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 catalytic activity in PC12 rat pheochromocytoma cells. After as early as 1 min from the beginning of the treatment with recombinant HIV-1 Tat protein, 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 induced a great increase in PI 3-kinase immunoprecipitated with an anti-phosphotyrosine antibody with a peak of activity (19-fold increase with respect to the basal levels) after a 15-min treatment. This increase in PI 3-kinase activity was significantly higher in PC12 cell cultures supplemented with Tat protein than in cultures stimulated by 100 ng/ml nerve growth factor (NGF; 8-fold increase with respect to the basal levels). Further experiments showed that Tat protein was able to specifically activate PI 3-kinase at picomolar concentrations. In fact: (i) maximal activation of PI 3-kinase was observed at concentrations as low as 1 ng/ml and was specifically blocked by anti-Tat neutralizing antibody; (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 blocked 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.
Milani
D
D
Institute of Human Anatomy, University of Ferrara, 44100 Ferrara, Italy.
Mazzoni
M
M
Borgatti
P
P
Zauli
G
G
Cantley
L
L
Capitani
S
S
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D004789:Enzyme Activation; D015696:Gene Products, tat / Q000276:immunology / Q000494:pharmacology*; D015497:HIV-1 / Q000737:chemistry*; D009474:Neurons / Q000187:drug effects* / Q000201:enzymology; D016716:PC12 Cells; D019869:Phosphatidylinositol 3-Kinases; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction; D014443:Tyrosine / Q000378:metabolism; D054322:tat Gene Products, Human Immunodeficiency Virus
D000911:Antibodies, Monoclonal; D015696:Gene Products, tat; D054322:tat Gene Products, Human Immunodeficiency Virus; D014443:Tyrosine; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor)