ULK1 is a Ser/Thr protein kinase and centrally involved in autophagy. Autophagy is an intracellular degradation process which contributes to the elimination of damaged or long-lived proteins and/or organelles. Autophagy occurs at basal levels in most cell types and ensures cellular homeostasis. However, autophagy can also be actively induced upon stress, including the withdrawal of nutrients or growth factors, treatment with chemotherapeutics, or intracellular infections.
In 1993, Tsukada and Ohsumi reported the isolation and characterization of 15 yeast mutants defective in the accumulation of autophagic bodies in the vacuoles. In a mutant termed apg1 (autophagy), nitrogen starvation did not induce protein degradation, and the apg1 mutant lost viability faster than wild-type cells during nitrogen starvation (Tsukada and Ohsumi 1993). In 1997, Matsuura et al. reported that the APG1 gene encodes a protein kinase, Apg1p. Of note, the authors observed that Apg1p shares significant homology with C. elegans UNC-51 protein (Matsuura et al. 1997).
In 1994, Thumm et al. reported three complementation groups termed aut1-3 (autophagocytosis), which were defective in the autophagic process (Thumm et al. 1994). In 1997, Straub et al. cloned the AUT3 gene and also described that the N-terminal part of Aut3p shows significant homologies to Ser/Thr kinases (Straub et al. 1997).
In 1996, Harding et al. reported that there is a genetic and phenotypic overlap between autophagy and the cytoplasm to vacuole protein targeting pathway. The authors found that AUT3 does not complement the identified CVT10 (Harding et al. 1996).
Finally, three other reports described the identification of GSA10, PAZ1, and PDD7 and analyzed their function in pexophagy, autophagy, micropexophagy, and microautophagy, respectively (Stromhaug et al. 2001; Mukaiyama et al. 2002; Komduur et al. 2003).
In 2003, a unified nomenclature for the so-called autophagy-related genes (ATGs)/proteins (Atgs) was proposed for yeast, and the former APG1/AUT3/CVT10/GSA10/PAZ1/PDD7 gene was designated ATG1 (Klionsky et al. 2003). If referring to humans, ATG and ATG are used for the gene and protein, respectively.
In 1974, Brenner described the unc-51 gene (uncoordinated movement) of the nematode C. elegans (Brenner 1974). Since axonal abnormalities had been observed in all the neurons examined in unc-51 mutants, Ogura et al. speculated that the corresponding gene product plays an important role during the construction of the neural network (Ogura et al. 1994). They found that the unc-51 gene encodes a Ser/Thr kinase whose activity is necessary for axonal elongation.
ULK1 and Autophagy
During autophagy, cytoplasmic cargo is enveloped within a double-membraned vesicle, called autophagosome. The biogenesis of autophagosomes includes different steps, i.e. nucleation, elongation and closure of the double-membraned vesicle. During nucleation, a phagophore is formed which expands and engulfs the cargo to be degraded. Different ATG proteins contribute to the execution of autophagy. These ATGs can be categorized into different modules, e.g. the ULK1 protein kinase complex, the class III PtdIns3K lipid kinase complex, the multi-spanning transmembrane protein ATG9A, or the ubiquitin-like ATG5/ATG12 or ATG8/LC3 conjugation systems. In 2007, Tooze’s group performed an siRNA screen of the kinome and identified ULK1 as an autophagy-modulating kinase (Chan et al. 2007). The authors observed that knockdown of ULK1 in HEK293 cells inhibited the autophagic response following amino acid starvation or mTOR inhibition by rapamycin, respectively. Additionally, they described distinct functional regions of ULK1. Overexpression of kinase-active or kinase-dead murine ULK1 (K46R) inhibited autophagy to a similar extent. Furthermore, truncated versions of kinase-active ULK1 lacking the CTD, the last 50 amino acids or the last three C-terminal amino acids VYA1051 (murine sequence), exhibited an increased dominant-negative effect. These last three C-terminal amino acids represent a PDZ domain-binding motif (named according to the first three proteins discovered to share this domain, i.e., PSD95, Dlg1, zo-1). The authors were able to map down the minimal region necessary for the dominant-negative effect to aa 1–351 of ULK1, encompassing the entire kinase domain (aa 1–278) and a region containing autophosphorylation sites (Yan et al. 1998; Chan et al. 2007). In contrast, the kinase domain alone did not display dominant-negative effects.
