Deciphering the evolution of composite-type GSKIP in mitochondria and Wnt signaling pathways.

We previously revealed the origin of mammalian simple-type glycogen synthase kinase interaction protein (GSKIP), which served as a scavenger and a competitor in the Wnt signaling pathway during evolution. In this study, we investigated the conserved and nonconserved regions of the composite-type GSKIP by utilizing bioinformatics tools, site-directed mutagenesis, and yeast two-hybrid methods. The regions were denoted as the pre-GSK3β binding site, which is located at the front of GSK3β-binding sites. Our data demonstrated that clustered mitochondria protein 1 (CLU1), a type of composite-type GSKIP that exists in the mitochondria of all eukaryotic organisms, possesses the protein known as domain of unknown function 727 (DUF727), with a pre-GSK3β-binding site and a mutant GSK3β-binding flanking region. Another type of composite-type GSKIP, armadillo repeat containing 4 (ARMC4), which is known for cilium movement in vertebrates, contains an unintegrated DUF727 flanking region with a pre-GSK3β-binding site (115SPxF118) only. In addition, the sequence of the GSK3β-binding site in CLU1 revealed that Q126L and V130L were not conserved, differing from the ideal GSK3β-binding sequence of simple-type GSKIP. We further illustrated two exceptions, namely 70 kilodalton heat shock proteins (Hsp70/DnaK) and Mitofilin in nematodes, that presented an unexpected ideal GSK3β-binding region with a pre-GSK3β sequence; this composite-type GSKIP could only occur in vertebrate species. Furthermore, we revealed the importance of the pre-GSK3β-binding site (118F or 118Y) and various mutant GSK3β-binding sites of composite-type GSKIP. Collectively, our data suggest that the new composite-type GSKIP starts with a DUF727 domain followed by a pre-GSK3β-binding site, with the subsequent addition of the GSK3β-binding site, which plays vital roles for CLU1, Mitofilin, and ARMC4 in mitochondria and Wnt signaling pathways during evolution.

Introduction

Glycogen synthase kinase interaction protein (GSKIP) was originally cloned and identified as a glycogen synthase kinase-3β (GSK3β) interacting protein through yeast two-hybrid screening using GSK3β [1-4]. Subsequently, GSKIP was also characterized as a small A-kinase anchoring protein (AKAP) [5, 6]. Studies have suggested that GSKIP is a cytosolic scaffolding protein that contains protein kinase A (PKA) RII- binding sites at the N-terminal residue V41/L45 and a GSK3β-binding domain at the C-terminal residue L130 [1–3, 5, 6].

In a previous study, transfection of the neuronal-like SH-SY5Y cell line with wild-type GSKIP inhibited neurite outgrowth, implying the possible role of GSKIP in the neuronal system [7]. GSKIP was demonstrated to modulate dynamin-related protein 1 (Drp1) phosphorylation and to provide neuroprotection against oxidative stress for SH-SY5Y cells [8]. These findings indicate that GSKIP functions as an anchor protein for GSK3β in the cAMP/PKA/Drp1 signaling axis. Moreover, it was reported that GSKIP could interact with PKA and GSK3β, which is required for Wnt signaling regulation, via a cytoplasmic destruction complex with β-catenin, leading to the negative regulation of Wnt signaling [6, 9]. Using knockout animal models, Deák et al. demonstrated that GSKIP regulates GSK3β activity and controls palatal shelf fusion during mice embryonic development [10]. GSKIP is associated with a predisposition to familial myeloid malignancies and poses a challenge for hematologists in germ-line gene duplication studies [11-13]. GSKIP is also involved in the PPARγ-related angiogenic potential of endothelial pulmonary microvascular endothelial cells [14]. A recent study showed that miR-150-5p significantly inhibited Wnt-β-catenin signaling by simultaneously targeting GSKIP and β-catenin in non-small-cell lung cancer (NSCLC) cells [15]. GSKIP was downregulated in disease-related cellular protein networks under different epidermal growth factor receptor (EGFR) mutations in NSCLC [16]. Moreover, long intergenic nonprotein coding RNA 173 (Linc00173) upregulated Etk by functioning as a competitive endogenous RNA “sponging” miRNA-218, leading to GSKIP upregulation and resulting in the translocation of β-catenin in small-cell lung cancer cells [17]. Additionally, miR-181c-5p was reported to mitigate the cancer cell characteristics and invasive properties of cervical squamous cell carcinoma by targeting the GSKIP gene [18]. In a previous study, we demonstrated that this protein PKA/GSKIP/GSK3β axis plays a role in Tau phosphorylation in Alzheimer’s disease [19]. Collectively, these findings indicate that (1) simple-type GSKIP may play a multifaceted role in several diseases in a context-dependent manner, and (2) it is involved in the regulation of mitochondria proteins and the Wnt signaling pathway.

GSKIP has two subtypes, simple and composite, which coexist in all prokaryotic and eukaryotic cells. In our previous study, we revealed the origin of mammalian simple-type GSKIP and its evolution and discovered that it is involved in the Wnt signaling pathway not only as a scavenger but also as a competitor [20]. However, the origin and evolution of composite-type GSKIP remain unknown. In the present study, we generated evidence to demonstrate that along with simple-type GSKIP-linked proteins, four groups of composite-type GSKIP, namely clustered mitochondria protein 1 (CLU1) and Mitofilin in mitochondria, heat shock protein 70 (Hsp70) chaperone, and Armadillo repeat-containing 4 (ARMC4), are involved in Wnt signaling. This can be used to uncover the origin of the GSK3-binding site during the evolution of the protein known as domain of unknown function 727 (DUF727) and four groups of composite-type GSKIP.

Materials and methods

Gene ontology and UniProt analysis

Gene Ontology (http://geneontology.org) and InterPro (http://www.ebi.ac.uk/interpro/) were used for data mining [21-23]. UniProt (https://doi.org/10.1093/nar/gkaa1100) was combined with InterPro [21-25] to compare the results available at the National Center for Biotechnology Information (NCBI) website for domain enrichment by using the E-Value for parameters. Gene-based tests were performed using the following keywords as indicators: GSKIP, DUF727, GSK3β, CLU1, and AMRC4 separately and in combination with GSKIP-related data (S1 Fig). We used FlyBase version FB2014_04 to identify conservative portions in InterPro. Panther data mining is a protein classification system that is applied for analysis based on evolutionary relationships. It is a large biology database of gene/protein families and their functionally related subfamilies. It can be used to classify and identify the function of gene products [26, 27].

Phylogenetic analysis

We used ClustalW [28] for multiple GSKIP sequence alignment for DUF727 (50–100 amino acids), pre-GSK3β (115–118 amino acids), and the GSK3β-binding domain (122–130 amino acids). The maximum likelihood method was used to select CLU1 to generate an unrooted phylogenetic tree of 20 species; a phylogenetic tree of 18 species was also found for one bacterium and ARMC4 orthologs [29-31]. ClustalW was used to ensure the tree structure [28]. The accession numbers of SSF103107, PF05303, IPR007967, and IPR023231 of all species in the UniProt search (S1 Table) and the sequence alignment and phylogenetic analysis of CLU1 and ARMC4 (S1 Fig) were used as parameters. The T-Coffee method was used to align full-length CLU1 and ARMC4 proteins and their fragmentary proteins spanning the SSF103107 unintegrated superfamily [32-34]. The MEGA-X program was used to produce the best-fitting amino acid substitution model [29-31]. We used the maximum likelihood method from MEGA-X to reconstruct phylogenetic trees with the LG substitution model [35, 36]. Boot strapping was used to evaluate the robustness of the phylogenetic trees.

Molecular modeling comparison

The initial three-dimensional (3D) NMR structural model of GSKIP (PDB ID: 1SGO) was obtained as previously described [20]. The NorthEast Structural Genomics consortium was used to obtain the human NMR structure of GSKIP (C14orf129, HSPC210; PDB ID: 1SGO). Next, the 3D structure of the ACT domain, which folds with a ferredoxin-like βαββαβ topology [37], was determined. The structure was minimized for 100,000 conjugate gradient steps and then subjected to 100-s isothermal, constant-volume MD simulation. The final structure was used in domain comparisons (GSKIP, ACT domain, and CLUH-KIAA0664-SSF103107) [1–4, 6, 7, 37].

Consensus sequences and sequence logos: Weblogo 3.6.0

To illustrate the consensus sequence logos of GSKIP, we presented typical simple-type logos and several composite types. The rules were adapted from those used by the sequence comparison website PROSITE (http://prosite.expasy.org/sequence_logo.html) [38], and standard one-letter codes were used for amino acids.

NetWheels: Helical wheel diagram of peptides and net projections maker

The helices found in peptides and proteins are commonly modeled in two dimensions [39]. They can offer a view of the central axis in a protein. Wheel and net projections have been used to represent the two dimensions of 3D helical structures, and they enable the observation of the helical structural properties, especially in terms of residue polarity and intramolecular bonding. We used the helical wheel diagram shown here to determine the distribution of amino acid residues in a helical segment within the sequence of simple-type GSKIP and various composite-type variants to distinguish the differences between the two types.

