The Tor and PKA signaling pathways independently target the Atg1/Atg13 protein kinase complex to control autophagy

Article (PDF Available)inProceedings of the National Academy of Sciences 106(40):17049-54 · October 2009with14 Reads
DOI: 10.1073/pnas.0903316106 · Source: PubMed
Abstract
Macroautophagy (or autophagy) is a conserved degradative pathway that has been implicated in a number of biological processes, including organismal aging, innate immunity, and the progression of human cancers. This pathway was initially identified as a cellular response to nutrient deprivation and is essential for cell survival during these periods of starvation. Autophagy is highly regulated and is under the control of a number of signaling pathways, including the Tor pathway, that coordinate cell growth with nutrient availability. These pathways appear to target a complex of proteins that contains the Atg1 protein kinase. The data here show that autophagy in Saccharomyces cerevisiae is also controlled by the cAMP-dependent protein kinase (PKA) pathway. Elevated levels of PKA activity inhibited autophagy and inactivation of the PKA pathway was sufficient to induce a robust autophagy response. We show that in addition to Atg1, PKA directly phosphorylates Atg13, a conserved regulator of Atg1 kinase activity. This phosphorylation regulates Atg13 localization to the preautophagosomal structure, the nucleation site from which autophagy pathway transport intermediates are formed. Atg13 is also phosphorylated in a Tor-dependent manner, but these modifications appear to occur at positions distinct from the PKA phosphorylation sites identified here. In all, our data indicate that the PKA and Tor pathways function independently to control autophagy in S. cerevisiae, and that the Atg1/Atg13 kinase complex is a key site of signal integration within this degradative pathway.
The Tor and PKA signaling pathways independently
target the Atg1/Atg13 protein kinase complex
to control autophagy
Joseph S. Stephan
a,b
, Yuh-Ying Yeh
a,b
, Vidhya Ramachandran
a,b
, Stephen J. Deminoff
a
, and Paul K. Herman
a,b,1
a
Department of Molecular Genetics and
b
Program in Molecular, Cellular and Developmental Biology, The Ohio State University, Columbus, OH 43210
Edited by Scott D. Emr, Cornell University, Ithaca, NY, and approved August 6, 2009 (received for review March 25, 2009)
Macroautophagy (or autophagy) is a conserved degradative pathway
that has been implicated in a number of biological processes, includ-
ing organismal aging, innate immunity, and the progression of
human cancers. This pathway was initially identified as a cellular
response to nutrient deprivation and is essential for cell survival
during these periods of starvation. Autophagy is highly regulated and
is under the control of a number of signaling pathways, including the
Tor pathway, that coordinate cell growth with nutrient availability.
These pathways appear to target a complex of proteins that contains
the Atg1 protein kinase. The data here show that autophagy in
Saccharomyces cerevisiae is also controlled by the cAMP-dependent
protein kinase (PKA) pathway. Elevated levels of PKA activity inhib-
ited autophagy and inactivation of the PKA pathway was sufficient
to induce a robust autophagy response. We show that in addition to
Atg1, PKA directly phosphorylates Atg13, a conserved regulator of
Atg1 kinase activity. This phosphorylation regulates Atg13 localiza-
tion to the preautophagosomal structure, the nucleation site from
which autophagy pathway transport intermediates are formed.
Atg13 is also phosphorylated in a Tor-dependent manner, but these
modifications appear to occur at positions distinct from the PKA
phosphorylation sites identified here. In all, our data indicate that the
PKA and Tor pathways function independently to control autophagy
in S. cerevisiae, and that the Atg1/Atg13 kinase complex is a key site
of signal integration within this degradative pathway.
cAMP-dependent protein kinase macroautophagy stationary phase
Tor protein kinase
M
acroautophag y (hereaf ter autophag y) is a highly-
c onserved membrane trafficking pathway that is respon-
sible for the turnover of bulk cytoplasmic protein and organelles
(1, 2). This pathway was initially identified as a cellular response
to nutrient deprivation (3, 4). However, recent studies indicate
that autophagy is involved in a wide variet y of physiological
processes, including tissue remodeling during development, the
removal of protein agg regates, and innate immune responses (5,
6). During autophagy, an isolation membrane emanates from a
nucleation site that is known as the preautophagosomal structure
(PAS) in Saccharomyces cerevisiae and the phagophore assembly
site in mammals (7, 8). This double membrane encapsulates
nearby cytoplasm and ultimately targets it to the vacuole/
lysosome for degradation. The breakdown products are then
rec ycled to allow for the synthesis of the macromolecules needed
for survival during the period of starvation (9). The cellular
c omponents mediating autophagy were initially described in S.
cerevisiae, and orthologs of many of these Atg proteins have since
been identified in other eukaryotes (10, 11).