Subsequently, Mizushima’s group reported that ULK1 and ULK2 co-localize with ATG16L1 and are accordingly targeted to the phagophore (Hara et al. 2008). Interestingly, these ULK1/ULK2 puncta could not be detected in Atg5-/- MEFs. However, this observation was later refined by the same group. It appears that ATG5 is not required for the recruitment of ULK1 to the phagophore but possibly important for keeping ULK1 at the membrane (Itakura and Mizushima 2010). Mizushima’s group also analyzed the importance of ULK1 kinase activity. They observed that overexpression of kinase-dead ULK1 (K46N) functions as a dominant-negative mutant (Hara et al. 2008). In contrast, overexpression of wild-type ULK1 led to morphologic alterations of the cell and was cytotoxic (Hara et al. 2008). These results are similar to observations made upon UNC-51/ATG1 overexpression in D. melanogaster, which results in apoptotic cell death (Scott et al. 2007).
In 2009, a work by Tooze’s group reassessed the dominant-negative effect of a different ULK1 kinase-dead version, K46I (Chan et al. 2009). The K46I mutant exhibited a stronger dominant-negative effect compared to wt ULK1 or the K46R mutant described above. Apparently, the kinase activity of ULK1 has to be significantly reduced to transform ULK1 into a dominant-negative protein (Chan et al. 2009). Accordingly, autophosphorylation was severely impaired in the K46I version. The dominant-negative potency of the kinase-dead ULK1 was unaltered when the PDZ-domain-binding motif was deleted but depended on a 7-residue motif within the CTD (aa sequence: I1038ERRLSA1044 in murine ULK1) (Chan et al. 2009). Collectively, these data led the authors to propose a model in which the reduced autophosphorylation of kinase-dead ULK1 causes a conformational change resulting in the exposure of the dominant-negatively acting CTD. This model was supported by the observation that overexpression of the CTD alone resulted in a strong dominant-negative effect, which was also dependent on the IERRLSA motif. Of note, the authors also found that the CTD domain of ULK1 mediates binding to membranes and ATG13 (see below) depending on aa 864–1001 or aa 829–1001, respectively, indicating that membrane association and ATG13 interaction are not critically involved in dominant-negative activity and that the IERRLSA motif might interact with additional autophagy-regulating proteins (Chan et al. 2009).
In contrast to ULK1 knockdown, silencing of ULK2 did not affect autophagy in HEK293 cells (Chan et al. 2007). This indicates that in this cellular system, the two kinases cannot compensate each other. However, compensatory roles of ULK1 and ULK2 can be deduced from the corresponding knockout mouse models. Both Ulk1-/- and Ulk2-/- mice are viable and do not show an overt autophagy phenotype (Kundu et al. 2008; Cheong et al. 2011; Lee and Tournier 2011). Interestingly, Ulk1-/- mice revealed delayed mitochondrial and ribosomal clearance during the final stages of erythroid maturation, indicating some ULK1-specific functions during selective autophagy processes which cannot be overtaken by ULK2 (Kundu et al. 2008). In contrast to the single knockout mouse models, ULK1/ULK2 double-deficient mice die shortly after birth, similar to mice deficient for other ATG proteins (Cheong et al. 2011, 2014). Furthermore, amino acid starvation-induced autophagy is blocked in MEFs of these double-deficient mice (Cheong et al. 2011; McAlpine et al. 2013). Of note, the dependency of glucose starvation-induced autophagy on ULK1/ULK2 was controversially reported (Cheong et al. 2011; McAlpine et al. 2013).