Cloning, site-directed mutagenesis, and DNA sequencing

The plasmids of pACT2-GSKIP and pAS2-1-GSK3β were constructed for the yeast two-hybrid assay as described previously [40-42]. Briefly, GSK3β was cloned in-frame with the Gal4 DNA-binding domain in the pAS2-1 vector (MATCHMAKER Two-HybridSystem 2, Clontech) to yield the pAS2-1-GSK3β bait plasmid. In addition, DNA fragments encoding GSKIP were amplified through PCR using Taq polymerase (TaKaRa). The PCR fragments were then inserted into the BamHI and XhoI sites of the pACT2 (Clontech) vector to construct the pACT2-GSKIP plasmid. GSKIP Y118P, Y118A, Y118F, F122P, F122A, F122Y, L126P, F126A, L126V, L126Q, L130P, L130A, L130I, and L130V mutants were created through a site-directed mutagenesis technique by using the QuikChange Lightning kit (GE Healthcare, Sunnyvale, CA, USA). Mutated nucleotides were verified using an ABI PRISM 3730 Genetic Analyzer (Perkin-Elmer) for DNA sequencing. All experimental procedures were performed in accordance with the manufacturer’s protocol.

Yeast two-hybrid system

Yeast two-hybrid screening was performed using the MATCHMAKER Two-Hybrid System 2 (Clontech) [40-42]. YRG-2 yeast host cells were purchased from Stratagene. pAS2-1 and pACT2 plasmids were cotransfected and selected on G2 plates deficient in tryptophan and leucine and on G3 plates deficient in histidine. The yeast host cells were MATa ura3–52 his3–200 ade2–101 lys2–801 trp1–901 leu2–3 112 gal4–542 gal80–538 LYS2::UASGAL1-TATA GAL1-HIS3 URA3::UASGAL4 17mers(x3)-TATACYC1-lacZ. A visible blue-color pattern in the colony filter lift assay on the G3 plates represented a positive interaction [41]. YRG-2 yeast cells were cotransfected with pACT2-GSKIP and an empty pAS2-1 vector and spread on G2 and G3 agar plates to determine the growth-inhibiting effect of GSKIP in yeast.

Results

Analysis of UniProt and GSKIP gene ontology: Identification of composite-type GSKIP

UniProt was used in combination with InterPro for domain enrichment analyses. The results were compared with the results in the NCBI database through stringent and extended gene-based tests for ranking the genes (Fig 1A, 1B and 1C). Our survey revealed that several domains were enriched for protein entries. The evolution of simple-type GSKIP and the GSK3β- and PKA RII-binding domains of 52 species has already been clearly demonstrated in previous reports [9, 20]. In this study, we extended the findings concerning four composite-type GSKIPs, namely CLU1 and ARMC4 (through the gene fusion mechanism presented in Fig 1A, 1B and 1C as well as the next subsection below) and another two proteins with sporadic occurrence (Mitofilin and Hsp70/Dnak), in nematodes (see the succeeding section on the hijack mechanism). CLU1 and ARMC4 contain DUF727; however, CLU1 contains other parts of CLU-N, CLU, CLU central, winged helix-like DNA-binding domains, and tetratricopeptide-like helical domains, but ARMC4 only contains 13X Armadillo repeats [43, 44]. DUF refers to a “domain of unknown function” [19–23, 45]. A short domain in the clustered mitochondria protein is involved in its mitochondrial cytoplasmic distribution [46, 47]. Moreover, ARMC4 in vertebrates has been identified as a multiprotein complex responsible for cilia movement; it is necessary for targeting and anchoring outer dynein arms [43, 44]. In the present study, data mining was performed to search the available protein entries, with the results yielding 210 of 936 (22%) with respect to CLU1, 198 of 936 (21%) with respect to CLU1/DUF727, and 210 of 936 (22%) with respect to CLU1/GSKIP. Additionally, the hidden (cryptic) superfamily of DUF727 (SSF103107) was indicated to be an unintegrated family. The hypothetical protein c14orf129 hspc210 superfamily entry could enrich entries for the GSKIP (SSF103107) domain to 1869/1872 (99.8%; Fig 1A, 4 vs. 1). When ARMC4 was retrieved (all entries must be searched to find the hidden code SSF103107), only the hidden (cryptic) superfamily of DUF727 (identified as the unintegrated family SSF103107) with 38/449 entries (8%) was shown to contain a pre-GSK3-binding site (115SPxF118) containing F118 instead of Y118 as compared with the normal GSK3-binding site (more details on the comparisons are provided in subsequent discussion). We occasionally found composite-type GSKIP in two bacterial species, one of which was gram-positive Desulfuribacillus alkaliarsenatis (A0A1E5G502_9BACL). The acetoin-utilization protein AcuB contained the CBS and ACT domains (CBS, 1–138 aa; ACT, 139–212 aa; GSKIP; DUF727, 154–204 aa), and DUF727 was located within the ACT domain as composite GSKIP. The ACT domain was identified in a PSI-BLAST search. Escherichia coli 3PGDH was discovered to be the first protein with an ACT domain that folds into a ferredoxin-like βαββαβ topology [37] (Fig 1D). The ACT domain is found in a variety of contexts and may be a conserved regulatory ligand-binding fold. However, DUF727 has antiparallel ββββ topology (PDB ID: 1SGO, the NorthEast Structural Genomics consortium was used for NMR analysis; Fig 1E), with a similar β-sheet homology (ferredoxin-like βαββαβ topology; Fig 1D right panel compared with Fig 1E) as the human CLUH-KIAA0664-SSF103107 superfamily domain (Fig 1E). We propose that during evolution, GSKIP may have originated from DUF727, as found in bacteria, and may have acquired the pre-GSK3β-binding motif and GSK3β-binding domain to become the ancestor of simple-type GSKIP. Through evolution, simple-type GSKIP later acquired the PKA RII-binding domain as GSKIP/AKAP in vertebrates, whereas simple-type GSKIP has been retained in invertebrates. For composite-type GSKIP, one bacterium was found to contain DUF727 with the ACT domain, whereas in all eukaryotes, DUF727 was incorrectly recognized as GSKIP. Although DUF727 lacks the ideal GSK3β-binding domain, it was still counted among the CLU/TIF31 and CLUH/KIAA0664 proteins. In some vertebrates, the GSKIP domain is also found in an ARMC4-containing protein with a pre-GSK3β-binding site only. Two sporadic Mitofilin and Hsp/DnaK proteins were found in invertebrates containing perfect pre-GSK3β-binding and GSK3β-binding sites (Fig 1F). We summarize four groups of composite-type GSKIP in Fig 1G.

Fig 1

Summary of UniProt and InterPro data mining to reveal two types of GSKIP.

A, Evolved simple-type GSKIP from DUF727 to pre-GSK3β and then evolution to the GSK3β-binding site. The results of UniProtKB data retrieval using keywords are provided. When using GSKIP as a keyword, a total of 2234 protein entries were found, but the records were reduced when other terms such as DUF727, GSK3β, and PKA were added. B, When using CLU1 as a keyword, 936 protein entries were found, and 210 protein entries were convergent with the keywords “DUF727” or “GSKIP.” C, Using ARMC4 as a keyword resulted in 449 protein entries, but only 38 records remained as the SSF103107 hidden code (marked with “*” as a cryptic code; see the text for details) when DUF727 or GSKIP was combined with AMRC4 for data mining. D, E. coli 3PGDH with a pair of ACT domains formed an eight-stranded antiparallel sheet whose 3D structure was determined (left) to fold into a ferredoxin-like βαββαβ topology (red box, right panel). E, NMR analysis of DUF727 (PDB ID: 1SGO) with the central part of antiparallel ββββ topology (red box, right panel) based on the Northeast Structural Genomics consortium (left). F, All queries of previous simple-type GSKIP were summarized using UniProt and Panther data mining (see the text for details). G, Four groups of composite-type GSKIP: AcuB, CLU1, ARMC4, and sporadic (Hsp70/Dnak and Mitofilin).

