Atg13 Binds ULK1, ULK2, and FIP200
From the genome database, the human gene KIAA0652 was predicted to encode a homologue of yeast ATG13
(Meijer et al., 2007
). We cloned its cDNA encoding 517 amino acids and tested if the protein encoded by the gene can interact with ULK1 and ULK2. Using recombinant constructs, we determined that Atg13 is coimmunoprecipitated with ULK1 or ULK2 but not with control proteins (, a–c). We generated polyclonal antibodies specific to the Atg13 N-terminus or its full length. Using the antibodies, we confirmed that endogenous ULK1 and Atg13 interact with each other (d and Supplemental Figure S1). We also observed that ULK2 is coimmunoprecipitated with Atg13, although the affinity of the interaction between Atg13 and ULK2 is weaker than the interaction between Atg13 and ULK1 (b).
Figure 1. Atg13 interacts with ULK1, ULK2, and FIP200. (a) ULK1 and ULK2 are coimmunoprecipitated with Atg13. HA-tagged ULK1 was expressed with myc-tagged Atg13 or control proteins (S6K1 and tubulin) in 293T cells. Anti-myc immune complexes were assessed for the (more ...)
Knowing that Atg13 interacts with ULK1 and ULK2, we wondered if Atg13 interacts with FIP200, which has been shown to interact with ULK1 and ULK2 (Hara et al., 2008
). FIP200 was detected in immune complexes containing Atg13 but not in control immune complexes (e). Furthermore, the amount of FIP200 in Atg13 immune complexes was higher than those in ULK1 and ULK2 immune complexes, supporting that Atg13 might bind FIP200 more tightly than ULK1 and ULK2 (f). It was noteworthy that coexpression with either ULK1 or ULK2 induced upward shifts of Atg13 and FIP200 bands on SDS-PAGE. This supports that ULK1 and ULK2 likely induce phosphorylation of Atg13 and FIP200 (, b and f).
Atg13 Directly Interacts with ULK1 and Mediates the Interaction between ULKs and FIP200
A polyclonal antibody generated against the full length of Atg13 allowed us to detect several isoforms in human cell lines (Supplemental Figure S1). We searched for Atg13 cDNA sequences that are available in the NCBI database and found several splicing variants. We cloned three of the isoforms and tested their interaction with ULKs. We found that deletion of the C-terminal 75 amino acids disabled the ability of Atg13 to bind ULK1 and ULK2 (, a and b, and Supplemental Figure S1). Consistent with an important role of the C-terminal residues of Atg13 for ULK binding, ULK1-, ULK2-, and FIP200-binding regions on Atg13 were all mapped to C-terminal residues 384-517 (c). Atg13-binding sites on ULK1 and ULK2 were mapped to C-terminal regions containing residues 829-1051 and 651-1036, respectively (d).
Figure 2. Atg13 interacts with ULK C-terminus and mediates the interaction between ULKs and FIP200. (a and b) Atg13 isoforms 1 and 2, but not isoform 3, bind ULK1 and ULK2. Myc-tagged Atg13 isoforms were expressed alone (a) or with HA-ULK2 (b) in 293T cells, and (more ...)
We next questioned if the interactions are direct. To determine if Atg13 can directly bind ULK1 or ULK2, we purified the full-length Atg13 and incubated it with a fragment of ULK1 containing residues 651-1051 or a fragment of ULK2 containing residues 651-1036. The C-terminal fragment of ULK1, but neither the control protein GST nor the ULK2 fragment, were bound to Atg13 (e), validating that the interaction between ULK1 and Atg13 is direct but the interaction between Atg13 and ULK2 might require other cellular factors.
To determine if Atg13, ULK1, and ULK2 directly bind FIP200, we prepared cell lysate from 293T cells expressing myc-tagged FIP200 and incubated it with GST-tagged Atg13, ULK1 (651-1051), and ULK2 (651-1036). Myc-FIP200 was pulled down only with GST-Atg13 but neither with ULK1 nor ULK2 constructs (f). However, coincubation with purified Atg13 allowed GST-ULK1 (651-1051) and GST-ULK2 (651-1036) to be pulled down with FIP200, supporting that Atg13 is required for the interaction between FIP200 and ULKs (f). Consistent with the important role of Atg13 for the interaction between FIP200 and ULK1, silencing of Atg13 in 293T cells reduced the amount of FIP200 bound to ULK1 (g). These results confirm that Atg13 directly binds FIP200 and mediates the interaction between FIP200 and ULKs. We also found that Atg13 is pulled down with GST-ULK2 (651-1036) when myc-tagged FIP200 is added to the incubation with Atg13 (f), indicating that the stable interaction between Atg13 and ULK2 requires FIP200.
