ACTIVATION OF SRC FAMILY KINASES BY HEPATITIS-B VIRUS HBX PROTEIN ANDCOUPLED SIGNALING TO RAS

The HBx protein of hepatitis B virus (HBV) is a small transcriptional transactivator that is essential for infection by the mammalian hepadn aviruses and is thought to be a cofactor in HBV-mediated liver cancer. HBx stimulates signal transduction pathways by acting in the cytoplas m, which accounts for many but not all of its transcriptional activiti es. Studies have shown that HBx protein activates Ras and downstream R as signaling pathways including Raf, mitogen-activated protein (MAP) k inase kinase kinase (MEK), and MAP kinases. In this study, we investig ated the mechanism of activation of Ras by HBx because it has been fou nd to be central to the ability of HBx protein to stimulate transcript ion and to release growth arrest in quiescent cells. In contrast to th e transient but strong stimulation of Ras typical of autocrine factors , activation of Ras by HBx protein was found to be constitutive but mo derate. HBx induced the association of Ras upstream activating protein s Shc, Grb2, and Sos and stimulated GTP loading onto Ras, but without directly participating in complex formation. Instead, HBx is shown to stimulate Ras-activating proteins by functioning as an intracellular c ytoplasmic activator of the Src family of tyrosine kinases, which can signal to Ras. HBx protein stimulated c-Src and Fyn kinases for a prol onged time, Activation of Src is shown to be indispensable for a numbe r of HBx activities, including activation of Ras and the Ras-Raf-MAP k inase pathway and stimulation of transcription mediated by transcripti on factor AP-1. Importantly, HBx protein expressed in cultured cells d uring HBV replication is shown to activate the Ras signaling pathway, Mechanisms by which HBx protein might activate Src kinases are discuss ed.