Two groups of composite-type GSKIP families may have evolved through a gene-fusion mechanism: CLU1 (mitochondria) and ARMC4 (Wnt pathway)

In different species, to determine the conserved sequences of GSK3β-binding regions and DUF727 in CLU1, we first selected one bacterial prototype and 20 GSKIP orthologs and used ClustalW and MEGA-X to construct a phylogenetic tree (Figs 2 and S1A). These 20 species comprised one bacteria [D. alkaliarsenatis (WP_069641749.1)] (prototype), four yeasts [Saccharomyces cerevisiae (NP_013725.1), Schizosaccharomyces pombe (NP_595319.2), Candida albicans (XP_710862.2), and Lodderomyces elongisporus (XP_001526951.1)], one slime mold [Dictyostelium discoideum (XP_629429.1)], one flatworm [Schistosoma Haematobium (XP_012796625.1)], one Nematoda [Caenorhabditis elegans (NP_499097.1)], four insects [Drosophila melanogaster (NP_646986) and Culex quinquefasciatus (XP_001843004.1)], including two species of mosquitoes [Aedes aegypti (XP_001651007.1) and Aedes albopictus (A0A182G693)], one brachiopod [Lingula anatina (XP_013383658.1)], and seven Chordata [Branchiostoma lanceolatum (XP_002595966.1), Myotis brandtii (Brandt’s bat, S7P578), Danio rerio (E7FE02), Gallus gallus (XP_015151355.1), Mus musculus (NP_001074627.1), Rattus norvegicus (NP_001292142.1), and Homo sapiens (NP_056044.3)]. Additionally, ClustalW was combined with the T-Coffee web-based program and MEGA-X to ensure a tree structure (Fig 3). Both a GSK3β-binding motif (122Fxxx126LxxR/K/QL130) and a pre-GSK3-binding motif (115SPxF118 rather than 115SPxY118 in simple-type GSKIP) were found in most of the GSKIP orthologs (Fig 3).

Fig 2

Phylogenetic analysis of four composite-type GSKIP proteins, namely ARMC4, CLU1, HSP70/Dnak and Mitofilin, among various species.

The evolutionary history of GSKIP was inferred using the maximum likelihood method. Initial trees for the heuristic search were obtained automatically by applying the neighbor-joining and BioNJ algorithms to a matrix of pairwise distances estimated using the JTT model and by then selecting the topology with the superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 43 amino acid sequences. The final dataset included a total of 161 positions. Phylogenetic tree generated using MEGA-X [31]. The bootstrap values represented as the likelihood function for each species as indicated.

Fig 3

Sequence alignment of 20 composite-type GSKIP/CLU1 proteins among various species.

Multiple sequence alignment with respect to the DUF727 region of GSKIP orthologs using T-Coffee. The conserved residues are indicated with asterisks, and residues with high similarity among the orthologs are marked with dots at the bottom. The 115SPxF118xxx122FxxxQxxRV130 motif in the pre-GSK3β- and GSK3β-binding regions is also indicated by blue and green boxes, respectively. T-Coffee also estimates alignment accuracy and improves phylogenetic tree reconstruction.

We found that two distinct bacteria contained composite-type GSKIP based on the observation of the aligned sequences without the GSK3β-binding site, which revealed that they are primitive [5, 6, 20]. Why and how composite-type GSKIP evolved a pre-GSK3β-binding site prior to the GSK3β functional domain should be investigated, and such an investigation could provide some indication of the role of the GSK3β-binding site in composite-type GSKIP evolution.

When DUF727 is regarded as an initial domain, the CLU1 mitochondria family [48, 49] and the ARMC4 family of proteins that emerge [43, 44] are composite-type GSKIPs. By contrast, in simple-type GSKIP, the GSK3β-binding region is conserved with some residues, indicating that the 122Fxxx126L/QxxR/KV/G/A130 region is essential. The Leu130 residue has been characterized as being essential for GSK3β binding in humans [1, 6, 20], indicating its inactivation when this site is modified with GSK3β. In the present study, this always occurred in CLU1 (Table 1), indicating that CLU1 is still in the process of evolution. Of note, the modification of the ARMC4-pre-GSK3 site 118F to 118Y abolished its interaction with GSK3 (Table 1), indicating the same evolutionary process of the ARMC4-pre-GSK3β site as that of the CLU1 family. When using the helical wheel diagram, the comparisons revealed various V126L residues, but no 130L residue, in CLU1 (Fig 4, GSKIP: 122–139 aa with its various mutants) and Logo 3.6.0 (Fig 5A–5D). These data enabled the evaluation of whether the consensus sequence is conserved in pre-GSK3- (115SPxF/Y118; Fig 5B and 5C compared with Fig 5A) and GSK3β-binding sites (122Lxxx126LxxK/RL130, Fig 5D compared with Fig 5A). Crucially, three composite GSKIP variants (126Q, 126V, and 130V) from different species were found to bind GSK3 by Y2H (Table 1). In addition, the DUF727 domain seemed to gradually become a pre-GSK3β-binding site (115SPxF118) as a flanking region with the consensus sequence site 115SPxF118xxx122Fxxx126LxxR/KL130, whereas in vertebrates, ARMC4 contained a hidden code SSF103107 (unintegrated DUF727 superfamily, Fig 5C) flanking region with pre-GSK3β-binding sites at 115SPxF118 only (see the next subsection below).

Table 1

Mutagenesis assay of the pre-GSK3β-binding site (118F or 118Y) and various mutant GSK3β-binding sites of full-length GSKIP and GSKIPtide (115–139).

GSKIP			GSKIPtide
	G2	G3		G2	G3
WT	+	+	WT	+++	+++
Y118P	+++	-	Y118P	+++	-
Y118A	+++	+++	Y118A	+++	-
Y118F	+++	+++	Y118F	+++	-*
F122P	+++	-	F122P	+++	-
F122A	+++	+++	F122A	+++	-
F122Y	+++	+++	F122Y	+++	-
L126P	+++	-	L126P	+++	-
L126A	+++	+++	L126A	+++	-
L126V	+++	+++	L126V	+++	-
L126Q	+++	+++	L126Q	+++	-
L130P	+++	-	L130P	+++	-
L130A	+++	+++	L130A	+++	-
L130I	+++	+++	L130I	+++	-
L130V	+++	+++	L130V	+++	-

The β-galactosidase filter assay was also used for semiquantitative analysis. (+) indicates positive interaction, (–) indicates the interaction is abolished, and (+++) indicates a strong interaction.

The red lines indicate the pre-GSK3β-binding site and various GSK3β-binding sites.

* Data are comparable to those of Hundsrucker et al., 2010 [6].

Fig 4

Helical wheel analysis of GSKIP wt and various GSKIP mutants at amino acid sequence 122–139, which harbors the GSK3β-binding site.

A, GSKIP wt. B, GSKIP L126Q mutant. C, GSKIP F122Y mutant. D, GSKIP Y118F mutant.

Fig 5

Differences in pre-GSK-binding site sequences.

Four consensus GSKIP sequences were represented using Weblogo 3.6.0. A, GSKIP (simple-type). B, CLU1 (CLUH). C, ARMC4. D, Sporadic (Mitofilin and Hsp70/Dnak).

A hidden code SSF103107 domain as a metaphor of DUF727 in the ARMC4 family

In our previous study, we used the keywords DUF727 (PF05303) and GSKIP (IPR007967) to search for ARMC4. Subsequently, DUF727 and GSKIP were eliminated as keywords from the website. Only the hidden code SSF103107 was used as a metaphor for DUF727 and GSKIP for searching for ARMC4. Although the hidden code has been classified into the DUF727 superfamily, the hidden code SSF103107 domain shares homology with DUF727 (PF05303) and GSKIP (IPR007967). Therefore, we checked all ARMC4 family queries, and 38 of 449 species (8%) were found to contain the hidden code SSF103107, with protein evolution through gene (domain) fusion. Because the pre-GSK3β-binding site (115SPxF118) was retained only in the ARMC4 family in vertebrates, the GSK3β-binding site is not yet present at this stage. The pre-GSK3β-binding site (115SPxF118) may exist as a primitive type in vertebrates. We selected 18 SSF103107 orthologs from vertebrates to determine their alignment and performed T-Coffee phylogenetic tree construction of ARMC4 proteins evolved through gene fusion in 8% of vertebrate species (S1B Fig). We selected SSF103107 orthologs, and ClustalW and MEGA-X were used for phylogenetic tree construction (S1C Fig). Proteins encoded by ARMC4, 10 armadillo repeat motifs (ARMs), and one HEAT repeat were found in ARMC4, which is thought to be involved in ciliary and flagellar movement [43, 44]. The term armadillo is derived from the historical name of the β-catenin gene in the fruit fly Drosophila, where the armadillo repeat was first discovered. Although β-catenin was previously thought to be a protein involved in linking cadherin cell adhesion proteins to the cytoskeleton, a recent study indicated that β-catenin regulates the homodimerization of alpha-catenin, which in turn controls act branching and bundling [50]. However, the armadillo repeat has been found in a wide range of proteins with other functions. This protein domain plays a vital role in transducing Wnt signals during embryonic development [51].

As described in the earlier text, we found that a substantial number of DUF727 domain insertions into CLU1 (in mitochondria) together with ARMC4 (in the Wnt pathway) may constitute the gene fusion recombination mechanism. We also detected ARMC4 in the pre-GSK3β-binding site (115SPxF118, Fig 5C), whereas the CLU1 family gradually formed from the pre-GSK3β-binding site to extend GSK3β-binding sites (115SPxF118xxxF122xxxQ/V126xxRV130, Fig 5B). However, no PKA-binding sites were found in composite-type GSKIP compared with simple-type GSKIP. We discovered evidence that the contribution of domain fusion to the evolution of multidomain proteins is bounded by the lower boundary of 63% in invertebrates and the upper boundary of 94% in vertebrates in the CLU1 family (Fig 3).

