RAF-1 KINASE TARGETS GA-BINDING PROTEIN IN TRANSCRIPTIONAL REGULATIONOF THE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 PROMOTER

The serine/threonine protein kinase Raf-1 is a component of a conserve d intracellular signaling cascade that controls responses to various e xtracellular stimuli. Transcription from several promoters, including the oncogene-responsive element in the polyomavirus enhancer, the c-fo s promoter, as well as other AP-1- and Ets-dependent promoters, can be induced by Raf-1 kinase. Previously, we have shown that activated Raf -1 kinase transactivates the human immunodeficiency virus type 1 (HIV- 1) long terminal repeat and have identified the NF-kappa B binding mot if as a Raf-1-responsive element (RafRE). We now report that Raf-1 kin ase-induced transactivation from the HIV RafRE involves the purine-ric h-repeat-binding protein (GABP), which is composed of two distinct sub units (alpha and beta). GABP alpha is an Ets oncogene-related DNA-bind ing protein, and GABP beta contains four ankyrin-like repeats that hav e been shown to be essential in protein-protein interactions. In elect rophoretic mobility shift assays using nuclear extracts from human Jur kat T cells, a protein-DNA complex which was supershifted with antiser um against GABP alpha and GABP beta was observed. Purified recombinant GABP alpha and beta interact with the HIV RafRE as judged from DNA bi nding assays. Cotransfection experiments with GABP alpha and beta and Raf-1 kinase demonstrate synergistic transactivation of the HIV-1 prom oter. Point mutations in the HIV RafRE abolished the Raf-1 kinase- as well as GABP alpha- and beta-induced transactivation. The observed Raf -1-GABP synergism presumably involves phosphorylation of GABP subunits , as treatment of cells with Raf-1 kinase activators serum and 12-O-te tradecanoylphorbol-13-acetate increases phosphorylation of GABP in viv o. However, GABP is not a target of Raf-1 kinase, instead, it is a sub strate of mitogen-activated protein kinase (MAPK/ERK), since in vitro phosphorylation of GABP alpha and beta was achieved by the reconstitut ed protein kinase cascade but not with purified Raf-1 or MEK. These re sults suggest that Raf-1 kinase-induced activation of the HIV-1 promot er is mediated by the classical cytoplasmic cascade resulting in MAPK/ ERK-mediated phosphorylation of GABP alpha and beta. Because the HIV R afRE corresponds to a region within the promoter which is essential fo r regulation of HIV-1 expression, the data indicate that in addition t o NF-kappa B, GABP transcription factors are important for induced exp ression of HIV.