Regulation of Autophagy by the Glycogen Synthase Kinase-3 (GSK-3) Signaling Pathway

Autophagy is a vital cellular mechanism that benefits cellular maintenance and survival during cell stress. It can eliminate damaged or long-lived organelles and improperly folded proteins to maintain cellular homeostasis, development, and differentiation. Impaired autophagy is associated with several diseases such as cancer, neurodegenerative diseases, and age-related macular degeneration (AMD). Several signaling pathways are associated with the regulation of the autophagy pathway. The glycogen synthase kinase-3 signaling pathway was reported to regulate the autophagy pathway. In this review, we will discuss the mechanisms by which the GSK-3 signaling pathway regulates autophagy. Autophagy and lysosomal function are regulated by transcription factor EB (TFEB). GSK-3 was shown to be involved in the regulation of TFEB nuclear expression in an mTORC1-dependent manner. In addition to mTORC1, GSK-3β also regulates TFEB via the protein kinase C (PKC) and the eukaryotic translation initiation factor 4A-3 (eIF4A3) signaling pathways. In addition to TFEB, we will also discuss the mechanisms by which the GSK-3 signaling pathway regulates autophagy by modulating other signaling molecules and autophagy inducers including, mTORC1, AKT and ULK1. In summary, this review provides a comprehensive understanding of the role of the GSK-3 signaling pathway in the regulation of autophagy.


Introduction
Glycogen synthase kinase-3 (GSK-3) has been identified to play a vital role in glucose metabolism [1]. Glycogen synthase kinase-3 (GSK-3) has two different paralogs, GSK-3α and GSK-3β [2,3]. GSK-3 is a serine/threonine kinase and was shown to suppress the activation of glycogen synthase, an important enzyme in glycogen metabolism [1]. The activation of GSK-3β is determined by the phosphorylation and dephosphorylation of GSK-3β on several sites [3,4]. Autophosphorylation at Tyr216 is known to constitutively activate GSK-3β and Tyr216 is also known to be phosphorylated by Src, a non-receptor tyrosine kinase [5,6]. Protein kinase B (AKT), protein kinase C (PKC) and the p90RSK signaling pathways are known to be involved in the phosphorylation of GSK-3β at serine 9 (Ser9) and its regulation [5].
Several transcription factors in the nucleus were identified as being regulated by GSK-3β. Cellular Myc (c-Myc) at threonine 58 (Thr58) and Snail at serine 96 (Ser96) and serine 100 (Ser100) can be phosphorylated by GSK-3β and trigger ubiquitin-dependent proteasomal degradation of c-Myc and Snail [4,[7][8][9]. Inhibition of mTORC1 was shown to stimulate GSK-3β nuclear localization [4,10]. Studies have shown that GSK-3β nuclear localization can be induced by inhibition of mTORC1 using mTORC1 inhibitor rapamycin, resulting in the reduction of c-Myc and Snail [4,10]. Inhibition of GSK-3β is also known to enhance autophagy and lysosomal acidification [11,12]. GSK-3β is known to be involved in neuronal development and suppression of GSK-3β showed the ability to reduce αsynuclein in cellular models of Parkinson's disease [2,[13][14][15]. GSK-3β was also reported to be associated with amyotrophic lateral sclerosis (ALS), elevated levels of active GSK-3β were found in the brain of ALS patients [2]. Inhibition of GSK-3β using lithium was shown to slow down the progression of ALS in ALS patients [2,16].