By contrast, in the vertebrate ARMC4 family, a cryptic (unintegrated) superfamily DUF727 (hidden code SSF103107) was also found to bind to the DUF727 domain in 8% of species with a pre-GSK3β-binding site (115SPxF118) (20 species compared with one bacterium). The association of DUF727 with the pre-GSK3β-binding site in vertebrate ARMC4 suggests that DUF727 (SSF103107) originates in a primitive stage of evolution. We suggest that the gene fusion mechanism is a major contributor to the evolution of the CLU1 (in mitochondria) and ARMC4 (in the Wnt pathway) families in composite-type GSKIP.

Hijack (recombination) mechanism: A noncanonical order (strange order) of Hsp70/DnaK and Mitofilin in evolution

Members of the Hsp70 family are ubiquitously expressed and highly conserved; for example, the major form of Hsp70 from E. coli, termed DnaK, is approximately 50% identical to human Hsp70s. Hsp70 chaperone-assisted folding involves repeated cycles of substrate binding and release. Hsp70 activity is ATP-dependent. Hsp70 proteins comprise two regions: the amino terminus, which is the ATPase domain, and the carboxyl terminus, which is the substrate-binding region [52, 53].

Unexpectedly, in this study, we demonstrated the noncanonical order of the sporadic Hsp70/DnaK (A0A261CFR1_9PELO, C. latens; A0A1I7XTM9_HETBA, Heterorhabditis bacteriophora; A0A368GFB0_ANCCA, Ancylostoma caninum; and A0A0D6M4D9_9BILA, A. ceylanicum) and Mitofilin (H3EKR7_PRIPA, Pristionchus pacificus) proteins found in the composite-type GSKIP of invertebrate nematodes; they exist in the mature forms 115SPxY118xxxFxxxLxxRL130 and 115SPxY118xxxFxxxV116xxKL130, respectively (Fig 5D). The pre-GSK3-binding site prefers 118Y instead of 118F in invertebrate nematodes compared with the CLU1 (all eukaryotes) and ARMC4 (vertebrate) families. These pre-GSK3β-binding sites utilizing 118Y instead of 118F to exhibit their GSK3β-binding activity were completely conserved in Mitofilin and Hsp70/DnaK in invertebrate nematodes but not vertebrates. The noncanonical order of these two composite-type GSKIPs in the evolutionary tree could be explained by a hijack (recombination) mechanism, as further evidenced by omega and T-Coffee comparisons. Apparently, the DUF727 domain gradually evolved as a pre-GSK3β-binding site (115SPxF118) flanking region with the consensus sequence site 115SPxF118xxx122FxxxLxxR/KL130 (Fig 5A), whereas in vertebrates, ARMC4 only contained the cryptic SSF103107 (unintegrated DUF727 superfamily, Fig 5C) flanking region with the pre-GSK3β-binding sites 115SPxF118 lacking the GSK3β-binding region (122FxxxLxxR/KL130). Altogether, these findings suggest that (1) at the pre-GSK3β-binding site, 118F is present prior to 118Y during evolution; (2) ARMC4 in higher organisms evolved through the GSK3-binding site of Hsp70/DnaK and Mitofilin; and (3) composite-type GSKIP evolved slower than simple-type GSKIP. We further performed site-directed mutagenesis and yeast two-hybrid assays to compare and reveal the importance of this pre-GSK3β-binding site with its flanking GSK3β-binding conserved sites in invertebrates and vertebrates (Table 1).

Human pre-GSK3β- and GSK3β-binding sites of the GSKIP domain in a budding yeast (S. cerevisiae) model

We used budding yeast (S. cerevisiae) as an ideal model organism for studying domains with pre-GSK3β- and GSK3β-binding regions to determine how a new composite-type GSKIP was conserved in multicellular organisms through natural selection and it did not interfere with endogenous simple-type GSKIPs. As described in the earlier text, the pre-GSK3β-binding site (118F or 118Y) and various mutant GSK3β-binding sites (from CLU1) were compared through site-directed mutagenesis, as in a previous study, using yeast two-hybrid methods. Our data revealed 118Y, 122F, 126F, and 130L to be essential for binding during evolution, which is consistent with our previous study results [20]. Taken together, these data imply that the pre-GSK3β-binding site with 118Y plays a crucial role for GSK3β-binding sites as well as 122F, 126L, and 130L (Table 1) [1, 3, 20].

Physiological implications and importance of GSKIP in Wnt signaling

Our study revealed that the bat CLUH protein of composite-type GSKIP possesses a dominant consensus sequence (115SPxF118xxxF122xxxQ126xxRV130) quite similar to all the CLUH species described in the earlier text (S2 Table).

Overexpression of ATG2B and GSKIP increased progenitor sensitivity to thrombopoietin, enhancing megakaryocyte progenitor differentiation [11, 13]. The presence of the AK7 gene results in a predisposition to ciliary dyskinesia [43, 44, 49]. The protein encoded by ARMC4 containing 10 ARMs has also been reported to be involved in ciliary movement [43, 44].

The molecular mechanism for the coevolution of these neighboring genes with both simple-type (GSKIP-AK7) and composite-type GSKIP (ARMC4) in vertebrates remains unclear. A study suggested that pangolins are natural hosts of beta-coronaviruses, and comprehensive surveillance of coronaviruses in pangolins could improve our understanding of the spectrum of COVID-19 (the pandemic arising from a type of coronavirus called Sars-CoV-2; see [54] and S2 Table). Urgent investigation of the bat genome is required and may reveal the orchestration of specific elements in the COVID-19 crisis. ARMC4 may function as a competitor to APC and β-catenin. In addition, the protein TCF4 in pangolins, as an intermediate host species of severe acute respiratory syndrome, indicates the involvement of the Wnt signaling pathway. This may be a coincidence but also raises the interesting question of what is the root cause of COVID-19 infection. Previously, we proposed that the ARMC4 family (as a composite-type GSKIP with a physiological role) together with simple-type GSKIP could function as competitors and is involved in the Wnt signaling pathway [20]. Both simple-type GSKIP and composite-type GSKIP (ARMC4) have been implicated in the Wnt signaling pathway (Fig 6) [50, 51, 55, 56].

Fig 6

Physiological implications and importance of both simple-type and composite-type GSKIP in Wnt signaling.

GSKIP may play different roles in the Wnt pathway in different species. For C. elegans and Drosophila, due to a lack of the Axin GSK3β-binding motif on either the APR1-PRY1 or APC-Axin complex, GSKIP orthologs (GSKIP-SGG and GSKIP-ZW3) may serve only as scavengers that prevent complex formation. For vertebrates such as Xenopus, D. rerio, and mammals, GSK3β is one of the proteins involved in the formation of the destruction complex with β-catenin. Thus, GSKIP as a negative regulator may play a dual role as a scavenger to prevent GSK3β binding to the destruction complex or as a competitor for the Axin GSK3β-binding site with GBP or FRAT involved in the canonical Wnt pathway. ARMC4 may function as a competitor to APC and β-catenin. In addition, the pangolin protein (TCF/LEF) is involved in the Wnt signaling pathway in pangolins, an intermediate host species for SARS (modified from [19, 51]).

Discussion

According to UniProt data, we found the domain/fusion mechanism of the CLU and ARMC4 families and the hijack (recombination) mechanism for two sporadic genes of Hsp70/DnaK [52, 53] and Mitofilin [46, 47] that were hijacked by the DUF727 domain. The evolution of these composite-type GSKIPs was also revealed by the gene location in the corresponding organelles.

In this study, we detected gene fusion events by using UniProt, T-Coffee, and ClustalW. A total of 1748 simple-type GSKIP proteins as components of gene fusion in composite-type GSKIP were also detected, many of which were from the CLUH and ARMC4 families. The predicted functional associations were with mitochondria and the Wnt signaling pathway. We demonstrated for the first time that gene fusion is a complex evolutionary process for CLUH and ARMC4, including phylogenetic distance. Gene fusion plays a key role in the evolution of gene architecture. We can observe its effect if gene fusion occurs in coding sequences [57]. When gene fusion occurs in the assembly of a new gene, new functions emerge through the addition of peptide modules to the multidomain protein.

The detection methods for gene fusion events on a large biological scale can provide insights into the multimodular architecture of proteins [26, 58, 59].

