Download Phosphorylation of Beclin 1 by DAP

[Autophagy 5:5, 1-3; 1 July 2009]; ©2009 Landes Bioscience
Article Addendum
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1Department
of Molecular Genetics; and 2Chemical Research Support; Weizmann Institute of Science; Rehovot, Israel
Abbreviations: DAP-kinase or DAPk, death-associated protein kinase
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various Bcl-2 family members, whereas its dissociation from Bcl-2
mediates autophagy.7
We recently discovered that Beclin 1 is a substrate for DAPk.1
DAPk, a calcium/calmodulin Ser/Thr kinase, functions as a tumor
suppressor gene and has been linked to several cell death pathways,
including autophagic cell death. Using pull-down assays, as well
as immunoprecipitation experiments, we show that DAPk physically interacts with Beclin 1. Interestingly, DAPk cannot bind a
mutant Beclin 1 lacking the Bcl-2 binding domain, implying that
this domain is required for the DAPk-Beclin 1 interaction. Using
in vitro kinase assays, we show that DAPk phosphorylates Beclin
1, and map the phosphorylation site to T119 by peptide phosphorylation assays, mass spectrometry mapping, and the use of
phospho-specific antibodies.
T119 is located within Beclin 1’s BH3 domain. We show that
DAPk significantly reduces the amounts of Bcl-XL, which are
immunoprecipitated by wild-type Beclin 1, leading to autophagy,
whereas it fails to reduce Bcl-XL binding to the T119A phosphosilencing mutant. Conversely, the T119E phospho-mimicking
mutant displays a weaker association with Bcl-XL, and induces
autophagy when overexpressed. These results suggest that phosphorylation on T119 by DAPk leads to Beclin 1’s dissociation
from its inhibitor, Bcl-XL, resulting in autophagy induction.
The interaction between BH3-only proteins and the anti-apoptotic Bcl-2 members is mediated by hydrophobic and electrostatic
interactions between the amphipathic α-helix of the BH3 domain,
and a hydrophobic pocket formed by the BH1-3 domains of
the anti-apoptotic Bcl-2 proteins. T119 is a residue unique to
Beclin 1’s BH3 domain. Other BH3-only proteins contain a
hydrophobic residue in the corresponding position, which acts
together with three additional hydrophobic residues present in
their BH3 amphipathic α-helix, to stabilize the interaction with
local hydrophobic pockets within the large target-binding groove.
The lack of a hydrophobic amino acid at position 119 in Beclin
1 might explain why Beclin 1’s binding to Bcl-XL is weaker than
that of other BH3-only proteins.10 Interestingly, substituting T119
in Beclin 1 to isoleucine enhances its association with Bcl-XL,
confirming that this is a critical site in the interface between Beclin
1 and its inhibitors.10 Our recently published data show that this
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Beclin 1, an essential autophagic protein, is a BH3-only
protein that binds Bcl-2 anti-apoptotic family members. The
dissociation of Beclin 1 from the Bcl-2 inhibitors is essential
for its autophagic activity, and therefore is tightly controlled.
We recently revealed a novel phosphorylation-based mechanism
by which death-associated protein kinase (DAPk) regulates this
process. We found that DAPk phosphorylates Beclin 1 on T119,
a critical residue within its BH3 domain, and thus promotes
Beclin 1 dissociation from Bcl-XL and autophagy induction.1
Here we report that T119 phosphorylation also reduces the
interaction between Beclin 1 and Bcl-2, in line with the high
degree of structural homology between the BH3 binding pockets
of Bcl-2 and Bcl-XL proteins. Our results reveal a new phosphorylation-based mechanism that reduces the interaction of Beclin
1 with its inhibitors to activate the autophagic machinery.
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Key words: autophagy, Bcl-2, Bcl-XL, beclin 1, DAP-kinase
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Einat Zalckvar,1 Hanna Berissi,1 Miriam Eisenstein2 and Adi Kimchi1,*
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Autophagy is an evolutionarily conserved process that has both
cytoprotective and cell death functions.2-5 Beclin 1 is a tightly
regulated haploinsufficient tumor suppressor, which is part of
a class III phosphatidlyinositol 3-kinase (PI3K) multiprotein
complex that participates in autophagosomes nucleation.6 The
autophagy-promoting activity of Beclin 1 is suppressed by antiapoptotic members of the Bcl-2 family through direct binding.
Beclin 1 is a bona fide BH3-only protein, and the α-helix of its
BH3 domain binds to the hydrophobic groove in Bcl-XL, similar to
the interactions previously shown for other BH3 only proteins.7-11
Under normal steady state growth conditions, Beclin 1 is bound to
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*Correspondence to: Adi Kimchi; Weizmann Institute of Science; Department of
Molecular Genetics; Hertzel St.; Rehovot 76100 Israel; Tel.: 972.8.9342428;
Fax: 972.8.9315938; Email: [email protected]
Submitted: 03/12/09; Revised: 03/31/09; Accepted: 04/02/09
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This manuscript has been published online, prior to printing. Once the issue is complete and page numbers have been assigned, the citation will change accordingly.
