Mixed lineage kinase 3 negatively regulates IKK activity and enhances etoposide-induced cell death.

Mixed lineage kinase 3 (MLK3) is a mitogen activated protein kinase kinase kinase (MAP3K) that activates multiple MAPK signaling pathways. Nuclear factor kappa B (NF-kappaB) is a transcription factor that has important functions in inflammation, immunity and cell survival. We found that silencing mlk3 expression with RNA interference (RNAi) in SKOV3 human ovarian cancer epithelial cells and NIH-3T3 murine fibroblasts led to a reduction in the level of the inhibitor of kappa B alpha (IkappaBalpha) protein. In addition, we observed enhanced basal IkappaB kinase (IKK) activity in HEK293 cells transiently transfected with MLK3 siRNA and in NIH3T3 cells stably expressing MLK3 shRNA (shMLK3). Furthermore, the basal level of NF-kappaB-dependent gene transcription was elevated in shMLK3 cells. Silencing mlk3 expression conferred resistance of cells to etoposide-induced apoptotic cell death and overexpression of wild type MLK3 (MLK3-WT) or kinase-dead MLK3 (MLK3-KD) promoted apoptotic cell death and cleavage of poly (ADP-ribose) polymerase (PARP). Overexpression of MLK3-WT or MLK3-KD enhanced etoposide-induced apoptotic cell death and cleavage of PARP. These data suggest that MLK3 functions to limit IKK activity, and depleting MLK3 helps protect cells from etoposide-induced cell death through activation of IKK-dependent signaling.

Cytokine-induced activation of mixed lineage kinase 3 requires TRAF2 and TRAF6.

Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates multiple mitogen-activated protein kinase (MAPK) pathways in response to growth factors, stresses and the pro-inflammatory cytokine, tumor necrosis factor (TNF). MLK3 is required for optimal activation of stress activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) signaling by TNF, however, the mechanism by which MLK3 is recruited and activated by the TNF receptor remains poorly understood. Here we report that both TNF and interleukin-1 beta (IL-1 beta) stimulation rapidly activate MLK3 kinase activity. We observed that TNF stimulates an interaction between MLK3 and TNF receptor associated factor (TRAF) 2 and IL-1 beta stimulates an interaction between MLK3 and TRAF6. RNA interference (RNAi) of traf2 or traf6 dramatically impairs MLK3 activation by TNF indicating that TRAF2 and TRAF6 are critically required for MLK3 activation. We show that TNF also stimulates ubiquitination of MLK3 and MLK3 can be conjugated with lysine 48 (K48)- and lysine 63 (K63)-linked polyubiquitin chains. Our results suggest that K48-linked ubiquitination directs MLK3 for proteosomal degradation while K63-linked ubiquitination is important for MLK3 kinase activity. These results reveal a novel mechanism for MLK3 activation by the pro-inflammatory cytokines TNF and IL-1 beta.