Characterization of the activation and regulation of the serine/threonine kinase, MEKK4

Abstract

MEKK4 is a recently identified serine/threonine kinase. Upon initiation of this study, MEKK4 had been shown to phosphorylate MKK6, an upstream kinase of the stress activated p38 MAP kinase, although no agonist or directly upstream activating kinase had been reported. To identify proteins that interact with MEKK4, a recombinant form of the protein was used as bait to bind interacting proteins from mammalian cell extracts. Using this method the calcium binding protein, annexin II, was identified as an interacting partner with MEKK4. The Far-Western immunoblot was another approach used to examine interacting proteins and revealed that a 120 kiloDalton protein interacts with MEKK4. It was then postulated and subsequently confirmed that this 120 kiloDalton protein was the calcium regulated tyrosine kinase, Pyk2. Studies proceeded to examine the interaction between Pyk2 and MEKK4 and revealed that MEKK4 was an angiotensin II-induced, extracellular calcium-dependent substrate for Pyk2 kinase activity. It was subsequently observed that MEKK4 functioned in the reported pathway of angiotensin II enhancing cyclooxygenase II transcription. Again using recombinant MEKK4 as bait, the MAP kinase, ERK1, was also found to interact with MEKK4. Additional studies demonstrated that ERK1 was not a substrate for MEKK4. Instead, the Pyk2-interacting tyrosine phosphatase, SHP-2, was found to bind to and regulate MEKK4 tyrosine phosphorylation. As studies by other groups had detailed SHP-2 as an important regulator for ERK activation, experiments shifted to examining a role for MEKK4 in scaffolding SHP-2 regulation of ERK activation. Studies confirmed that the SH2 domains of SHP-2 were important in regulating the subset of the total cellular ERK population bound to MEKK4. Collectively these results demonstrate a dual function for MEKK4; as an active kinase that functions upstream of the p38 MAP kinase in the Pyk2 pathway; and as a scaffold for SHP-2 regulation of ERK activity. This study then defines two novel, potentially overlapping, cellular roles for MEKK4 in regulating the cardiovascular response to angiotensin II.