In bacteria, a novel ligand-binding domain (ATP-binding motif), named the ACT domain, was identified through a PSI-BLAST search. E. coli 3PGDH was the first protein with an ACT domain that was found to fold into a ferredoxin-like (potential ATP in photosynthetic bacteria) βαββαβ topology. The ACT domain is found in a variety of contexts and is proposed to be a conserved regulatory ligand-binding fold. However, DUF727 has an antiparallel ββββ topology (NMR PDF file), sharing a similar sheet homology (Fig 1D and [2]) as the human CLUH-KIAA0664-SSF103107 superfamily domain (Fig 1E). Thus, Hsp70/Dnak, which requires ATP, chaperones, mitochondria, and Mitofilin, requires an ATP source for binding to CLU1. In particular, this standalone ACT domain protein might form complexes upon binding to other proteins, such as kinases, which interact with and regulate ARMC4, β-catenin, and APC in the Wnt pathway. We also observed that DUF727 fused to CLU1 and ARMC4. Moreover, domain accretion through a gene fusion mechanism may be a major contributor to gene evolution [60].

Three GSKIP-containing isoforms in humans are located in different chromosomes: GSKIP in chromosome 14, CLUH/KIAA0664 in chromosome 17, and ARMC4 in chromosome 10, and GSKIPs, CLUH, and ARMC4 still retain a DUF727. This may elucidate certain events in the evolutionary process. In particular, the unique order of Hsp70/Dnak and Mitofilin evolution may be evidence of another mechanism (hijacking). Our queries resulted in several new findings. First, we found a highly primitive form of GSKIP in lower organisms, such as gram-positive bacteria, and neither a PKA-binding domain nor a GSK3β-binding site was found when tracing GSKIP homologs in vertebrates and invertebrates [27, 37]. Second, the DUF727 domain in CLU1 and ARMC4, regarded as the central fragment of GSKIP, can exist alone or with other domains as part of multidomain structures. Third, DUF727 first extended to a pre-GSK3β site at 115SPXF118 in vertebrate ARMC4 during evolution. Fourth, two sporadic composite-type GSKIPs (Mitofilin and Hsp70/Dnak) resulting from a hijacking mechanism were found in invertebrate nematodes. As shown in Fig 1F, at an evolutionarily later time, simple-type GSKIP acquired the PKA RII-binding domain as GSKIP/AKAP in vertebrates. Simple-type GSKIP is still retained in invertebrates. For composite-type GSKIP, one bacterium containing DUF727 with the ACT domain was found, whereas in all eukaryotes, DUF727 was improperly recognized as GSKIP. Although DUF727 lacks the ideal GSK3β-binding domain, it is still counted in CLU/TIF31 and CLUH/KIAA0664 sequences. In some vertebrates, the GSKIP domain was found in an ARMC4-containing protein with a pre-GSK3β-binding site only. The two sporadic proteins of Mitofilin and Hsp/DnaK were found in invertebrates containing perfect pre-GSK3β-binding and GSK3β-binding sites.

In sum, this study generated evidence demonstrating that the composite-type GSKIP of CLU1 and Mitofilin in mitochondria and the Hsp70 chaperone and ARMC4 have functions in Wnt signaling and simple-type GSKIP-linked proteins have roles in the origin of GSK3β-binding sites during DUF727/GSKIP evolution. Both simple-type and composite-type GSKIP (ARMC4) attract the most attention due to their degree of involvement in the Wnt signaling pathway (Fig 1F and Fig 6). Moreover, the entire genome of Rhinolophus ferrumequinum (the greater horseshoe bat) should be sequenced because doing so can provide insights into the importance of the bat genome. The likely insights into the bat genome might contribute to the efforts to end the current COVID-19 outbreak in so far as the insights may elucidate how GSKIPs function in bat mitochondria and Wnt signaling pathways; particularly in immunology studies may also help to understand how mother nature of bat genome harboring coronavirus could be used to combat emerging variants of this pandemic virus.

Two questions remain unanswered: first, under what conditions did this recombination occur to form composite-type GSKIP (to mitochondria, chaperone proteins, or armadillo repeats)? Second, in which organism did this recombination occur? For evolutionary biologists, these composite-GSKIP proteins can reveal the key steps in the evolution of GSKIP, particularly for composite-type GSKIP.

Conclusions

Composite-type GSKIPs demonstrated the coevolution of pre-GSK3β- and GSK3β-binding sites that extended to DUF727 in the CLU1 and ARMC4 families, and the study findings may provide insights into the importance of both simple-type and composite-type GSKIP for the mitochondrial and Wnt signaling pathways.

Quote terms: SSF103107 (T3), PF05303 (T1), IPR007967 (G1), and IPR023231 (G3) of all species in the UniProt search.

T: type, G: group.

(DOCX)

Click here for additional data file.

Quote terms: “Clustered mitochondria protein” (CLUH) of Bat in UniProt search.

(DOCX)

Click here for additional data file.

Phylogenetic analysis of composite-type GSKIP, CLU1, and ARMC4.

Sequence alignment and phylogenetic analysis of ARMC4 from 18 vertebrate species along with a bacterium are shown. A and B, Phylogenetic tree of CLU1 and ARMC4 generated using MEGA-X, respectively. Boot strapping values represented the likelihood function for each other species as indicated. The boot strapping test measures the internal consistency of data produced above than 0.5 (50%) of the bootstrap replicates are consistent. C, Multiple sequence alignment with respect to the DUF727 region of GSKIP orthologs was conducted using ClustalW. D, T-Coffee estimates of alignment accuracy improved phylogenetic tree reconstruction. The conserved residues are indicated with asterisks, and residues with high similarity among the orthologs are marked with dots at the bottom. The label * indicates the possible region of DUF727 in ARMC4.

(TIF)

Click here for additional data file.

22 Jul 2021

PONE-D-21-20264

Deciphering the evolution of composite-type GSKIP in mitochondria and Wnt signaling pathways

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Reviewer #1: GSKIP paper

The authors provide information about the evolution of GSKIP. The bioinformatics used is probably suitable for the analysis of evolutionary events. However, to this reviewer who is not a specialist in evolution and analysis of evolution, it is almost impossible to follow the arguments throughout the manuscript.

The authors need to define certain technical terms, most important what they mean by simple and composite types. What is meant by pre-GSK3beta?

The introduction nicely introduces GSKIP. However, then in lines 97-99 the link to context-dependency and Wnt signalling is completely obscure. Please explain better.

Fig. 1 provides structural information. It would be most valuable to overlay the different structure to visualize overlap/differences.

The authors suggest throughtout the manuscript that GSKIP gained the AKAP function in vertebrates. However, Hundsrucker et al, JBC 2010, had shown that GSKIP of C. elegans already binds PKA R subunits. Please address the discrepancy.

Line 405 sentence starting “In addition, Mitofilin…” is unconnected to the preceeding text. Please embed better.

Please do not term mutant versions of any binding domains or proteins “flawed”, please use mutant.

Starting with line 437, Hundsrucker had already addressed the question of whether lower GSKIP of lower organisms binds GSK3beta (Hundsrucker JBC 2010). Please consider in your analysis.

Line 462, what is meant by dominant consensus sequence?

Line 472, the link to Covid is unclear, please clarify and explain better.

Fig. 6, the title is misleading since – to the understanding of this reviewer- Dema et al., JBC 2016, had clearly shown a role of GSKIP in Wnt signalling.

Line 527, discussion, the paragraph is unconnected to the previous discussion. Please provide clear links.

Similary, line 562, the paragraph about Covid. What is the reason for speculating on Covid and GSKIP. Please substantiate.

In the conclusion, line 577, the thoughts about target-based drug discovery are not at all embedded into the discussion.

Reviewer #2: Tsai et al. investigated the role of Glycogen synthase kinase interaction protein (GSKIP) in mitochondria and the Wnt signaling pathways.

The manuscript needs to be re-written. Both, English must be improved including the structure of most sentences and the thread, which guides the reader through the information given by the authors.

The introduction is a numeration of findings. It is not clear, how the findings from the introduction should guide to the research question answered by the authors.

Methods: The authors mention a few times, that information were given previously, without giving a reference. Information about the used material is incomplete (e.g. line 173; 174; 179; …).

Into the discussion, there is redundancy of the results, e.g. from page 33.

It is not clear, why the authors stat that the yawn to sequence the whole genome of Rhinolophus ferrumequinum. Please, explain in more detail. However, such ideas are not sufficient to be present in a discussion. In addition, the link from the finding presented here, to Sars-CoV-2 (The virus is called Sars-CoV-2 and the disease arising from an infection is called COVID19) only can be assumed, but it is not clearly stated by the authors.

The conclusion is not clear. Please, only conclude from your results and do not speculate bats and viruses. The results do not support this.

Reviewer #3: The authors set the foundations of some interesting thoughts and ideas in their manuscript, but those ideas are clouded by poor judgment of their results and a lack of clarity in the writing that weighs the manuscript down. For example, the authors wrote (line 272) “to determine the conserved sequence of GSK3β-binding regions and DUF727 in CLU1, we selected one bacterial prototype and 20 GSKIP orthologs and used ClustalW and MEGA-X to construct a phylogenetic tree”. However, a phylogenetic tree is not the best option to determine conservation between sequences, a sequence alignment would be a more adequate choice. Moreover, the values represented in the phylogenetic tree (Fig. 2), if they mean bootstrap, are too low to be considered significant (e.g., 0.37; 0.25). The authors should include more information that clarifies the meaning of those values and reconsider if the phylogenetic tree is relevant. Secondly, the authors used the yeast two-hybrid system to investigate different GSK3β-binding sites and claim several positions as crucial for the binding when they use a fragment of the protein, however, the authors overlook the results when they use the entire protein. Thirdly, the manuscript suggests a certain anthropocentric tone with concepts like lower and higher animals. I recommend the authors to read the following short paper (https://doi.org/10.1002/bies.201370093) and I encourage them to reconsider the use of lower and higher animals.

