SIKE’s Phosphorylation Sites Alter Quaternary Structure

• DOI: 10.1096/fasebj.2022.36.s1.r5420
• Publication Date: 2022-05-03T08:34:55Z
• Authors: Daniel Etiel; Alyssa Pham; Sally Ryu; Cade Lyons; Marissa Dzotsi; Diya Patel; Thereasa Nguyen; Ryan S. McCool; Jessica K. Bell
• Keywords: 0301 basic medicine; 03 medical and health sciences

Protein-protein interactions (PPIs) are a crucial part of intracellular communication and function. The "rules" for specificity and/or selection protein-protein interaction not completely understood, but protein modifications, such as phosphorylation, have been implicated in this process. Suppressor IKKepsilon (SIKE) is associated with multiple, distinct proteins including TANK-binding kinase 1 (TBK1), STRIPAK (striatin-interacting phosphatase kinase), cytoskeletal tubulin actinin. Although SIKE's function fully defined these complexes, modification has observed SIKE (phosphorylated at six serine residues: 133, 185, 187, 188, 190, 198) that may direct PPI formation: Co-immunoprecipitation revealed the was enhanced phosphomimetic (S133/185/187/188/190/198E). We hypothesize quaternary state SIKE, regulated by dictates formation. Using mutant, size exclusion chromatography chemical crosslinking studies showed monomeric species, whereas native separated dimer. These support undergoing phosphorylation-induced change state. Prior computational assessment dimer interface stability single substitutions identified S187E, S190E, S198E significantly different from WT on per residue basis. Phosphomimetic mutants sites were created sequence confirmed. In SEC, point mutations shift elution pattern primarily to containing more species. To assess tubulin, constructs labeled using maleimide-thiol conjugation BODIPY TMR. BODIPY-labeled mutations, S6E monitored fluorescence polarization. assays effect phosphorylation partners. Together, work advances our understanding role changes play regulating interactions.