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 extracellul ar HIV-1 Tat protein in lymphoid CD4 T cells. Methods: CD4 Jurkat T cells were treated with a series of glutathione S-tra nsferase (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); t wo-exon Tat, in which the seven arginine residues have been changed to:alan ine residues (GST-TatArg(mut)), GST-Tat Delta N, which shows a deletion of the N-terminal 21 amino acids. The cells were either treated with soluble C ST-Tat proteins or seeded on plates coated with CST-Tat proteins immobilize d 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(m ut) and GST-Tat Delta N, activated JNK in a dose-dependent manner, induced a rapid phosphorylation of c-Jun on Ser(63) and promoted the de novo synthe sis of c-Jun protein. Moreover, both GST-Tat2E and GST-Tat1E also stimulate d ERK/MAPK. However, the activation of JNK was maximal at concentrations of 100 nM of GST-Tat2E and was blocked by the SG-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 INK requires the second exon of Tat , which is dispensable for the activation of ERK/MAPK. The ability to stimu late INK and ERK/MAPK does not require Tat internalization. (C) 1999 Lippin cott Williams & Wilkins.