MOLECULAR AND CELLULAR BIOLOGY, Jan. 2009, p. 157–171
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 29, No. 1
Kinase-Inactivated ULK Proteins Inhibit Autophagy via Their Conserved
C-Terminal Domains Using an Atg13-Independent Mechanism?
Edmond Y. W. Chan,† Andrea Longatti,‡ Nicole C. McKnight,‡ and Sharon A. Tooze*
Secretory Pathways Laboratory, London Research Institute, Cancer Research UK, 44 Lincoln’s Inn Fields,
London WC2A 3PX, United Kingdom
Received 10 July 2008/Returned for modification 11 August 2008/Accepted 14 October 2008
The yeast Atg1 serine/threonine protein kinase and its mammalian homologs ULK1 and ULK2 play critical
roles during the activation of autophagy. Previous studies have demonstrated that the conserved C-terminal
domain (CTD) of ULK1 controls the regulatory function and localization of the protein. Here, we explored the
role of kinase activity and intramolecular interactions to further understand ULK function. We demonstrate
that the dominant-negative activity of kinase-dead mutants requires a 7-residue motif within the CTD. Our
data lead to a model in which the functions of ULK1 and ULK2 are controlled by autophosphorylation and
conformational changes involving exposure of the CTD. Additional mapping indicates that the CTD contains
other distinct regions that direct membrane association and interaction with the putative human homologue
of Atg13, which we have here characterized. Atg13 is required for autophagy and Atg9 trafficking during
autophagy. However, Atg13 does not bind the 7-residue dominant-negative motif in the CTD of ULK proteins
nor is the inhibitory activity of the CTDs rescued by Atg13 ectopic expression, suggesting that in mammalian
cells, the CTD may interact with additional autophagy proteins.
During macroautophagy in mammalian cells, a membrane
cisterna wraps around cytoplasmic material to form a nas-
cent autophagosome, which then fuses with late endosomal
structures to initiate the degradation of autophagosomal
contents. The targets of macroautophagy (herein referred to
as autophagy) can include long-lived proteins, organelles,
ubiquitinated cellular substrates, and aberrant protein ag-
gregates (7, 13, 15, 24, 26). Autophagy has been implicated
in a number of medical contexts, such as cancer, neurode-
generation, and immunity (as recently reviewed in refer-
ences 16, 19, and 21), raising interest in understanding its
basic regulatory mechanisms.
The serine-threonine protein kinase Atg1 was originally
identified as a critical autophagy regulator in genetic screens
performed with the yeast Saccharomyces cerevisiae (17, 29, 31,
34). Autophagy in yeast is induced by nitrogen starvation or
rapamycin treatment, and studies with yeast have shown that
Atg1 functions at a regulatory step downstream of the nutrient-
sensing signaling kinase TOR (target of rapamycin). Atg1
forms part of a complex that includes additional autophagy
(Atg) proteins, such as Atg13, Atg17, Atg29, Atg31, Atg11,
Atg20, and Atg24 (9–12, 22). While the proteins Atg17, Atg29,
and Atg31 have autophagy-specific functions, Atg11, Atg20,
and Atg24 function in the yeast autophagy-related CVT (cyto-
plasm-to-vacuole targeting) pathway, raising the possibility
that a number of different Atg1-containing subcomplexes may
exist in vivo to regulate distinct pathways. Atg1 also regulates
the recycling of Atg9 between the preautophagosomal struc-
ture (PAS) and a peripheral pool via a mechanism involving
Atg2, Atg18, and the phosphatidylinositol-3 kinase complex I
(25). In addition, the localization of yeast Atg1 to the PAS can
be regulated by cyclic AMP-dependent protein kinase-medi-
ated phosphorylation in a starvation-dependent manner (2).
Although the role of Atg1 kinase activity has been con-
troversial (1, 8), recent data have demonstrated that Atg1
acts in distinct kinase-dependent and kinase-independent
roles (5, 11). Kinase-inactivated Atg1 was capable of direct-
ing the assembly of the PAS containing Atg8, Atg17, and
Atg29, consistent with the proposal that Atg1 plays a struc-
tural role. However, the kinase activity of Atg1 was required
to drive protein or membrane dynamics through disassembly
or dissociation of Atg proteins from the PAS, a step which
is required to produce fully formed autophagosomes.
Full kinase activity of Atg1 in yeast requires its binding
partners Atg13 and Atg17 (10). Interestingly, the kinase activ-
ity of Atg1 is stimulated when autophagy is induced in yeast,
and this correlates with increased binding of Atg1 to Atg13 and
Atg17 upon autophagy induction (10, 11). Furthermore, the
efficiency of Atg1-Atg13 binding is inversely correlated with
levels of phosphorylation on Atg13 (10), leading to the model
in which Atg13 is dephosphorylated upon autophagy induction,
thereby promoting its ability to bind to and act as a cofactor for
Atg1. The autophagy-dependent kinases and phosphatases
controlling yeast Atg13 phosphorylation remain to be deter-
mined. However, Atg13 dephosphorylation has been shown to
be TOR dependent (10).
The single Atg1 homologues of Dictyostelium discoideum,
Caenorhabditis elegans, and Drosophila melanogaster have been
confirmed to be key autophagy regulators (20, 23, 27). In
contrast, mammals have at least two Atg1 homologues (6),
Unc-51-like kinase 1 (ULK1) and ULK2, that share strong
* Corresponding author. Mailing address: Secretory Pathways Lab-
oratory, London Research Institute, Cancer Research UK, 44 Lin-
coln’s Inn Fields, London WC2A 3PX, United Kingdom. Phone: (44)
207 269 3122. Fax: (44) 207 269 3417. E-mail: sharon.tooze@cancer
† Present address: Strathclyde Institute of Pharmacy and Biomedical
Sciences, University of Strathclyde, Glasgow, United Kingdom.
‡ These authors contributed equally to this work.
?Published ahead of print on 20 October 2008.
homology with the C. elegans Atg1 homologue Unc (uncoor-
dinated)-51 (32). Whether ULK1 and ULK2 play similar roles
for autophagy induction remains unclear.
We previously found that, in HEK293A cells, small inter-
fering RNA (siRNA)-mediated knockdown of ULK1 was
sufficient to reduce starvation-induced autophagy and in-
hibit the starvation-dependent redistribution of mammalian
Atg9 (mAtg9) to a dispersed, peripherally localized pool (3,
37). In this cell system, knockdown of ULK2 had no effect
on the induction of autophagy or mAtg9 traffic, suggesting a
preferential role of ULK1 in autophagy. ULK1 and -2 have
overlapping widespread mRNA expression patterns (35, 36).
However, only ULK1 mRNA was upregulated in maturing
reticulocyte cultures to promote autophagic clearance of
mitochondria, indicating that some specificity exists in vivo
(15). These authors went on to show that mice lacking
ULK1 displayed abnormal erythrocyte maturation but were
viable and without a developmental phenotype, in contrast
with other models of mice deficient in autophagy genes (13,
14). These data suggest that ULK2 can support autophagic
function in the absence of ULK1, implying a more special-
ized role for ULK1 in vivo (15). Although the precise roles
of ULK1 and ULK2 require further clarification, our data
on ULK1 and recent other data show that both of these
proteins can localize to mammalian PASs (called isolation
membranes, or phagophores) in a starvation-dependent manner
All Atg1 homologs share similar domain structures in which
the kinase catalytic domain comprises the N-terminal one-
third of the protein and the remaining two-thirds contain reg-
ulatory sequences. Comparison of the mouse ULK1 and ULK2
sequences with that of C. elegans Unc-51 has allowed the def-
inition of a C-terminal domain (CTD) (222 residues long in
mouse ULK1) that shows relatively high levels of conservation
(32), suggestive of an important biological function. We pre-
viously described how the deletion of a three-residue PDZ
binding motif at the CTD C terminus transforms ULK1 into a
potent dominant inhibitor of autophagy (3). In D. discoideum,
defects in development and autophagy found in Atg1 null cells
could not be rescued with a mutant D. discoideum Atg1 con-
taining a deletion of the last 40 amino acids of the CTD (30).
A recent study has identified a novel ULK1-binding protein
called FIP200 (focal adhesion kinase family-interacting protein
of 200 kDa), which was required for starvation-induced autoph-
agy, proper ULK1 phosphorylation, and ULK1 stability (6).
FIP200 binding to ULK1 required the CTD, further support-
ing a role of the CTD in autophagy regulation.
In this study, we aimed to gain insight into the function of
ULK1 and ULK2 for the regulation of autophagy by studying
the role of their kinase activities and the regulation of these
activities by the CTD. We demonstrated that complete abla-
tion of ULK1 kinase activity results in decreased protein auto-
phosphorylation and a potent dominant-negative effect on au-
tophagy. Our further analysis has led to a working model in
which autophosphorylation is critical for promoting a closed
molecular conformation that regulates interactions of the
CTD. In further support of this model, expression of the CTD
from either ULK1 or ULK2 was sufficient to inhibit autophagy,
and additional analysis has identified a 7-residue motif critical
for this effect. We determined that this motif was distinct from
other signals within the CTD that direct membrane binding of
ULK1 and ULK2 and incorporation into large protein com-
To explore other mechanisms involved in the function of the
CTD, we identified a putative human Atg13 orthologue that
we confirmed is essential for autophagy by using siRNA de-
pletion. In addition, we found that loss of human Atg13 af-
fected the trafficking of mAtg9, as was previously observed
after the loss of ULK1 (37). Although mAtg13 bound the
CTDs of both ULK1 and ULK2, this interaction utilized se-
quences that were distinct from the dominant-negative 7-resi-
due motif, and overexpression of Atg13 did not rescue the
dominant-negative activity of the overexpressed CTDs. Thus,
our data have identified a 7-residue motif in the ULK1 and
ULK2 CTDs that engages a novel dominant-negative mecha-
nism which is independent of Atg13 and membrane-associated
MATERIALS AND METHODS
Cell culture. HEK293A cells and their derivative cell line stably expressing
enhanced green fluorescent protein (EGFP)-rat LC3 (293/GFP-LC3) have been
described previously (3). Cells were maintained in Dulbecco’s modified Eagle’s
medium with 10% fetal bovine serum, which also served as the full nutrient
medium in the analytical experiments. Starvation medium consisted of Earle’s
balanced saline solution (EBSS), containing 0.5 mM leupeptin where indicated.
DNA constructs. Expression plasmids for N-terminal Myc epitope-tagged wild-
type and kinase-dead (K46R) murine ULK1 (ULK1-K46R) have been described
previously (3). ULK2 containing an N-terminal Myc tag was constructed by PCR
from a full-length clone containing murine ULK2 (IMAGE:5709559) as the
template. The CTDs of ULK1 and -2 were amplified from full-length cDNAs
using PCR and cloned with an N-terminal Myc tag into a modified pcDNA3.1 or
pRK5 backbone, respectively. Substitutions in the kinase domains of ULK1 and
-2 were generated using a PCR site-directed mutagenesis kit (Stratagene), and
additional deletion constructs were generated by isolating internal regions using
PCR. We obtained a full-length cDNA containing the human putative Atg13
homolog KIAA0652 (pF1KSDA0652; Kazusa DNA Research Institute,
Kisarazu, Japan). A C-terminal FLAG epitope tag was added onto the Atg13
open reading frame using PCR and cloned into a modified pcDNA3.1 vector. For
biochemical analyses, cells were transfected using Lipofectamine 2000 (Invitro-
gen) according to the manufacturer’s protocols and analyzed 24 h later.
siRNAs and knockdown determination. A pool of four siRNA duplexes (On-
Target plus SMARTpool) targeting the putative human Atg13 (KIAA0652,
Entrez nucleotide accession no. NM_014741) was obtained (Dharmacon). Two
duplexes within this pool specific for KIAA0652 that were confirmed in multiple
assays correspond to Duplex1 (5?-AGA CCA UCU UUG UCC GAA A-3?)
(sense sequence 989 to 1007) and Duplex2 (5?-GAA GAA UGU CCG CGA
GUU U-3?) (sense sequence 1398 to 1416). The siRNAs targeting human ULK1
have been described previously (3). Experimental and control siRNAs were
transfected into HEK293 and 293/GFP-LC3 cells by use of a wet/reverse-knock-
down protocol in which trypsinized cells in suspension are plated directly into an
siRNA-Lipofectamine 2000 (Invitrogen) mixture. Fresh medium is replenished
24 h before the day of the experiment.
Confirmation of siRNA knockdown by reverse transcription-PCR was carried
out by assaying message RNA levels in cell samples following 72 h of knockdown,
as described previously (3), using Sybr green real-time PCR. QuantiTect (Qiagen
Biotech) primer sets were obtained for detection of the putative Atg13
(KIAA0652) transcript and the beta-actin control.
Immunoprecipitations and in vitro kinase assays. Singly or doubly transfected
cells were lysed in ice-cold TNTE (20 mM Tris, pH 7.5, 150 mM NaCl, 0.3%
[vol/vol] Triton X-100 [TX-100], 5 mM EDTA) containing Complete EDTA-free
protease inhibitor cocktail (Roche) and PhosStop phosphatase inhibitor cocktail
(Roche). Lysates cleared by centrifugation were incubated with protein
G-Sepharose beads coupled to anti-Myc monoclonal 9E10 or anti-FLAG mono-
clonal M2 (Sigma-Aldrich) antibody for 1 h and then washed three times with
TNTE. For coimmunoprecipitation studies, proteins were eluted using 3? so-
dium dodecyl sulfate (SDS) sample buffer (with heating to 65°C for 5 min) and
analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
For in vitro kinase assays, following the three TNTE washes, immunoprecipi-
158 CHAN ET AL.MOL. CELL. BIOL.
tates were further washed once with kinase reaction buffer (KRB) (20 mM
HEPES, pH 7.5, 20 mM MgCl2, 25 mM beta-glycerophosphate, 2 mM dithio-
threitol, 100 ?M sodium orthovanadate) and then incubated at a final volume of
20 ?l in KRB containing 20 ?M ATP, 5 ?g myelin basic protein (MBP), and 5
?Ci [?-32P]ATP at 30°C for 15 min. For mixed-bead kinase reactions, immuno-
precipitates of ULK1 and -2 and Atg13-FLAG were combined after the TNTE
wash step, further washed in KRB, and then incubated in a 20-?l final volume of
KRB containing 20 ?M ATP at 30°C for 30 min. Reactions were stopped with the
addition of 3? SDS sample buffer during heating to 65°C for 5 min. [32P]-labeled
reaction products were resolved on NuPAGE 4 to 12% bis-Tris gels in morpho-
linepropanesulfonic acid (MOPS)-SDS running buffer (Invitrogen). Mixed-bead
reaction products were resolved by Laemmli SDS-PAGE. Proteins were trans-
ferred to polyvinylidene difluoride (PVDF) membranes and then analyzed using
a phosphorimager scanner and anti-Myc immunoblotting.
Immunoblotting. To detect GFP-LC3 lipidation, 293/GFP-LC3 cells were
lysed following various treatments in 1? SDS sample buffer. Lysates were then
heated to 65°C for 5 min and passed through a 27-gauge needle five times to
reduce viscosity before analysis by 10% Laemmli SDS-PAGE. To detect lipida-
tion of endogenous LC3, HEK293A cells were lysed following treatments in
ice-cold TNTE containing Complete EDTA-free protease inhibitor cocktail
(Roche). Lysates cleared by centrifugation were mixed with 3? SDS sample
buffer, heated to 100°C for 5 min, and then analyzed on either 12% Laemmli
SDS-PAGE gels or 4 to 12% bis-Tris NuPAGE gels (morpholineethanesulfonic
acid [MES]-SDS running conditions) (Invitrogen). Proteins were transferred to
PVDF membranes. GFP-LC3 and endogenous LC3 were detected with 5F10
anti-LC3 monoclonal antibody (Nanotools, Teningen, Germany). Additional
immunoblotting antibodies included anti-ULK1 polyclonal H-240 (Santa Cruz),
anti-Myc 9E10 monoclonal, ?-tubulin polyclonal (6046; Abcam, Cambridge,
United Kingdom), anti-FLAG M2 monoclonal (Sigma-Aldrich), and antiactin
monoclonal AC-40 (Sigma-Aldrich). Rabbit polyclonal antibodies toward human
Atg13 (KIAA0652) were generated using the peptide sequence LAVHEKNVR
EFDAFVETLQ. Following incubation with primary antibodies, signals were
detected and quantified using secondary antibodies coupled to infrared chro-
mophores and a two-channel scanning method (Licor Odyssey), as described
previously (3). Statistical analyses for various pairwise comparisons were per-
formed using Student’s two-tailed t test on sample sets with equal variances.
Limited proteolysis. Cells were lysed in ice-cold TNT (50 mM Tris, pH 8.0, 150
mM NaCl, 0.5% TX-100) (without protease inhibitors) and then incubated with
protein G-Sepharose beads coupled to 9E10 anti-Myc monoclonal antibody for
1 h. Immunoprecipitates were washed three times with TNT and then incubated
in a 20-?l final reaction volume of TNT containing the concentrations of se-
quencing-grade chymotrypsin (Roche) indicated in Fig. 3 on ice for 15 min.
Reactions were stopped (addition of 3? SDS sample buffer and heating to
100°C) and analyzed by SDS-PAGE and anti-Myc immunoblotting.
Long-lived protein degradation. Autophagy-dependent degradation of [14C]va-
line-labeled cellular proteins in transfected 293/GFP-LC3 cells was measured as
previously described (3).
Membrane association. Following transfection and treatments as indicated,
293/GFP-LC3 cells were scraped into ice-cold homogenization buffer (HB) (20
mM HEPES, pH 7.2, 1 mM EGTA, 5 mM MgCl2, 150 mM KCl) containing
Complete EDTA-free protease and PhosStop phosphatase inhibitors (Roche).
Cells were disrupted by being passed 20 times through a 27-gauge needle and
then cleared using a low-speed (1,000 ? g) centrifugation. These cleared lysates
were then centrifuged at 100,000 ? g for 1 h (4°C) to obtain a crude membrane
pellet and the resulting supernatant fraction. Where indicated, membrane pellets
were further washed/extracted in HB containing either high salt concentrations
or 1% TX-100 and then recentrifuged at 100,000 ? g. Membrane pellets were
finally resuspended in HB containing 1% TX-100. Membrane and supernatant
fractions were mixed with 3? SDS sample buffer, heated to 65°C for 5 min, and
resolved by SDS-PAGE for immunoblotting.
Native gel electrophoresis. Following transfection and treatments as indicated,
HEK293A cells were lysed in ice-cold 1? native PAGE sample buffer (Invitro-
gen) containing 1% TX-100 and Complete EDTA-free protease and PhosStop
phosphatase inhibitors. Cell lysates were precleared by centrifugation and then
mixed with Coomassie G-250 dye additive (0.25% final) (Invitrogen) before
being run on 3 to 12% Novex NativePAGE bis-Tris gels (Invitrogen) according
to the manufacturer’s protocols. Resolved proteins were transferred to PVDF
membranes and detected with an anti-Myc monoclonal antibody, horseradish
peroxidase-coupled secondary antibodies, and chemiluminescence.
Microscopic analysis of cell structures. For analysis of GFP-LC3-labeled
autophagosomes using a Cellomics ArrayScan VTI high-content screening sys-
tem (Thermo Scientific), 293/GFP-LC3 cells were seeded into 96-well tissue
culture plates coated with poly-D-lysine-containing Lipofectamine-siRNA mix-
tures. Seventy-two hours after transfection, cells were starved, fixed, and ana-
lyzed using an automated calibrated quantitation algorithm (spot total intensity
per object) (Thermo Scientific).
For confocal analyses, 293/GFP-LC3 cells or parental HEK293 cells were
either plated onto glass coverslips and transfected with expression constructs or
plated onto glass coverslips in an siRNA-Lipofectamine mixture. Following var-
ious treatments, cells were fixed and then further stained using 9E10 monoclonal
Myc antibody, M2 monoclonal FLAG antibody, or a hamster anti-Atg9 mono-
clonal antibody generated by immunizing animals with a previously described
C-terminal peptide (37). Samples were further analyzed using a confocal laser
scanner fitted onto a Zeiss Axioplan 2 microscope.
Kinase function of ULK1. It was recently shown that in NIH
3T3 cells kinase-inactivated ULK1-K46N had dominant-
negative properties on starvation-induced autophagy (6).
Our previous data using a HEK293 cell system showed that
the kinase-inactivated ULK1-K46R mutant behaved simi-
larly to wild-type ULK1 in modulating autophagy (3). Our
results also contrasted with the reported dominant-negative
properties of ULK1-K46R in neurite outgrowth assays (32).
Since ULK1-K46R displayed normal electrophoretic mobil-
ity (3), we hypothesized that ULK1-K46R had remaining
kinase activity and we generated an additional kinase-inac-
tivated mutant, ULK1-K46I. We examined the effects of
these mutations by assaying the efficiency of immunopre-
cipitated ULK1 proteins to undergo autophosphorylation
and to phosphorylate the generic substrate MBP. The K46R
substitution inactivated these in vitro kinase activities only
partially, while the K46I mutation produced stronger inac-
tivation (Fig. 1A).
Kinase function in ULK1?CTD. We previously demon-
strated that deletion of the ULK1 CTD generates a potent
dominant-negative molecule, ULK1?CTD (3). We questioned
whether the CTD modulated kinase activity, possibly via an
intramolecular interaction. We observed that deletion of the
CTD in ULK1 increased autophosphorylation levels (approx-
imately 2.8-fold), while phosphorylation of MBP was reduced
approximately 80% following deletion of the CTD (Fig. 1B).
From these data, we speculated that loss of the CTD produced
a kinase with an altered conformation in which the kinase
domain has increased accessibility to the spacer domain for
autophosphorylation and a decreased ability to phosphorylate
Kinase function in full-length ULK1. Since our in vitro data
indicated that the K46R and K46I substitutions had various
severities, we retested these mutants for their effect on starva-
tion-induced autophagy in 293/GFP-LC3 cells (3). In this cell
system, we can assess autophagy activation by measuring the
conversion of its unmodified (GFP-LC3-I) form to its modi-
fied, lipidated species (GFP-LC3-II). Strikingly, we observed
that ULK1-K46I exhibited strong dominant-negative proper-
ties compared to both the wild-type control and ULK1-K46R
(Fig. 2A). In addition, ULK1-K46I displayed faster electro-
phoretic mobility, consistent with decreased autophosphor-
ylation in vivo, than wild-type ULK1 and ULK1-K46R.
These data demonstrate that strong inactivation of catalytic
activity is required to transform ULK1 into a dominant-
To understand further this dominant inhibitory effect, we
performed a deletion analysis of ULK1-K46I. We determined
VOL. 29, 2009 FUNCTION AND BINDING PARTNERS OF ULK CTD 159
that ULK1-K46I lost approximately 50% of its potency to
inhibit GFP-LC3-II upon deletion of its CTD (Fig. 2B). An
equivalent loss of potency was seen with ULK1-K46I lacking
the C-terminal 50 amino acids [ULK1-K46I(1–1001)]. Finer
mapping determined that dominant-negative potency was sim-
ilarly lost after the deletion of 10 residues from the ULK1 C
terminus (ERR-STOP mutant). However, deletion of the PDZ
binding motif from ULK1-K46I (LSG-STOP) had no effect on
dominant-negative function. Thus, the ability of kinase-inac-
tive, nonautophosphorylated ULK1 to inhibit autophagy de-
pended upon a small region just inside its C terminus. In
conjunction with our in vitro kinase data showing altered au-
tophosphorylation efficiency following CTD deletion, we pro-
posed a working model in which the CTD is normally folded
back to keep the protein in a more closed conformation. Full
kinase inactivation of ULK1 leads to a loss of autophosphor-
ylation and a conformational change that causes the CTD to be
abnormally exposed, revealing cryptic binding sites within the
CTD that then recruit and titrate out other essential autophagy
Limited proteolysis reveals a conformational change in
ULK1-K46I. To probe for possible conformational changes, we
performed limited proteolysis on immunoprecipitated ULK1
compared to ULK1-K46I and ULK1-K46R. Treatment of im-
munoprecipitated wild-type Myc-ULK1 with low concentra-
tions of chymotrypsin resulted in the loss of the full-length
protein and generated distinct protected fragments ranging
between 60 and 70 kDa in apparent molecular mass (Fig.
3B). Using the relative mobilities of previously described
ULK1 deletion constructs, such as ULK1?CTD [ULK1(1–
828)], ULK1(1–427), and ULK1(1–351), for comparison (Fig.
3A), we observed that the larger predominant digestion prod-
ucts are approximately 420 to 500 residues in length. Digestion
of wild-type Myc-ULK1 with higher concentrations of enzyme
generated smaller protected digestion products of approxi-
mately 350 to 380 residues. Digestion of Myc-ULK1-K46I also
resulted in the loss of the full-length protein. However, distinct
protected fragments were not detected efficiently, which is
consistent with the idea that internal regions of the kinase-
inactivated mutant were more accessible to protease. Diges-
tion of Myc-ULK1-K46R yielded an intermediate phenotype,
suggesting that the closed molecular conformation of our
working model is altered only mildly when the kinase is par-
Kinase inactivation in full-length ULK2. Hara et al. (6)
recently reported that kinase-inactivated ULK2 also exhibited
dominant-negative properties on autophagy. Given our ob-
served differential effects of kinase inactivation in the context
of ULK1, we performed a similar analysis using ULK2. We
confirmed that the mutation of K39 in ULK2 to R or I resulted
in the partial or full inactivation, respectively, of in vitro auto-
phosphorylation and MBP phosphorylation activities (Fig.
4A). We next tested the effects of wild-type ULK2 and these
kinase mutants on starvation-induced autophagy in 293/GFP-
LC3 cells (Fig. 4B). Surprisingly, overexpression of wild-type
ULK2 enhanced the levels of GFP-LC3-II following amino
acid starvation. This enhancing effect was not seen with the
ULK2-K39R mutant (which contains partially inactivated ki-
nase activity). In agreement with our ULK1 results, ULK2-
K39I produced strong inhibitory effects on starvation-induced
GFP-LC3 lipidation. Strong and weak effects on autophos-
phorylation could also be observed as differences in electro-
phoretic mobilities for overexpressed ULK2-K39I and -K39R,
respectively (Fig. 4B).
Analysis of ULK2 reveals both differences and similarities
to ULK1. Consistently with recent observations showing the
localization of ULK2 on Atg16L-positive-forming autophago-
somes (also termed isolation membranes, or phagophores) (6)
and our results with ULK1 (3), we could detect a proportion of
ULK2 on GFP-LC3-labeled autophagosomes in starved 293/
GFP-LC3 cells (Fig. 4C). Overexpressed ULK2 did not pro-
mote GFP-LC3 lipidation under full nutrient conditions (zero
time point) but appeared to enhance the response following
amino acid starvation (Fig. 4D). Using deletion analysis, we
observed that ULK2 lacking its C-terminal three residues
(ATV) similarly enhanced starvation-induced GFP-LC3 lipida-
tion (Fig. 4E). Thus, while deletion of the C-terminal PDZ
motif modulated ULK1 function (3), residues at the extreme C
terminus of ULK2 were not required to enhance GFP-LC3
lipidation. A ULK2?CTD mutant had no effect on GFP-LC3
lipidation, in contrast with ULK1?CTD (Fig. 4F), which has
dominant inhibitory properties (3). These data on wild-type
and ?CTD deletion mutants indicate that overexpression of
ULK1 and ULK2 may have distinct effects on autophagy. In
contrast, kinase-inactivated forms of ULK1 and ULK2 are
both strong dominant-negative inhibitors of autophagy, as
evidenced by their ability to block starvation-induced GFP-
LC3 puncta (Fig. 4G). Finally, similarly to ULK1, limited pro-
FIG. 1. In vitro kinase activities of ULK1 mutants. (A and B)
HEK293A cells were transfected with Myc-tagged ULK1 constructs,
lysed, and used for immunoprecipitation reactions. Immunoprecipi-
tates were incubated in an in vitro kinase reaction mixture containing
[32P]ATP and the generic substrate MBP. Reaction products were
resolved on SDS-PAGE gels (top). Anti-Myc immunoblotting was
performed as a control to quantify the amount of ULK1 protein pre-
cipitated. Phosphorimager analysis detected levels of ULK1 autophos-
phorylation (P32-auto) and MBP phosphorylation (P32-MBP). Spe-
cific activities (in relative units) are expressed as the amount of
phosphorylation normalized to the amount of Myc-tagged ULK1 per
reaction (corrected for background activities detected in a parallel
control reaction performed with untransfected cells [Untr] [not shown
in panel B]). Each bar represents results from three independent
samples ? standard deviations, and each experiment was reproduced
a minimum of two times.
160 CHAN ET AL.MOL. CELL. BIOL.
teolysis of immunoprecipitated wild-type Myc-ULK2 gener-
ated protected fragments of roughly 60 to 75 kDa in apparent
molecular mass (Fig. 4H). Immunoprecipitated Myc-ULK2-
K39I could also be digested by chymotrypsin (note the loss of
the full-length protein), but smaller protected fragments did
not accumulate, consistent with our model that kinase-inacti-
vated ULK2, like ULK1, has an altered protease-sensitive con-
Dominant-negative activity of the ULK CTDs depends on a
short conserved sequence within the C terminus. In our model,
as a result of a decreased kinase activity, an abnormally ex-
posed CTD engages in uncontrolled interactions with essential
autophagy regulatory components, thereby halting the path-
way. Supporting this model, overexpression of the CTD alone
from either ULK1 (amino acids 829 to 1051) or ULK2 (amino
acids 811 to 1037) resulted in a strong dominant-negative effect
on starvation-induced GFP-LC3 lipidation (Fig. 5A). The
CTD of ULK1 or ULK2 also inhibited starvation-induced pro-
tein degradation (Fig. 5B). Overexpression of the ULK1 CTD
inhibited lipidation of endogenous LC3 to similar extents, as
previously described for N-terminal ULK1 dominant-negative
fragments (3), such as ULK1(1–351) (Fig. 5C).
To investigate this dominant-negative mechanism, we per-
formed a deletion analysis of the ULK1 CTD. Similarly to
observations from a deletion analysis of full-length ULK1-
K46I, removal of the PDZ binding motif (LSG-STOP) and the
FIG. 3. Differential sensitivities of ULK1 kinase mutants to limited
proteolysis. (A) Schematic showing the domain structure of ULK1,
including the N-terminal Myc tag (M), the kinase domain, the serine-
and proline-rich spacer domain (S/P spacer), and the conserved CTD.
Positions of stop codons used by Chan et al. (3) to generate various
deletion mutants are indicated at the bottom. (B) ULK1 constructs
transfected into 293A cells were immunoprecipitated and then incu-
bated with the indicated concentrations of chymotrypsin for 15 min on
ice. Reaction products were resolved on SDS-PAGE gels and detected
by anti-Myc immunoblotting. Molecular mass markers are indicated on
the left. The relative mobilities of the uncleaved proteins, previously
described ULK1 truncation mutants, and the heavy and light immu-
noglobulin G (IgG) chains are shown on the right.
FIG. 2. Inhibition of GFP-LC3 lipidation by ULK1-K46I. (A and B) 293/GFP-LC3 cells were transfected with a control plasmid expressing
luciferase (pLUC) or the indicated Myc-tagged ULK1 constructs. Twenty-four hours after transfection, cells were either left alone or starved for
2 h in EBSS containing leupeptin and lysed for SDS-PAGE analysis of GFP-LC3 lipidation (conversion of GFP-LC3-I to GFP-LC3-II) by use of
anti-LC3 antibodies. The membrane was also probed with anti-Myc and -?-tubulin (?Tub) antibodies. GFP-LC3 lipidation is quantified as follows:
GFP-LC3-II/(GFP-LC3-I ? GFP-LC3-II). Each bar represents results from three independent samples ? standard deviations. (A)*, P ? 0.05;
NS, P ? 0.20 (in pairwise comparisons with wild-type ULK1). (B)*, P ? 0.05; NS, P ? 0.18 (in pairwise comparisons with full-length ULK1-K46I).
(C) Alignments show C-terminal amino acid residues from CTDs of ULK1 and ULK2. Positions of stop codons used to generate various
C-terminal truncations are indicated at the top. ULK1(1–1001) is missing its C-terminal 50 residues; ?CTD is a deletion of the entire CTD and
corresponds to amino acids 1 to 828 of ULK1. The EGL-STOP mutant is further studied in Fig. 6F.
VOL. 29, 2009 FUNCTION AND BINDING PARTNERS OF ULK CTD161
next 4 amino acid residues (LSA-STOP) had no effect on the
dominant-negative potency of the CTD (Fig. 5D). However,
removal of the C-terminal 10 residues (ERR-STOP) began to
rescue the dominant-negative activity, and further truncation
(KLC-STOP) produced a stronger reversion of the dominant-
negative activity. This region contains a 7-residue motif (IER
RLSA) that is entirely conserved between the CTDs of ULK1
and ULK2 from human and mouse (Fig. 2C). Using the CTD
of ULK2 (which is likely more ancestral than ULK1 [E. Y. W.
Chan and S. A. Tooze, unpublished observations]) as a refer-
ence, we determined that the IERRLSA motif is perfectly
conserved in a predicted avian ULK2 orthologue but displays
clearly less conservation in the predicted ULK2 orthologues of
fish and is unrecognizable in the Atg1 orthologues of D. mela-
nogaster and C. elegans. Thus, our deletion analysis demon-
strated that the ability of the ULK1/2 CTDs to act as dominant-
negative molecules is highly dependent upon a well-conserved
7-residue motif at the C terminus.
Membrane binding signal within the CTD. We next aimed
to elucidate the dominant-negative mechanism of the ULK
CTDs, which by deletion analysis appeared to require the same
amino acid residues as kinase-inactivated ULK1 and -2. Our
FIG. 4. Autophagy regulatory roles of wild-type and kinase-dead ULK2. (A) In vitro autophosphorylation and MBP kinase activities for ULK2
constructs were measured as described in the legend for Fig. 1. (B, D, E, and F) Modulation of GFP-LC3 lipidation by ULK constructs was
detected as described in the legend for Fig. 2. (B)*, P ? 0.05; NS, P ? 0.94 (in pairwise comparisons with pLUC-transfected, starved cells). (D)*,
P ? 0.03 in pairwise comparisons with pLUC-transfected cells at the same time point. (E)*, P ? 0.06 in pairwise comparisons with pLUC-
transfected, starved cells. (F)*, P ? 0.02; NS, P ? 0.42 (in pairwise comparisons with pLUC-transfected, starved cells). (C) In 293/GFP-LC3 cells
starved in EBSS-leupeptin for 2 h, Myc-tagged ULK2 could be observed on multiple cytoplasmic structures, a portion of which colocalized with
GFP-LC3, as indicated by arrows and the boxed inset. Bar ? 10 ?m. (G) Myc-tagged ULK1-K46I or ULK2-K39I inhibited the formation of
cytoplasmic GFP-LC3-labeled autophagosomes in starved 293/GFP-LC3 cells. Bar ? 10 ?m. (H) Limited proteolysis of wild-type ULK2 and
ULK2-K39I, assayed as described in the legend for Fig. 3. Untr, untransfected; P32-auto, ULK1 autophosphorylation; P32-MBP, MBP phosphor-
ylation; ?Tub, ?-tubulin; IgG, immunoglobulin G.
162 CHAN ET AL.MOL. CELL. BIOL.
previous data showed that localization of ULK1 to GFP-LC3-
labeled structures was dependent on the CTD (3), so we in-
vestigated the relationship between dominant-negative activity
and membrane association by using biochemical assays. We
first confirmed that approximately 30% of the total overex-
pressed Myc-tagged ULK1 was associated with the crude
membrane pellet in starved 293/GFP-LC3 cells (Fig. 6A),
which was consistent with previous fractionation data from
brain extracts (33). For control purposes, we observed that
lipidated GFP-LC3-II was associated exclusively with crude
membranes (relative to the soluble supernatant). Some GFP-
LC3-I was also detected on the membranes, but this associa-
tion could be disrupted by an additional salt wash of the pellet.
Importantly, ULK1 and GFP-LC3-II were stably membrane
bound even after a 0.5 M salt wash. We next confirmed that the
association of ULK1 to crude membrane fractions was lost
upon deletion of the CTD (Fig. 6B). However, deletion of the
PDZ binding motif (LSG-STOP) or deletion of the last 50
amino acids [ULK1(1–1001)] of ULK1 did not disrupt mem-
brane binding. Thus, ULK1 could still associate with mem-
branes even after removal of C-terminal residues that are re-
quired for dominant-negative activity, suggesting that these
functions were independent. Supporting the idea that altered
membrane binding was not involved in the dominant-negative
mechanism, kinase-inactivated forms of ULK1 and ULK2
were detected in the membrane fraction with an efficiency
similar to that of wild-type proteins (Fig. 6C).