The mammalian target of rapamycin (mTOR) is a protein kinase comprised of two different complexes, mammalian target of rapamycin complex 1 (mTORC1) and mammalian target of rapamycin complex 2 (mTORC2) [17,18]. mTORC1 and mTORC2 have different components, structure, and functions [17,18]. mTORC1 and mTORC2 also show a difference in sensitivity to rapamycin; mTORC1 is highly responsive to rapamycin compared to mTORC2 which is not responsive to rapamycin [19]. mTORC1 complex contains several proteins that regulate its activity [20]. mTORC1 translocates to the lysosomal surface and is activated in an amino acid-rich environment [20]. Multiple studies have shown that mTORC1 plays a crucial role in the regulation of the autophagy pathway [19,21]. Inhibition of mTORC1 was shown to upregulate autophagy [19,21]. In the absence of amino acids, mTORC1 is translocated from the lysosomal surface to the cytosol [21]. In the presence of amino acids, Rag GTPases are activated, which are involved in the transportation of mTORC1 to the surface of the lysosomes [22,23]. Ragulator activates Rag GTPases in an amino acid-dependent manner and facilitates the interaction of Rag GTPases with Raptor, one of the components of mTORC1 complex [24]. The interaction of Rag GTPase with Raptor can induce mTORC1 transportation from cytosol to lysosomal surface, where Ras homolog enriched in brain (Rheb) switches mTORC1 into an active form [23,24]. The functions of mTORC1 are mainly associated with protein synthesis and autophagy [24]. mTORC1 regulates cellular functions by regulating the expression of downstream targets, ribosomal S6 kinases (S6K) and eukaryotic translation initiation factor 4E (eIF4E]-binding protein 1 (4E-BP1) [24][25][26]. 4E-BP1 inhibits translation initiation via binding to the eukaryotic initiation factor 4E (eIF4E) and prevents the interaction of eIF4E and other eukaryotic initiation factors [24,26]. Activated mTORC1 can phosphorylate 4E-BP1 resulting in the dissociation of 4E-BP1 from eIF4E and interaction of elF4E with elF4G leading to translation initiation [24,26]. Activated mTORC1 also phosphorylates S6K at threonine 389 (Thr389) and activated S6K enhances protein synthesis [27]. Thus, 4E-BP1 and S6K are often described as mTOR downstream mediators. mTORC1 was also reported to regulate mitochondrial function [28]. Studies have shown activation of mTOR in mouse models of mitochondrial myopathy (MM) [29]. MM causes respiratory chain (RC) dysfunction due to mutation of mitochondrial replicative helicase twinkle [29]. mTORC1 has shown to be responsible for several stress responses caused by mitochondrial dysfunction in mouse models of MM [29]. mTORC1 inhibitor rapamycin was shown to alleviate mitochondrial unfolded protein response and other stress responses to reverse the progression of MM [29].
Autophagy is a cellular degradation process that delivers intercellular cargo to the lysosomes [30]. Autophagy can be triggered by cellular stress including nutrient deprivation and hypoxia [31]. Furthermore, autophagy can degrade long-lived proteins and organelles and recycle them to support cell survival [32]. The process of autophagy involves several steps including autophagy induction, autophagosome formation, and the fusion of autophagosome and lysosomes [33]. UNC-51-like kinase (ULK1) is a serine/threonine kinase and was identified as one of the components to initiate the autophagy pathway [34]. The activation of mTORC1 was shown to inhibit ULK1 activity [35]. Inhibition of mTORC1 results in a promotion of ULK1 activity followed by autophagosome formation and induction of the autophagy pathway [34,35]. Autophagosome is a double-membrane vesicle containing intercellular material [36]. In the last steps of the autophagy pathway, autophagosomes fuse with lysosomes to form autolysosomes for the degradation of cellular material that is inside the autophagolysosome [37]. Inhibition of mTORC1 was shown to enhance autophagy pathway [38,39]. Studies have shown that repression of mTORC1 by hayatine, an mTORC1 inhibitor, can enhance autophagy flux in tumor cells [39]. Other studies have shown that activation of mTORC1 by palmitic acid (PA) impaired autophagy flux and repressed lysosomal associated membrane protein-2 (LAMP2) expression in mouse Hepa-1c1c7 cells [40]. Furthermore, mTORC1 inhibition by rapamycin showed an induction of autophagy in C57BL/6J mice fed with an alcohol-containing diet for 8 weeks [40]. mTORC1 hyperactivation was reported in the isolated islet of type 2 diabetes patients and type 2 diabetes animal models [41]. Impaired autophagy is one of the features of type 2 diabetes, mTORC1 hyperactivation induced by large-tumor suppressor 2 (LATS2), suppressed autophagy and further triggered pancreatic β-cell apoptosis [42]. The activation of mTORC1 was shown to be potentially associated with RPE degeneration in the pathogenesis of age-related macular degeneration (AMD) [43][44][45]. A study has shown that upregulation of ULK1 and 4E-BP1 phosphorylation is observed in RPE/choroid tissues from dry AMD patients [43]. These findings also suggest that hyper activation of the mTORC1 pathway is associated with the suppression of autophagy in dry AMD patients [43]. Studies have also shown activated mTORC1 results in RPE degeneration [44,45]. In this review, we will discuss the mechanisms by which the GSK-3 signaling pathway regulates autophagy in an mTORC1-dependent and independent manner.