ULK1 has been implicated in the organization of the autophagosome formation site. For example, Karanasios et al. recently reported that nucleation occurs in regions where the ULK1 complex coalesces with the ER and the ATG9 compartment (Karanasios et al. 2016). Additionally, it has been reported that C9orf72 controls the initiation of autophagy by regulating the Rab1a-dependent recruitment of the ULK1 complex to the phagophore (Webster et al. 2016).
The ULK1 Complex
Generally, the autophagy-initiating kinase ULK1 functions within a protein complex. The other core components of this complex are ATG13, focal adhesion kinase family-interacting protein of 200 kDa (FIP200; alternatively termed RB1-inducible coiled-coil 1, RB1CC1), and ATG101.
In 2008, Hara et al. reported that FIP200 represents a ULK1-binding protein, which is involved in the regulation of diverse cellular functions (Hara et al. 2008). The authors found that the CTD of ULK1 is required for its association with FIP200 and that FIP200 – like ULK1 – localizes to the phagophore upon starvation. Finally, they described that autophagy was inhibited in FIP200-deficient cells and that both stability and phosphorylation of ULK1 were impaired in these cells.
In 2007, Meijer et al. proposed that the human protein KIAA0652 represents the ortholog of yeast Atg13 (Meijer et al. 2007). Chan et al. showed that an siRNA-mediated knockdown of ATG13 inhibited the formation of starvation-induced GFP-LC3 puncta similar to the ULK1 knockdown (Chan et al. 2009). As described above, the authors could map the ATG13-binding site within ULK1 to aa 829–1001. Furthermore, they observed that ATG13 becomes phosphorylated by ULK1 and that ectopic expression of ATG13 cannot rescue inhibition of autophagy caused by dominant-negative ULK1 variants (Chan et al. 2009).
In 2009, three groups independently reported mechanistic details how the ULK1-ATG13-FIP200 complex mediates mTOR signaling (see below) to the autophagic machinery (Ganley et al. 2009; Hosokawa et al. 2009a; Jung et al. 2009). Like Chan et al., Kim’s group mapped ATG13-binding sites on ULK1 and ULK2 to C-terminal regions containing residues 829–1051 and 651–1036, respectively (Jung et al. 2009). Vice versa, they observed that the last 75 amino acids of ATG13 are required for binding of ULK1/ULK2 and the C-terminal residues 384–517 of ATG13 are required for binding of ULK1/ULK2 and FIP200. Our group fine mapped the ULK1- and FIP200-binding regions of ATG13. It appears that the last three amino acids of ATG13 are required for ULK1 binding and that the residues 348–373 (human sequence, isoform 2) mediate FIP200 binding [(Hieke et al. 2015) and unpublished results]. Generally it appears that the interaction between ULK1 and FIP200 depends on ATG13, but one group also reported the direct association of ULK1 with FIP200 (Ganley et al. 2009). As determined by size exclusion chromatography, Mizushima’s group reported that ULK1, ATG13, and FIP200 are included in a large protein complex of an apparent molecular weight of ∼3 MDa, and this complex was not sensitive to starvation. Using recombinant proteins, Jiang’s group observed a protein complex >1 MDa. All three proteins localize to the phagophore upon autophagy induction. Both ATG13 and FIP200 are required for maximal ULK1 kinase activity, ULK1 stability, and ULK1 localization to the phagophore (Ganley et al. 2009; Hosokawa et al. 2009a; Jung et al. 2009). In turn, ATG13 and FIP200 are substrates for ULK1. Finally, all three groups reported that ULK1 and ATG13 are phosphorylated by mTOR (Ganley et al. 2009; Hosokawa et al. 2009a; Jung et al. 2009). Along these lines, Hosokawa et al. reported that mTOR complex 1 (mTORC1) associates with the ULK1 complex under nutrient-rich conditions and dissociates under starvation conditions. Collectively, these observations led to the following model: under nutrient-rich conditions, mTORC1 associates with the ULK1 complex and phosphorylates ULK1 and ATG13 at inhibitory sites. Under starvation conditions, mTORC1 dissociates, and the mTOR-dependent inhibitory sites become dephosphorylated. Subsequently, active ULK1 autophosphorylates and transphosphorylates both ATG13 and FIP200, thus initiating autophagosome formation.
