Extracellular HIV-1 Tat protein differentially activates the JNK and ERK/MAPK pathways in CD4 T cells.
OBJECTIVE
To investigate the intracellular signals elicited by extracellular HIV-1 Tat protein in lymphoid CD4 T cells.


METHODS
CD4 Jurkat T cells were treated with a series of glutathione S-transferase (GST)-Tat fusion proteins: full-length two-exon GST-Tat (GST-Tat2E); one-exon Tat, in which the second exon of Tat was deleted (GST-Tat1E); two-exon Tat, in which the seven arginine residues have been changed to alanine residues (GST-TatArg(mut)), GST-TatdeltaN, which shows a deletion of the N-terminal 21 amino acids. The cells were either treated with soluble GST-Tat proteins or seeded on plates coated with GST-Tat proteins immobilized on plastic. At various time points, Jurkat cells were lysed and examined for c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) activity.


RESULTS
Soluble and immobilized GST-Tat2E, but not GST-Tat1E, GST-TatArg(mut) and GST-TatdeltaN, activated JNK in a dose-dependent manner, induced a rapid phosphorylation of c-Jun on Ser63 and promoted the de novo synthesis of c-Jun protein. Moreover, both GST-Tat2E and GST-Tat1E also stimulated ERK/MAPK. However, the activation of JNK was maximal at concentrations of 100 nM of GST-Tat2E and was blocked by the S6-kinase inhibitor rapamycin, whereas the activation of ERK/MAPK was already maximal at 1 nM of GST-Tat2E and was enhanced by rapamycin.


CONCLUSIONS
Tat-mediated activation of JNK requires the second exon of Tat, which is dispensable for the activation of ERK/MAPK. The ability to stimulate JNK and ERK/MAPK does not require Tat internalization.
Mischiati
C
C
Department of Morphology and Embryology, University of Ferrara, Italy.
Pironi
F
F
Milani
D
D
Giacca
M
M
Mirandola
P
P
Capitani
S
S
Zauli
G
G
D015496:CD4-Positive T-Lymphocytes / Q000201:enzymology*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004791:Enzyme Inhibitors; D005091:Exons; D015696:Gene Products, tat / Q000235:genetics / Q000494:pharmacology*; D005982:Glutathione Transferase / Q000235:genetics; D015497:HIV-1; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases; D019169:Jurkat Cells; D048670:MAP Kinase Kinase 4; D020935:MAP Kinase Signaling System; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010766:Phosphorylation; D016755:Proto-Oncogene Proteins c-jun / Q000096:biosynthesis; D020123:Sirolimus / Q000494:pharmacology; D054322:tat Gene Products, Human Immunodeficiency Virus
D004791:Enzyme Inhibitors; D015696:Gene Products, tat; D016755:Proto-Oncogene Proteins c-jun; D054322:tat Gene Products, Human Immunodeficiency Virus; D005982:Glutathione Transferase; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D020929:Mitogen-Activated Protein Kinase Kinases; D020123:Sirolimus