EXTRACELLULAR HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 TAT PROTEIN ACTIVATES PHOSPHATIDYLINOSITOL 3-KINASE IN PC12 NEURONAL CELLS

We have here investigated the effect of the regulatory Tat protein of the human immunodeficiency virus type 1 (HIV-1) on the PI 3-kinase cat alytic activity in PC12 rat pheochromocytoma cells. After as early as 1 min from the beginning of the treatment with recombinant HIV-1 Tat p rotein, a significant increase in the tyrosine phosphorylation levels of the p85 regulatory subunit of PI 3-kinase was noticed in 48 h serum -starved PC12 cells. Moreover, the addition of Tat to PC12 cells induc ed a great increase in PI 3-kinase immunoprecipitated with an anti-pho sphotyrosine antibody with a peak of activity (19-fold increase with r espect to the basal levels) after a 15-min treatment. This increase in PI 3-kinase activity was significantly higher in PC12 cell cultures s upplemented with Tat protein than in cultures stimulated by 100 ng/ml nerve growth factor (NGF; 8-fold increase with respect to the basal le vels). Further experiments showed that Tat protein was able to specifi cally activate PI 3-kinase at picomolar concentrations, In fact: (i) m aximal activation of PI 3-kinase was observed at concentrations as low as 1 ng/ml and was specifically blocked by anti-Tat neutralizing anti body; (ii) a Tat-dependent activation was also observed in experiments in which PI 3-kinase activity was evaluated in either anti-Tyr(P) or anti-p85 immunoprecipitates; (iii) 100 nM wortmannin completely blocke d the Tat-mediated increase in PI 3-kinase activity both in vitro and in vivo. Our data strongly support the concept that extracellular Tat acts as a cell stimulator, inducing intracellular signal transduction in uninfected cells.