In 2011, two groups reported the identification and characterization of the fourth core component of the ULK1 complex. Since this protein has no obvious homolog in yeast, it was named ATG101 (Hosokawa et al. 2009b; Mercer et al. 2009). Both groups reported that (1) ATG101 is most likely recruited to the ULK1 complex through ATG13, (2) suppression of ATG101 expression leads to decreased autophagy, and (3) ATG101 is required for ATG13 stability (Hosokawa et al. 2009b; Mercer et al. 2009).
Upstream Regulation of ULK1
ULK1 phosphorylation at Ser556, Ser638, and Ser758
Enhanced phosphorylation by AMPK-activating compounds
4x Ser-to-Ala mutant (Ser467, Ser555, Thr574, Ser637 in mouse ULK1) does not support autophagy
Egan et al. (2011)
Phosphorylated under nutrient-rich conditions and at 0.5 h of starvation important for enhanced binding of ULK1 to GST-14-3-3ζ upon AMPK activation
Mack et al. (2012)
Phosphorylated by AMPK in vitro
Mutation of this site to Ala abrogates 14-3-3 binding to ULK1
Bach et al. (2011)
Important for translocation of ULK1 to mitochondria and for mitophagy
Tian et al. (2015)
Dephosphorylated upon starvation
Shang et al. (2011)
Enhanced phosphorylation by AMPK-activating compounds
4x Ser-to-Ala mutant (Ser467, Ser555, Thr574, Ser637 in mouse ULK1) does not support autophagy
Egan et al. (2011)
Phosphorylated under nutrient-rich conditions and at 0.5 h or 2 h of starvation
Phosphorylated upon treatment with 2-deoxyglucose predominant AMPK-dependent site in vitro and in vivo
Mack et al. (2012)
Dephosphorylated upon starvation
Faster dephosphorylated than Ser758
Phosphorylated by mTOR and phosphorylation is maintained by AMPK
Shang et al. (2011)
Phosphorylated under nutrient-rich conditions
Phosphorylation disrupts ULK1-AMPK interaction
Kim et al. (2011)
Dephosphorylated upon starvation
Phosphorylation required for ULK1-AMPK interaction
Shang et al. (2011)
Identified under nutrient-rich conditions
Dorsey et al. (2009)
Phosphorylated under nutrient-rich conditions and at 2 h of starvation
Mack et al. (2012)
Next to phosphorylation, other posttranslational modifications have been shown to regulate ULK1 activity, e.g., acetylation and ubiquitination. Lin et al. found that glycogen synthase kinase-3 (GSK3) activates acetyltransferase TIP60, which in turn directly acetylates and stimulates ULK1 (Lin et al. 2012). Several groups characterized ULK1 ubiquitination. In 2007, Zhou et al. reported that nerve growth factor can induce the interaction of ULK1 with TrkA receptor complexes through promoting K63-polyubiquitination of ULK1 and binding of ULK1 to p62/SQSTM1 (Zhou et al. 2007). Our group observed that deubiquitinase inhibition results in ULK1 aggregation and blockade of autophagy (Driessen et al. 2015). Jiao et al. reported that p32 depletion leads to increased K48- but decreased K63-polyubiquitination, ultimately resulting in proteasome-mediated degradation of ULK1 (Jiao et al. 2015). So far, several E3 ubiquitin ligases have been implicated in ULK1 ubiquitination, including TRAF6, MUL1, and Cul3-KLHL20 (Nazio et al. 2013; Li et al. 2015; Liu et al. 2016).
