Extracellular HIV-1 Tat protein induces the rapid Ser133 phosphorylation and activation of CREB transcription factor in both Jurkat lymphoblastoid T cells and primary peripheral blood mononuclear cells.
Extracellular HIV-1 Tat protein (0.1-100 ng/ml) induced a rapid (peak at 30 min) increase in the Ser133 phosphorylation levels of the transcription factor CREB in serum-starved Jurkat cells, as revealed by Western blot and indirect immunofluorescence analyses. Nuclear cAMP-responsive element (CRE) binding activity in electrophoretic mobility shift assays was constitutive in unstimulated Jurkat cells, showing only a small increase upon Tat treatment. However, transient transfection experiments performed with various chloramphenicol acetyl-transferase (CAT) constructs showed that Tat produced a fourfold induction of CAT activity only in the presence of a CRE-dependent CAT construct. Moreover, the use of plasmids encoding for GAL4-CREB fusion proteins demonstrated that Tat induction of pG4-CAT reporter gene required the CREB moiety of the GAL4-CREB fusion protein and that Ser133 CREB was essential for Tat activity. Extracellular Tat also stimulated Ser133 CREB phosphorylation in freshly isolated PBMC; this effect was completely blocked by either staurosporin, a broad-spectrum inhibitor of various protein kinases, or PD 98059, a specific inhibitor of mitogen-activated protein kinases (MAPK). Furthermore, extracellular Tat induced a rapid (peak at 5-15 min) stimulation of the MAPK catalytic activity in primary PBMC. Altogether, these findings suggest that HIV-1 Tat protein activates CREB in lymphoid cells through a signal cascade involving the MAPK pathway.
Gibellini
D
D
Institute of Microbiology, University of Bologna, Italy.
Bassini
A
A
Pierpaoli
S
S
Bertolaso
L
L
Milani
D
D
Capitani
S
S
La Placa
M
M
Zauli
G
G
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D017362:Cyclic AMP Response Element-Binding Protein / Q000737:chemistry / Q000378:metabolism*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D015696:Gene Products, tat / Q000494:pharmacology*; D017930:Genes, Reporter; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D019169:Jurkat Cells; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D019311:Staurosporine / Q000494:pharmacology; D014162:Transfection; D054322:tat Gene Products, Human Immunodeficiency Virus
D017362:Cyclic AMP Response Element-Binding Protein; D004791:Enzyme Inhibitors; D005419:Flavonoids; D015696:Gene Products, tat; D054322:tat Gene Products, Human Immunodeficiency Virus; D012694:Serine; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019311:Staurosporine; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one