Characterization of the stress-activated protein kinase, MEKK3

Abstract

The cAMP-dependent protein kinase, PKA, mediates diverse cellular processes including proliferation and differentiation, as well as the phosphorylation of proteins involved in cell death and survival such as BAD and GSK-3beta. In this work, I demonstrate that PKA phosphorylates the stress-activated protein kinase, MEKK3 in vivo and in vitro. When (His)6FLAG·MEKK3 was expressed in Sf9 insect cells and purified with Ni-Sepharose, we identified 14-3-3 protein by liquid chromatography and electrospray tandem mass spectrometry (LC-MS) as co-purifying with recombinant MEKK3. The yeast two hybrid system was also utilized to identify 14-3-3 as interacting proteins with MEKK3 as well as Bcl-xL. The interaction between MEKK3 and Bcl-xL was specific for the caspase-cleaved Bcl-x L which suggests a role for MEKK3 in apoptotic signaling pathways. However, the physiological significance of this interaction is unclear since caspase-cleaved Bcl-xL did not act as a MEKK3 substrate or alter MEKK3 kinase activity. Since 14-3-3 proteins have been reported to interact with proteins through phosphoserine, we sequenced (His)6FLAG·MEKK3 by LC-MS to identify phosphorylated amino acids. Of the tryptic peptides sequenced, two consisted of amino acids 164--174 and 335--349 and serines 166 and 337 were phosphorylated within the respective peptides. Phosphorylation of both serines was localized within two consensus PKA phosphorylation sites, RXX(S/T). PKA activators such as serum and forskolin increased phosphorylation of endogenous MEKK3 at Ser166 as well as the preferential recruitment of phospho-MEKK3 with 14-3-3. These results connect the stress-activated pathways regulated by MEKK3 with pathways regulated by PKA.