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Glycogen synthase kinase-3 (GSK3) plays important roles in numerous signaling pathways that regulate a variety of cellular processes including cell proliferation, differentiation, apoptosis and embryonic development. In the canonical Wnt signaling pathway, GSK3 phosphorylation mediates proteasomal targeting and degradation of β-catenin via the destruction complex. We recently reported a biochemical screen that discovered multiple additional protein substrates whose stability is regulated by Wnt signaling and/or GSK3 and these have important implications for Wnt/GSK3 regulation of different cellular processes.1 In this article, we also present a bio-informatics based screen for proteins whose stability may be controlled by GSK3 and β-Trcp, the SCF E3 ubiquitin ligase that is responsible for β-catenin degradation in the Wnt signaling pathway. Furthermore, we review various GSK3 regulated proteolysis substrates described in the literature. We propose that GSK3 phosphorylation dependent proteolysis is a widespread mechanism that the cell employs to regulate a variety of cell processes in response to signals.
Glycogen synthase kinase-3 (GSK3) was originally identified in the 1980's as a key regulator of glycogen metabolism.2 When blood glucose level is low, GSK3 constitutively phosphorylates glycogen synthase and inhibits its activity. This inhibition is released by increased insulin signaling resulting from elevated blood glucose levels in which case PI3K-Akt signaling cascade inhibits GSK3 activity and glycogen synthase is de-phosphorylated and activated, resulting in the synthesis of glycogen from glucose (reviewed in ref. 3).
Since its discovery, GSK3 has been found to phosphorylate many proteins and play important roles in a variety of cellular processes such as cell proliferation, differentiation, microtubule dynamics, cell cycle and apoptosis.4 In fact, a consensus motif and context based computational analysis of identified in vivo protein phosphorylation sites indicates that GSK3 is one of the kinases with most substrates in the cell.5 In turn, multiple cell signaling pathways are known to inhibit GSK3 activity, such as the PI3K-Akt pathway, the Wnt signaling pathway and the MAPK pathway. The general function and regulation of GSK3 has been the subject of numerous excellent reviews (refs. 4 and 6) and therefore will not be the focus of this article.
An interesting characteristic of GSK3 is that it is usually constitutively active in the cell and inhibited by upstream signals. Phosphorylation by GSK3 usually negatively regulates its downstream substrates, as exemplified by the classic GSK3 regulation of glycogen synthase that was described earlier. In many cases GSK3 phosphorylation earmarks target proteins for ubiquitination and proteolysis, one famous example being the regulation of β-catenin stability in the canonical Wnt signaling pathway. In this article, we will discuss potential Wnt and/or GSK3 regulated protein degradation substrates revealed by a biochemical screen1 and a bioinformatics based search. We will also review other GSK3 regulated proteolytic substrates from literature and propose protein stability control as a major mechanism that GSK3 employs to regulate cellular processes.
Originally identified using genetic studies in Drosophila and developmental studies in Xenopus,7 the canonical Wnt signaling pathway plays a very important role in numerous processes such as embryonic development, tissue regeneration and carcinogenesis. In the absence of Wnt signaling, casein kinase I (CKI) phosphorylation of β-catenin primes it for subsequent GSK3β phosphorylation at multiple sites at the N-terminus. Phosphorylated β-catenin is then recognized by β-Trcp and rapidly degraded by the 26S proteasome. Other components of the multi-protein “destruction complex” that targets β-catenin for degradation, the tumor suppressor genes APC (adenomatous polyposis coli) and Axin, serve as scaffolding proteins for this reaction. The level of the cytoplasmic and nuclear β-catenin that mediates Wnt signaling is thus kept low in resting cells, resulting in the silence of downstream target genes. In the presence of Wnt ligand the destruction complex is inactivated through a mechanism that is not fully understood (likely dissociation of the complex), allowing β-catenin levels to increase in the cytoplasm. The accumulated β-catenin then translocates into the nucleus and binds to LEF/TCF transcription factors to activate downstream target genes. GSK3 dependent control of β-catenin stability is thus the central regulatory mechanism of the canonical Wnt signaling pathway (reviewed in refs. 8–10).
