SIKE’s Phosphorylation Sites Alter Quaternary Structure

Abstract

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