Patients infected with HIV-1 display marked abnormalities in endothelial morphology and function. Derangements in intimal organization, proliferation, apoptosis, and differentiation are seen in a number of AIDS-related pathologic conditions involving aberrant vascular function and angiogenic behavior. The viral accessory protein Tat has been implicated as a potential endothelial protocytokine, given its ability to ligate endothelial integrins and Vascular Endothelial Growth Factor Receptor-2. While the pro-angiogenic effects of HIV-1 Tat have been closely scrutinized, the signal pathways involved are poorly understood. Studies in the previous funding period showed that Tat caused rapid activation of an endothelial cell NADPH oxidase, leading to downstream INK MAPK activation. During characterization of a key activating subunit of the oxidase, p47phox, in endothelial cells, we found constitutive association of this protein with the endothelial cytoskeleton, suggesting physical targeting of the oxidase to oxidant-sensitive signaling complexes such as those containing tyrosine kinases and phosphatases. A search for p47-binding partners yielded the orphan adapter TRAF4, and the association ofp47phox with TRAF4 appeared necessary for activation of INK by Tat. A secondary screen for TRAF4-interacting proteins yielded the Proline-rich Tyrosine Kinase-2 (Pyk2)scaffold Hic-5. A third screen using an open form ofp47phox as bait revealed an additional protein, Endothelial-derived Gene-1 (EG-1), which may be associated with tumor angiogenesis. We hypothesize that Tat initiates signaling through site-directed production of oxidants to Hic-5 and EG-1-related signaling complexes. The broad objectives of this grant are to further define the nature and significance of Tat dependent oxidant signaling through Hic-5 and EG-1 in endothelial cells.