Tumor necrosis factor (TNF) receptor-associated factors, such as TRAF7, are signal transducers for members of the TNF receptor superfamily. Fluorescence microscopy demonstrated TRAF7 localization in a vesicular pattern in the cytosol in the presence or absence of MEKK3. SDS-PAGE analysis suggested that MEKK3 phosphorylates sites in the N-terminal region of TRAF7 and induces ubiquitination. In the presence of E1 (UBE1) and E2 activating and conjugating enzymes, a TRAF7 fragment containing the RING domain was ubiquitinated, indicating that TRAF7 may have E3 ubiquitin ligase activity. Luciferase reporter and RNAi analysis established that coexpression of wildtype TRAF7 and MEKK3 resulted in a synergistic activation of NFKB and AP1.