Atg13, ULK1, and ULK2 Are Important for Autophagosome Formation
Knowing that Atg13 interacts with ULK1 and ULK2, we questioned if Atg13 is involved in the regulation of autophagy induction. First, we knocked down Atg13, ULK1, or ULK2 in HEK293T cells through transduction using a lentiviral shRNA and conducted the autophagy flux assay by comparing the levels of the lipid modified form of LC3 (microtubule-associated protein 1 light chain 3), LC3-II, in the absence and presence of lysosomal inhibitors such as pepstatin A and E-64 (Mizushima and Yoshimori, 2007
). Rapamycin, in combination with the lysosomal inhibitors, induced an increase of LC3-II levels in scrambled control shRNA cells, indicating an accumulation of LC3-II due to the inhibition of LC3-II degradation in lysosomes (, a and b). On the other hand, the accumulation was significantly suppressed in Atg13- or ULK2-silenced cells or ULK1-deficient MEF cells. This result suggests that disruption of the ULK-Atg13 complex suppresses the autophagic flux of LC3-II, thereby inhibiting LC3-II accumulation in autophagosomes. To further clarify the effects of Atg13, ULK1, or ULK2 silencing on autophagy induction, we monitored the amounts of p62, a protein that is degraded by autophagy (Mizushima and Yoshimori, 2007
). Supporting the important roles of Atg13, ULK1, and ULK2 in autophagic degradation of p62, rapamycin induced p62 degradation in scrambled control cells, whereas the degradation was suppressed in Atg13-, ULK1-, and ULK2-silenced cells (c).
Figure 3. Atg13, ULK1, and ULK2 are important for autophagy. (a) Knockdown of Atg13 or ULK2 or knockout of ULK1 inhibits rapamycin-induced autophagic flux of LC3-II. HEK293T cells stably transduced by lentiviral shRNAs or MEF cells were treated with rapamycin (100 (more ...)
Lastly, to confirm that Atg13, ULK1, and ULK2 are involved in the regulation of autophagy, we analyzed the rapamycin-induced autophagosome formation. HeLa cells transduced by Atg13 or ULK1 shRNA exhibited a reduction in the formation of autophagosomes decorated with LC3 after rapamycin treatment relative to control cells transduced with scrambled shRNA (d and Supplemental Figure S2). About 19% of Atg13-silenced cells and 18% of ULK1-silenced cells exhibited the LC3-positive autophagosome structures compared with 44% of control cells (e). Rapamycin increased the number of LC3-positive cells by 2.0-fold for Atg13- and 2.7-fold for ULK1-silenced cells relative to 7.1-fold for scrambled cells. Similarly, ULK2 silencing reduced the number of cells having LC3-positive autophagosome structures from 73 to 37% at 2 h and from 42 to 28% at 5 h of rapamycin treatment (, f and g). Rapamycin increased the number of LC3-positive cells by only 2.3- and 1.3-fold in ULK2-silenced cells relative to 17.5- and 3.2-fold in scrambled cells for 2 and 5 h, respectively.
Another noticeable change was an increase in S6K1 phosphorylation at Thr389 in Atg13-, ULK1-, or ULK2-silenced cells (c and Supplemental Figure S2). This result agrees with the previous findings that overexpression of Atg1 in Drosophila
fat body has a negative effect on S6K1 phosphorylation and knockdown of ULK1 or ULK2 in mammalian cells increases S6K1 phosphorylation (Lee et al., 2007
; Scott et al., 2007
). The up-regulation of S6K1 phosphorylation by Atg13, ULK1, or ULK2 knockdown occurred in both 293T and HeLa cells (c and Supplemental Figure S2). This result suggests that the ULK complexes may have functions for the regulation of mTORC1 activity as well as autophagy.