The flux through the autophagy pathway is tightly controlled
by multiple signaling pathways, including the Tor pathway, that
are responsible for coordinating cell growth with nutrient avail-
abilit y. One of the key targets of this control appears to be a
c omplex of proteins that contains the Atg1 protein kinase
(12–15). Atg1 is specifically recruited to the PAS, and activated,
in response to conditions that induce autophagy (7, 16). In
c ontrast, most Atg proteins are constitutively localized to this
nucleation str ucture (17). Recent work suggests that the mam-
malian Atg1 proteins, ULK1 and ULK2, are also recruited to the
phagophore assembly site, and activated, upon nutrient depri-
vation (18, 19). A key question that remains is how do these
signaling pathways work together through Atg1 to ensure the
appropriate autophagic response.
In S. cerevisiae, the cAMP-dependent protein kinase (PKA)
signaling pathway has also been implicated in the control of
autophagy (13, 20). However, the precise nature of this control, and
how it is integrated with that of the Tor pathway, is still somewhat
controversial (13, 21). In this report, we show that the PKA pathway
is critical for the appropriate control of autophagy in S. cerevisiae.
Inhibition of PKA signaling is sufficient to induce robust autophagy
activity, and this control appears to occur independently, or in
parallel to, that exerted by the Tor pathway. For example, both of
these pathways target Atg13, a key regulator of Atg1 protein kinase
activity, but appear to affect distinct sets of phosphorylation sites on
this protein. In all, the data here indicate that both the PKA and Tor
pathways are important, but independent, regulators of autophagy,
and that the Atg1 protein kinase complex is a key site of signal
integration within this pathway.
Results
Inactivation of the PKA Pathway Is Sufficient to Induce Autophagy.
Elevated levels of PKA activity have been shown to inhibit the
autophagy process (13). Here, we tested whether the inactivation
of this signaling pathway was also sufficient to induce autophagy.
To shutdown PKA signaling, we used an inducible form of a
dominant negative allele of RAS2, known as RAS2
ala22
(22). In
S. cerevisiae, the Ras proteins, Ras1 and Ras2, regulate cAMP
production, and thus PKA activity, by directly stimulating ad-
enylyl cyclase (23, 24). The RAS2
ala22
allele used here was under
the control of the promoter from the MET3 gene, a locus that is
repressed when methionine is in the growth medium (25). We
found that autophagy was efficiently induced upon expression of
the RAS2
ala22
protein and that the kinetics of induction were
similar to that observed with rapamycin treatment (Fig. 1A).
Moreover, this RAS2
ala22
-mediated induction was dependent
upon the presence of Atg1 (Fig. 1B ). To directly compare the
ef fects of inhibiting the Tor and PKA pathways, we used a
c oncentration of rapamycin that produced a growth arrest
similar to that observed in the MET3-RAS2
ala22
strain. [Note that
rapamycin specifically inhibits the TORC1 complex and that we
will be referring to this complex when we discuss Tor signaling
Author contributions: J.S.S., Y.-Y.Y., V.R., S.J.D., and P.K.H. designed research; J.S.S., Y.-Y.Y.,
V.R., and S.J.D. performed research; J.S.S., Y.-Y.Y., V.R., and S.J.D. contributed new re-
agents/analytic tools; J.S.S., Y.-Y.Y., V.R., S.J.D., and P.K.H. analyzed data; and J.S.S. and
P.K.H. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1
To whom correspondence should be addressed. E-mail: herman.81@osu.edu.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0903316106/DCSupplemental.
www.pnas.orgcgidoi10.1073pnas.0903316106 PNAS
October 6, 2009
vol. 106
no. 40
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CELL BIOLOGY
in this report (26).] Therefore, a decrease in PKA function that
was suf ficient to arrest S. cerevisiae growth resulted in a con-
c omitant increase in autophagy activity. These results are con-
sistent with a previous study that used a galactose-inducible form
of this RAS2
ala22
allele (13).
The second method to inactivate PKA activity made use of an
‘‘analog-sensitive’’ version of this enzyme. This approach for
inactivating protein kinases was pioneered by Kevan Shokat and
his colleagues, and involves altering a specific residue within the
k inase active site (27, 28). Particular substitutions at this ‘‘gate-
keeper’’ position render these enzy mes sensitive to membrane-
soluble inhibitors, like 1NM-PP1 (29). Here, we used a yeast
strain that has an ‘‘analog-sensitive’’ allele of TPK1, tpk1-as,as
the sole source of PKA activity (27, 28). S. cerevisiae has three
functionally redundant PKA catalytic subunits that are encoded
by the TPK1–3 genes (30). The tpk1-as strain used here, gener-
ously provided by Dr. James Broach, lacks TPK2 and TPK3, and
c ontains an allele of TPK1, tpk1-M164G, that is sensitive to the
dr ug, 1NM-PP1. Interestingly, we found that 1NM-PP1 concen-
trations that inhibited cell growth in the tpk1-as strain also
resulted in a robust induction of the autophagy pathway (Fig. 1
CE and Figs. S1 and S2). For these experiments, autophagy
activity was assessed with three different assays that are de-
scribed in the Materials and Methods. Therefore, the inhibition
of PKA signaling by two different means resulted in an induction
of autophagy activity similar to that observed upon inactivation
of the Tor pathway. These results are significant because they
c ontradict a recent study that suggested that the loss of PKA
activity was not sufficient to induce autophagy (21). This latter
study also used a yeast strain that contained analog-sensitive
versions of PKA; this strain is in the same genetic backg round as
the tpk1-as strain used here. However, this previous study used
a 1NM-PP1 concentration of only 0.1
M, a concentration that
did not have a significant effect upon the g rowth rate of the
tpk1-as strain (Fig. 1C). Therefore, the low level of autophagy
observed was likely due to residual PKA activity remain ing in the
analog-sensitive cells. In all, our data here indicate that the
inactivation of the PKA pathway is sufficient to induce autoph-
agy activ ity in S. cerevisiae cells.
