ABSTRACT
In previous studies we showed that an oxido-reductase is involved in the restoration of contact inhibition in fibrosacoma cells, implicating the redox state in growth control. In the present report we demonstrate that autophosphorylation of casein kinase II (CKII) is redox-dependent. We have also shown that phosphorylation of the transcription factor Jun by CKII is regulated by the redox state. In vitro kinase assays revealed that CKII-catalyzed phosphorylation increased the affinity of Jun for the DNA. In conformational analyses, CKII maintained an intact structure only within the redox range permissive to its autophosphorylation. Collectively, these data suggest that the redox state profoundly influences the ability of CKII to phosphorylate its substrate Jun and may do so by affecting the autophosphorylation as well as the structure of CKII. To demonstrate the biological relevance of these observations, redox potentials of fibroblast nuclei from different stages of growth were measured. Results indicated that as cell density increased, the intranuclear environment gradually became less reducing. Redox-dependent behaviors of growth-associated proteins such as Jun and CKII, together with evidence for in vivo changes in the nuclear redox state suggest that a redox mechanism may be involved in the regulation of cell growth.
Subject(s)
DNA/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-jun/metabolism , Casein Kinase II , Cell Division , Cell Line , Cell Nucleus/metabolism , DNA/genetics , Electrophoresis, Polyacrylamide Gel , Enzyme Activation , Fibroblasts , Glutathione/metabolism , Glutathione Disulfide/metabolism , Humans , Oligodeoxyribonucleotides/metabolism , Oxidation-Reduction , Phosphorylation , Protein Binding , Protein Conformation , Protein Serine-Threonine Kinases/chemistry , Proto-Oncogene Proteins c-jun/chemistryABSTRACT
HIV-host infection systems in vitro are important in the pre-clinical assessment of anti-retroviral drug activity. The present report describes the development of a new HIV-host model comprised of an epithelial cell line of HeLa lineage (HeLa-1), transfected with expression vectors bearing tat and rev (TART) genes of HIV-1 as well as the CD4 receptor gene, and HIV-1(delta Tat/Rev), a biologically contained strain of HIV-1 deleted in tat and rev. Measurement of infectivity, by syncytium formation and reverse transcriptase assay, revealed that HeLa-1 is infected with HIV-1(deltaTat/Rev). This virus failed to productively infect the TART-deficient CD4-positive HeLa cells, confirming its contained, non-infectious nature. The HeLa-1/HIV-1deltaTat/Rev system was used to measure the anti-retroviral activity of a human leukocyte-derived interferon (IFN-alphan3) preparation, several nucleoside analogs, and protease inhibitors. The HeLa-1/ HIV-1(deltaTat/Rev model provides a biologically contained system for the study of the HIV pathogenesis and the relative and combined therapeutic effects of anti-retroviral agents in vitro.
Subject(s)
Anti-HIV Agents/pharmacology , Drug Evaluation, Preclinical/methods , HIV Protease Inhibitors/pharmacology , HIV-1/drug effects , Interferon-alpha/pharmacology , Nelfinavir/pharmacology , Reverse Transcriptase Inhibitors/pharmacology , CD4 Antigens/genetics , Gene Expression , Gene Products, rev/genetics , Gene Products, tat/genetics , HeLa Cells , Humans , In Vitro Techniques , Ritonavir/pharmacology , Saquinavir/pharmacology , Zidovudine/pharmacology , rev Gene Products, Human Immunodeficiency Virus , tat Gene Products, Human Immunodeficiency VirusABSTRACT
Several protein kinase C (PKC) isoforms are expressed in human platelets. We report that PKC-delta is tyrosine phosphorylated within 30 s of platelet activation by thrombin. This correlated with a 2-3-fold increase in the kinase activity of PKC-delta relative to unstimulated platelets. The tyrosine phosphorylated PKC-delta isoform was associated with the platelet particulate (100,000 x g insoluble) fraction. Alpha(IIb)beta3 integrin mediated platelet adhesion to fibrinogen did not significantly affect PKC-delta activity. Tyrosine phosphorylation of PKC-delta was similarly not detected in fibrinogen adherent platelet lysates. Treatment of the platelets with mAb 7E3 prior to the addition of thrombin blocked aggregation having no effect on the thrombin induced PKC-delta activation. We conclude that PKC-delta is activated in platelets by an alpha(IIb)beta3 independent pathway.