In addition to β-catenin, levels of Snail, a repressor of E-cadherin gene transcription and Smad1, the BMP signaling mediator, were recently reported to be regulated by the canonical Wnt signaling pathway and GSK3.11–15 Very similar to β-catenin, stability of the Snail protein is controlled by GSK3 dependent phosphorylation followed by β-Trcp mediated ubiquitination and proteosomal degradation in the absence of Wnt signaling. Wnt signal inhibits the GSK3 mediated phosphorylation and consequently increases Snail protein levels, which in turn triggers the epithelial-mesechymal transition (EMT) in certain cell types.12–14 The BMP signaling mediator Smad1 is regulated by Wnt signaling in a similar fashion. However, instead of total Smad1 protein levels, only the BMP/MAPK activated form of Smad1 (C-terminal phosphorylated Smad1) is recognized and regulated by Wnt/GSK3, resulting in a fine control of BMP signaling strength and longevity by Wnt signaling.15 Interestingly, a recent study revealed that the protein stability of another Smad, Smad3, is regulated by Axin and GSK3β but possibly not Wnt signaling.16 Further studies may help to confirm whether Wnt signaling can regulate Smad3 stability in certain contexts.
We recently reported a screen for potential Wnt and/or GSK3 regulated protein degradation substrates using in vitro expression cloning technique and biochemical reconstitution in a Xenopus egg cytoplasmic extract.1 Using LiCl as a general GSK3 inhibitor or GID (the GSK3 interacting domain of Axin) protein as specific inhibitor of GSK3 activity in the Wnt signaling pathway, we screened pools of cDNAs encoding ~10,000 polypeptides and identified 35 potential GSK3 regulated proteolytic substrates that responded to LiCl in the screen. 12 of the 35 candidates responded to both LiCl and GID, and to other destruction complex inhibitors such as Dsh (dishevelled) and AxinΔRGS (axin that misses the APC-interacting RGS domain), and are thus strong candidates for Wnt regulation. Wnt regulation of selected candidates was verified by expression in vivo in Xenopus embryos. The other 23 candidates responded to LiCl but not GID in the screen, and are thus potential GSK3 protein substrates but not Wnt regulated (Fig. 1 and ref. 1).
This study provided evidence that Wnt signaling regulates the stability of multiple proteins, and therefore various cellular processes, in additional to β-catenin mediated gene expression. Many of the identified substrates interact with each other or have similar cellular functions, indicating Wnt and/or GSK3 regulation of those cellular functions. One example includes MRLC (myosin regulatory light chain) and TMEM4 (or MASP for MIR-interacting saposin-like protein). MRLC is a main regulator of myosin contractility and cell motility and its activation has been known to be regulated by multiple signaling pathways.17–19 Wnt signaling has also been known to regulate MRLC activity and cell motility, although through the noncanonical Wnt pathways.20 Little is known about TMEM4, but it has been reported to interact with MRLC and positively regulate its activity and stability.21,22 Finding MRLC and TMEM4 in the screen indicates that the canonical Wnt signaling pathway may also control cell motility in some cell contexts through the destruction complex mediated regulation of MRLC and TMEM4 stability. Interestingly, components of the destruction complex, GSK3 and APC, have been implicated in regulation of cell polarity and cell migration through their regulation of microtubule polarity and dynamics.23,24
Another group of interesting targets identified in the screen include the RNA binding proteins TIAR (T-cell restricted intracellular antigen-related protein) and Sam68, a STAR (signal transduction and activation of RNA) family protein. TIAR has been shown to bind to U-rich sequences near 5' splice sites of pre-mRNAs and modulates alternative splicing.25,26 Indeed, one bioinformatics study estimates that ~15% of alternative cassette exons in the genome are regulated by TIA1/TIAR, suggesting a widespread role of TIAR in the regulation of alternative splicing.27 Sam68 has been reported to regulate the splicing of genes such as CD44 and Bcl-x, and its activity is regulated by cellular signaling and phosphorylation.28,29 Identifying TIAR and Sam68 as potential Wnt signaling regulated proteolysis substrates therefore raise the exciting possibility that Wnt signaling may regulate mRNA splicing and processing through regulation of the stability of these proteins.