Atg13 Is a Substrate for PKA in Vitro and in Vivo. The Atg13 protein
physically interacts with Atg1 and is required for full Atg1
protein kinase activity in vitro (14, 31–35). Although the mech-
an istic basis of this activation is not yet understood, the Atg1-
Atg13 interaction in S. cerevisiae appears to be regulated by Tor
signaling activity. A recent study also identified Atg13 as a
candidate substrate for PKA in this budding yeast (20). This
identification was based on the presence of evolutionarily-
c onserved matches in Atg13 to the PKA consensus phosphory-
lation site. Two sites were very similar to the consensus of
R-R-x-S/T-B, where x refers to any amino acid and B to a
hydrophobic residue (Sites 2 and 3; Fig. 2A) (36). A third
c onserved site that dev iates more from the consensus was also
identified (Site 1). We found that each of these sites was
phosphorylated by PKA in vitro, and alteration of all three sites
resulted in a greater than 95% decrease in Atg13 phosphoryla-
tion (Atg13-AAA; Fig. 2 B and C and Fig. S3). Similar results
were observed in vivo with an assay that makes use of an antibody
that specifically recognizes phosphorylated PKA sites (Fig. 2D)
(37, 38). For these experiments, Atg13 was precipit ated from cell
extracts and the level of PKA phosphorylation was assessed by
Western blotting with this
-substrate antibody. This Atg13
signal was lost upon phosphatase treatment, and was restored by
a subsequent incubation with PKA and ATP (Fig. 2D). In
addition, this in vivo signal was elevated in cells that possessed
elevated levels of PKA activity (Fig. 2E). Finally, Atg13 recog-
n ition by this
-substrate antibody was lost following the inac-
tivation of PKA activity (Fig. 2F). In all, these data indicated that
Atg13 was a direct substrate for PKA in S. cerevisiae.
The Loss of PKA Phosphorylation on Atg13 Was Correlated with the
Induction of Autophagy. Atg13 has been shown to be required for
Atg1 kinase activity in vitro (14). Here, we tested whether this
protein was also required for Atg1 activ ity in vivo by taking
advant age of a previous observation concerning the mobility of
this protein in SDS-polyacrylamide gels (14, 20). In particular,
autophosphorylation was found to retard the mobility of Atg1 in
these gels, and the presence of this slower-migrating band can
Fig. 1. Inactivation of the Ras/PKA signaling pathway was sufficient to
induce autophagy. (A) Autophagy induction upon rapamycin treatment
and/or expression of the dominant-negative RAS2
ala22
allele. Wild-type cells
(TN125) were grown to mid-log phase and treated with 20 ng/mL rapamycin
for4hat30°C.Cells carrying the MET3-RAS2
ala22
allele were transferred to an
SC minimal medium lacking methionine for4hat3Ctoinduce expression
from the MET3 promoter. Autophagy levels were assessed with an alkaline
phosphatase (ALP)-based assay as describedin the Materials and Methods. The
white bars indicate the relative levels of ALP activity in the untreated controls
and the black bars the activity following the indicated treatments. (B) The
autophagy activity induced upon inactivation of the Ras/PKA pathway was
dependent upon the presence of the Atg1 protein. Autophagy levels were
assessed with the ALP-based assay in isogenic wild-type and atg1 cells after
4 h of rapamycin treatment (R) or exposure to the RAS2
ala22
protein (A). (C)
Growth curves for isogenic TPK1 and tpk1-as strains in YPAD at 30 °C are
shown. The drug 1NM-PP1 was present at the indicated concentrations. (D)
Inactivation of PKA signaling in the tpk1-as strain resulted in the induction of
autophagy. Autophagy levels were assessed with the ALP-based assay in
tpk1-as cells (PHY4710) that were treated for 5 h with either 200 ng/mL
rapamycin or the indicated concentrations of 1NM-PP1. (E) Cells over-
expressing Ape1 were treated for 4 h with either 200 ng/ml rapamycin or 25
M 1NM-PP1, and the relative level of Ape1 processing was assessed by
Western blotting. Cont, untreated cells.
