Reciprocal Regulation of MAPK on upstream MKK

Abstract

Phosphorylation is a process in which kinase proteins add phosphate groups to proteins. The addition of a phosphate group modifies the activity and function of the proteins. Mitogen-activated protein kinases (MAPKs), including C-Jun N-terminal kinases (JNK), are kinases that play a role in many cellular functions including apoptosis, differentiation, and survival. Mitogen-activated protein kinase kinases (MKKs) are substrates of MAPKs. Our group recently found that downstream MAP kinases can enhance the activation of upstream MKKs. This interesting regulation can be explained by two alternative mechanisms: (i) MAPK directly phosphorylate MKK, and (ii) binding of MAPK enhances the auto-phosphorylation of MKK. To determine the phosphorylation mechanism, a kinase-dead JNK3 mutant in which the lysine is replaced with an Arginine (J3K93R) was isolated and purified. The phosphorylation of MKK7 was then tested separately in the presence of both the wild-type JNK3 and the mutant J3K93R through kinase assays and western blotting techniques. We found that phosphorylation does not transpire in the presence of J3K93R while phosphorylation does occur in the presence of the wild-type JNK3. The phosphorylation pattern strongly suggests that the presence of JNK3 is required for MKK7 phosphorylation instead of auto-phosphorylation. A better understanding of the phosphorylation process will facilitate additional research to more fully characterize the role that the MAPK pathway plays in differentiation, apoptosis, and survival as well as the many other cellular responses that have contributions from MAPKs.