Human T-cell lymphotropic virus type I (HTLV-I) is associated with two human diseases, adult T-cell leukemia (ATL), and tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM). HTLV-I Tax1 is released from infected lymphocytes and functions as an extracellular cytokine to stimulate gene expression in, and proliferation of, uninfected lymphocytes. Human T-cell lymphotropic virus type I (HTLV-I) Tax1 induces the activation and nuclear localization of the cellular transcription factor, NF-kappaB. Treatment of cells with calphostin C, a protein kinase C (PKC) inhibitor, blocked induction of NF-kappaB DNA binding activity in HTLV-I-transformed C81 cells and Tax1-stimulated murine pre-B cells, suggesting that PKC was an important intermediate in the NF-kappaB induction pathway. It was further demonstrated that Tax1 associates with, and activates, PKC. PKC was coimmunoprecipitated with anti-Tax1 sera from Tax1-expressing MT4 extracts and Jurkat extracts in the presence of exogenous Tax1 protein. In addition, Glutathione-S-transferase (GST)-Tax1 protein bound specifically to the alpha, delta and eta PKC isoenzymes synthesized in rabbit reticulocyte lysates. The addition of Tax1 to in vitro kinase reactions leads to the phosphorylation of Tax1 and an eighteen-fold increase in the autophosphorylation of PKC. Transfection of Jurkat cells with wild-type Tax1 stimulated membrane translocation of PKC. In contrast, Tax1 mutant M22, which fails to stimulate NF-kappaB-dependant transcription, failed to stimulate membrane translocation of PKC. Tax1 did not directly increase PKC phosphorylation of IkappaBalpha. Our results are consistent with a model in which Tax1 interacts with PKC and stimulates membrane translocation and triggering of the PKC pathway. Subsequent steps in the PKC cascade likely stimulate phosphorylation of IkappaBalpha. NF-kappaB regulates expression of several viral and cellular genes including the HIV LTR, MHC class I and IL-2Ralpha cytokine genes. It has been demonstrated that Rb stimulates binding of the NF-kappaB p50 homodimer. Addition of Rb protein to an in vitro gel shift binding assay stimulated p50 binding greater than ten-fold. Interestingly, by analyzing NF-kappaB-dependent transcription activity in vitro, we demonstrate that Rb suppresses transcriptional activity of p50. Chymotrypsin analysis suggests that Rb induces a conformational change in the NF-kappaB-DNA complex resulting in binding of a transcriptionally inactive complex. Finally, we demonstrate by coimmunoprecipitation analysis that the Rb-p50 complex is present in Jurkat cell extracts. Our results suggest that Rb may play an important role in regulation of NF-kappaB transcriptional activity.
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