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www.pnas.orgcgidoi10.1073pnas.0903316106 Stephan et al.
serve as an indicator of Atg1 kinase activ ity in vivo (Fig. 3A). We
found that the proportion of Atg1 in the slower migrating form
increased upon rapamycin treatment (Fig. 3A). This activation of
Atg1 did not occur in cells that lacked either Atg13 or Atg17;
these mutants were the only atg strains tested that exhibited a
sign ificant defect in this Atg1 autophosphorylation. Therefore,
Atg13 was required in vivo for Atg1 protein kinase activity.
We used the tpk1-as strain described above to examine the
temporal relationship between the PKA phosphorylation of
Atg13, and the induction of both Atg1 kinase activity and
autophagy. We found that the PKA-dependent phosphorylation
of Atg13 was largely gone after 30 min of treatment w ith the
dr ug, 1NM-PP1 (Fig. 3B). The first signs of Atg1 kinase activity
were apparent at this time, but the fraction of Atg1 in the
slower-migrating form continued to increase at later time points
(Fig. 3B). Autophagy activity was found to increase in a similar
manner (Fig. 3C). Finally, we found that this inactivation of the
PKA pathway also resulted in the re-localization of Atg1 from
the cytoplasm to the PAS (Fig. 3D). These data therefore
indicated that the loss of Atg13 phosphorylation preceded the
activation of Atg1 and the induction of the autophagy pathway.
PKA Phosphorylation Regulates the Association of Atg13 with the PAS.
As observed previously, we found that Atg13 was largely cyto-
plasmic in growing cells and was associated with the PAS upon
nutrient limitation (Fig. 4A). Here, we tested whether this
localization to the PAS was regulated by PKA phosphorylation.
A clear precedent exists as PKA phosphorylation has been
shown to inhibit the PAS association of Atg1 (20). We found that
the Atg13-AAA variant was localized to the PAS in both growing
and nitrogen-starved cells (Fig. 4A and Fig. S4). Moreover, the
association of the wild-type Atg13 (Atg13-SSS) with the PAS was
inhibited by the presence of the RAS2
val19
allele that results in
c onstitutively-elevated levels of PKA activity (39). This inhibi-
tion was dependent upon the PKA sites as the Atg13-AAA
protein was still constitutively localized to the PAS in RAS2
val19
cells (Fig. 4A and Fig. S4). Finally, we found that the w ild-type
Atg13 protein was recruited to the PAS following the inactiva-
tion of the Ras/PKA pathway (Fig. 4B). These data are therefore
c onsistent with PKA phosphorylation regulating the association
of Atg13 with the PAS.
Previous studies have shown that Atg13 interacts with a
number of proteins, most notably Atg1 and Atg17 (4042). Since
Atg17 has been suggested to play a role in organizing the PAS,
we tested whether the PAS association of Atg13-AAA was
dependent upon the presence of Atg17 (43–45). Indeed, we
found that the Atg13-AAA variant was less efficiently targeted
to the PAS in cells lacking Atg17 (Fig. 4 C and D). In addition,
Atg13-AAA exhibited a stronger interaction with Atg17 than the
wild-type Atg13 protein and this interaction was no longer
influenced by rapamycin treatment (Fig. 4E) (41). Instead,
Atg13-AAA appeared to be c onstitutively associated with
Fig. 2. The Atg13 protein was a substrate for PKA. (A) The three conserved
sites of PKA phosphorylation in Atg13 are shown. (B and C) The in vitro
phosphorylation of Atg13 was dependent upon the presence of the above
three PKA sites. The indicated Atg13 variants were precipitated from yeast
cells and incubated with [
-
32
P] ATP and either bovine PKA (bPKA) (in B)orthe
S. cerevisiae Tpk1 (C). S, serine; A, alanine. (D) The three PKA sites were
required for the in vivo phosphorylation of Atg13. The indicated Atg13
proteins were immunoprecipitated from yeast cell extracts with an
-HA
antibody and treated with
phosphatase and then incubated with bPKA and
3 mM ATP, as indicated. The level of PKA phosphorylation was assessed by
Western blotting with an
-substrate antibody that recognizes phosphory-
lated PKA sites. (E) The in vivo level of PKA phosphorylation on Atg13 was
elevated in a strain over-expressing Tpk1. (F) Recognition by the
-substrate
antibody was lost following inactivation of PKA. The tpk1-as strain was
incubated with 10
M 1NM-PP1 for 4 h and the PKA phosphorylation level of
Atg13 was assessed by Western blotting with the
-substrate antibody.