Some proteins identified as Wnt regulated destruction complex targets in the screen also play roles in the Wnt signaling pathway. Two of the LiCl and GID positive targets, Trim29 and TACSTD1, have recently been reported to be positive regulators of β-catenin signaling. TACSTD1, also called EpCAM, is a membrane protein that is overexpressed in cancer cells and was one of the first identified cancer antigens.30–32 It undergoes proteolytic cleavage upon extracellular domain homophilic binding and releases the intracellular domain (EpICD). EpICD then enters the nucleus and binds to transcription factors including β-catenin and Lef-1, and activates downstream gene expression.33 Trim29, also known as ATDC (ataxiatelangiectasia group D complementing gene), was found to positively regulate Dishevelled levels and inhibit the destruction complex, thus stabilizing β-catenin.34 Potential Wnt regulation of the stability of EpCAM and ATDC therefore suggests that there exists feedback control of the canonical Wnt/β-catenin signaling pathway, probably resulting in fine control of the signaling strength and length in various cellular contexts.
Both β-catenin and Snail contain a “DSGxxS/TxxxS/T” motif that is necessary for their regulation by Wnt signaling.12 DSG(X)2+nS is the recognition site for the E3 ubiquitin ligase β-Trcp: when the serine in the DSG motif is phosphorylated, β-Trcp binds to this motif and adds ubiquitin onto a lysine usually located upstream (reviewed in ref. 35). “S/TxxxS/Tp” is the GSK3 consensus phosphorylation site, in which the second (c-terminal) serine or threonine is usually primed by phosphorylation by another kinase before GSK3 phosphorylates the N-terminal serine/threonine. In many cases there are tandem repeats of this consensus motif, resulting in processive phosphorylation as in the case of β-catenin (reviewed in ref. 6).
We hypothesized that proteins containing the DSGxxS β-Trcp binding motif followed by tandem GSK3 consensus sites are likely regulated by GSK3 and β-Trcp in a similar fashion as β-catenin and Snail. With this hypothesis, we searched the UniProtKB/Swiss-Prot protein database for a “D/ESGxxS/TxxxS/TxxxS/T” motif. We identified 605 hits in 579 sequences in the database that contains ~500,000 sequence entries (release 57.4, eukaryota). As important regulatory amino acid motifs are usually evolutionarily conserved, we blasted each hit sequence against its homolog and identified ones that contained motifs that were evolutionarily conserved among numerous species. This narrowed the list down to 38 potential protein substrates regulated by GSK3 and β-Trcp. They can be included in functional groups as shown in Table 1, indicating potential GSK3/β-Trcp regulation of the respective cellular processes.
Both β-catenin and Snail were, by design, identified in the search. Interestingly, the search identified FGD3 (faciogenital dysplasia 3), a putative Cdc42 guanine nucleotide exchange factor (GEF) whose stability was reported to be regulated by GSK3 and β-Trcp36 and plakoglobin, which was known to be regulated by Wnt signaling in a similar manner as β-catenin.37–39 These two positive hits serve as validation for the screen.
The search also identified many interesting potential GSK3/β-Trcp targets. NACα has been reported to be regulated by GSK3 phosphorylation dependent ubiquitination but not the DSG motif and β-Trcp (Table 2). Further studies may help clarify this apparent discrepancy. Additionally, AKAP220 (A-kinase anchor protein 220) and Ninein have been found to bind GSK3β, and EPO-R (Erythropoietin receptor precursor) is known to be β-Trcp substrate, which makes them strong candidates for GSK3 and β-Trcp regulated proteolysis.40–42 There are also two hits, ARD1 (N-terminal acetyltransferase complex subunit) and LZTS2 (Leucine zipper putative tumor suppressor 2), which interact with β-catenin and play roles in the canonical Wnt signaling pathway.43,44
GSK3 has been found to regulate the proteolysis of an increasing number of proteins (Table 2). In the absence of upstream inhibitory signals, GSK3 phosphorylation of serine or threonine creates binding sites for phosphorylation-dependent E3 ubiquitin ligases such as Fbw7 and β-Trcp, allowing subsequent ubiquitination and proteolysis of the substrates. In many cases GSK3 needs a priming phosphorylation by another kinase, thus allowing additional regulatory input from other signaling pathways. The regulated proteins include transcription factors and signaling proteins with various cellular functions including cell proliferation, differentiation, apoptosis and response to stress. Growth signals and mitogens typically inhibit GSK3 activity, resulting in the increase of substrate protein levels and activation of respective downstream cellular events.