Regarding the lack of clarity of the manuscript, I would like to point out that it is easy to find sentences out of context (e.g. line 139: “It can occur as a result of translocation, interstitial deletion, or chromosomal inversion.”; line 304; “Its inactivation with GSK3β was found when this site was modified.”), without enough clarity (e.g. line 132: “Accession number (S1 Table) and amino acid sequence (S1 Fig) were used as parameters.”; line:359: “The protein encoded by ARMC4, 10 armadillo repeat motifs (ARMs), and one HEAT repeat were contained in ARMC4, which is thought to be involved in ciliary and flagellar movement”), or hardly readable (e.g. line 542: “Second, in CLU1 and ARMC4, the DUF 727 domain, only as central fragment of GSKIP, can exist alone or with other domains as part of multi-domain structures.”) among others. Moreover, several statements lack a reference (e.g., line 125 after “gene products”; line 143 after “previously described”; line 169 “previously described”; line 207 after “Armadillo repeats”; line 304 after “humans”) or some references are not the correct ones (e.g., line 209; line 211; line 295; line 367; line 369). Authors should check that no error has occurred during the reference formatting process. Considering previous publications, from the same authors, this manuscript is not as clear as the others. The authors should give more context in the abstract and introduction to avoid confusion. Besides, a scheme of the workflow that they employed in their domain survey will help the reader follow the process. In relation to that, the authors need to make the results section “Analysis of UniProt and GSKIP gene ontology: Identification of composite-type GSKIP” more straightforward. The authors could, for example, refer more to their own figures (i.e., adding Fig. 1G in line 207 after “Armadillo repeats”; adding Fig. 1G in line 229 after GSKIP; adding Fig. 1F in line 236 after “invertebrates”) and maybe represent figures 1A, B and C as Venn diagrams. In line 253, the authors say “Using GSKIP as a keyword, a total of 1849 protein entries were shown” and in lines 255 authors say “Using CLU1 as a keyword, 879 protein entries were shown”, but the figure shows 2234 entries for GSKIP and 936 for CLU1. The authors need to check why those are different. Furthermore, in the results section “Two groups of composite-type GSKIP families may have evolved through a gene-fusion mechanism: CLU1 mitochondria) and ARM4 (Wnt pathway)” the list of accession numbers (lines 276 to 288) does not help the readability of the manuscript. Authors could add those numbers in figure 2 or in the supplemental material. Also, the authors should be more precise when they name the groups for the different species (i.e., line 280 “worm” is very unspecific, authors should consider “round-worm” or "nematode"; in lines 284 and 285, the authors wrote that the phylogenetic tree have one chordate and six vertebrates, however, vertebrates are chordates too, so the phylogenetic tree has seven chordates among them one cephalochordate [Branchiostoma lanceolatum] and six vertebrates.

Finally, some typographical errors that should be corrected:

Line 100: add “that” before co-exist

Line 116 and line 117: redundant reference, also this is not the format to cite Uniprot. The correct cite is https://doi.org/10.1093/nar/gkaa1100

Figure 1E: In the secondary structure drawing the letters are cut

Line 277: “Schizosac charomyces” should be one word

Line 289: “S3 Fig” should be “Fig 3”

Line 339: “light blue” should be “green”

Line 371 and line 373: “MARC4” should be “ARMC4”

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Reviewer #2: No

Reviewer #3: Yes: Josep Marti-Solans

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5 Sep 2021

Reviewer #1: GSKIP paper

Q1: The authors provide information about the evolution of GSKIP. The bioinformatics used is probably suitable for the analysis of evolutionary events. However, to this reviewer who is not a specialist in evolution and analysis of evolution, it is almost impossible to follow the arguments throughout the manuscript.

A1: We thank the reviewer for your time and consideration in reviewing our article. Your constructive comments are encouraging, for which we are very grateful. We have addressed the comments as thoroughly as possible in the revised version of the manuscript. In particular, we have rewritten our abstract and introduction to improve our rationales.

Q2: The authors need to define certain technical terms, most important what they mean by simple and composite types. What is meant by pre-GSK3beta?

A2: Thank you for your comment. For the simple-type GKSIP, please see C.Hundsrucker, et al. JBC. 2010, figure 5. For the composite-type GSKIP, refer to Chou CH, et al. BBA. 2018, figure 2C. Both types of GSKIP have been described in the text.

For the mean of pre-GSK3beta binding site, during the evolution process of DUF727/GSKIP, the pre-GSK3beta binding site is prior to the GSK3beta binding site. However, it is particularly for the evolutionarily conserved pre-GSK3 binding site (115SPxF118) of composite-type GSKIP in both vertebrate and invertebrate, compared to evolved simple-type GSKIP (most of the species are 115SPxY118).

Q3: The introduction nicely introduces GSKIP. However, then in lines 97-99 the link to context-dependency and Wnt signalling is completely obscure. Please explain better.

A3: Thanks for your comment. We have revised those sentences in lines 93-96.

Q4: Fig. 1 provides structural information. It would be most valuable to overlay the different structure to visualize overlap/differences.

The authors suggest throughtout the manuscript that GSKIP gained the AKAP function in vertebrates. However, Hundsrucker et al, JBC 2010, had shown that GSKIP of C. elegans already binds PKA R subunits. Please address the discrepancy.

A4: Thank you for your comment. Hundsrucker et al, JBC 2010, indicated that the PKA binding site only exists in simple-type GSKIP as an AKAP, but not in composite-type GSKIP. In this manuscript, all the composite-type GSKIP (CLU1 and ARMC4) lack the PKA binding site, thus, the composite-type GSKIP cannot be recognized as an AKAP.

Q5: Line 405 sentence starting “In addition, Mitofilin…” is unconnected to the preceeding text. Please embed better.

A5: Thanks for your suggestion. We have already revised this paragraph (lines 406-412).

Q6: Please do not term mutant versions of any binding domains or proteins “flawed”, please use mutant.

A6: Thank you for your suggestion. We have replaced flawed to mutant.

Q7: Starting with line 437, Hundsrucker had already addressed the question of whether lower GSKIP of lower organisms binds GSK3beta (Hundsrucker JBC 2010). Please consider in your analysis.

A7: In Hundsrucker’s study (JBC 2010), they only showed that the simple-type GSKIP of lower organisms binds GSK3beta.

In our study, we performed mutagenesis assay of various mutant GSK3β-binding sites from both invertebrate (lower organisms) and vertebrate (higher organisms) of composite-type CLU1, particularly for the pre-GSK3β-binding site (118F or 118Y).

Q8: Line 462, what is meant by dominant consensus sequence?

A8: Dominant consensus sequence referring to 115SPxF118xxxF122xxxQ126xxRV130 was found in composite-type of CLU1, which exists in many species, particularly for several sites at F118, Q126, V130 are not perfect compared to the ideal consensus sequence (115SPxY118xxxF122xxxL126xxRL130) for simple-type GSK3beta binding site (Please see text in lines 244, 553).

Q9: Line 472, the link to Covid is unclear, please clarify and explain better.

A9: Indeed, we have already discussed your concern in detail for COVID-19 and possible ARMC4 in Wnt signaling pathway in our result section (lines 458-477). Moreover, we expect to obtain the available entire genome sequence of Rhinolophus ferrumequinum (the Greater horseshoe bat) because it can provide insights into the importance of the bat genome (lines 558-566). This might contribute to the efforts of the current COVID-19 outbreak for guiding GSKIPs in bat mitochondria and signaling pathways along with immunology (how mother nature of bat genome harboring coronavirus) to combat emerging pandemic viruses.

Q10: Fig. 6, the title is misleading since – to the understanding of this reviewer- Dema et al., JBC 2016, had clearly shown a role of GSKIP in Wnt signalling.

A10: We agreed. Dema et al., JBC 2016 and Chou CH, et al. BBA. 2018 only addressed the role of simple-type GSKIP in Wnt signalling.

In this study, we found both simple-type and composite-type in the title of fig 6. Particularly, we uncovered and emphasised that ARMC4 (composite-type GSKIP) and pangolin (beta-catenin, armadillo domain) might also be involved in the Wnt signaling pathway.

Q11: Line 527, discussion, the paragraph is unconnected to the previous discussion. Please provide clear links.

A11: Thank you for your suggestion. We have eliminated the unconnected sentence and linked the part of ATP (lines 523-524).

