In vivo and in vitro modulatory effect of human immunodeficiency virus type-1 (HIV-1) Tat protein on protein kinase C...

Extracellular HIV-1 Tat protein shows a pleiotropic activity on the survival/proliferation of different cell types, which may be relevant to the pathogenesis of the immune suppression as well as of the frequent neoplastic disorders observed during the course of HIV-1 disease. Therefore, we investigated the effect of recombinant Tat on the protein kinase C (PKC) activity in Jurkat CD4(+) T lymphoma cells by using a serine substituted specific PKC peptide substrate, which allowed the evaluation of the whole catalytic activity of both Ca++-dependent and Ca++-independent PKC isoforms. High concentrations of recombinant Tat (1 mu g/ml) induced an early (5 min) stimulation followed by a secondary (30-60 min) inhibition of PKC in whole Jurkat cell homogenates. Immuno-localization experiments showed that recombinant Tat protein was rapidly taken up by Jurkat cells within the first 5 min from the addition in culture, thus suggesting the possibility that the secondary inhibitory phase of Tat on PKC activity in Jurkat cells could be due to a direct interaction between the two proteins. Consistently, PKC immunoprecipitated from Jurkat cells or purified from rat brain was significantly inhibited by the addition of high (0.1-1 mu g) but not low (1-10 ng) doses of Tat in a cell-free in vitro assay. The inhibition of PKC catalytic activity mediated by 1 mu g of Tat was at least partially due to competition among substrates. The present data may help in understanding the opposite effects on the survival/proliferation of different cell types observed in the presence of picomolar (stimulation) vs nanomolar (inhibition) concentrations of recombinant Tat.