Fig. 3. The loss of PKA phosphorylation on Atg13 preceded the activation of
Atg1 and the induction of autophagy. (A) Atg13 and Atg17 were required for
Atg1 autophosphorylation in vivo. Cell extracts were prepared from the
indicated yeast strains and the levels of autophosphorylated Atg1 were
assessed by Western blotting. The cells were treated with 200 ng/mL rapamy-
cinfor0or2hat30°C.Theatg1
KD
strain has a kinase-defective allele of ATG1,
atg1-K54A. In the bottom panel, Atg1 was immunoprecipitated from yeast
cell extracts and then treated with
phosphatase, as indicated. (B) The relative
levels of Atg13 phosphorylation by PKA (top panel) and of the ‘‘activated’’
form of Atg1 (bottom) were assessed by Western blotting at the indicated
times after the addition of 25
M 1NM-PP1 to a culture of tpk1-as cells. (C)
Autophagy activity was assessed with the ALP-based assay at the indicated
times after the addition of 25
M 1NM-PP1 to tpk1-as cells. (D) The localization
of an Atg1-YFP protein was assessed by fluorescence microscopy 4 h after the
addition of 25
M 1NM-PP1 to a culture of tpk1-as cells.
Stephan et al. PNAS
October 6, 2009
vol. 106
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CELL BIOLOGY
Atg17. In contrast, alteration of the PKA sites did not signifi-
cantly influence the Atg1-Atg13 interaction (Fig. S5A). Finally,
Atg17 was found to be associated with the PAS in both growing
and nitrogen-starved cells, and this localization was not inhibited
by the presence of RAS2
val19
(Fig. S5B) (40, 45). Therefore, PKA
was apparently regulating the PAS association of Atg13, at least
in part, by interfering with its interaction with Atg17.
The PKA and Tor Pathways Independently Target the Atg13 Protein.
The simultaneous inactivation of both the PKA and Tor path-
ways produced a more rapid and greater induction of autophagy
than was observed with the loss of either pathway alone (Figs. 1A
and 5A). This result is consistent with these pathways work ing
independently of each other to control autophagy. To examine
this possibility, we tested how shutting down one of these
pathways would influence the activity of the other. For these
ex periments, we used the PKA- and Tor-dependent phospho-
rylations of Atg13 as reporters for the activity of the respective
signaling pathways. We found that the in vivo level of Atg13
phosphorylation by PKA was not diminished upon rapamycin
treatment (Fig. 5B). In addition, the inactivation of the Ras/PKA
pathway did not result in a loss of the Tor-dependent phosphor-
ylation present on Atg13 (Fig. 5C). This latter phosphorylation
causes Atg13 to run as a broad smear on SDS-polyacrylamide
gels. This smear rapidly collapses into a tight, faster-migrating
band upon rapamycin treatment (Fig. 5C) (14, 46). Finally, the
in vitro phosphorylation of Atg13 by PKA did not alter the
mobilit y of this protein in SDS-polyacrylamide gels (see Fig. 2).
Therefore, although the precise locations of the Tor-dependent
phosphorylation sites on Atg13 have yet to be identified, these
positions appear to be distinct from the PKA sites described
here. In all, these data are consistent with the Tor and PKA
pathways working independently to control autophagy.
Discussion
Autophagy is a nonspecific deg radative process that must be
tightly controlled to prevent the inappropriate turnover of
material needed for cell growth. The work here adds to our
current understanding of this process by demonstrating that the
Fig. 4. The PAS localization of Atg13 was regulated by PKA phosphorylation.
(A) Fluorescence microscopy was performed with cells that contained the
indicated YFP-Atg13 fusion proteins. The Atg13 proteins had either wild-type
(Atg13-SSS) or nonphosphorylatable versions (Atg13-AAA) of the three PKA
sites. The CFP-Atg11 fusion protein was present in all cells and served as a
marker for the PAS. The RAS2
val19
allele was present in the indicated strains.
Nitrogen starvation was achieved by transferring the cells from SC glucose
minimal medium to the SD-N medium for1hat3C.(B) The localization of
a wild-type Atg13-YFP protein in the indicated cells was assessed by fluores-
cence microscopy. A22, RAS2
ala22
.(C) Atg17 was required for the efficient
localization of Atg13-AAA to the PAS in log phase cells. The fraction of cells
with Atg1-AA or Atg13-AAA present in a perivacuolar punctate spot in the
indicated strains is shown (20). At least 50 cell images were examined for each
strain. (D) Representative images for the indicated strains in C.(E) Alteration
of the PKA phosphorylation sites in Atg13 influenced the interaction with
Atg17. The indicated Atg13 proteins were immunoprecipitated from yeast cell
extracts and the relative levels of the associated Atg17 were assessed by
Western blotting. The cell extracts were prepared from either mid-log phase
cultures (Log) or from cells that were treated with rapamycin (Rap). S, Atg13-
SSS; A, Atg13-AAA.