GSK-3-Mediated Regulation of TFEB
Transcriptional factor EB (TFEB) is identified as an activator of autophagy through the upregulation of autophagy and lysosomal genes [46]. TFEB-regulated downstream genes are involved in different steps of the autophagy pathway, including autophagy initiation, autophagosome formation, and the fusion of autophagosome and lysosome [46]. Furthermore, TFEB in the nucleus activates the expression of genes in the coordinated lysosomal expression and regulation (CLEAR) network to enhance autophagy and lysosomal biogenesis [46,47]. TFEB is a transcription factor belonging to the MITF/TFE family [46]. The subcellular localization of TFEB is regulated by mTORC1 [46]. Activated mTORC1 phosphorylates two residues of TFEB, serine 142 (Ser142) and serine 211 (Ser211), leading to TFEB cytosolic retention and further inhibiting TFEB activation [48]. TFEB in the cytosol can be translocated to the lysosomal surface to be phosphorylated by mTORC1, leading to cytosolic retention of TFEB [49]. The activation of mTORC1 phosphorylates TFEB at Ser211 and promotes TFEB binding to the 14-3-3 protein, which masks the nuclear localization signal (NLS) and prevents TFEB nuclear localization [50]. TFEB can be dephosphorylated by calcineurin, a phosphatase resulting in nuclear translocation of TFEB [51]. TFEB nuclear translocation was shown to be induced by constitutively active calcineurin in cells grown in a nutrient-rich environment [52]. The activation of calcineurin is correlated with the concentration of calcium in the cytosol of cells [53]. Mucolipin 1 (MCOLN1), also known as TRPML1, is a calcium channel on the lysosomal membrane and activation of MCOLN1 triggers calcium release from the lysosomes and enhances the activation of calcineurin [54,55]. mTORC1 phosphorylates MCOLN1 at serine 572 (Ser572) and serine 576 (Ser576) on the lysosomal surface to inactivate MCOLN1 [56]. Nutrient deprivation in cells results in mTORC1 dissociation from the lysosomes and mTORC1 is no longer able to phosphorylate MCOLN1 [52,56]. Activated calcineurin dephosphorylates Ser142 and Ser211 of TFEB and triggers TFEB nuclear translocation ( Figure 1) [55]. serine 576 (Ser576) can be phosphorylated by activated mTORC1 located on the lysosomal surface to suppress MCOLN1 activity [56]. However, the inhibition of mTORC1 via starvation activates MCOLN1 and release calcium from lysosomes [52,57]. The increase in calcium in cytosol activates calcineurin and further leads to calcineurin-mediated dephosphorylation of TFEB. Dephosphorylated TFEB translocates to the nucleus to activate gene expression of CLEAR network genes to promote lysosomal biogenesis and autophagy.
GSK-3β was reported to phosphorylate TFEB at serine 138 (Ser138) and facilitate TFEB nuclear export [60]. Phosphorylation of TFEB at Ser138 by GSK-3 was shown to depend on the activation of mTORC1 [61]. Studies have suggested an involvement of GSK-3β-mTORC1 and ERK signaling pathways in the nuclear export of TFEB [60,61]. mTORC1 and the MAPK extracellular signal-regulated kinase (ERK) were reported to phosphorylate TFEB at Ser142 [60,61]. Phosphorylation of TFEB at Ser142 stimulate GSK-3β-mediated phosphorylation of TFEB at Ser138 [60,61]. Studies have shown that mTORC1 regulates TFEB subcellular localization from the nucleus to cytoplasm in a chromosomal maintenance 1 (CRM1)-dependent manner in HeLa cells [61]. The nuclear export signal (NES) is recognized by CRM1, also known as Exportin-1 (XPO1), which is known to regulate the nuclear export of several proteins including TFEB [62,63]. The inhibition of CRM1 prevents nuclear export of TFEB and enhances TFEB levels in the nucleus and induces autophagy flux in vitro in rat primary cortical neurons and HeLa cells [62,63]. TFEB phosphorylation at Ser142 and Ser138 are required for TFEB translocation from nucleus to cytoplasm [60,61]. These residues are in close proximity to the NES of TFEB and possibly involved in the binding of CRM1 to the NES site of TFEB and facilitate TFEB nuclear export [60,61,64]. Mutation of Ser142 and Ser138 to alanine was shown to suppress the nuclear export of TFEB, leading to TFEB nuclear retention [60,61]. Mutation of Ser138 only affected the ability of GSK-3 to phosphorylate TFEB at Ser138 but not Ser142 [61]. These studies implied that TFEB and CRM1 interaction possibly requires Ser142 and Ser138 phosphorylation, suggesting mTORC1 and GSK-3 affect the interaction of TFEB and CRM1 [61]. Inhibition of mTORC1 by Torin was also shown to abrogate phosphorylation of nuclear TFEB at Ser138 and Ser142 [61]. These suggest that GSK-3 regulates TFEB phosphorylation and subcellular localization in an mTORC1-dependent manner (Figure 2).

