphorbol Acetate

The chronically infected promonocytic clone U1 expresses low-to-undetectable constitutive levels of human immunodeficiency virus (HIV). Virus replication in these cells can be increased up to 25-fold by phorbol esters (phorbol-12-myristate-13-acetate), recombinant cytokines such as tumor necrosis factor-alpha, and cytokine-enriched mononuclear cell supernatants. We have tested specific activators of protein kinases (PK) and PK inhibitors (isoquinolinesulfonamide derivatives), as well as calcium-mobilizing agents, for their effect on constitutive and induced virus expression in U1 cells. Virus expression was measured by reverse transcriptase, Western blot, and nuclear run-on analysis. Activation of PKC by 1-oleyl,2-acetylglycerol, a synthetic analog of the natural ligand 1,2-diacylglycerol, and bryostatin 1 (a recently described specific PKC activator) resulted in a two- to eightfold increase in virus production. In contrast, activators of cyclic-nucleotide-dependent PKs were not effective in inducing virus expression. PK inhibitors were tested for their effect on HIV upregulation by cytokines and other inducing agents. The isoquinolinesulfonamide derivative H7, a potent inhibitor of PKC activation, effectively blocked (70 to 90%) HIV induction by cytokines and phorbol-12-myristate-13-acetate. The derivative HA1004, which is more selective for cyclic-nucleotide-dependent kinases, did not suppress viral induction. In addition, increases in intracellular calcium levels dramatically enhanced HIV production induced by both specific PKC activators and cytokines. These results indicate that activation of PKC is a common pathway involved in the upregulation of HIV expression in chronically infected cells stimulated by cytokines and other inducing agents.

Topics: Biological Factors; Bryostatins; Cell Line; Cell Nucleus; Cytokines; Diglycerides; Enzyme Activation; Guanosine Triphosphate; HIV; Humans; Lactones; Leukemia, Promyelocytic, Acute; Macrolides; Mitogens; Protein Kinase C; Recombinant Proteins; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha; Viral Proteins; Virus Replication