Fig. 5. The PKA and Tor pathways independently target Atg13 to control
autophagy activity. (A) Simultaneous inactivation of the PKA and Tor path-
ways resulted in an elevated autophagy response relative to the loss of either
pathway alone. Autophagy levels were assessed with the ALP-based assay in
tpk1-as cells that had been treated for the indicated time with either 25
M
1NM-PP1, 200 ng/mL rapamycin, or both reagents. The data shown are from
a single experiment that was representative of at least three independent
replicates. (B) The PKA phosphorylation of Atg13 was not diminished upon
inactivation of the Tor pathway. Atg13 was immunoprecipitated from cells
followinga2htreatment with either 20 (Lo) or 200 (Hi) ng/mL rapamycin. The
level of PKA phosphorylation was subsequently assessed by Western blotting
with the
-PKA substrate antibody. (C) Inactivation of the Ras/PKA pathway
did not influence the Tor-dependent phosphorylation of Atg13. Cell extracts
were prepared from the indicated cells and the level of Tor-dependent
phosphorylation of Atg13 was assessed by Western blotting. In the left-hand
lanes, cells containing either MET3-RAS2
ala22
(A22) or a control plasmid (-)
were incubated in methionine-free medium for6htoallow for expression
from theMET3 promoter. In the middlelanes, the tpk1-as strain, PHY4710,was
incubated for4hwith0or5
M 1NM-PP1. The bottom panel in the middle
lanes shows the level of PKA phosphorylation on Atg13 as assessed by Western
blotting with the
-PKA substrate antibody. Note that the relative spread of
the Atg13 ‘‘smear’’ is dependent upon the running conditions of the gel. (D)
A model depicting the proposed roles of the PKA and Tor pathways in the
control of autophagy. The dashed lines indicate the Atg1 and Atg13 interac-
tions with each other or the PAS (and perhaps Atg17), and the solid lines
indicate the regulatory effects of the PKA and/or Tor pathways on these
interactions. See the text for additional details.
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www.pnas.orgcgidoi10.1073pnas.0903316106 Stephan et al.
Ras/PKA pathway is an important regulator of autophag y in S.
cerevisiae. Inactivation of this signaling pathway by two dif ferent
methods was sufficient for a robust induction of autophagy
activity. This control appears to be exerted through the Atg1
c omplex as both Atg1 itself and Atg13, a key regulator of this
k inase, were shown to be direct substrates for PKA (20) (Fig. 2).
For both proteins, this phosphorylation appears to regulate the
association with the PAS. Interestingly, the data here indicate
that this control by PKA is independent of that exerted by the
Tor pathway. For example, although both pathways regulate
Atg13 phosphorylation, each appears to control a distinct set of
phosphorylation events. In all, this report suggests that the
Atg1/Atg13 complex serves as an important point of signal
integration within the autophagy pathway. The relevance of this
c omplex as a regulatory c ontrol point may be evolutionarily
c onserved as both Atg1 and Atg13 have been found to be targets
for the Tor pathway in other eukaryotes (32, 33, 35, 47).
The autophagy pathway in S. cerevisiae therefore appears to be
under the c ontrol of at least two independent regulatory inputs.
A model that attempts to summarize our current understanding
of this regulation is presented in Fig. 5D. In this model, PKA
activity specifically inhibits the PAS association of both Atg1 and
Atg13. In contrast, the Tor pathway appears to influence both
this localization and the Atg1-Atg13 interaction that is necessar y
for the activation of Atg1 kinase activity (14). Therefore, inac-
tivation of the Tor pathway would result in the for mation of an
active Atg1-Atg13 complex at the PAS and the ensuing induction
of autophag y (1). Upon the loss of PKA activity, however, it is
less clear how the Atg1 enzyme is activated. One possibility is
that the increased local concentrations of Atg1 and Atg13 at the
PAS are sufficient to override the inhibitory effects of Tor
activity on the Atg1-Atg13 interaction. Alternatively, Atg13
c ould be selectively dephosphorylated at the PAS. Although
further experimentation is needed to distinguish between these
possibilities, the work here shows that the loss of PKA signaling
results in increased Atg1 activity and the induction of autophag y.
A n additional question that arises is why more than one signaling
pathway might be needed for the regulation of autophagy. A n
interesting possibility is that these pathways are responding to
distinct nutritional cues and that different types of starvations
might elicit distinct autophagy responses in the cell (48). The
existence of these multiple inputs would therefore provide the
cell with greater flexibility in its response to changing environ-
ment al conditions. Deter mining how these pathways influence
this degradative process, and how these signaling activities are
c oordinated, are therefore important questions for future work.
Materials and Methods
Additional methods, including a description of growth conditions and plasmid
construction, are included in SI Text.
