Academic & Professional Updates
I recently completed the PhD program and work as a postdoctoral research fellow studying protein phosphatases at Vanderbilt University.
My dissertation focused on the serine/threonine protein kinase, MEKK3. Much effort has focused on the identification of MAPK cascades that are activated by the MEKK family of protein kinases. However, direct phosphorylation and regulation of the MEKK proteins has not been extensively studied.
To address this question, we expressed recombinant (His)6FLAG·MEKK3 in Sf9 insect cells and tethered the purified protein to Ni-Sepharose so that we could purify interacting proteins and then identify such proteins by liquid chromatography and mass spectrometry (LC-MS). 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. 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; serines 166 and 337 were phosphorylated within the respective peptides. Phosphorylation of both serines was localized within a consensus AGC kinase phosphorylation site, RXRXX(S/T). Antibodies were developed that specifically recognize the phosphorylated serine residues.
The availability of these antibodies allowed us to demonstrate that various stimuli (tumor necrosis factor, arsenite, forskolin, and serum) promote phosphorylation of Ser166 and Ser337. Interestingly, neither of these phosphorylated amino acids is required for association with 14-3-3 protein or regulation of MEKK3-dependent ERK and JNK activity. Together, these studies suggested that MEKK3 is a convergence point of multiple upstream signaling pathways.