In some cases GSK3 phosphorylation dependent ubiquitination does not lead to full degradation but rather proteolytic cleavage processing of the substrate, as in the case of transcription activator Ci (Cubitus interruptus) in the Hedgehog (Hh) signaling pathway (described in more detail in the next section).45–48 Although less well-studied, GSK3 phosphorylation and subsequent ubiquitination may also regulate p105 to p50 protein cleavage processing in the NFκB pathway.49
In some signaling events, phosphorylation by GSK3 plays both positive and negative roles on the same substrates, as in the case of MafA and SRC-3. MafA is a basic leucine zipper (bZip) family transcription factor that promotes oncogenic transformation in embryonic fibroblasts and insulin expression in β cells.50 SRC-3 is a steroid receptor co-activator that regulates cell growth.51 Phosphorylation by GSK3 induces both activation and degradation of MafA and SRC-3. Another GSK3 regulated proteolysis substrate, BCL-3, may also be regulated through a similar mechanism.52,53 This tightly coupled activation and degradation creates a `rapid spike' of downstream substrate activity by limiting the temporal length of activation that may be necessary for strict regulation.54–56
GSK3 regulation of β-catenin in the canonical Wnt signaling pathway has long been known to play important roles in embryonic development and tumorigenesis.83,84 GSK3 has also been found to function similarly in the Hedgehog (Hh) signaling pathway, which plays important roles in many developmental processes and diseases (reviewed in ref. 85). In the absence of the Hh signal, GSK3 phosphorylation results in the Slimb/β-Trcp dependent ubiquitination of transcription activator Ci. As we mentioned earlier, ubiquitination does not lead to full degradation but rather cleavage of the full-length Ci protein (Ci155) into a truncated form (Ci75), which acts as a repressor of Hh downstream genes. Hh signaling blocks this process through an unknown mechanism, resulting in the activation of downstream gene expression. The similar roles of GSK3 in Wnt signaling, TGFβ/BMP signaling and Hh signaling pathways suggest that GSK3 regulated protein proteolysis plays important roles in the regulation of cell proliferation and differentiation in embryonic development.
Interestingly, we identified Vent/Vox family protein members as potential GSK3 but not Wnt signaling regulated protein degradation substrates in the biochemical screen.1 Vent/Vox family homeobox transcription repressors are known antagonists of dorsalizing signals in early embryonic patterning in Xenopus and zebrafish, and their transcription has been reported to be regulated by BMP and Wnt signaling.86–88 Another study also reported that the stability of one Vent/Vox family protein, Xom, is regulated during Xenopus embryonic development, although the evidence suggested it was through a kinase different from GSK3.89 If GSK3 is confirmed to regulate the stability of Vent/Vox family proteins, it will be very interesting to identify the upstream signaling pathways that regulate GSK3 activity in this process.