Yeast Strain Construction and Growth Conditions. The yeast strains used in this
study were PHY1220 (MAT
his3-200 leu2–3,112 lys2– 801 trp1–101 ura3–52
suc2-9), PHY1942 (PHY1220 prc1::HIS3 pep4::LEU2 prb1::hisG), TN125
(MATa ade2 his3 leu2 lys2 trp1 ura3 pho8::pho860), YYK126 (TN125
atg1::LEU2), YYK130 (TN125 atg13::TRP1), PHY3687 (TN125 atg1::LEU2
atg13::kanMX), Y3175 (ade2–1 can1–100 his3–11,15 leu2–3,112 trp1–1
ura3–1 tpk2::KAN tpk3::TRP1 tpk1-M164G), and PHY4710 (Y3175
pho8::pho860). This latter strain was generated by integration of a previ-
ously described plasmid, PTN9, into Y3175 (49). Strains carrying the MET3-
RAS2
val19
or MET3-RAS2
ala22
alleles were grown in medium containing 500
M
methionine to keep the MET3 promoter in its repressed state. Expression from
the MET3 promoter was induced by transferring cells to a medium that lacked
methionine. Expression from the CUP1 promoter was induced by the addition
of 100
M CuSO
4
to the growth medium. The drug, 1NM-PP1, was generously
provided by Kevan Shokat.
Western Blotting and Immunoprecipitations. Protein extracts for Western
blotting were prepared by a glass bead lysis protocol described previously (13).
The resulting protein extracts were separated on SDS-polyacrylamide gels and
transferred to nitrocellulose membranes (Hybond ECL, Amersham Bio-
sciences) at 4 °C. The membranes were probed with the appropriate primary
and secondary antibodies and the Supersignal chemiluminescent substrate
(Pierce) was used to illuminate the reactive bands. The immunoprecipitation
experiments were performed as described (37, 38).
Autophagy Assays. The alkaline phosphatase (ALP) assay for autophagy activ-
ity was performed as described (13, 49). This assay measures the delivery and
subsequent activation of an altered form of the Pho8 phosphatase by the
autophagy pathway. Two additional assays assessed the processing of ami-
nopeptidase I (Ape1) and the accumulation of autophagic bodies, and are
discussed in SI Text.
PKA Phosphorylation Assays. In general, the in vitro phosphorylation assays
were performed with HA epitope-tagged proteins that were under the con-
trol of the yeast CUP1 promoter in the yeast strain, PHY1942. The strains were
grown to mid-log phase in selective SC minimal medium containing 2%
glucose, and induced with 100
M copper sulfate for 90 mins. The HA
epitope-tagged Atg13 variants were isolated on an
-HA antibody resin
(Roche), and incubated with [
-
32
P] ATP (PerkinElmer) and 5 U bovine PKA
catalytic subunit (Sigma), as described previously (20, 38). A Western immu-
noblot control was performed with an
-HA antibody (Sigma) to assess the
relative amount of Atg13 present in each sample.
The in vivo level of PKA phosphorylation was assessed with an
-PKA
substrate antibody (Cell Signaling) as described (37, 38). Briefly, the substrate
proteins were immunoprecipitated from yeast cell extracts, separated on
SDS-polyacrylamide gels and the relative level of occupancy at the PKA sites
was assessed by Western blotting with the
-PKA substrate antibody used at
a concentration of 1:2,000.
Fluorescence Microscopy. The CFP-Atg11, YFP-Atg13, and YFP-Atg17 fusions
were under the control of the inducible promoter from the yeast CUP1 gene.
Expression of these fusion proteins was induced by the addition of 100
M
CuSO
4
for1hat3C.Thesamples were imaged as described (20).
ACKNOWLEGEMENTS. We thank James Broach, Daniel Klionsky, Takeshi Noda,
Yoshinori Ohsumi, Kevan Shokat, and Jeremy Thorner for reagents used in this
study, and members of the Herman lab for helpful discussions and comments
on the manuscript. J.S.S. was supported, in part, by a graduate student
fellowship from the Jeffrey Seilheimer Lung Cancer Foundation. This
work was supported by a grant from the National Institutes of Health
(GM65227) to P.K.H.
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www.pnas.orgcgidoi10.1073pnas.0903316106 Stephan et al.
    • "of Atg13, enabling it to form a complex with Atg1 and thereby initiating the autophagy process. Both Atg1 and Atg13 are direct substrates for PKA, but its phosphorylation sites are distinct from those targeted by Tor (Stephan et al., 2009). The concerted regulation of autophagy by Tor and PKA is rather complex and currently not fully understood. "
    [Show abstract] [Hide abstract] ABSTRACT: Most filamentous fungi have a saprophytic lifestyle and proliferate on organic materials from plants. They contain a large arsenal of enzymes that are expressed and secreted in response to available carbon sources. Regulatory networks in which carbon-specific transcription factors and wide-domain transcription factors play essential roles tightly control the expression of these enzymes. While a suitable carbon source is present, the entire metabolism of the fungus is directed to grow as fast as possible via hyphal tip extension. At a certain point however, the available exogenous carbon source will be limited, resulting in a carbon starvation response. Filamentous fungi react to carbon starvation with some very specific responses including the induction of glycosyl hydrolases involved in fungal cell wall degradation (autolysis) and the onset of asexual spore formation. Recent advances in genome-wide transcriptomic and proteomic approaches have revealed new insights into molecular mechanisms involved in these fungal-specific carbon starvation responses. Together with the genetic accessibility of filamentous fungi, the omics technologies have made a large contribution in broadening our understanding of the carbon starvation response in recent years. In this review, we have focussed on summarizing and integrating the most important cellular responses of filamentous fungi towards carbon starvation. As most studies to date have been carried out with Aspergillus nidulans and Aspergillus niger, the focus will be further on these two species and to compare their responses to carbon starvation.