As mentioned above, ULK1 autophosphorylates and transphosphorylates the associated proteins ATG13, FIP200, and ATG101 (Hara et al. 2008; Chan et al. 2009; Dorsey et al. 2009; Ganley et al. 2009; Hosokawa et al. 2009a; Jung et al. 2009; Alers et al. 2011; Bach et al. 2011; Joo et al. 2011; Egan et al. 2015; Lazarus et al. 2015). So far, only the ULK1 autophosphorylation at Thr180 and Ser1047 and the phosphorylation of ATG13 at Ser318 (human isoform 2) have been shown to be functionally relevant. Lazarus et al. suggested that Thr180 is an autophosphorylation site, and Bach et al. reported that this site is required for kinase activity (Bach et al. 2011; Lazarus et al. 2015). Autophosphorylation at ULK1 Ser1047 (murine sequence) was observed by Dorsey et al., and the authors speculated that autophosphorylation at Ser1047 is required for efficient phosphorylation of the putative PKA site Ser1043, which is located within the 7-residue motif I1038ERRLSA1044 described above (Dorsey et al. 2009). Joo et al. reported that ATG13 Ser318 phosphorylation by ULK1 is required for efficient clearance of damages mitochondria (Joo et al. 2011). The functional relevance of ULK1-dependent phosphorylation of FIP200 and ATG101 awaits further clarification.
The phosphatidylinositol 3-phosphate (PI3P)-generating VPS34/Beclin 1 complex is also centrally involved in the initiation of autophagy, since PI3P recruits further downstream effectors such as DFCP1 and proteins of the Atg18/WIPI-family. With regard to the components of the VPS34/Beclin 1 complex, several ULK1-dependent phospho-acceptor sites have been identified, including VPS34, Beclin 1, ATG14, and AMBRA1. Di Bartolomeo et al. reported that ULK1 phosphorylates AMBRA1 and thus releases the VPS34/Beclin 1 complex from dynein (Di Bartolomeo et al. 2010). The subsequent relocalization of the VPS34/Beclin 1 complex to the endoplasmic reticulum enables autophagosome nucleation. Russell et al. reported that the ULK1-dependent phosphorylation of Beclin 1 at Ser15 (human sequence) enhances the activity of the VPS34 complex (Russell et al. 2013). Similarly, Park et al. observed that ULK1-dependent phosphorylation of ATG14 at Ser29 increases the activity of the VPS34 complex (Park et al. 2016). Egan et al. reported that also the catalytic subunit VPS34 is phosphorylated at Ser249 by ULK1 (Egan et al. 2015). In contrast to AMBRA1, Beclin 1, and ATG14, the functional relevance of ULK1-dependent VPS34 phosphorylation remains unclear so far.
Next to the two autophagy-initiating complexes, several additional autophagy-relevant proteins have been identified as ULK1 substrates, including Raptor, AMPK, p62/SQSTM1, FUNDC1, DAPK3/ZIPK, FLCN, and enzymes involved in glucose metabolic fluxes (Dunlop et al. 2011; Löffler et al. 2011; Tang et al. 2011; Dunlop et al. 2014; Wu et al. 2014; Lim et al. 2015; Li et al. 2016b). It has been demonstrated that ULK1 phosphorylates Raptor and all three subunits of AMPK. These phosphorylations mediate negative feedback loops toward the ULK1-regulating kinases MTOR and AMPK, respectively (Dunlop et al. 2011; Löffler et al. 2011). Additionally, the autophagy receptor p62/SQSTM1 and the mitophagy receptor FUNDC1 have been identified as ULK1 substrates (Wu et al. 2014; Lim et al. 2015). Recently, Li et al. could show that ULK1 phosphorylates the glycolytic enzymes hexokinase, phosphofructokinase 1, and enolase 1, and the gluconeogenic enzyme fructose-1,6-bisphosphatase upon nutritional deprivation (Li et al. 2016b). Collectively, these phosphorylations result in sustained glucose uptake, inhibited gluconeogenesis, and increased glucose flux to pentose phosphate way. Apparently, ULK1 is a bifurcate signaling node controlling both autophagy and glucose metabolic fluxes upon nutritional stress (Li et al. 2016b). Furthermore, in yeast several other Atg proteins have been identified as Atg1 substrates, e.g., Atg2, Atg8, Atg9, and Atg18 (Ptacek et al. 2005; Papinski et al. 2014). However, it awaits further clarification whether these phosphorylation events can be transferred to the mammalian system.
