HIV-1 TAT INDUCES TYROSINE PHOSPHORYLATION OF P125(FAK) AND ITS ASSOCIATION WITH PHOSPHOINOSITIDE 3-KINASE IN PC12 CELLS

Objective: To evaluate the signal transduction potential of HIV-1 Tat in a neuronal cell model. Methods: The tyrosine phosphorylation levels of the focal adhesion kinase p125(FAK) and its association with phosp hoinositide 3-kinase (PI 3-K) were evaluated in serum-starved rat pheo chromocytoma PC12 cells, either treated with low concentrations (0.1-1 nM) of extracellular HIV-1 Tat protein or stably transfected with tar cDNA. Results: Extracellular Tat induced a rapid increase of p125(FAK ) tyrosine phosphorylation and p125(FAK)-associated PI 3-K activity. B y using recombinant mutated Tat proteins, it was found that deletion o f amino acids 73-86 encoded by the second exon of the tar gene resulte d in a significant decrease of the ability of Tar to induce p125(FAK) tyrosine phosphorylation. Paradoxically, mutations in the basic region encoded by the first exon of tat, which is essential for nuclear loca lization and HIV-1 LTR transactivation, increased the ability of Tat t o stimulate p125(FAK) tyrosine phosphorylation. Moreover, in compariso n with cells transfected with a control vector, PC12 cells stably tran sfected with Lat cDNA showed greater amounts of p125(FAK) protein, an increase in p125(FAK) tyrosine phosphorylation and higher levels of p1 25(FAK)-associated PI 3-K activity. The addition of anti-rat neutraliz ing antibody to tat-transfected PC12 cells in culture blocked both the p125(FAK) tyrosine phosphorylation and its association with PI 3-K bu t did not affect the total amount of p125 (FAK). Conclusion: HIV-1 Tat protein enhanced both the expression and the functionality of p125(FA K) in PC12 neuronal cells. Whereas the first event required intracellu lar Tat, the increased p125(FAK) phosphorylation was strictly dependen t upon extracellular Tat. (C) 1998 Lippincott-Raven Publishers.