Since deletion of the entire CTD largely abolished mem-
brane association, we questioned whether this region alone
could bind membranes. Indeed, the CTD of ULK1 could be
detected in the membrane pellets derived from extracts of
starved 293/GFP-LC3 cells, and this association was salt resis-
tant (Fig. 6D). As expected, an overexpressed ULK1 CTD
reduced the amount of membrane-associated GFP-LC3-II. Ex-
pression of the ULK1 CTD clearly inhibited the formation of
starvation-induced GFP-LC3-positive autophagosomes (Fig.
6E). Besides existing as a diffuse cytosolic pool, the ULK1
CTD could be observed on discreet puncta that colocalized
with the remaining GFP-LC3 puncta, consistent with the idea
that the CTD contains autophagosomal targeting signals.
Membrane association was also confirmed biochemically for
the ULK2 CTD (data not shown).
In agreement with deletion analysis of full-length ULK1, the
last 50 residues of the ULK1 CTD C terminus were not re-
quired for membrane binding (Fig. 6F), suggesting that mem-
brane targeting signals were distinct from the region involved
in dominant-negative interactions. To map further the minimal
membrane targeting region, we analyzed the hydropathy pro-
FIG. 5. CTDs of ULK1 and ULK2 inhibit autophagy. (A) Control (pLUC) and expression constructs for Myc-tagged ULK1 and ULK2 CTDs
were transfected into 293/GFP-LC3 cells. Inhibition of starvation-induced GFP-LC3 lipidation was measured as described in the legend for Fig.
2.**, P ? 0.006 in pairwise comparisons with pLUC-transfected, starved cells. (B) 293/GFP-LC3 cells transfected as described for panel A were
labeled overnight with [14C]valine. Cells were then either left untreated or starved in EBSS for 2 h, and autophagic degradation of long-lived
proteins (prot deg) was analyzed. Cell samples transfected in parallel were used as a control to detect overexpressed constructs. Each bar represents
the average from three independent samples ? the standard deviation. (C) HEK293A cells were transfected for 24 h with control pLUC plasmid,
a Myc-tagged ULK1(1–351) deletion mutant as an internal control, or Myc-tagged ULK1 CTD. Cells were then left untreated or starved in
EBSS-leupeptin for 2 h and then lysed for SDS-PAGE analysis of LC3 lipidation. The ratio of LC3-II/LC3-I measured for each sample is indicated
at the bottom. (D) Myc-tagged ULK1 CTD constructs with C-terminal truncations were transfected into 293/GFP-LC3 cells, and GFP-LC3
lipidation was measured as described in the legend for Fig. 2.*, P ? 0.05; NS, P ? 0.4 (in pairwise comparisons with full-length-CTD-transfected,
starved cells). A schematic of the truncations is shown in Fig. 2. ?Tub, ?-tubulin.
VOL. 29, 2009 FUNCTION AND BINDING PARTNERS OF ULK CTD163
file of the ULK1 CTD to identify regions amenable for dele-
tion. Up to 35 amino acid residues (containing both a hydro-
phobic and a hydrophilic stretch) (MYC-LKG) could be
removed from the N terminus of the ULK1 CTD without
disruption of membrane binding (Fig. 6G). Thus, deletion
analysis of the CTD N and C termini has further defined the
membrane association region to amino acids 864 to 1001.
Although membrane binding did not appear to be critically
involved in dominant-negative activity, we explored whether
membrane association of ULK proteins was regulated upon
induction of autophagy. Overexpressed ULK1 binding to
membranes was unaltered following amino acid starvation
(Fig. 6H). In contrast, overexpressed ULK2 exhibited both
faster electrophoretic mobility and increased membrane asso-
ciation following amino acid starvation. These data suggest
that, upon autophagy induction, ULK2 becomes partially de-
phosphorylated and more strongly associated with autophago-
somal membranes, providing more evidence that ULK1 and
ULK2 may have distinct roles in autophagy.
Further investigation unexpectedly revealed that the associ-
ation of ULK1 to membrane fractions was resistant to extrac-
tion with 1% TX-100 (in contrast to lipidated GFP-LC3-II,
which was no longer membrane bound) (Fig. 6I). This tight
association of ULK1 to detergent-resistant membranes was
confirmed for endogenous ULK1 (Fig. 6J). Note that mem-
brane binding of endogenous ULK1 was not altered following
amino acid starvation (data not shown).
ULK1 and -2 form distinct protein complexes. Since altered
membrane binding did not appear to explain the dominant-
negative effects of the ULK1/2 CTD or kinase-dead proteins,
we investigated whether these mutant ULK proteins displayed
altered protein complex formation. Using native polyacryl-
amide gels, we detected overexpressed ULK1 in two major
species: a smaller population with an apparent molecular mass
FIG. 6. Membrane targeting signal within the CTDs of ULK1 and ULK2. (A) 293/GFP-LC3 cells were transfected with Myc-ULK1 and starved
in EBSS-leupeptin for 2 h. Cell homogenates were centrifuged at 100,000 ? g to isolate membrane (Memb) and supernatant (Sup) fractions.
Aliquots of the supernatant (representing 2% of the cell sample) and membrane (representing 10% of the cell sample) fractions were analyzed
by SDS-PAGE. The top half of the blot was probed with anti-Myc and the bottom with anti-LC3. Where indicated, the membrane fraction was
washed with HB containing 0.15 M or 0.5 M KCl. (B and C) Myc-tagged ULK1 and -2 constructs were transfected into cells and analyzed for
membrane association as described above. (D) After transfection with Myc-tagged ULK1 CTD, cells were left untreated or starved in EBSS-
leupeptin for 2 h. Where indicated, the membrane pellet was washed in HB containing 0.5 M KCl and analyzed as described above. (E) 293/
GFP-LC3 cells were transfected with Myc-ULK1 CTD for 24 h and then starved in EBSS-leupeptin for 2 h before fixation and immunostaining
with anti-Myc monoclonal antibody. In a Z section close to the substratum (Z ? 0), ULK1 CTD strongly inhibited GFP-LC3 punctum formation.
In the Z ? 1 section, Myc-ULK1 CTD could be detected colocalizing with GFP-LC3-positive structures (inset). Bar ? 10 ?m. (F) Membrane
associations of ULK1 CTD (Full CTD) and various C-terminal deletion constructs in starved 293/GFP-LC3 cells. See the schematic in Fig. 2 for
the positions of inserted stop codons. The expression of ULK1 CTD was detected with anti-Myc, and GFP-LC3 was detected with anti-LC3.
(G) Membrane associations of ULK1 CTD and two N-terminal deletion constructs, Myc-LHS (845–1051) and Myc-LKG (864–1051), in starved
293/GFP-LC3 cells. (H) 293/GFP-LC3 cells were transfected with full-length Myc-ULK1 or -ULK2 and then left untreated or starved in
EBSS-leupeptin for 2 h before isolation of supernatant and membrane fractions. Expressed proteins were detected as described above. (I) Mem-
brane association of transfected Myc-ULK1 in starved 293/GFP-LC3 cells. Membrane pellets were analyzed before washes (none) or following
extraction in HB (0) or HB supplemented with 1.0% TX-100. (J) Membrane association of endogenous ULK1 was analyzed in untransfected,
starved 293/GFP-LC3 cells following the fractionation and wash procedures described for panel I. WT, wild type; Untrans, untransfected.
164 CHAN ET AL.MOL. CELL. BIOL.
of 400 to 500 kDa, and a larger complex migrating with an
apparent molecular mass of ?1,200 kDa (Fig. 7A). By com-
parison, overexpressed ULK2 in native gels was detected pri-
marily as one protein species migrating at ?1,000 kDa. Al-
though these protein complexes were not altered following
amino acid starvation, these results suggest that ULK1 and
ULK2 enter into distinct molecular complexes. Interestingly,
and in contrast with wild-type ULK1, kinase-inactivated
ULK1-K46I and ULK1-K46R existed primarily as a larger
1,200-kDa species (data not shown), raising the possibility that
altered protein complexes were related to our dominant-neg-
ative mechanism. However, ULK1 lacking its C-terminal 50
residues still entered into two molecular complexes, similarly
to the wild type, indicating that the C-terminal dominant-
negative IERRLSA motif was not involved in these molecular
interactions (Fig. 7B). Further deletion of the entire CTD
prevented ULK1 from entering into the high-molecular-mass
complex, indicating a requirement for sequences within the
CTD between amino acids 829 and 1001.
Putative human homologue of Atg13. Our deletion analyses
indicated a requirement of amino acid residues within the
CTD C terminus for dominant-negative activity. Recent find-
ings have shown that C-terminal residues of yeast Atg1 direct
binding to Atg13 (5). Thus, we questioned whether an Atg13
orthologue was involved in the dominant inhibitory mechanism
of the ULK1/2 CTDs. Using both Schizosaccharomyces pombe
and Saccharomyces cerevisiae Atg13 protein sequences, we
searched the genomes of higher organisms using repetitive
rounds of PSI-BLAST for an mAtg13 orthologue. In the first
round of iteration, we identified a putative orthologue in D.
melanogaster (CG7331) in addition to orthologues in mice
(Entrez protein database accession no. NP_663503.1) and hu-
mans (Entrez protein database accession no. NP_055556.2/
KIAA0652), which have both been annotated as the gene
products of harbinger transposase derived 1 (HARBI1). Harbi1
(Entrez protein database accession no. AAH02378) had pre-
viously been identified as the putative human Atg13 ortho-
logue (18). The D. melanogaster CG7331 protein was found in
a systematic characterization of protein interactions to bind D.
melanogaster Atg1 (28), supporting its role as a putative Atg13
orthologue. Bioinformatic analysis of current genome infor-
mation revealed that the putative vertebrate Atg13 ortho-
logues are tightly conserved (94% amino acid identity for
mouse versus human), while the putative orthologues in D.
melanogaster and C. elegans are clearly divergent.