    Chapter · Aug 2016 · Molecular Brain
    • "Gene deletions were generated using one-step deletion protocol as described in [61]. The tpk1-as allele was realized by deleting tpk2, tpk3, and by making a point mutation in tpk1M164G as described in [14, 33]. The C-terminal auxin induced degron (AID) tag (used for the KOG1-AID and TUP1-AID alleles), which also includes three copies of the V5 epitope, was generated using one step PCR integration (the plasmid was a gift from Leon Chan, UC Berkeley). "
    [Show abstract] [Hide abstract] ABSTRACT: Cell fate choices are tightly controlled by the interplay between intrinsic and extrinsic signals, and gene regulatory networks. In Saccharomyces cerevisiae, the decision to enter into gametogenesis or sporulation is dictated by mating type and nutrient availability. These signals regulate the expression of the master regulator of gametogenesis, IME1. Here we describe how nutrients control IME1 expression. We find that protein kinase A (PKA) and target of rapamycin complex I (TORC1) signalling mediate nutrient regulation of IME1 expression. Inhibiting both pathways is sufficient to induce IME1 expression and complete sporulation in nutrient-rich conditions. Our ability to induce sporulation under nutrient rich conditions allowed us to show that respiration and fermentation are interchangeable energy sources for IME1 transcription. Furthermore, we find that TORC1 can both promote and inhibit gametogenesis. Down-regulation of TORC1 is required to activate IME1. However, complete inactivation of TORC1 inhibits IME1 induction, indicating that an intermediate level of TORC1 signalling is required for entry into sporulation. Finally, we show that the transcriptional repressor Tup1 binds and represses the IME1 promoter when nutrients are ample, but is released from the IME1 promoter when both PKA and TORC1 are inhibited. Collectively our data demonstrate that nutrient control of entry into sporulation is mediated by a combination of energy availability, TORC1 and PKA activities that converge on the IME1 promoter.
    Full-text · Article · Jun 2016
    • "When nutrients are abundant, mTOR (mammalian target of rapamycin) is active for maintaining cell growth. However, when rapidly growing cells are treated with rapamycin, hypoxia or amino starvation, the protein synthesis is generally downregulated and autophagy is activated through the Atg1 dependent pathway505152. p70S6K1 (p70 ribosomal protein S6 kinases 1), a kind of ribosomal protein kinase, controls cell growth and migration. "
    [Show abstract] [Hide abstract] ABSTRACT: Background Our previous finding showed that brain ischemic preconditioning mediates neuroprotection through endoplasmic reticulum (ER) stress-induced autophagy. This study was aimed at exploring the role of ER chaperone GRP78 in IPC induced autophagy activation in neural cells. Results Ischemic preconditioning (IPC) and oxygen glucose deprivation (OGD) models were established in rat pheochromocytoma (PC12) cells and primary cultured murine cortical neurons. IPC exerted neuroprotection against subsequent OGD injury in both PC12 cells and primary cortical neurons. IPC increased GRP78 expression and activated autophagy, as evidenced by upregulated LC3 and Beclin1, increased autophagic flux and formation of autophagosomes. BAPTA(dibromo-1,2-bis(aminophenoxy)ethane N,N,N9,N9 - tetra acetic acid, 0.125-2 μM) and small interfering RNA targeted GRP78 abrogated IPC induced neuroprotection and decreased the expression of GRP78, LC3II/LC3I and Beclin1. In contrast, lentiviral vector mediated GRP78 overexpression (LV-GRP78) strengthened resistance of PC12 cells to OGD injury and increased LC3 and Beclin1 expression. Moreover, knockdown of GRP78 in stable GRP78 overexpressing PC12 cells abolished the upregulation of LC3II/LC3I. GRP78 might activate autophagy through AMPK - mTOR pathway. Conclusion These results suggest that IPC- induced GRP78 upregulation is involved in autophagy activation, and hence exerts protection against ischemic injury in neural cells. Electronic supplementary material The online version of this article (doi:10.1186/s13041-015-0112-3) contains supplementary material, which is available to authorized users.
    Full-text · Article · Dec 2015
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