The origin of GSKIP, a multifaceted regulatory factor in the mammalian Wnt pathway

Abstract

GSK3β interacting protein (GSKIP) is a naturally occurring negative regulator of GSK3β and retains both the Protein Kinase A Regulatory subunit binding (PKA-RII) domain and GSK3β interacting domain. Of these two domains, we found that PKA-RII is required for forming a working complex comprising PKA/GSKIP/GSK3β/Drp1 to influence phosphorylation of Drp1 Ser637. In this study, bioinformatics and experimental explorations re-analyzing GSKIP's biofunctions suggest that the evolutionarily conserved Domain of Unknown Function (DUF727) is an ancestral prototype of GSKIP in prokaryotes, and acquired the C-terminal GSK3β binding site (tail) in invertebrates except for Saccharomyces spp., after which the N-terminal PKA-RII binding region (head) evolved in vertebrates. These two regions mutually influence each other and modulate GSKIP binding to GSK3β in yeast two-hybrid assays and co-immunoprecipitation. Molecular modeling showed that mammalian GSKIP could form a dimer through the L130 residue (GSK3β binding site) rather than V41/L45 residues. In contrast, V41/L45P mutant facilitated a gain-of-function effect on GSKIP dimerization, further influencing binding behavior to GSK3β compared to GSKIP wild-type (wt). The V41/L45 residues are not only responsible for PKA RII binding that controls GSK3β activity, but also affect dimerization of GSKIP monomer, with net results of gain-of-function in GSKIP-GSK3β interaction. In addition to its reported role in modulating Drp1, Ser637 phosphorylation caused mitochondrial elongation; we postulated that GSKIP might be involved in the Wnt signaling pathway as a scavenger to recruit GSK3β away from the β-catenin destruction complex and as a competitor to compete for GSK3β binding, resulting in accumulation of S675 phosphorylated β-catenin.

Keywords: Bioinformatics; DUF727; GSK3β; GSKIP; PKA; Wnt pathway.