GSK3 has been known to play an inhibitory role in cell cycle progression and cell proliferation, at least partly through its regulation of the stability of cyclin E, cyclin D1, cdc25A and c-Myc.74,75,77 Cyclin D1 and cyclin E protein levels dictate the G1 to S transition and has been known to be tightly regulated by mitogens and cell cycle signals (reviewed in ref. 90). GSK3 phosphorylation mediates rapid degradation of both cyclin D1 and cyclin E. Ras signal inactivates GSK3 through the PI3K-Akt pathway and results in accumulation of stabilized cyclins, triggering cell cycle progression.74,75 It may also lead to the accumulation of Cdc25A phosphatase, another GSK3 regulated protein degradation substrate, and activate cyclin-dependent protein kinases (Cdks).77 At the same time, mitogen signaling also inhibits the GSK3 mediated degradation of c-Myc, resulting in the activation of its target genes, including cyclin D1, cyclin E and other cell cycle mediators.70,72 GSK3 thus has both direct and indirect roles in regulation of cell cycle progression. Interestingly, GSK3 activity is high in quiescent cells and cells in G1 phase, but lowers as cells progress into S phase, reflecting upstream regulation of its activity in cell cycle.77
In line with its role in cell cycle regulation, GSK3 regulated proteolysis also plays important roles in the cellular response to stress stimuli such as nutrient deprivation or DNA damage. One example is the regulation of p21cip1 (p21) turnover in response to UV irradiation. Following low dose UV irradiation p21 is rapidly degraded through ubiquitination, which releases its inhibition of PCNA (proliferating cell nuclear antigen, a cofactor of DNA polymerase δ) and allows DNA repair.61 Increase of GSK3 activity after UV irradiation was found to be responsible for triggering p21 degradation in this process.60 GSK3 mediated degradation of cdc25A may also play a role in inducing cell cycle arrest after UV irradiation, thus permitting more time for DNA repair.77
GSK3 plays pro-apoptotic roles in the cell, as its overexpression induces apoptosis and inhibition protects cells against apoptotic stimuli.91,92 It has recently been found to negatively regulate the protein stability of Mcl-1, a Bcl-2 like anti-apoptotic protein that antagonize the effects of pro-apoptotic Bcl-2 family proteins on mitochondrial outer membrane permeabilization (MOMP) and cytochrome c release.93 Cell stimuli such as growth factors or increased glucose level inhibits GSK3 activity through PI3K-Akt and promotes cell survival, while cellular stress such as UV irradiation increases GSK3 activity and triggers apoptosis.65,66,68 It is conceivable that GSK3 serves as a central kinase that integrates multiple positive and negative stimuli for cell survival, and triggers different cellular response through regulation of the turnover of various signaling protein substrates.
The upstream signals that control GSK3 activity in regulation of many of these established or potential GSK3 regulated proteolysis protein substrates are still unknown. The protein targets that responded to both LiCl and GID protein in the biochemical screen are likely to be regulated by Wnt signaling (Fig. 1). On the contrary, the targets that responded to LiCl but not GID in the screen are regulated by GSK3 but not Wnt signaling, as LiCl acts as general GSK3 inhibitor while GID is specific to the destruction complex in our assay.1
Different upstream signals may regulate GSK3 activity through different mechanisms. For example, the PI3K-Akt pathway induces the inhibitory Serine 9 phosphorylation of GSK3β (Serine 21 in GSK3α) while Wnt signaling does not act through this phosphorylation. Instead, it may inhibit the GSK3 activity by dissociating the destruction complex, thus prohibiting GSK3 from reaching its targets.4,6,94 It will be very interesting to see how specific upstream signals and cell stimuli regulate GSK3 activity and affect the stability of specific substrates, and how different upstream signals may converge on GSK3 and mediate crosstalk between each other to orchestrate cellular response to external and internal signals.
GSK3 phosphorylates a great number of proteins and resides in the middle of many important signaling pathways that respond to cellular stimuli such as growth factors and stress. GSK3 has been found to regulate the ubiquitination and proteolysis of a number of important signaling proteins or transcription factors. The results from our recent screens indicate that there are many more proteins whose stability may be regulated by GSK3 phosphorylation. Protein stability control is thus emerging as a major mechanism that GSK3 employs to modulate cellular processes. Moreover, many different signaling pathways utilize this GSK3 dependent mechanism to target proteins for turnover. We speculate that GSK3 is a conserved kinase that regulates protein turnover in response to cellular signals, and in turn regulates a variety of cell processes such as cell proliferation, differentiation, apoptosis, embryonic patterning and tumorigenesis.