Regulation of TFEB by PKC-GSK-3β Signaling Pathway
In addition to mTORC1, GSK-3β is also reported to be regulated by other upstream targets that in turn regulate TFEB nuclear export [65,66]. Protein kinase C (PKC) is serine/threonine protein kinases and was reported to regulate autophagy [67]. The phosphorylation of ULK1 at serine 423 (Ser423) by protein kinase C alpha (PKCα) was shown to prevent ULK1 and Syntaxin 17 (STX17) interaction leading to inhibition of autolysosome formation [67]. Moreover, a study showed that the activation of PKCα and δ can suppress GSK-3β, resulting in repression of TFEB phosphorylation at Ser134 and Ser138 and an increase in TFEB nuclear localization and activation [65]. Furthermore, HEP14 (5β-O-angelate-20-deoxyingenol), a compound that can induce the activity of PKCα and protein kinase C delta (PKCδ), was used to enhance PKCα and PKCδ activity and further triggered TFEB activation [65]. The activation of mTORC1 was not affected by the HEP14 treatment, which indicated that PKCα and δ-mediated TFEB nuclear localization occurs through GSK-3β in an mTORC1-independent manner [65].

Regulation of TFEB by elF4A3-GSK-3β Signaling Pathway
The expression of GSK-3β was downregulated by depletion of eukaryotic translation initiation factor 4A-3 (eIF4A3) [66]. eLF4A3 was identified as a component of the exon junction complex, which has several functions including mRNA export and splicing of mRNA [68,69]. Thus, eIF4A3 also plays a key role in regulating mRNA splicing and mRNA quality control [69]. Reduction of eIF4A3 was shown to induce TFEB nuclear localization and further leads to enhanced autophagy flux [66]. Suppression of eIF4A3 resulted in exon-skipping of GSK-3β, which leads to suppression of GSK-3β activity [66]. Inhibition of GSK-3β has been known to suppress phosphorylation of TFEB [65]. In order to examine whether GSK-3β is involved in the nuclear translocation of TFEB induced by suppression of eIF4A3, cells that overexpressed GSK-3β were co-treated with eIF4A3 siRNA and showed abrogation of TFEB nuclear translocation suggesting the role of GSK-3β in the eIF4A3-TFEB pathway [66].