Finally, there are several ULK1 substrates with no or at least no apparent function in autophagy. These include Syntenin-1, MAPK p38α, STING, and SEC16A and are discussed below (Rajesh et al. 2011; Konno et al. 2013; Saleiro et al. 2015; Joo et al. 2016).
Non-autophagic Functions of ULK1
Early reports of ULK1 function have assigned a role of this kinase for neuronal development. As described above, ULK1 was named according to its homology to C. elegans unc-51 (Kuroyanagi et al. 1998; Yan et al. 1998). Mutations in the unc-51 gene of C. elegans result in various abnormalities in axonal elongation and axonal structures (Ogura et al. 1994), and ultrastructural analyses revealed significant enlargements in axon diameter as well as abnormal vesicles and cisternae-like structures within axons of unc-51 and unc-14 mutants (McIntire et al. 1992). In subsequent studies, it was reported that UNC-14 protein interacts with UNC-51 (Ogura et al. 1997) and that VAB-8 and UNC-14 are substrates for UNC-51 and mediate its function in axon outgrowth (Lai and Garriga 2004). In C. elegans, the gene vab-8 encodes a protein with kinesin motor similarity (Wolf et al. 1998), and UNC-14 is a RUN domain protein that binds to the kinesin light chain protein 2 (KLC2) and regulates synaptic vesicle localization (Sakamoto et al. 2005). Furthermore, it was shown that UNC-51 and UNC-14 regulate the subcellular localization of UNC-5, which is the receptor for the guidance protein Netrin/UNC-6 (Ogura and Goshima 2006).
UNC-51/ATG1 has been implicated in axonal vesicle transport processes also in D. melanogaster. Toda et al. reported that UNC-51/ATG1 regulates the interaction between synaptic vesicles and motor complexes. UNC-51/ATG1 phosphorylates UNC-76, a kinesin heavy chain adaptor protein, and then phosphorylated UNC-76 binds Synaptotagmin-1, a synaptic vesicle protein (Toda et al. 2008). Mochizuki et al. demonstrated that Unc-51/ATG1-mediated kinesin-dependent vesicle transport is important for the targeted localization of guidance molecules and organelles in developing neurons (Mochizuki et al. 2011).
With regard to mammalian ULK1, Tomoda et al. reported that ULK1 functions in the formation of granule cell axons (Tomoda et al. 1999). Okazaki et al. observed that ULK1 interacts with the ATG8 isoform GABARAP, and the authors suggested that this interaction is important for vesicle transport and axonal elongation in mammalian neurons (Okazaki et al. 2000). Two other ULK1-interacting proteins important for axon extension were identified by Tomoda et al. in 2004, i.e., SynGAP and Syntenin-1 (Tomoda et al. 2004). SynGAP was shown to bind via its C-terminus to the C-terminal domain of ULK1, whereas the PDZ domains of Syntenin-1 presumably bind the C-terminal PDZ-binding consensus motif VYA1051 of ULK1. The authors suggest that ULK1 downregulates the GTPase-activating protein (GAP) activity of SynGAP, which normally attenuates Ras and Rab5 signaling. This allows Ras and Rab5 to function in axon formation, partially through Rab5-mediated endocytic pathways (Tomoda et al. 2004). Syntenin-1 in turn was suggested as scaffold, bringing together ULK1/SynGAP and Rab5 (Tomoda et al. 2004). Rajesh et al. demonstrated that ULK1 phosphorylates Syntenin-1 and thus prevents the interaction of Syntenin-1 with ubiquitinated proteins (Rajesh et al. 2011).
