HIV-1 Tat induces tyrosine phosphorylation of p125FAK 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 p125FAK and its association with phosphoinositide 3-kinase (PI 3-K) were evaluated in serum-starved rat pheochromocytoma PC12 cells, either treated with low concentrations (0.1-1 nM) of extracellular HIV-1 Tat protein or stably transfected with Tat cDNA.


RESULTS
Extracellular Tat induced a rapid increase of p125FAK tyrosine phosphorylation and p125FAK-associated PI 3-K activity. By using recombinant mutated Tat proteins, it was found that deletion of amino acids 73-86 encoded by the second exon of the tat gene resulted in a significant decrease of the ability of Tat to induce p125FAK tyrosine phosphorylation. Paradoxically, mutations in the basic region encoded by the first exon of tat, which is essential for nuclear localization and HIV-1 LTR transactivation, increased the ability of Tat to stimulate p125FAK tyrosine phosphorylation. Moreover, in comparison with cells transfected with a control vector, PC12 cells stably transfected with tat cDNA showed greater amounts of p125FAK protein, an increase in p125FAK tyrosine phosphorylation and higher levels of p125FAK-associated PI 3-K activity. The addition of anti-Tat neutralizing antibody to tat-transfected PC12 cells in culture blocked both the p125FAK tyrosine phosphorylation and its association with PI 3-K but did not affect the total amount of p125FAK.


CONCLUSIONS
HIV-1 Tat protein enhanced both the expression and the functionality of p1 25FAK in PC12 neuronal cells. Whereas the first event required intracellular Tat, the increased p125FAK phosphorylation was strictly dependent upon extracellular Tat.
Department of Morphology and Embriology, University of Ferrara, Italy.
D
D
Milani
M
M
Mazzoni
G
G
Zauli
C
C
Mischiati
D
D
Gibellini
M
M
Giacca
S
S
Capitani
D000818:Animals; D002384:Catalysis; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D005091:Exons; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D015696:Gene Products, tat / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D016716:PC12 Cells; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism*; D054322:tat Gene Products, Human Immunodeficiency Virus
D015815:Cell Adhesion Molecules; D015696:Gene Products, tat; D054322:tat Gene Products, Human Immunodeficiency Virus; D014443:Tyrosine; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; C496052:Ptk2 protein, rat