ULK1 and ULK2 Phosphorylate Atg13 and FIP200
Knowing that Atg13 is an important element for autophagosome formation, we wondered about the mechanism involving the Atg13-ULK complexes in the regulation of autophagy. From B, we knew that ULK induces phosphorylation of Atg13. Treatment of immunoprecipitated Atg13 with lambda phosphatase reversed the mobility shift of Atg13 and coexpression of ULK1 with Atg13 led to incorporation of 32P into Atg13 in 293T cells, thus confirming that ULK1 induces Atg13 phosphorylation (, a and b). Furthermore, the band of Atg13 on SDS-PAGE was shifted up when Atg13 was isolated from cells expressing wild-type ULK1 or ULK2 but not a kinase-dead mutant of ULK1 or a control protein (c). To determine if ULKs directly phosphorylate Atg13, we isolated ULK1 from 293T cells expressing myc-tagged ULK1 by immunoprecipitation using anti-myc antibody and incubated it with E. coli–purified Atg13 in vitro in the presence of 32P-ATP. ULK1 wild type, but not its kinase-dead mutant, induced incorporation of 32P into Atg13 (d). We also observed a drastic increase of 32P incorporation into ULK1 wild type but not mutant, implying that ULK1 undergoes autophosphorylation. We also determined that ULK2 phosphorylates Atg13 in vitro and undergoes autophosphorylation (d).
Figure 4. ULK1 and ULK2 phosphorylate Atg13 and FIP200. (a) ULK1 induces phosphorylation of Atg13. Myc-tagged Atg13 was coexpressed with HA-tagged ULK1 in 293T cells. Myc immunoprecipitate was obtained and treated with lambda phosphatase for 30 min. The migration (more ...)
also showed that FIP200 band on SDS-PAGE is shifted up when ULK1 or ULK2 is coexpressed (f and e). To investigate if ULKs phosphorylate FIP200, we purified a fragment of FIP200 containing C-terminal residues 860-end from E. coli and incubated it with ULK1 or ULK2 immunoprecipitate in the presence of 32P-ATP. Both ULK1 and ULK2, but not the ULK1 kinase-dead mutant, showed a high kinase activity toward phosphorylation of the FIP200 fragment (f). Knowing that ULK1 and ULK2 phosphorylate Atg13 and FIP200 in vitro, we analyzed if the phosphorylations occur in living cells. Knockdown of ULK1 or ULK2 in 293T cells or knockout of ULK1 in MEF cells induced drastic downshifts of Atg13 and FIP200 bands, supporting that ULK1 and ULK2 determine the phosphorylation status of Atg13 and FIP200 in cells (g). Collectively, these results suggest that ULK1 and ULK2 are the kinase phosphorylating their binding proteins Atg13 and FIP200.
mTOR Phosphorylates Atg13, ULK1, and ULK2
The above results suggest that ULK1 and ULK2 form the protein complexes with Atg13 and FIP200 and phosphorylate the proteins. Now, we question how the complex machinery is regulated by mTOR. We first investigated if rapamycin or leucine deprivation could cause any change in the phosphorylational status of the proteins. Rapamycin or leucine deprivation for 1 h induced downshifts of ULK1, ULK2, and Atg13 bands on SDS-PAGE (a). Rapamycin almost completely inhibited incorporation of 32
P into ULK1 in 293T cells but only marginally for Atg13 (, b and c). We reasoned that the marginal inhibition of Atg13 phosphorylation by rapamycin might be due to a low basal level of Atg13 phosphorylation. Consistent with this reasoning, overexpression of Ras homolog enriched in brain (Rheb), the GTPase that stimulates the kinase activity of mTOR (Saucedo et al., 2003
; Stocker et al., 2003
; Tee et al., 2003
; Zhang et al., 2003
), caused a drastic upward shift of Atg13 band and incorporation of 32
P into Atg13 and rapamycin suppressed the changes (c). Rheb overexpression also caused an upward shift of the ULK1 band, but rapamycin inhibited it. Further support shows that Atg13 bands were shifted up when Atg13 was isolated from cells expressing mTOR wild type but not its kinase dead mutant (Supplemental Figure S3). The mTOR-mediated phosphorylation of Atg13 occurred even in ULK1- or ULK2-silenced cells, indicating that the Atg13 phosphorylation may occur independently of ULK1 and ULK2 (Supplemental Figure S3). Taken together, these results suggest that mTOR induces phosphorylations of Atg13, ULK1, and ULK2.