To confirm the role of this putative Atg13 orthologue in
autophagy, we obtained an siRNA pool targeting the human
transcript NM_014741. In 293/GFP-LC3 cells, knockdown of
Atg13 inhibited the formation of starvation-induced GFP-LC3
punctum formation to an extent similar to that of ULK1
knockdown (Fig. 8A). We identified two individual siRNA
duplexes within this pool that could be confirmed to knock
down Atg13 mRNA (data not shown) and inhibit starvation-
induced lipidation of GFP-LC3 (Fig. 8B). We further con-
firmed that knockdown of Atg13 in parental HEK293 cells
inhibited lipidation of endogenous LC3 during unstarved and
starvation conditions, both without and with lysosomal pro-
tease inhibition by leupeptin (Fig. 8C).
As an independent means to confirm the function of Atg13
in autophagy, we investigated its role in mAtg9 trafficking
under starvation conditions (Fig. 8D). mAtg9 is a multispan-
ning membrane protein required for autophagy, and after
amino acid starvation or rapamycin treatment, mAtg9 redis-
tributes from a juxtanuclear clustered pool to a dispersed pe-
ripheral cytosolic pool (37). We have previously shown that
siRNA knockdown of ULK1 inhibits the starvation-induced
redistribution of mAtg9 (37). Using this assay, we find that
knockdown of human Atg13 similarly prevented the starvation-
induced dispersal of mAtg9, which supports the proposed iden-
tification of KIAA0652 as human Atg13 and demonstrates the
function of Atg13 in a ULK1-dependent autophagic pathway.
We expressed the putative human Atg13 orthologue with a
C-terminal FLAG tag in 293/GFP-LC3 cells (Fig. 9A). Ectopi-
cally expressed Atg13-FLAG existed primarily as a diffuse cy-
toplasmic pool in both unstarved (data not shown) and amino
acid-starved conditions. In some cells, Atg13-FLAG could be
observed on small cytoplasmic puncta that were often juxta-
posed, but not substantially colocalized, with GFP-LC3-la-
beled structures. Consistently with this immunofluorescence
analysis, approximately 5% of Atg13-FLAG (migrating at a
70-kDa apparent molecular mass by Laemmli SDS-PAGE)
was membrane associated (Fig. 9B) in unstarved conditions.
Amino acid starvation enhanced the membrane association of
Atg13-FLAG. We generated polyclonal antisera toward Atg13
and confirmed the loss of the endogenous protein following
knockdown (Fig. 9C). Endogenous Atg13 bound to mem-
branes, although this association was sensitive to 1% TX-100
extraction (Fig. 9D), which contrasts with the TX-100-resistant
membrane association of ULK1.
Overexpression of Atg13 is not sufficient to rescue inhibition
of autophagy caused by ULK dominant-negative proteins.
Data from yeast indicate that Atg13 binds Atg1 in a phosphor-
ylation-dependent manner (10). We found that, in cotrans-
fected HEK293 cells, Atg13-FLAG could readily coimmuno-
precipitate both Myc-tagged ULK1 and ULK2 (Fig. 10A).
FIG. 7. Molecular complexes of ULK1 and ULK2 detected using
native PAGE. HEK293A cells were transfected with ULK1 and ULK2
and starved for 2 h in EBSS where indicated (A) or ULK1 constructs
(B) before lysis in native gel sample buffer. Complexes were resolved
on native PAGE gels and then detected by immunoblotting with anti-
Myc antibody (top). Positions of native-gel molecular mass markers
are indicated on the left. As a control, aliquots of lysates were resolved
by conventional SDS-PAGE to detect overexpressed proteins (anti-
Myc) and total protein (?-tubulin [?Tub]) (bottom). WT, wild type.
VOL. 29, 2009FUNCTION AND BINDING PARTNERS OF ULK CTD 165
With control immunoblots of the lysates, we consistently ob-
served that Atg13-FLAG singly overexpressed migrated as a
sharp band close to 70 kDa. Strikingly, coexpression with either
ULK1 or ULK2 led to the reduced mobility of Atg13, suggest-
ing that ULK1 and ULK2 were regulating the phosphorylation
of human Atg13. In yeast, both Atg13 phosphorylation and
Atg1-Atg13 binding could be regulated by rapamycin treat-
ment (10). In contrast, amino acid starvation in our over-
expression cell system did not robustly affect either the
electrophoretic mobility of Atg13 or its ability to coimmunopre-
cipitate with ULK1 or ULK2 (data not shown). In contrast to
wild-type ULK proteins, coexpressed ULK1-K46I or ULK2-
K39I did not promote a hypershift of Atg13-FLAG. However,
Atg13-FLAG still coimmunoprecipitated with both ULK1-
K46I and ULK2-K39I, suggesting that binding was not regu-
lated by Atg13 phosphorylation (Fig. 10B and C).
Since the last 20 residues of yeast Atg1 are critical for
binding to yeast Atg13 (5), we predicted that the ULK CTD
would be involved in the binding of human Atg13, and we
tested this idea using coimmunoprecipitation experiments.
While coexpression with ULK1?CTD resulted in the loss of
the Atg13-FLAG protein, this effect was less pronounced
when the ULK2?CTD mutant was used. As shown in Fig.
10D, when ULK2?CTD and Atg13 were coexpressed, Atg13
did not hypershift or coimmunoprecipitate with ULK2?CTD.
Importantly, the CTDs of both ULK1 and ULK2 readily
coimmunoprecipitated with Atg13-FLAG (Fig. 10E). Sur-
prisingly, the ULK1 CTD lacking 50 residues from its C
terminus [ULK1CTD(829–1001)] still coimmunoprecipi-
tated with Atg13-FLAG with an efficiency similar to that of
the full-length CTD (Fig. 10F). We similarly observed that
the C-terminal 50 residues of ULK2 were dispensable for
Atg13 binding (data not shown). Thus, the C-terminal por-
tions of the ULK1 and -2 CTDs (including the dominant-
negative IERRLSA motif) could be removed without affect-
ing Atg13 binding.
Earlier, we hypothesized that the dominant-negative
mechanism initiated by the CTDs and kinase-dead versions
of ULK1 and -2 might involve the titration of an essential
Atg13-dependent function, perhaps through Atg13 recruit-
ment. However, our mapping data suggested that the region of
the CTD binding Atg13 was separable from the residues that
participated in dominant-negative interactions. In agreement
with this conclusion, coexpression with Atg13-FLAG did not
produce any rescuing effects toward the dominant-negative
activities of the ULK1 and -2 CTDs on GFP-LC3 lipidation
(Fig. 10G). Atg13-FLAG coexpression similarly could not res-
cue the inhibition of autophagy caused by ULK1-K46I or
ULK2-K39I (data not shown). Thus, we conclude that the
FIG. 8. A putative human Atg13 homologue is required for autoph-
agy. (A) 293/GFP-LC3 cells were transfected with control siRNA
(Ctrl) or siRNA targeting ULK1 or a SMARTpool targeting
KIAA0652 (Atg13) for 72 h. Cells were then left unstarved or starved
in EBSS-leupeptin for 2 h before fixation and morphological analysis.
The quantification represents the total GFP-LC3 spot intensity per cell
(arbitrary units), and each bar represents the average of 60 image fields
(from six independent cell samples) ? the standard deviation.**, P ?
0.0007 in pairwise comparisons with Ctrl knockdown starved cells.
(B) 293/GFP-LC3 cells were transfected for 72 h with control siRNA,
the Atg13 SMARTpool, or individual duplexes (#1 or #2) specific for
Atg13. Cells were left unstarved or starved as described for panel A
and then lysed for immunoblot analysis of GFP-LC3 lipidation, as
described in the legend for Fig. 2.*, P ? 0.02 in pairwise comparisons
with siRNA Ctrl starved cells. (C) HEK293A cells were transfected
with control siRNA or Atg13 duplex 2 for 72 h. Cells were then left
unstarved (Unst) or starved (st) for 2 h in EBSS (with or without
leupeptin [Leu]) before lysis for immunoblot analysis of endogenous
LC3 lipidation. The quantified ratio of LC3-II/LC3-I is shown below
each sample. ?Tub, ?-tubulin. (D) 293/GFP-LC3 cells were trans-
fected for 72 h with control siRNA (siCtrl) or siRNAs targeting ULK1
(siULK1) or Atg13 (duplex 2) (siAtg13), starved in EBSS-leupeptin for
2 h, and then fixed and immunostained to detect endogenous mAtg9
localization (red). After starvation in siCtrl-treated cells, mAtg9 be-
comes redistributed predominantly to a diffuse cytoplasmic pool. After
knockdown of Atg13, mAtg9 redistribution is blocked and mAtg9-
positive vesicles remain in juxtanuclear clusters (arrows) to an extent
similar to that after knockdown of ULK1. Inhibition of starvation-
induced GFP-LC3 autophagosome formation after knockdown of
ULK1 and Atg13 is shown as a control. Bar ? 10 ?m.
