Mixed-lineage kinase 1 (MLK1) is a mitogen-activated protein kinase kinase kinase capable of activating the c-Jun NH(2)-terminal kinase (JNK) pathway. Full-length MLK1 has 1104 amino acids and a domain structure identical to MLK2 and MLK3. Immunoblot and mass spectrometry show that MLK1 is threonine (and possibly serine) phosphorylated in or near the activation loop. A kinase-dead mutant is not, consistent with autophosphorylation. Mutation to alanine of any of the four serine or threonine residues in the activation loop reduces both the activity of the recombinant kinase domain and JNK pathway activation driven by full-length MLK1 expressed in mammalian cells. Furthermore, the gel mobility of the mutant MLK1s is closer to that of the kinase-dead than wild type, consistent with reduced phosphorylation. Thr312 is the key residue: MLK1[T312A] retains only basal activity (about 1-2% of wild type), and its gel mobility is indistinguishable from kinase-dead. Thr312 does not suffice, however; phosphorylation of multiple sites is necessary for full activation of MLK1. An activation mechanism consistent with these data involves phosphorylation of multiple sites in the activation loop, with phosphorylation of Thr312 required for full phosphorylation. This mechanism is broadly similar to that previously reported for MLK3 [Leung, I. W., and Lassam, N. (2001) J. Biol. Chem. 276, 1961-1967], but the key residue differs.

CEP-1347 (KT7515) promotes neuronal survival at dosages that inhibit activation of the c-Jun amino-terminal kinases (JNKs) in primary embryonic cultures and differentiated PC12 cells after trophic withdrawal and in mice treated with 1-methyl-4-phenyl tetrahydropyridine. In an effort to identify molecular target(s) of CEP-1347 in the JNK cascade, JNK1 and known upstream regulators of JNK1 were co-expressed in Cos-7 cells to determine whether CEP-1347 could modulate JNK1 activation. CEP-1347 blocked JNK1 activation induced by members of the mixed lineage kinase (MLK) family (MLK3, MLK2, MLK1, dual leucine zipper kinase, and leucine zipper kinase). The response was selective because CEP-1347 did not inhibit JNK1 activation in cells induced by kinases independent of the MLK cascade. CEP-1347 inhibition of recombinant MLK members in vitro was competitive with ATP, resulting in IC50values ranging from 23 to 51 nm, comparable to inhibitory potencies observed in intact cells. In addition, overexpression of MLK3 led to death in Chinese hamster ovary cells, and CEP-1347 blocked this death at doses comparable to those that inhibited MLK3 kinase activity. These results identify MLKs as targets of CEP-1347 in the JNK signaling cascade and demonstrate that CEP-1347 can block MLK-induced cell death.