A connection between ULK1-regulated axon elongation and endocytic processes was also suggested by Zhou et al. (Zhou et al. 2007). They observed that knockdown of ULK1 resulted in reduced endocytosis of nerve growth factor (NGF), excessive axon arborization, and reduced axon elongation. Additionally, they showed that NGF induces the interaction with TrkA receptor complexes via ULK1 polyubiquitination and p62/SQSTM1 binding. The authors proposed that the above-described interactions between ULK1 and SynGAP/Syntenin-1 allow ULK1 to traffic NGF-bound TrkA receptors into endocytic compartments, thereby suppressing excessive filopodia extension and axon branching (Zhou et al. 2007).
Collectively, it appears that ULK1 regulates axon elongation/guidance, presumably via the regulation of endocytosis and/or synaptic vesicle trafficking processes. However, it remains unclear whether autophagy signaling is involved in the processes described above. Of note, the uncoordinated phenotype of unc-51 mutants is not phenocopied by epg-1 (atg13) or epg-9 (atg101) mutants (Tian et al. 2009; Liang et al. 2012). Nevertheless, it is well established that autophagy is important for cellular homeostasis in neurons, and the disruption of autophagy has been associated with the pathogenesis of neurodegeneration (Hara et al. 2006; Komatsu et al. 2006; Liang et al. 2010). For example, the neural-specific deletion of FIP200 results in cerebellar degeneration caused by increased neuronal loss and axon degeneration (Liang et al. 2010). The authors also observed increased p62/SQSTM1 accumulation in the cerebellum and reduced autophagosome formation in mice with a neural-specific deletion of the Fip200 gene. These observations suggest a role for FIP200 in the regulation of neuronal homeostasis mainly through its function in autophagy (Liang et al. 2010). Another interesting observation was made by Wang et al. (Wang et al. 2013). They observed that FIP200 is required for maintenance and differentiation of postnatal neural stem cells (NSCs). The loss of NSCs was accompanied by an increase in ROS, indicating that FIP200-dependent autophagy contributes to the maintenance of NSCs through the regulation of the oxidative state. Noteworthy, embryonic NSCs were not affected by FIP200 deletion (Wang et al. 2013). Finally, a strong case for a non-canonical function of ULK1 in the brain was recently made by Joo et al. (Joo et al. 2016). They also used a conditional knockout strategy in order to analyze the function of ULK1/ULK2 in the brain. The corresponding mice showed neuronal degeneration, but this was not associated with p62/SQSTM1 accumulation or abnormal membranous structures (Joo et al. 2016). Instead, the authors observed that neuronal death was associated with the activation of the unfolded protein response (UPR) pathway (Joo et al. 2016). Additionally, they found that ULK1 phosphorylates SEC16A, and this phosphorylation regulated the assembly of ER exit sites (ERES) and ER-to-Golgi trafficking (Joo et al. 2016). In the absence of ULK1, these trafficking defects activated the UPR pathway. Since this ULK1-dependent regulation of ER-to-Golgi trafficking might be conserved in C. elegans and did not require ATG13, the authors speculated that these trafficking alterations might contribute to the uncoordinated phenotype in unc-51 mutants (Joo et al. 2016).
Next to its function during neuronal development, there are indications that ULK1 regulates interferon signaling. The stimulator of interferon genes (STING) is activated by cytosolic DNA species as well as cyclic dinucleotides. Activated STING associated with TBK1 traffics to endosomal/lysosomal compartments and activates the transcription factors interferon regulatory factor 3 (IRF3) and NF-κB. Konno et al. found that STING is phosphorylated by ULK1 and that STING-dependent IRF3 activation is suppressed (Konno et al. 2013). Of note, ULK1 activation occurred following its disassociation from AMPK and was induced by cyclic dinucleotides generated by cGAMP synthase, cGAS. Accordingly, it appears that cyclic dinucleotides both initiate STING function and trigger a negative feedback loop (Konno et al. 2013). Saleiro et al. demonstrated that ULK1 mediates type I IFN-inducible activation of mitogen-activated protein kinase (MAPK) p38α either directly or via intermediate kinases (Saleiro et al. 2015). This in turn leads to the subsequent transcriptional activation of IFN-regulated genes. Collectively, ULK1 appears to control both IFN production and IFN-dependent biological responses (Konno et al. 2013; Saleiro et al. 2015).