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Phosphorylation of Beclin 1 by DAP-kinase promotes autophagy by
weakening its interactions with Bcl-2 and Bcl-XL
Previously published online as an Autophagy E-publication:
http://www.landesbioscience.com/journals/autophagy/article/8625
Addendum to: Zalckvar E, Berissi H, Mizrachy L, Idelchuk Y, Koren I, Eisenstein M,
Sabanay H, Pinkas-Kramarski R, Kimchi A. DAP-kinase-mediated phosphorylation
on the BH3 domain of beclin 1 promotes dissociation of beclin 1 from Bcl-XL and
induction of autophagy. EMBO Rep 2009; 10:285–92; PMID: 19180116; DOI:
10.1038/embor.2008.246.
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2009; Vol. 5 Issue 5
Interaction of Beclin 1 with Bcl-2/Bcl-XL is regulated by DAPk
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Figure 2. Phosphorylation on Beclin 1’s T119 reduces its interaction with
Bcl-2. HEK293T cells were cotransfected with Flag-tagged wild-type (WT),
T119A or T119E Beclin 1 mutants (described in ref. 1) and the Bcl-2
expression vector (OriGene). Beclin 1 was immunoprecipitated using antiFlag antibodies (Sigma F3165), and the co-immunoprecipitated proteins,
as well as the total cell extracts, were blotted using rabbit anti-Beclin 1
and mouse anti-Bcl-2 antibodies (Santa Cruz Biotechnology sc-11427 and
sc-7382, respectively).
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critical site is in fact a target of tight regulation by phosphorylation, causing a severe clash in the interaction with Bcl-XL.
We next examined whether phosphorylation on T119 also
affects the interaction of Beclin 1 with Bcl-2. We first compared
in silico the amino acids in Bcl-XL that form the binding pocket
of T119 to the corresponding residues in Bcl-2 (Fig. 1). We found
that these residues are conserved in Bcl-2, except for the solventexposed A104 in Bcl-XL, which is replaced by the acidic D108
in Bcl-2. Therefore, the T119 binding pocket is predicted to be
very similar in Bcl-XL and Bcl-2, and phosphorylation of T119
will most likely lead to similar disruption of the binding between
Beclin 1 and Bcl-2. Moreover, the presence of an additional
negative charge (D108) in Bcl-2 suggests that it may repel phosphorylated Beclin 1 even more than Bcl-XL.
To further investigate whether the T119 phosphorylation
reduces association of Beclin 1 and Bcl-2, we used the phosphomimicking (T119E) and phospho-silencing (T119A) mutants of
Beclin 1 and tested their binding to Bcl-2, as compared to the
wild-type Beclin 1. HEK293T cells were cotransfected with Flagtagged Beclin 1 and Bcl-2, and the anti-Flag immunoprecipitates
were immunoblotted with anti-Bcl-2 antibodies. The threonine
to glutamic acid substitution at position 119 strongly reduced the
binding of Beclin 1 to Bcl-2 (Fig. 2). These results suggest that
phosphorylation on T119 also reduces the association of Beclin
1 with Bcl-2.
Notably, it has been recently reported that phosphorylation
of Bcl-2 by JNK1, at residues T69, S70 and S87 located within
the unstructured loop of Bcl-2, is another mechanism which
reduces the interaction between Beclin 1 and its inhibitor.12 Taken
together with our results, it is suggested here that the interaction
between Beclin 1 and its inhibitors is dynamic, and subjected
to regulation by phosphorylation of either one of the two partners in this complex (Fig. 3). One event is the DAPk-mediated
phosphorylation on the T119 residue of Beclin 1, and the other
is the JNK-1-dependent phosphorylation at residues T69, S70
and S87 of Bcl-2. Future experiments should test whether these
post-translational modifications on Beclin 1 and on its inhibitors occur simultaneously or are mutually exclusive. To this end,
we will examine the state of the two phosphorylation events in
response to different autophagic inducers. In case both proteins are
phosphorylated simultaneously, possible synergistic effects leading
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Figure 1. The binding cavity of T119 as observed in the Bcl-XL-Beclin 1
complex. (Protein Data Bank code 2p1l). Beclin 1 is colored in green,
blue and red for carbon, oxygen and nitrogen atoms, respectively. T119
is emphasized (ball and stick presentation) and the stabilizing hydrogen
bond between T119/Oγ and the BH3 helix backbone is indicated by a
red line. Bcl-XL is shown in yellow with the side chains of the residues
that form the hydrophobic T119 binding pocket indicated. All these
residues are conserved in Bcl-2, except for the exposed A104, which is
replaced by D.
Figure 3. A model showing two phosphorylation events which regulate the
interaction between Beclin 1 and its inhibitors. DAPk-mediated phosphorylation on Beclin 1’s T119 residue, and JNK1-mediated phosphorylation on
residues T69, S70 and S87 on Bcl-2, each reduces Beclin 1’s interaction
with its inhibitors leading to autophagy.
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Autophagy
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Interaction of Beclin 1 with Bcl-2/Bcl-XL is regulated by DAPk
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to a more severe effect on the interaction between Beclin 1 and its
inhibitors will be tested using the phospho-mimicking mutants of
Bcl-2 and Beclin 1. Answers to these questions will significantly
advance our understanding of the regulation of Beclin 1 and
autophagy induction.
Acknowledgements
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We thank B. Levine for the Flag-Beclin 1 plasmid. This
work was supported by the Kahn Fund for System Biology at
the Weizmann Institute of Science, and by the Israel Science
Foundation (ISF). A.K. is the incumbent of Helena Rubinstein
Chair of Cancer Research.
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