AuthorAdams, Deanna Grace
AdvisorVaillancourt, Richard R.
MetadataShow full item record
PublisherThe University of Arizona.
RightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
AbstractThe 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.
Degree ProgramGraduate College
Pharmacology & Toxicology