Regulation of Autophagy by AKT-GSK-3β Signaling Pathway
Protein kinase B (AKT) is known to be involved in several signaling pathways and cellular functions including apoptosis and gene transcription [70]. More than 50 proteins were identified as being regulated by AKT, including mTORC1 and GSK-3 [71,72]. AKT phosphorylation is key to the regulation of AKT activity, for example, GSK-3α phosphorylates AKT at threonine 312 to inactivate AKT [72,73]. The phosphoinositide-dependent protein kinase 1 (PDK1) and mTORC2 were identified to phosphorylate AKT and trigger AKT activity [72]. AKT is also identified to modulate TFEB by phosphorylating TFEB at serine 467 (Ser467), leading to suppression of TFEB nuclear localization [74]. Inhibition of AKT using AKT inhibitor, trehalose and MK-2206, showed induction of TFEB nuclear localization in both WT and lysosomal associated membrane protein-2 (LAMP2) knockout (KO) mouse RPE cells [75]. Furthermore, oral trehalose administration induced the expression of CLEAR genes and stimulated TFEB nuclear localization [74]. Trehalose also showed an increase in the LC3-II/I ratio in WT and LMP2 KO RPE [75]. The induction of autophagy by AKT inhibitor, trehalose, was shown to rescue several disease phenotypes in cell and animal models [76,77]. Exposure to cigarette smoke or hydroquinone, present in cigarette smoke, result in oxidative damage to the RPE [76]. The study shows that oxidative damage induced by hydroquinone can be inhibited by upregulation of TFEB and CLEAR network gene expression by AKT inhibitor, trehalose [76]. Other studies have also shown induction of LC3 II expression in trehalose treatment, suggesting autophagy was activated by trehalose in the acute kidney injury (AKI) mouse model [77]. Additionally, the mitochondrial dysfunction and fragmentation in AKI mice were also shown to be rescued by trehalose [77]. The AKT signaling pathway is known to phosphorylate GSK-3β at Ser9 to inactivate GSK-3β [78]. Interestingly, knockdown of GSK-3β also suppresses AKT and further stimulated autophagy [79]. It was shown that repression of GSK-3β decreased AKT activity and enhanced AMPK activity, leading to the induction of forkhead box protein O1 (FOXO1) [79]. FOXO1 is a transcription factor, which plays an important role in the induction of autophagosome formation in Human Aortic Endothelial Cells (HAECs) [79]. Furthermore, this study showed that the activity of mTORC1 was not altered in GSK-3β knockdown HAECs, suggesting suppression of GSK-3β induces autophagy in an mTORC1-independent manner in specific cell types [79].

GSK-3β-Mediated Regulation of ULK1
ULK1 is one of the downstream targets of mTORC1 and plays a crucial role in the initiation of the autophagy pathway [34,80]. The activation of ULK1 is controlled by phosphorylation and dephosphorylation [81]. ULK1 is part of the ULK1 complex formed with autophagy-related protein 101 (ATG101), autophagy-related protein 13 (ATG13), and focal adhesion kinase family interacting protein of 200 kD (FIP200) in the cells [34,81]. The activation of ULK1 is regulated by mTORC1. mTORC1 phosphorylates ULK1 and suppresses its catalytic activity [34,80,81]. It was reported that mTORC1 phosphorylates ULK1 at serine 757 (Ser757) and inhibits its activity in nutrient-rich conditions [35,38]. The inactive form of mTORC1 in starvation and cellular stress can dissociate from the ULK1 complex [38]. Thus, ULK1 can be dephosphorylated by protein phosphatase 2A (PP2A) and protein phosphatase 1D magnesium-dependent delta isoform (PPM1D) [38,82]. Autophosphorylation at Thr180 of ULK1 triggers ULK1 activation [38,82]. Inhibition of mTORC1 can enhance ULK1 activity to trigger the initiation of autophagy by phosphorylating the autophagyassociated downstream targets of ULK1 including autophagy-related protein 9 (ATG9) and Beclin 1 (BECN1) [83]. ATG9 functions as a transmembrane protein and participates in autophagosome formation [84,85]. An ATG9 vesicle formed by ATG9 is mobilized to the pre-autophagosomal structure (PAS) in starvation conditions and functions as a seed to the growing phagophore (also known as isolation membrane), which can be expanded to form the autophagosome [84,85]. BECN1 forms a class III phosphatidylinositol 3-kinase (PI3K-III) complex by interacting with VPS 34 and other factors for autophagosome formation [86]. Furthermore, it was reported that ULK1 can phosphorylate BECN1 at serine 15 (Ser15) and serine 30 (Ser30) to enhance BECN1 interaction with other autophagy-associated proteins to facilitate autophagosome maturation and autophagosome biogenesis [86]. It was also reported that ULK1 is not only involved in the steps of autophagy initiation but also in the fusion of autophagosome and lysosome [67]. Activated ULK1 interacts with Syntaxin 17 (STX17) and mobilizes STX17 to autophagosomes, which facilities the interaction between STX17 and synaptosomal-associated protein 29 (SNAP29) to form a complex [67]. This STX17 complex is known to be involved in the steps of autophagosome and lysosome fusion [87]. Inhibition of ULK1 by ULK1 inhibitor, ULK-101, was shown to suppress autophagic nucleation and autophagy flux in U2OS cells [88].
