MLK3 phosphorylation by ERK1/2 is required for oxidative stress-induced invasion of colorectal cancer cells.

Mixed lineage kinase 3 (MLK3) functions in migration and/or invasion of several human cancers; however, the role of MLK3 in colorectal cancer (CRC) invasion is unknown. MLK3 is a mitogen-activated protein kinase (MAPK) kinase kinase (MAP3K) which activates MAPK pathways through either kinase-dependent or -independent mechanisms. Human colorectal tumors display increased levels of reactive oxygen species (ROS) or oxidative stress. ROS, such as HO, are important for carcinogenesis and activate MAPK signaling pathways. In human colorectal carcinoma (HCT116) cells treated with HO, extracellular signal-regulated kinases 1 and 2 (ERK1/2) were activated and MLK3 exhibited reduced electrophoretic mobility (shift) in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which was eliminated by phosphatase treatment. Pretreatment with the ROS scavenger N-acetyl-L-cysteine, the ERK1/2 inhibitor UO126, or ERK1/2 siRNA knockdown blocked the HO-induced shift of MLK3, while MLK3 inhibition with Cep1347 did not. In co-immunoprecipitation experiments performed on HO-treated HCT116 cells, endogenous MLK3 associated with endogenous ERK1/2 and B-Raf. Active ERK1 phosphorylated kinase dead FLAG-MLK3 in vitro, whereas ERK1 phosphorylation of kinase dead FLAG-MLK3-S705A-S758A was reduced. Both MLK3 siRNA knockdown and FLAG-MLK3-S705A-S758A expression decreased ERK1/2 activation in HO-treated cells. Prolonged HO treatment activated ERK1/2 and promoted invasion of colon cancer cells, which was attenuated by MLK3 siRNA knockdown. Furthermore, S705A-S758A-FLAG-MLK3 demonstrated decreased oxidative-stress induced colon cancer cell invasion, but increased interaction with GST-B-Raf as compared with wild-type-FLAG-MLK3 in HO-treated cells. These results suggest oxidative stress stimulates an ERK1/2-dependent phosphorylation of MLK3 on Ser and Ser, which promotes MLK3-dependent B-Raf and ERK1/2 activation; this positive feedback loop enhances the invasion of colon cancer cells.