Figure 5. mTOR phosphorylates Atg13, ULK1, and ULK2. (a) Rapamycin or leucine deprivation induces dephosphorylation of ULK1, ULK2, and Atg13. HEK293T cells were treated with rapamycin or vehicle for 1 h or deprived of leucine for 2 h (−leu) or deprived (more ...)
Next, we questioned if mTOR can directly phosphorylate the proteins. We isolated mTOR immunoprecipitate from 293T cells and incubated it with E. coli–purified Atg13 in the presence of 32P-ATP. The mTOR immunoprecipitate induced high incorporation of 32P into Atg13 (d). Supporting that the Atg13 phosphorylation is due to the kinase activity of mTOR, only wild-type mTOR but not a kinase-dead mutant was able to phosphorylate Atg13 in vitro (e). We also tested an active fragment of mTOR that contains residues 1362-end as an enzyme source and confirmed that the active form of mTOR phosphorylates Atg13 (e). On the other hand, Atg13 was barely phosphorylated by the protein kinase S6K1 in vitro, indicating that S6K1 may not be the kinase phosphorylating Atg13 (unpublished data).
To investigate if mTOR can directly phosphorylate ULK1, we prepared two ULK1 constructs as substrates: 1) the kinase-dead mutant M92A and 2) a fragment containing 281-end that lacks the N-terminal kinase domain, from 293T cells by immunoprecipitation using anti-myc antibody. Both substrates were highly phosphorylated by the active fragment of mTOR (f). To further confirm the direct phosphorylation of ULK1 by mTOR, we purified a soluble fragment of ULK1 containing residues 651-end from E. coli. Endogenous or recombinant mTOR wild type, but not the kinase-dead mutant, induced the phosphorylation of the ULK1 fragment (g). In a similar manner, we confirmed that mTOR wild type, but not its kinase-dead mutant, phosphorylates a fragment of ULK2 containing C-terminal residues 651-end in vitro (h). On the basis of all these results from in vitro kinase assay and analysis of the phosphorylation in living cells, we conclude that mTOR is the kinase phosphorylating Atg13, ULK1, and ULK2.
mTOR Inhibits ULK1 and ULK2 Kinase Activity
Now that we have identified that mTOR phosphorylates the Atg13-ULK complexes, we questioned the functional consequence of the phosphorylations. Because rapamycin induces autophagy, we reasoned that rapamycin might activate ULK. We isolated endogenous and recombinant ULK1 from 293T cells treated with either rapamycin or vehicle for 1 h and analyzed the kinase activity of ULK1. ULK1 isolated from rapamycin-treated cells, relative to the vehicle-treated cells, exhibited a higher kinase activity toward phosphorylation of MBP (, a and b). Like rapamycin, leucine deprivation also led to a high kinase activity of ULK1 (, c and d). Consistent with the negative effects of mTOR on the ULK activity, Rheb overexpression reduced the kinase activity of ULK1 (, e and f). We also confirmed that ULK2, like ULK1, is stimulated by rapamycin using two different substrates, MBP and the FIP200 fragment containing C-terminal residues 860-1591 (, g and h).
Figure 6. mTOR negatively regulates the kinase activity of ULK. (a) Rapamycin increases the kinase activity of ULK1. 293T cells were treated with rapamycin or vehicle for 1 h. ULK1 immunoprecipitate was isolated using anti-ULK1 antibody and incubated with MBP (1 (more ...)