Activated GSK-3β phosphorylates ULK1 at serine 405 (Ser405) and serine 415 (Ser415) in GABA Type A Receptor-Associated Protein (GABARAP)-interacting region of ULK1, leading to the induction of autophagy in adult hippocampal neural stem (HCN) cells [89]. In addition to ULK1 phosphorylation directly by GSK-3, GSK-3 was also reported to regulate ULK1 through HIV-1 Tat interactive protein, 60 kD (TIP60), an acetyltransferase [90]. The activation of GSK-3 phosphorylates TIP60 at serine 86 (Ser86) and stimulates the activation of TIP60, which leads to acetylation of ULK1 [90]. The phosphorylation of TIP60 at Ser86 by GSK-3 can be suppressed by GSK-3 inhibitor, suggesting that GSK-3 is an upstream regulator of TIP60 [90]. The endoplasmic reticulum (ER) plays a vital role in protein synthesis and maturation, thus, endoplasmic reticulum (ER) stress is involved in the pathogenesis of several diseases [91]. ER stress was shown to induce autophagy for cell survival under cellular stress such as oxidative stress and hypoxia [91][92][93]. The activation of GSK-3β is reported to be induced by ER stress and further triggers autophagy through the GSK-3β-TIP60-ULK1 pathway [91]. The ER stress inducer, Tunicamycin (TM), was shown to decrease phosphorylation of GSK-3β at serine 9 (Ser9), which activates GSK-3β-mediated phosphorylation of TIP60 at Ser86 [91]. Phosphorylation of TIP60 by GSK-3β, in turn, leads to activation of ULK1 by acetylation resulting in an enhancement of ER stress-induced autophagy activation [91].

GSK-3-Mediated Regulation of the mTORC1 Signaling Pathway
GSK-3 was also reported to function upstream of mTORC1 by directly phosphorylating the regulatory associated protein of mTOR (Raptor) at serine 859 (Ser859) [94]. Inhibition of GSK-3 showed decreased phosphorylation of Raptor on Ser859, which prevents Rap-tor and mTOR interaction, leading to inhibition of mTORC1 [94]. GSK-3 suppression showed the ability to meditate lysosomal acidification through an mTORC1-dependent manner [12]. On the other hand, both GSK-3 inhibition and GSK-3 activation were shown to regulate mTORC1 via tuberous sclerosis complex 2 (TSC2) [12,95,96]. Inhibition of GSK-3 is shown to activate TSC2, a negative regulator of mTORC1, further leading to the repression of mTORC1 [12]. It is also shown that the effect of GSK-3 inhibition on mTORC1 was abolished in TSC knockout MEF cells [12]. TSC2, a GTPase-activating protein (GAP), regulates mTORC1 through Rheb, which converts Rheb-guanosine triphosphate (GTP) into Rheb-guanosine diphosphate (GDP) form to inactivate Rheb, leading to mTORC1 inactivation [97,98]. The inhibition of GSK-3 α/β using the CHIR99021 GSK-3 α/β inhibitor was shown to enhance the ratio of LC3 A/B-II to LC3 A/B-I and effectively decrease p62 expression in epithelioid sarcoma cells [11], suggesting that induction of autophagy occurs in epithelioid sarcoma cells [11]. Moreover, this study also examined the mTORC1 expression and found downregulation of mTOR and p-mTOR expression in CHIR99021 GSK-3 α/β inhibitor treatment [11]. On the other hand, some reports show that activation of GSK-3 phosphorylates and activates TSC2, leading to downregulation of mTORC1 [95,96]. However, TSC2 needs to be phosphorylated at serine 1345 (Ser1345) by AMPK in order for GSK-3 to phosphorylate TSC2 [96].

