Human mitogen-activated protein kinase kinase kinase mediates the stress-induced activation of mitogen-activated protein kinase cascades

The mitogen-activated protein kinase (MAPK) cascades represent one of the important signalling mechanisms in response to environmental stimuli. We report the identification of a human MAPK kinase kinase, MAPKKK4, via sequence similarity with other MAPKKKs, When truncated MAPKKK4 (Delta MAPKKK4) was overexpressed in HEK293 cells, it was constitutively active and induced the activation of endogenous p38 alpha, c-Jun N-terminal kinase (JNK)1/2 and extracellular signal-regulated kinase (ERK)2 in vivo. Kinase-inactive Delta MAPKKK4 partly inhibited the activation of p38a, JNK1/2 and ERK2 induced by stress, tumour necrosis factor alpha or epidermal growth factor, suggesting that MAPKKK4 might be physiologically involved in all three MAPK cascades, Co-expressed MAP kinase kinase (MKK)-1, MKK-4, MKK-3 and MKK-6 were activated in vivo by Delta MAPKKK4. All of the above MKKs purified from Escherichia coli were phosphorylated and activated by Delta MAPKKK4 immunoprecipitates in vitro. When expressed by lower plasmid doses, Delta MAPKKK4 preferentially activated MKK-3 and p38 alpha in vivo. Overexpression of Delta MAPKKK4 did not activate the NF-kappa B pathway. Immunoprecipitation of endogenous MAPKKK4 by specific antibodies showed that MAPKKK4 was activated after the treatment of K562 cells with various stress conditions. As a broadly distributed kinase, MAPKKK4 might serve as a stress responder. MAPKKK4 is 91% identical with the recently described murine MEKK-4 beta and might be its human homologue. It is also identical with the recently cloned human MAP three kinase 1 except for the lack of an internal sequence homologous to the murine MEKK-4 alpha isoform. Differences in the reported functional activities of the three kinases are discussed.