Tumor necrosis factor α rapidly activates the mitogen-activated protein kinase (MAPK) cascade in a MAPK kinase kinase-dependent, c-Raf-1-independent fashion in mouse macrophages

Tumor necrosis factor α (TNFα) is bound by two cell surface receptors, CD120a (p55) and CD120b (p75), that belong to the TNF/nerve growth factor receptor family and whose signaling is initiated by receptor multimerization in the plane of the plasma membrane. The initial signaling events activated by receptor crosslinking are unknown, although activation of the mitogen- activated protein kinase (MAPK) cascade occurs shortly after ligand binding to CD120a. In this study, we investigated the upstream kinases that mediate the activation of the 42-kDa MAPK p42(mapk/erk2) following crosslinking of CD120a in mouse macrophages. Exposure of mouse macrophages to TNFα stimulated a time-dependent increase in the activity of MAPK/ERK kinase (MEK) that temporally preceded peak activation of p42(mapk/erk2). MEKs, dual- specificity threonine/tyrosine kinases, act as a convergence point for several signaling pathways including Ras/Raf, MEK kinase (MEKK), and Mos. Incubation of macrophages with TNFα was found to transiently stimulate a MEKK that peaked in activity within 30 sec of exposure and progressively declined toward basal levels by 5 min. By contrast, under these conditions, activation of either c-Raf-1 or Raf-B was not detected. These data suggest that the activation of the MAPK cascade in response to TNFα is mediated by the sequential activation of a MEKK and a MEK in a c-Raf-1- and Raf-B- independent fashion.