mTORC1 Regulates Foxk1 through GSK-3
Several studies also show that inhibition of mTORC1 regulates the transcription factor forkhead/winged-helix family k1 (Foxk1) phosphorylation through the GSK-3 signaling pathway [99,100]. Foxk1 was shown to suppress autophagy as a transcriptional repressor [101]. Foxk1 was reported to participate in several cellular mechanisms including cellular metabolism [102]. The translocation of FoxK1 from the cytoplasm to the nucleus was shown to be regulated by mTORC1 [101]. Treatment of mTORC1 inhibitor rapamycin abolished the nuclear translocation of Foxk1 in the presence or absence of insulin treatment in the alpha mouse liver 12 (AML12) cells [100]. Activation of mTORC1 can promote Foxk1 nuclear localization and suppression of autophagy in the nutrient-rich environment [101]. Inhibition of mTORC1 causes phosphorylation of Foxk1 by GSK3 and phosphorylated Foxk1 binds to the 14-3-3 interacting protein resulting in its cytosolic retention, further leading to derepression of autophagy genes [99,103]. Studies have shown that suppression of Foxk1 was shown to upregulate the expression of LC3 II and downregulate the expression of p62 [102]. These results further confirmed that suppression of Foxk1 expression can enhance autophagy in MGC803 and AGS cells [102]. However, the upregulation of Foxk1 phosphorylation by mTORC1 repression can be blocked via inhibition of GSK-3 using GSK-3 inhibitor, CHIR99021 and knockdown of GSK-3α and GSK-3β, suggesting the involvement of the GSK-3 signaling pathway in the regulation of FoxK1 [99].

Calcium Regulates the Activation of GSK-3β
The phosphorylation of GSK-3β is reported to be regulated by intercellular calcium levels [104][105][106]. Induction of intercellular calcium levels by the activation of ion-channel protein transient receptor potential cation channel subfamily V member 4 (TRPV4) triggers GSK-3β phosphorylation and inactivation [105]. Moreover, overexpression of transient receptor potential cation channel subfamily M member 4 (TRPM4) in LNCaP cells promoted GSK-3β phosphorylation at ser9 [106]. Induction of AKT1 phosphorylation and activation was also observed in the LNCaP cells overexpressing TRPM4 [106]. Epidermal growth factor (EGF) stimulates AKT activation through calcium and calmodulin [107]. The induction of AKT1 and GSK-3β phosphorylation by EGF can be suppressed by the inhibition of Ca 2+ /CaM signaling [106]. Furthermore, cells treated with TCN, an AKT inhibitor, showed that the phosphorylation of GSK-3β in EGF treated PC3 cells was reduced [106]. Studies have shown that rotenone, a pesticide associated with α-synuclein aggregation, induces intercellular calcium levels and suppresses AKT and GSK-3β phosphorylation [104,108]. In this study, they showed that BAPTA, a chelator of intracellular calcium, can alleviate the downregulation of AKT and GSK-3β phosphorylation induced by rotenone treatment [104]. Furthermore, rotenone caused impairment of autophagy, which can be prevented by inhibition of GSK-3β [104]. On the other hand, calcium also can regulate GSK-3β activity through calpain; calpain truncates the N-terminal regulatory domain of GSK-3β [109].

Conclusions
In this review, we discussed the mechanisms by which the GSK-3 signaling pathway regulates autophagy. The GSK-3 signaling pathway is implicated in the pathogenesis of several diseases such as neurodegenerative diseases and cancer [73,110]. Autophagy plays a vital role in maintaining cellular homeostasis and cell survival [33]. The GSK-3 signaling pathway was reported to regulate autophagy flux via the mTORC1, PKC and AKT signaling pathways [30,61,65,78,111]. mTORC1, known as a major autophagy regulator, is involved in the phosphorylation and inhibition of GSK-3β via its substrate, S6K [58,59]. GSK-3β also has the ability to regulate mTORC1 by phosphorylating Raptor on Ser859 directly [94]. Studies have also shown that GSK-3 can regulate mTORC1 by modulating the activity of TSC2 [12,95,96]. mTORC1 can not only regulate TFEB activity through GSK-3β-mediated TFEB phosphorylation but also regulate Foxk1 phosphorylation via GSK-3β [99]. ER stress is also known to induce GSK-3β activation and phosphorylate ULK1 leading to induction of autophagy [91]. On the other hand, several studies have also shown that inhibition of GSK-3β upregulates autophagy flux [11,12]. Moreover, GSK-3β can be also regulated by PKC and elF4A3 to trigger TFEB nuclear localization and activation [65,66]. In conclusion, the studies suggest that the GSK-3 signaling pathway regulates autophagy by modulating several signaling pathways in an mTORC1-dependent and independent manner.