Q12: Similary, line 562, the paragraph about Covid. What is the reason for speculating on Covid and GSKIP. Please substantiate.

A12: Thank you for your comment. Please see our answer 9. From lines 466 to 481 and figure 6 “Physiological implications and importance of both simple-type and composite-type GSKIP in Wnt signaling”, we have already provided many concepts for COVID and GSKIPs. Furthermore, the genome of the bat (Rhinolophus ferrumequinum, the Greater horseshoe bat), which might be the possible source of COVID, has not been well-established yet. It can provide insights into the importance of the simple-type GSKIP in the bat’s genome. The likely insights into the bat genome might contribute to the efforts to end the current COVID-19 outbreak in so far as the insights may elucidate how GSKIPs function in bat mitochondria and Wnt signaling pathways; particularly in immunology studies may also help to understand how mother nature of bat genome harboring coronavirus could be used to combat emerging variants of this pandemic virus. (lines 558-566)

Q13: In the conclusion, line 577, the thoughts about target-based drug discovery are not at all embedded into the discussion.

A13: We agree. We have modified that paragraph in the conclusion.

Reviewer #2: Tsai et al. investigated the role of Glycogen synthase kinase interaction protein (GSKIP) in mitochondria and the Wnt signaling pathways.

Q1: The manuscript needs to be rewritten. Both, English must be improved including the structure of most sentences and the thread, which guides the reader through the information given by the authors.

A1: Your comment is well-taken. We have already rewritten the whole manuscript. Also, we have addressed the comments as thoroughly as possible in the revised version of the manuscript.

Q2: The introduction is a numeration of findings. It is not clear, how the findings from the introduction should guide to the research question answered by the authors.

A2: Thanks for your suggestions. We have rewritten the rationale of your concerns in lines 93-96 “Collectively, simple-type GSKIP was believed to play a multifaceted role in several diseases oriented and a context-dependent manner, involved in mitochondria and the regulation of Wnt signaling pathway”. Based on above finding, we further explored the evolution of composite-type GSKIP (CLU1 and ARMC4) involving in mitochondria and the regulation of Wnt signaling pathway.

Q3: Methods: The authors mention a few times, that information were given previously, without giving a reference. Information about the used material is incomplete (e.g. line 173; 174; 179; …).

A3: Thank you for your comment. We have added references in line 166.

Q4: Into the discussion, there is redundancy of the results, e.g. from page 33. It is not clear, why the authors stat that the yawn to sequence the whole genome of Rhinolophus ferrumequinum. Please, explain in more detail. However, such ideas are not sufficient to be present in a discussion.

A4: In fact, we have already discussed your concern in detail for COVID-19 and possible ARMC4 in Wnt signaling pathway in our result section (lines 468-481) “A study suggested that pangolins are natural hosts of beta-coronaviruses, and comprehensive surveillance of coronaviruses in pangolins could improve our understanding of the spectrum of COVID-19…”

To avoid the redundancy of the results and discussion here, we have modified the paragraph from lines 558-566. In this study, we are eagerly waiting for the whole genome sequence of Rhinolophus ferrumequinum. While the GSKIPs (simple-type and composite-type: Clu1; ARMC4; pangolin as a protein or intermediated host?) are somehow feeble, and the mechanisms are yet to be proved.

Q5: In addition, the link from the finding presented here, to Sars-CoV-2 (The virus is called Sars-CoV-2 and the disease arising from an infection is called COVID19) only can be assumed, but it is not clearly stated by the authors.

A5: We agree. We have added the sentence describing COVID-19 in lines 470-471 and cited in the references 54.

Q6: The conclusion is not clear. Please, only conclude from your results and do not speculate bats and viruses. The results do not support this.

A6: We agree. We have eliminated the part of bat and COVID-19 in the conclusion section.

Reviewer #3: The authors set the foundations of some interesting thoughts and ideas in their manuscript, but those ideas are clouded by poor judgment of their results and a lack of clarity in the writing that weighs the manuscript down.

Q1: For example, the authors wrote (line 272) “to determine the conserved sequence of GSK3β-binding regions and DUF727 in CLU1, we selected one bacterial prototype and 20 GSKIP orthologs and used ClustalW and MEGA-X to construct a phylogenetic tree”. However, a phylogenetic tree is not the best option to determine conservation between sequences, a sequence alignment would be a more adequate choice.

A1: We agree. In fact, our data of T-coffee have already shown a multiple sequence alignment with respect to the DUF727 region of CLU1 orthologues (also ARMC4). Additionally, T-Coffee in figure 3 was performed to estimate alignment accuracy and improve phylogenetic tree reconstruction. Therefore, we revised the content of line 272 that we “firstly” selected one bacterial prototype and 20 GSKIP orthologs and used ClustalW and MEGA-X to construct a phylogenetic tree….. and the sentence from lines 288-291” Both a GSK3β-binding motif (122Fxxx126LxxR/K/QL130)….”.

Q2: Moreover, the values represented in the phylogenetic tree (Fig. 2), if they mean bootstrap, are too low to be considered significant (e.g., 0.37; 0.25). The authors should include more information that clarifies the meaning of those values and reconsider if the phylogenetic tree is relevant.

A2: Thanks for your comment, the low bootstrap values represented in the phylogenetic tree (e.g., 0.37; 0.25) could possibly due to several reasons: firstly, we mixed all the phylogenetic analysis of four composite-type GSKIP, including ARMC4, CLU1, HSP70/Dnak and Mitofilin, among different species (figure 2). Secondly, the generating of the phylogenetic tree is dependent on the sequence homology among different species, while the 3D structure of DUF727 (figure 1 D) folds into a central part of anti-parallel ββββ topology (figure 1), indicating that the similarity of the DUF727 structure among species. Finally, when we performed the bootstrap test in MEGA, we set the number of bootstrap replicates as 500 in CLU1 and ARMC4 analysis, respectively (Supplement figure 1A and B). As MEGA finishes the bootstrap test, the flap shows the bootstrap consensus tree, which is a consensus of the bootstrap replicate trees. The bootstrap test measures the internal consistency of data produced above than 0.5 (50%) of the bootstrap replicates are consistent.

Q3: Secondly, the authors used the yeast two-hybrid system to investigate different GSK3β-binding sites and claim several positions as crucial for the binding when they use a fragment of the protein, however, the authors overlook the results when they use the entire protein.

A3: Your comment is well-taken. We used simple-type of GSKIP as an entire protein (1-139, full-length) GSKIP and GSKIPtide (115-139, fragment) as a template to perform mutagenesis assay of various flawed GSK3β-binding sites from different species of composite-type CLU1 particularly for pre-GSK3β-binding site (118F or 118Y). We also added the above paragraph to the text in the method section: Cloning, site-directed mutagenesis and DNA sequencing.

Q4: Thirdly, the manuscript suggests a certain anthropocentric tone with concepts like lower and higher animals. I recommend the authors to read the following short paper (https://doi.org/10.1002/bies.201370093) and I encourage them to reconsider the use of lower and higher animals.

A4: We agree. We eliminated all the terms of lower and higher in this article. Except for bacteria, we used “highly primitive form of GSKIP in lower organisms” in lines 535-536.

Q5: Regarding the lack of clarity of the manuscript, I would like to point out that it is easy to find sentences out of context (e.g. line 139: “It can occur as a result of translocation, interstitial deletion, or chromosomal inversion.”; line 304; “Its inactivation with GSK3β was found when this site was modified.”), without enough clarity (e.g. line 132: “Accession number (S1 Table) and amino acid sequence (S1 Fig) were used as parameters.”; line:359: “The protein encoded by ARMC4, 10 armadillo repeat motifs (ARMs), and one HEAT repeat were contained in ARMC4, which is thought to be involved in ciliary and flagellar movement”), or hardly readable (e.g. line 542: “Second, in CLU1 and ARMC4, the DUF 727 domain, only as central fragment of GSKIP, can exist alone or with other domains as part of multi-domain structures.”) among others.

A5: Thanks for your comment.

1. The sentence has been deleted.

2. The sentence in lines 302-303 had been modified to indicate that its inactivation with GSK3β when this site was modified.

3. We added more descriptions in lines 129-132 for the S1 table and S1 figure.

4. Please take a look of the following sentence (line 362) for ARMC4 “The term armadillo is derived from the historical name of the β-catenin gene in the fruit fly Drosophila, where the armadillo repeat was first discovered.”

5. We have modified lines 538-540.

Q6: Moreover, several statements lack a reference (e.g., line 125 after “gene products”; line 143 after “previously described”; line 169 “previously described”; line 207 after “Armadillo repeats”; line 304 after “humans”) or some references are not the correct ones (e.g., line 209; line 211; line 295; line 367; line 369). Authors should check that no error has occurred during the reference formatting process.

A6: Thank you for your suggestions. We have already cited and checked all the references.

Q7: Considering previous publications, from the same authors, this manuscript is not as clear as the others. The authors should give more context in the abstract and introduction to avoid confusion.

