Raf-1 is a serine/threonine protein kinase that plays a central role in signal transduction from the membrane to the nucleus. Activated Raf-1 activates transcription from AP-1, Ets, and NF-~B binding sites. Raf- induced transactivation from the NF-~B binding sites in the human immunodeficiency virus (HIV) long terminal repeat (LTR) appears to result from Raf-activating GABP, an ets family transcription factor that is composed of two subunits (alpha and beta). Overexpression of GABP induced expression from the HIV LTR and activated Raf synergized with GABP for HIV-LTR-driven expression. The GABP alpha mutant carrying a single amino acid substitution in the phosphorylation site and IkappaB alpha appear to reduce synergistic transcriptional activity. In contrast, point mutations in two different GABP beta phosphorylation sites appear to have no effect on synergistic activity. Using a gel shift assay, we have determined that purified GABP binds to the NF-kappaB binding sites in the HIV-LTR in vitro. This binding was specific and required both NF-kappaB binding sites since a single site failed to compete for binding. GABP alpha and GABP beta appear to associate in a heterodimeric complex after Raf activation in vivo as judged by co-immunoprecipitation experiments. To determine if activated Raf phosphorylates GABP directly, we used activated Raf produced by mixed infections in Sf9 cells with baculoviruses expressing Raf-1, v-Ras and Lck. Although this activated Raf efficiently phosphorylated mitogen- activated protein (MAP) kinase kinase initiating a kinase cascade which resulted in Jun phosphorylation by MAP kinase, direct GABP phosphorylation was not observed. Activated MAP kinase, however, did phosphorylate GABP alpha and beta1 subunits in vitro. Moreover, activated Raf-1 appears to induce the phosphorylation of GABP beta1 in vivo.