The second line of evidence in support of mTOR inhibition of ULK1 activity came from the observation of the mobility shift of ULK1 bands on SDS-PAGE. When cells were treated with rapamycin for a long period of time, ULK1 bands were converted from fast-migrating forms to slow-migrating ones (i and Supplemental Figure S4). This supports that the dephosphorylated form of ULK1 generated during rapamycin treatment might have a kinase activity and undergo autophosphorylation. The third line of evidence was that rapamycin or leucine deprivation induced a drastic upward shift of the FIP200 band on SDS-PAGE (, i and j). We interpreted this as a result of activation of ULKs that are the kinases phosphorylating FIP200. Using ULK1 null MEF cells and ULK2-silenced 293T cells, we confirmed that the rapamycin- and starvation-induced phosphorylation of FIP200 depends on ULK1 and ULK2 (j). We could not determine contribution of each ULK to FIP200 and Atg13 phosphorylation, which may require double knockout or knockdown cells. Given that ULK1 null mice is viable (Kundu et al., 2008
), it may be possible that ULK1 and ULK2 have redundant functions. However, based on the mobility shift assay in j, it is likely that ULK1 is the major kinase phosphorylating FIP200 and Atg13 in response to rapamycin and nutrient starvation at least in MEF and 293T cells.
In S. cerevisiae
, nutrient deprivation or rapamycin alters the affinity of the interaction between Atg1 and Atg13 (Kamada et al., 2000
). We investigated whether rapamycin has similar effects on the interaction between Atg13 and ULK. Although we observed that the affinity of the interaction increases at 24 h of rapamycin treatment, the expression levels of ULK1 and Atg13 were also increased (i). Furthermore, the affinity of the interaction was not drastically altered by leucine deprivation for 1, 2, or 24 h. We also found that the interaction between ULK2 and Atg13 is not significantly altered by rapamycin or leucine deprivation (data not shown). Given that rapamycin or leucine deprivation for 1 h is sufficient to have the stimulatory effect on ULK activity, we conclude that the activity of ULKs is not altered by any drastic change in the affinity of the interaction with Atg13.
Atg13 Is Important for the ULK1 Kinase Activity and FIP200 Phosphorylation by ULKs
Knowing that rapamycin increases the kinase activities of ULK1 and ULK2, we wondered whether Atg13 plays any role in the ULK activation. We isolated myc-tagged ULK1 from 293T cells and incubated it with different amounts of Atg13 isolated from E. coli and analyzed the kinase activity of ULK1. The levels of the substrate MBP incorporated with 32P increased when higher amounts of Atg13 were added to the reaction (, a and b). The 32P-labeled MBP level was no longer increased when the amount of Atg13 was over a certain concentration (lane 5), which might be because the amount of Atg13 has exceeded the binding capacity of ULK1. Agreeing with the positive role of Atg13 for ULK1 activity, the kinase activity of ULK1 was increased when ULK1 was isolated from cells expressing Atg13 (, c and d, and Supplemental Figure S4). Similar to the results in , a and b, when the expression levels of Atg13 were higher than a certain concentration, the kinase activity and the isolated amount of ULK1 were reduced. This might be due to sequestration of a cellular component crucial for the stability and the activity of ULK1 by Atg13 at a high level of expression. Another possibility is that Atg13 might be a competitive inhibitor for the phosphorylation of MBP because Atg13 itself is a substrate of ULK.
Figure 7. Atg13 positively regulates ULK activity and is required for starvation-induced phosphorylation of FIP200. (a) Atg13 increases the kinase activity of ULK1 in vitro. Myc-tagged ULK1 was isolated from 293T cells and incubated with purified Atg13 at various (more ...)
Consistent with the dependence of ULK stability on Atg13, we observed that knockdown of Atg13 induces a drastic reduction in the expression level of ULK1 in HeLa cells (e). The stability of ULK1 was only marginally affected by Atg13 knockdown in 293T cells (, a and c, and e), indicating that the regulation of ULK stability by Atg13 may depend on other cellular factors or cellular contexts. Another noticeable change was observed in Atg13-silenced cells with regard to the phosphorylation of FIP200. Knockdown of Atg13 suppressed phosphorylation of FIP200 and disabled rapamycin or leucine deprivation to induce FIP200 phosphorylation (e). This result suggests that Atg13 is required for the mTOR-mediated activation of ULK or at least for the phosphorylation of FIP200 by ULKs. Starvation-induced phosphorylation of ULK1 still occurred in Atg13-silenced cells, suggesting that mTOR can phosphorylate ULK1 independently of Atg13 (e). Taken together, these results suggest that Atg13 plays a positive role in mTOR-regulated autophagy processes by enhancing the catalytic activity of ULK and allowing ULK to phosphorylate FIP200, the protein crucial for autophagosome formation (Hara et al., 2008