A7: Thank you for your suggestions. We have rewritten our abstract and introduction, particularly for the evolutionarily conserved pre-GSK3 binding site (115SPxF118) of composite-type GSKIP in both vertebrate and invertebrate, compared to evolved simple-type GSKIP (most of the species are 115SPxY118).

Q8: Besides, a scheme of the workflow that they employed in their domain survey will help the reader follow the process. In relation to that, the authors need to make the results section “Analysis of UniProt and GSKIP gene ontology: Identification of composite-type GSKIP” more straightforward. The authors could, for example, refer more to their own figures (i.e., adding Fig. 1G in line 207 after “Armadillo repeats”; adding Fig. 1G in line 229 after GSKIP; adding Fig. 1F in line 236 after “invertebrates”) and maybe represent figures 1A, B and C as Venn diagrams.

In line 253, the authors say “Using GSKIP as a keyword, a total of 1849 protein entries were shown” and in lines 255 authors say “Using CLU1 as a keyword, 879 protein entries were shown”, but the figure shows 2234 entries for GSKIP and 936 for CLU1. The authors need to check why those are different.

A8: Thank you for your suggestions.

1. In fact, in figure 1G, we have already demonstrated all the domains of four groups of composite-type GSKIP, including AcuB, CLU1, ARMC4, and sporadic (Hsp70/Dnak and Mitofilin). To consider the result of searching for those evolved GSK3 binding sites and PKA binding sites in composite-type GSKIP of CLU1 and ARMC4 is zero, indicating PKA domain is not yet involved in the evolution. We therefore use the number but not Venn diagram to represent all the queries.

2. It is our mistake. In line 252, the protein entries of GSKIP is not 1849, the correct one is 2234. In line 254, the protein entries of CLU1 is not 879, the correct one is 936. All the correct numbers have shown in lines 254-256.

Q9: Furthermore, in the results section “Two groups of composite-type GSKIP families may have evolved through a gene-fusion mechanism: CLU1 mitochondria) and ARM4 (Wnt pathway)” the list of accession numbers (lines 276 to 288) does not help the readability of the manuscript.

A9: Thank you for your comment. In this study, we found interestingly that DUF727/GSKIP domain was combined with CLU1 gene (mitochondria) and ARMC4 gene (Wnt pathway), designated as composite-type GSKIP. Therefore, all the accession numbers represent and ensure these two groups of composite-type GSKIP families (lines 274-286).

Q10: Authors could add those numbers in figure 2 or in the supplemental material. Also, the authors should be more precise when they name the groups for the different species (i.e., line 280 “worm” is very unspecific, authors should consider “round-worm” or "nematode"; in lines 284 and 285, the authors wrote that the phylogenetic tree have one chordate and six vertebrates, however, vertebrates are chordates too, so the phylogenetic tree has seven chordates among them one cephalochordate [Branchiostoma lanceolatum] and six vertebrates.

A10: Thank you for your suggestions. We have already corrected the name of the species and the number (in lines 279, 283).

Q11: Finally, some typographical errors that should be corrected:

Line 100: add “that” before co-exist

Line 116 and line 117: redundant reference, also this is not the format to cite Uniprot. The correct cite is https://doi.org/10.1093/nar/gkaa1100

Figure 1E: In the secondary structure drawing the letters are cut

Line 277: “Schizosac charomyces” should be one word

Line 289: “S3 Fig” should be “Fig 3”

Line 339: “light blue” should be “green”

Line 371 and line 373: “MARC4” should be “ARMC4”

A11: Thanks for your time and kindly reminder. We have already corrected all the errors.

Submitted filename: Response to reviewer.docx

Click here for additional data file.

6 Oct 2021

PONE-D-21-20264R1Deciphering the evolution of composite-type GSKIP in mitochondria and Wnt signaling pathwaysPLOS ONE

Dear Dr. Hong,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Note that, although the reviewers agree that the manuscript has been significantly improved, there are still a few important issues that need to be addressed before the manuscript is acceptable for publication.

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Reviewers' comments:

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Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

**********

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: N/A

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Reviewer #3: Yes

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Although the authors have addressed the concerns of this reviewer there are still a few things to clarify. It still has not been properly defined what is meant by simple-type and composite forms of GSKIP. In line, in the introduction the authors list all known functions of GSKIP but do not elaborate on which GSKIP form, simple-type or composite, can be allocated to which function. Please do so in order to put the reader into a position to follow.

Reviewer #2: All suggestions and comments were addressed by the authors. I have no further comment sor suggestions.

Reviewer #3: Deciphering the evolution of composite-type GSKIP in mitochondria and Wnt signaling pathways

A considerable improvement has already taken place in the comprehensibility of the manuscript, however, the manuscript is still difficult to follow and needs to rethink again. Besides, some issues should be addressed.

For example, the list of organisms from lines 275 to 287 is referring only to CLU1, but in the tree, there are more species. Maybe the authors would like to refer to Fig S1A instead of Fig 2 in line 274. Secondly, because the four composite-type GSKIP differ a lot in their domains the bootstraps in figure 2 are not be reliable. A bootstrap support above 95% is very good and very well accepted and a bootstrap support between 75% and 95% is reasonably good, anything below 75% is very poor support and anything below 50% is of no use. If authors want to show phylogenetic relationships between the four composite-type GSKIP I recommend them to use only the orthologous domains to construct the tree.

Another issue is that the authors claim in line 305 the mutation of Leu130 always causes abolition of the interaction, however in full-length GSKIP the interaction is positive for 3 out of 4 mutations tested.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Josep Marti-Solans

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25 Oct 2021

Reviewer #1: Although the authors have addressed the concerns of this reviewer there are still a few things to clarify.

Q1. It still has not been properly defined what is meant by simple-type and composite forms of GSKIP. In line, in the introduction the authors list all known functions of GSKIP but do not elaborate on which GSKIP form, simple-type or composite, can be allocated to which function. Please do so in order to put the reader into a position to follow.

A1. Sorry for the confusion of “simple-type and composite forms of GSKIP”. GSKIP has two subtypes, there is only one simple-type GSKIP, all the functions described in the introduction part belong to this type. However, there are four groups of composite-type GSKIP (clustered mitochondria protein 1 (CLU1) and Mitofilin in mitochondria, heat shock protein 70 (Hsp70), and Armadillo repeat-containing 4 (ARMC4). In this article, we try to decipher and emphasize the origin and evolution of these four groups of composite-type GSKIP (Referring lines 102-109).

Reviewer #2: All suggestions and comments were addressed by the authors. I have no further comments or suggestions.

Thank you for all the suggestions and efforts.

Reviewer #3: Deciphering the evolution of composite-type GSKIP in mitochondria and Wnt signaling pathways A considerable improvement has already taken place in the comprehensibility of the manuscript, however, the manuscript is still difficult to follow and needs to rethink again. Besides, some issues should be addressed.

Q1. the list of organisms from lines 275 to 287 is referring only to CLU1, but in the tree, there are more species. Maybe the authors would like to refer to Fig S1A instead of Fig 2 in line 274.

A1. Thank you for your comment. We have referred to figure S1A in line 274.

Q2. Because the four composite-type GSKIP differ a lot in their domains the bootstraps in figure 2 are not be reliable. A bootstrap support above 95% is very good and very well accepted and a bootstrap support between 75% and 95% is reasonably good, anything below 75% is very poor support and anything below 50% is of no use. If authors want to show phylogenetic relationships between the four composite-type GSKIP I recommend them to use only the orthologous domains to construct the tree.

A2. We agree. In general, we believe that a reliable bootstrap between 75% and 95% is for individual orthologous domains to construct the tree. Our data is for four groups of orthologous domains (from human to bacteria should be more diverse) to construct the tree, therefore, a bootstrap 50% could be acceptable.

Q3. Another issue is that the authors claim in line 305 the mutation of Leu130 always causes abolition of the interaction, however in full-length GSKIP the interaction is positive for 3 out of 4 mutations tested.

A3. Thank you for your comment. Since Leu130 plays a crucial role in GSKIP binding to GSK3beta for simple-type GSKIP, our mutagenesis assay (table 1) of the full length of GSKIP (1-139) and GSKIPtide (115-139) 3 out of 4 mutations due to the conformational change (particularly for Pro130 causes distort helix in both full-length and GSKIPtide). (Please see references 1,3,6 and 20 in this study.)

Submitted filename: Response to reviewer.docx

Click here for additional data file.

17 Dec 2021

Deciphering the evolution of composite-type GSKIP in mitochondria and Wnt signaling pathways

PONE-D-21-20264R2

Dear Dr. Hong,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: The concerns of this reviewer have been addresses. This makes it easier to follow the paper. Thank you.

Reviewer #2: I have no further comments and suggestions. All questions ans issues have been rased by the authors.

**********

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5 Jan 2022

PONE-D-21-20264R2

Deciphering the evolution of composite-type GSKIP in mitochondria and Wnt signaling pathways

Dear Dr. Hong:

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