166 CHAN ET AL.MOL. CELL. BIOL.
dominant inhibitory mechanism involves an essential factor
independent of Atg13.
Phosphorylation of Atg13 by ULK proteins. Last, because
the kinase that phosphorylates Atg13 in yeast remains unclear,
we investigated whether ULK1 and -2 could directly phosphor-
ylate Atg13. We asked whether immunoprecipitated ULK pro-
teins could phosphorylate immunoprecipitated Atg13-FLAG
in a mixed-bead in vitro kinase reaction. Immunoprecipitated
Atg13-FLAG incubated alone under reaction conditions did
not undergo a mobility hypershift in SDS-PAGE, demonstrat-
ing that there was no associated kinase activity in the immu-
noprecipitate (Fig. 10H). However, incubation of Atg13 to-
gether with immunoprecipitated ULK1 or ULK2 did result in
a decreased mobility of Atg13. This mobility change was not
observed when Atg13 was incubated with kinase-dead ULK1
and -2 or ULK proteins lacking their CTDs. These data sup-
port the conclusion that Atg13 binds ULK1 and -2 via their
CTDs, promoting phosphorylation of Atg13 via the catalytic
kinase domain. Immunoblotting of lysates from cells under
normal medium conditions revealed that endogenous Atg13
migrates as a doublet, suggestive of distinct species with dif-
ferent levels of phosphorylation (Fig. 10I). Expression of
ULK1 or ULK2 promoted substantially more Atg13 with re-
duced mobility, further supporting the phosphorylation of
Atg13 by ULK1 and -2 in vivo.
ULK proteins adopt a closed conformation to prevent the
CTD from forming abnormal interactions. Our previous data
showed unexpectedly that the ULK1-K46R kinase mutant (32)
did not show a strong dominant inhibitory effect in our autoph-
agy cell system (3). Here, we confirmed that while the K46R
substitution significantly impaired catalytic function in vitro,
the charge-disruptive K46I substitution fully ablated activity
and produced a mutant protein that displayed less autophos-
phorylation and inhibited starvation-induced autophagy. These
data agree with a recent report that showed a ULK1-K46N
mutant to inhibit autophagy in NIH 3T3 fibroblasts (6). Our
data also showed that the CTD was required for kinase-inac-
tive ULK1 to inhibit autophagy. Similarly, in D. discoideum,
deletion of CTD sequences rescued the dominant-negative
effects of the Atg1-K36A kinase mutant on autophagy-depen-
dent survival (30). Deletion of the CTD also stimulated in vitro
autophosphorylation but decreased the ability of the kinase to
phosphorylate exogenous substrates.
These findings and our other results led us to propose a
working model in which the CTD is normally folded back onto
the N-terminal region to keep the protein in a closed molec-
ular conformation (Fig. 11). It is still unclear whether the
interaction of the CTD with N-terminal regions is controlled
by the kinase domain (perhaps via a protein-protein interac-
tion) or via an interaction at the autophosphorylation sites.
The K46I mutation triggers a robust conformational change
leading to exposure of the CTD, potentially revealing a binding
site in the CTD that interacts with essential autophagy regu-
Our deletion analysis has identified a 7-residue motif (IER
RLSA) within the last 14 C-terminal amino acids of the CTD
that was required for dominant-negative function, which we
propose constitutes a binding site for a critical autophagy reg-
ulatory factor (“Y” in Fig. 11). Interestingly, the IERRLSA
motif is well conserved in ULK1/2 proteins from mice, humans,
and other vertebrates but not in Atg1 homologs of S. cerevisiae,
D. melanogaster, and C. elegans. Based on this sequence infor-
mation, we speculate that the factor binding to the IERRLSA
motif might be specific to vertebrates.
Could conformational changes represent a physiological
mechanism for the regulation of ULK proteins? The in vitro
limited proteolysis assay that we employed could not detect
significant changes after amino acid starvation (Chan and
Tooze, unpublished), although this could represent a limita-
tion of our system. However, our findings have established that
an almost complete ablation of the kinase activity is required to
produce readily detectable effects in protease cleavage and
autophagy assays. It was recently demonstrated that the kinase
activity of endogenous ULK1 in mouse embryonic fibroblasts
was modestly activated following amino acid starvation (6),
which suggests that subtle conformational alterations could
possibly represent a normal means of ULK1 regulation.
Signals within the ULK1 CTD direct membrane binding
and protein complex formation. We and others have seen that
ULK1 localizes to GFP-LC3- or Atg16L1-positive autophago-
somal structures (3, 6) and that this localization depends upon
the CTD. In order to define the downstream effectors of the
FIG. 9. Human Atg13 is membrane associated. (A) 293/GFP-LC3 cells expressing Atg13-FLAG were starved for 2 h in EBSS-leupeptin, fixed,
and then immunostained using anti-FLAG antibody. Atg13-FLAG can be found both in a diffuse cytosolic pool and localized to punctate structures
that do not colocalize with GFP-LC3. Bar ? 10 ?m. (B) Membrane association of Atg13-FLAG was analyzed as described in the legend for Fig.
6. (C) HEK293A cells were transfected with control or Atg13 duplex 2 (Dup#2) for 72 h. Cell lysates were then immunoblotted with a polyclonal
antibody for endogenous Atg13 or actin as a loading control. (D) Membrane fractions analyzed as described for Fig. 6J from untransfected
293/GFP-LC3 cells were probed for endogenous Atg13. Sup, supernatant; Memb, membrane fraction; Ctrl, control.
VOL. 29, 2009 FUNCTION AND BINDING PARTNERS OF ULK CTD167
CTD (i.e., to identify factor “Y”), we used biochemical frac-
tionation and imaging techniques to characterize the ULK
CTD. The CTDs of both ULK1 and ULK2 contained signals
that could direct membrane binding, and further deletion map-
ping identified N- and C-terminal sequences that were dispens-
able for membrane association. Importantly, CTD deletion
mutants lacking the IERRLSA dominant-negative motif still
retained normal membrane binding efficiencies, which suggests
that the critical inhibitory interactions initiated by the CTD are
not membrane associated (Fig. 11B). Our data on overex-
pressed and endogenous ULK1 also suggest that association to
membranes was resistant to detergent extraction, unlike results
for other autophagy proteins, such as the lipidated form of
LC3, and Atg13. Our preliminary data using floatation in de-
tergent-containing gradients suggest that ULK1 cofractionates
with markers of lipid rafts (A. Longatti and S. A. Tooze,
unpublished observations), although the molecular basis of
these observations requires further investigation.
In S. cerevisiae, Atg1 enters into a number of protein
complexes (9–12, 22). Using native gel electrophoresis, we
observed ULK1 in two distinct populations suggestive of
different subcomplexes. Inactivation of kinase function
shifted the ULK1-K46I protein toward the higher-molecular-
weight complex, with a corresponding loss of the low-molecu-
lar-weight species (Chan and Tooze, unpublished). Impor-
tantly, while entry of ULK1 into the higher-molecular-weight
population required the entire CTD, up to 50 C-terminal res-
idues, including the IERRLSA dominant-negative motif, could
be removed from the CTD C terminus without affecting entry
into either protein complex. Thus, the dominant-negative mo-
FIG. 10. Binding of Atg13 to the CTD is required for phosphorylation by ULK1 and ULK2. (A, B, and C) HEK293A cells were transfected
with Atg13-FLAG alone (?) or in combination (?) with Myc-ULK1, Myc-ULK2, Myc-ULK1-K46I, or Myc-ULK2-K39I, as indicated. An aliquot
of cell lysate was analyzed by SDS-PAGE and immunoblotting with Myc (top) and FLAG (bottom) antibodies. Lysates of doubly transfected cells
were incubated with anti-FLAG beads to coimmunoprecipitate Atg13 and ULK proteins. (D) Myc-ULK2 and Myc-ULK2?CTD were coexpressed
with FLAG-Atg13 in HEK293A cells. As a control, Atg13-FLAG was expressed alone (?). Cell lysates were immunoprecipitated with anti-Myc
or anti-FLAG antibody and then immunoblotted as described above. (E) Coimmunoprecipitation of the Myc-tagged CTDs of ULK1 and ULK2
with Atg13-FLAG. Lysates and immunoprecipitates were analyzed as described above. (F) Coimmunoprecipitation of Myc-ULK1CTD(829–1001)
with Atg13-FLAG. Lysates and immunoprecipitates were analyzed as described above. The asterisks in panels D, E, and F indicate nonspecific
bands arising from the antibody used for immunoprecipitation, as demonstrated by the antibody-plus-beads-alone control (Ab ctrl). (G) 293/
GFP-LC3 cells were cotransfected with Myc-ULK1 CTD or Myc-ULK2CTD with or without Atg13-FLAG for 24 h and then either left unstarved
or starved in EBSS-leupeptin for 2 h before lysis for analysis of GFP-LC3 lipidation, as described in the legend for Fig. 2. Protein expression
controls (top) using anti-FLAG and anti-Myc antibodies are shown. (H) Immunoprecipitated Atg13-FLAG protein was used as a substrate for
immunoprecipitated Myc-ULK proteins in a mixed-bead in vitro kinase reaction. Phosphorylation of Atg13-FLAG is detected as a decrease in
mobility. A control reaction without the addition of ULK proteins (?) shows no mobility shift. (I) HEK293A cells were either left untransfected
(?) or transfected with Myc-ULK1 or Myc-ULK2 before cell lysates were immunoblotted to detect hypershifting on endogenous Atg13. Lys,
lysates; IP, immunoprecipitate.