ULK1 also regulates cell death and proliferation processes independent of its autophagic function. Joshi et al. reported that ULK1 sensitizes cells to H2O2-induced necrotic cell death (Joshi et al. 2015). They observed that ULK1 localizes to the nucleus and enhances the activity of the poly(ADP-ribose) polymerase 1 (PARP1) in a kinase-dependent manner. Thus, ULK1 contributes to ATP depletion and H2O2-induced cell death (Joshi et al. 2015). Li et al. observed that UNC-51/ATG1 in Drosophila is involved in apoptosis-induced compensatory proliferation in eye and wing imaginal discs (Li et al. 2016a).
ULK1 as Therapeutic Target
Generally, autophagy and its dysregulation have been implicated in several diseases or pathophysiological processes, such as cancer, neurodegenerative diseases, or infections. Accordingly, the modulation of autophagic pathways might be a promising therapeutic intervention. With regard to cancer, high ULK1 expression levels have been suggested as biomarker in predicting poor prognosis in colorectal cancer, nasopharyngeal carcinoma, and hepatocellular carcinoma (Xu et al. 2013; Yun et al. 2015; Zou et al. 2015). Additionally, high expression of ULK1 is associated with shorter overall survival in patients with metastatic prostate cancer after androgen deprivation therapy (Zhang et al. 2016). In contrast, low ULK1 expression is associated with operable breast cancer progression and is an adverse prognostic marker of survival for patients (Tang et al. 2012). Similarly, low expressions of ULK1 were correlated with lymph node metastasis, advanced TNM stage, and poor prognosis in gastric cancer (Cao et al. 2016) and with lymph node metastasis and poor survival in esophageal squamous cell carcinoma patients (Jiang et al. 2014). Accordingly, it appears that ULK1 expression levels and/or activity might be differentially regulated in different cancer types or progression of disease.
Since ULK1 is a Ser/Thr protein kinase, it represents an attractive druggable target. To date, some ULK1-specific inhibitors have been reported, e.g., aminoquinazoline or aminopyrimidine class of compounds, MRT67307 and MRT68921, or SBI-0206965 (Egan et al. 2015; Lazarus et al. 2015; Lazarus and Shokat 2015; Petherick et al. 2015). In 2015, Lazarus et al. reported the first structure of the mammalian ULK1 kinase domain (Lazarus et al. 2015). Next to the standard kinase fold, ULK1 possesses several unique features, including a large loop between the N- and C-terminal lobes, an autophosphorylation site at Thr180, and a large basic patch on the opposite side of the ATP binding site (Lazarus et al. 2015). It can be anticipated that additional ULK1 kinase inhibitors will be discovered in the future. Additionally, the protein-protein interactions within the ULK1 complex represent another attractive target for the modulation of autophagic processes.
ULK1 is a Ser/Thr protein kinase centrally involved in the initiation of autophagy. It is associated with ATG13, FIP200, and ATG101 in order to form the ULK1 complex. ULK1 is regulated by a network of nutrient- and energy-sensing kinases such as mTOR or AMPK. In turn, several downstream substrates of ULK1 have been identified and characterized, including several components of the ULK1- and VPS34/Beclin 1 complex. Next to its central function in autophagy signaling, non-canonical substrates and functions of ULK1 have been merged, including neuronal development, interferon signaling, and cell death. As a protein kinase, ULK1 represents a suitable druggable target, and future therapeutic interventions may rely on the modification of ULK1 function/activity.
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