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.

GSKIP-Mediated Anchoring Increases Phosphorylation of Tau by PKA but Not by GSK3beta via cAMP/PKA/GSKIP/GSK3/Tau Axis Signaling in Cerebrospinal Fluid and iPS Cells in Alzheimer Disease

“…We recently identified residue L130 of GSKIP as a critical point for binding with GSK3β, and the L130P GSKIP mutant resulted in loss of inhibition of neurite outgrowth in human neuroblastoma SH-SY5Y cells [5]. Further studies have demonstrated that mammalian GSKIP favors dimer instead of monomer because the V41/L45 sites are distal to the L130 residue in the GSKIP monomer formation, thus preventing mutual interactions between PKA RII and the GSK3β binding region, indicating that L130 point mutation is essential for the GSK3β binding function [7,9]. To determine whether GSKIP/GSK-3β/Tau could form an assembly, co-IP assay using GFP–Tau was used to pull down GSK3β, GSKIP, and PKA RII, as shown in Figure 1.…”

“…The latter is known to act synergistically with cAMP/PKA signaling to inhibit GSK3β activity [1,7]. GSKIP that directly interacts with GSK3β and negatively regulates GSK3β signaling is required for negative Wnt signaling regulation via a cytoplasmic destruction complex that targets β-catenin for degradation [5,6,8,9]. GSKIP can compete for GSK3β binding, resulting in control of the β-catenin stabilizing phosphorylation at Ser675 by PKA [9].…”

“…GSKIP that directly interacts with GSK3β and negatively regulates GSK3β signaling is required for negative Wnt signaling regulation via a cytoplasmic destruction complex that targets β-catenin for degradation [5,6,8,9]. GSKIP can compete for GSK3β binding, resulting in control of the β-catenin stabilizing phosphorylation at Ser675 by PKA [9]. Its interaction with GSK3β facilitates control of the phosphorylation at Ser-33/37/Thr-41, which destabilizes β-catenin.…”

“…We have revealed that PKA enzyme activity is required for the formation of a complex comprising PKA/GSKIP/GSK3β that influences the phosphorylation of dynamin related protein 1 (Drp1) at Ser637 and β-catenin at Ser675 [7,9]. Recently, Ser409 phosphorylation of Tau—a known PKA/GSK3β substrate—was revealed to be associated with the termination of neurons in AD [27].…”

Disease-related cellular protein networks differentially affected under different EGFR mutations in lung adenocarcinoma

“…Thus, Rock has been a promising therapeutic target for NSCLC. GSKIP is a glycogen synthase kinase-3 (GSK3) β-interacting protein, which is involved as a negative regulator of GSK3-β in the Wnt signalling pathway 52 . Interestingly, the AURK (A, B and C) network was mostly downregulated in lung adenocarcinoma patients with the Ex19del mutation.…”