168 CHAN ET AL.MOL. CELL. BIOL.
tif proposed to bind the critical factor “Y” is separate from
other CTD sequences that direct formation of higher-molecu-
lar-weight ULK1 protein complexes.
Our data further suggest the existence of two types of ULK1
complexes: a small one containing proteins that bind to ULK1
in a CTD-dependent manner, and a large complex containing
proteins that can still bind kinase-inactive ULK1. It has been
demonstrated that in S. cerevisiae Atg1 plays two roles in bulk
nonselective autophagy: one being structural, facilitating re-
cruitment of Atg proteins to the PAS, and the second being
responsible for recycling proteins from the PAS during forma-
tion (4, 5). The former role requires the CTD, while the latter
requires the kinase domain. While our data do not provide any
support for a dual role of the mAtg1 orthologues, further
analysis of the small and large complexes may shed light on
whether ULK1 or ULK2 behaves similarly to yeast Atg1. Fur-
ther investigation is also required to understand the differences
between ULK1 and ULK2 complexes revealed by native gel
Human Atg13 binds to the CTD independently of the IER
RLSA motif. Since binding of yeast Atg13 depends upon the
C-terminal 20 residues of yeast Atg1 (5), we asked whether the
ULK1 and ULK2 CTDs could bind the human homologue of
Atg13 and whether this binding occurred via the IERRLSA
motif. We identified and characterized a putative human
Atg13 orthologue and showed that it was required for autoph-
agy by using LC3 lipidation and the mAtg9 subcellular redis-
tribution assays. Consistent with a role in autophagy, tagged
versions of Atg13 became more enriched on membranes fol-
lowing the activation of autophagy. Alteration of mAtg9 traf-
ficking caused by the loss of Atg13 could be due to either
decreased Atg13 function or an indirect modulation of ULK1
function or stability. In yeast, Atg1 kinase activity is not re-
quired for Atg9 trafficking, so it is unlikely that the loss of
ULK1 kinase activity underlies the aberrant mAtg9 trafficking.
Rather, as we previously showed, siRNA-mediated depletion
of ULK1 inhibits mAtg9 trafficking (37), and our preliminary
data suggest that siRNA depletion of Atg13 causes a decrease
in ULK1 levels (N. C. McKnight and S. A. Tooze, unpublished
observations). Further work is required to clarify the role of
human Atg13 in mAtg9 trafficking.
Despite their sequence differences, ULK1 and ULK2 both
bound human Atg13 via their CTDs. The IERRLSA motif that
we identified here, while not well conserved in yeast, includes
an R residue (position 1041 in ULK1) which corresponds to
the conserved R885 in yeast Atg1 that constitutes part of an
Atg1-Atg13 interaction motif (5). Furthermore, within the C-
terminal 20 amino acids of yeast Atg1 is a conserved Y residue
at position 878, and substitution of both R885 and Y878 with
alanine (Y878A/R885A) in Atg1 results in the loss of recruit-
ment of Atg17 to the PAS (5). Interestingly, this Y residue is
conserved in mouse and human ULK2 but not ULK1. How-
ever, our data using CTD deletions of ULK1 and ULK2 show
that the IERRLSA motif (and also the last 20 amino acids) is
not required for human Atg13 binding. We speculate either
that another binding site has evolved in the ULKs for human
Atg13 or possibly that the Y878A/R885A mutations, which
were made in the context of the whole yeast Atg1 protein,
altered the conformation or accessibility of the true Atg13
In support of our model and binding data, coexpression with
Atg13 did not rescue the inhibition of autophagy caused by
ULK CTDs (or kinase-dead mutants [data not shown]), sug-
gesting that Atg13 is not the protein being titrated out by the
7-residue CTD motif. A possible candidate for this binding
protein (and perhaps factor “Y”) is FIP200, which recently has
been shown to bind ULK1 and ULK2 in a CTD-dependent
manner and to regulate ULK1 kinase function and protein
stability (6). Interestingly, Hara et al. suggest that FIP200 may
be the human homologue of yeast Atg17 and that, although
there is no sequence conservation between Atg17 and FIP200,
both proteins have coiled-coil domains, suggesting a conserva-
tion of function as a scaffold protein (6).
Phosphorylation of Atg13 by ULK1 and ULK2. In the course
of our studies, we observed that Atg13 displayed a de-
creased electrophoretic mobility when coexpressed with
ULK1 or ULK2, suggestive of a modification by phosphor-
ylation. Consistent with direct phosphorylation of Atg13 by
ULK1 and ULK2, Atg13 did not show an altered migration
FIG. 11. Conformational changes of ULK proteins modulated by
autophosphorylation status. (A) Domain structure of ULK1 showing
the N-terminal kinase domain and the critical lysine 46 residue (K46)
targeted in our mutant constructs. Our data are consistent with the
existence of multiple, but so far unidentified, autophosphorylation (P)
sites within residues 278 to 351 of the serine- and proline-rich spacer
domain (S/P spacer). Our previous findings indicate a region spanning
residues 278 to 351 that is critical for inhibition of autophagy, presum-
ably via interaction with an unknown protein (X). Our previous data
also indicated that the last three residues of ULK1 (VYA) are critical
for regulatory function, possibly by binding a PDZ domain-containing
protein. Our findings here indicate multiple functions directed by se-
quences within the conserved CTD. A region between residues 829
and 1001 contains signals for binding to membranes and interaction
with human Atg13. Additional sequences between residues 1038 and
1044 (Fig. 2) contain a conserved motif (IERRLSA, black diamond)
that is required for dominant inhibitory activity by interacting with
another unknown factor (Y). (B) Proposed model showing phosphor-
ylation-dependent conformational changes. In normal resting cells,
ULK proteins are autophosphorylated, and these modifications help
maintain a closed conformation that (i) brings Atg13 in closer prox-
imity to the kinase domain for phosphorylation and (ii) keeps the
dominant-negative motif hidden or inaccessible. Full ablation of kinase
activity results in a low degree of autophosphorylation and a more
open molecular conformation that exposes the dominant inhibitory
CTD motif, allowing it to bind the unknown factor (Y) that is critical
VOL. 29, 2009 FUNCTION AND BINDING PARTNERS OF ULK CTD169
when coexpressed with ULK proteins lacking Atg13 binding
sites (?CTD) or kinase-inactivated ULK proteins. Further-
more, both ULK1 and ULK2 could promote Atg13 phos-
phorylation in a kinase-dependent fashion following an in
vitro kinase reaction. These data suggest that Atg13 binds to
ULK1 and ULK2 via the CTDs and is phosphorylated by the
kinase domain (Fig. 11). In contrast with findings for yeast,
binding of Atg13 to ULK proteins in our HEK293 cell
model was not modulated by starvation of cells or by the
overall phosphorylation status of Atg13. The definition of
Atg13 phosphorylation sites and their regulatory function
requires further investigation.
Differences between ULK1 and ULK2. Several lines of evi-
dence have shown that ULK1 and ULK2 behave similarly.
Kinase mutants of both ULK1 and ULK2 have dominant-
negative effects, and our findings suggest that this is via expo-
sure of a conserved motif within their CTDs. In addition, both
ULK1 and ULK2 localize to autophagosomal structures and
both proteins bind Atg13 and FIP200.
However, multiple aspects of our findings also highlight
that ULK1 and ULK2 may have independent functions or
modes of regulation. Deletion of CTD sequences in overex-
pressed ULK1 and ULK2 appears to promote different down-
stream effects. ULK1 and ULK2 can enter into different mo-
lecular complexes, and the binding of ULK2 to membranes
was increased following amino acid starvation, in contrast to
results with ULK1. Finally, we previously observed that knock-
down of ULK1 but not ULK2 in HEK293 cells inhibited star-
vation-induced autophagy and starvation-induced mAtg9 re-
distribution. We also previously observed that overexpression
agy, while here we show that overexpression of ULK2 in the
same cell system enhanced GFP-LC3 lipidation following star-
vation. We have not yet been able to confirm the ability of
overexpressed ULK2 to modulate other measures of autoph-
agic flux by use of assays such as degradation of long-lived
proteins or p62/SQSTM-1 stability (Chan, Longatti, and Tooze,
unpublished), possibly due to the limited capabilities of
downstream pathways. While additional experiments are re-
quired to fully understand the functional differences be-
tween ULK1 and ULK2, our biochemical data presented
here are in general agreement with recent knockout mouse
data that suggest ULK1 and ULK2 could be performing
distinct roles in specialized and general autophagy. In addi-
tion, to further understand the mammalian equivalent of the
yeast Atg1 signaling complex, it will be important to dissect
the binding site within the ULK CTD for FIP200 and Atg13
as well as to examine the relationship between FIP200 and
Atg13 for regulating the kinase activity and function of ULK
We thank Mike Howell (LRI High Throughput Screening Unit) for
advice and assistance in the Cellomics array scan experiments and
Mike Mitchell (CRUK Bioinformtics Unit) for help with Atg13 bioin-
formatics. We thank the Secretory Pathways Laboratory for advice and
encouragement and Cancer Research UK for support.
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