CST Guide:

Section I: Research Areas
These protein targets represent key
nodes within autophagy signaling
pathways and are commonly studied
in autophagy research. Primary
antibodies, antibody conjugates, and
antibody sampler kits containing
these targets are available from CST.
Listing as of September 2014. See our
website for current product information.
M Monoclonal Antibody
P Polyclonal Antibody
S SignalSilence ® siRNA
207
2012–2014 citations
CST antibodies for LC3B have been
cited over 207 times in high-impact,
peer-reviewed publications from the
global research community.
Antibody
Validation
Principles
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Commonly Studied Autophagy Targets
Target M P S Target M P S
Ambra1
• Phospho-Beclin-1
Atg3
(Ser93/96) •
•
Atg4A
Bif-1
•
•
Atg4B
BNIP3
• • •
•
Atg4C
BNIP3L/Nix
• •
•
Atg5
FIP200
• • •
•
Atg7
GABARAP
• • •
•
Atg9A GABARAPL2
• •
•
Atg12
LC3A
• •
• •
Atg13 LC3A/B
• •
• •
Atg14
LC3B
• •
• • •
Atg16L1
MTMR3
•
•
Atg101
MTMR14
•
•
Beclin-1
NBR1
• • •
•
Phospho-Beclin-1 (Ser15)
Rubicon
•
•
SQSTM1/p62 • • •
Select Citations:
Mancias, J.D. et al. (2014) Quantitative proteomics identifies
NCOA4 as the cargo receptor mediating ferritinophagy.
Nature 509, 105–109.
Bejarano, E. et al. (2014) Connexins modulate autophagosome
biogenesis. Nat. Cell Biol. 16, 401–414.
Jang, Y.H. et al. (2014) Phospholipase D-mediated autophagic
regulation is a potential target for cancer therapy. Cell
Death Differ. 21, 533–546.
Kim, J. et al. (2014) Differential regulation of distinct Vps34
complexes by AMPK in nutrient stress and autophagy. Cell
152, 290–303.
Mealer, R.G. et al. (2014) Rhes, a striatal-selective protein
implicated in Huntington disease, binds beclin-1 and activates
autophagy. J. Biol. Chem. 289, 3547–3554.
Efeyan, A. et al. (2014) Regulation of mTORC1 by the Rag
GTPases is necessary for neonatal autophagy and survival.
Nature 93, 679–683.
Brot, S. et al. (2014) Collapsin response mediator protein 5
(CRMP5) induces mitophagy, thereby regulating mitochondrion
numbers in dendrites. J. Biol. Chem. 289, 2261–2276.
Martins, I. et al. (2014) Molecular mechanisms of ATP
secretion during immunogenic cell death. Cell Death Differ.
21, 79–91.
Lu, B. et al. (2014) JAK/STAT1 signaling promotes HMGB1
hyperacetylation and nuclear translocation. Proc. Natl. Acad.
Sci. USA 111, 3068–3073.
Li, Q. et al. (2014) Cited2, a transcriptional modulator
protein, regulates metabolism in murine embryonic stem
cells. J. Biol. Chem. 289, 251–263.
Papa, L. et al. (2014) SirT3 regulates the mitochondrial
unfolded protein response. Mol Cell Biol. 34, 699–710.
Hong, S.W. et al. (2013) SVCT-2 in breast cancer acts
as an indicator for L-ascorbate treatment. Oncogene 32,
1508–1517.
Zhai, H. et al. (2013) Inhibition of autophagy and tumor
growth in colon cancer by miR-502. Oncogene 32,
1570–1579.
Brot, S. et al. (2014) Collapsin response mediator protein 5
(CRMP5) induces mitophagy, thereby regulating mitochondrion
numbers in dendrites. J. Biol. Chem. 289, 2261–2276.
Target M P S
Phospho-SQSTM1/p62
(Thr269/Ser272) •
Phospho-SQSTM1/p62
(Ser403)
•
TECPR1 •
TMEM49/VMP1 •
ULK1 • • •
Phospho-ULK1 (Ser317) • •
Phospho-ULK1 (Ser467) •
Phospho-ULK1 (Ser555) •
Phospho-ULK1 (Ser638) • •
Phospho-ULK1 (Ser757) • •
UVRAG • •
WIPI1
• •
WIPI2
•
Phospho-WIPI2 (Ser413) •
Mealer, R.G. et al. (2014) Rhes, a striatal-selective protein
implicated in Huntington disease, binds beclin-1 and activates
autophagy. J. Biol. Chem. 289, 3547–3554.
Huck, B. et al. (2014) Elevated protein kinase D3 (PKD3)
expression supports proliferation of triple-negative breast
cancer cells and contributes to mTORC1-S6K1 pathway
activation. J. Biol. Chem. 289, 3138–3147.
Conacci-Sorrell, M. et al. (2014) Stress-induced cleavage of
Myc promotes cancer cell survival. Genes Dev. 28, 689–707.
Huck, B. et al. (2014) Elevated protein kinase D3 (PKD3)
expression supports proliferation of triple-negative breast
cancer cells and contributes to mTORC1-S6K1 pathway
activation. J. Biol. Chem. 289, 3138–3147.
Talaber, G. et al. (2014) HRES-1/Rab4 promotes the formation
of LC3(+) autophagosomes and the accumulation of
mitochondria during autophagy. PLoS One 9, 84392.
Lin, G. et al. (2014) Reduced Warburg effect in cancer cells
undergoing autophagy: steady- state 1H-MRS and real-time
hyperpolarized 13C-MRS studies. PLoS One 9, 92645.
Nemati, F. et al. (2014) Targeting Bcl-2/Bcl-XL induces antitumor
activity in uveal melanoma patient-derived xenografts.
PLoS One 9, e80836.
Ren, X.S. et al. (2014) Activation of the PI3K/mTOR pathway
is involved in cystic proliferation of cholangiocytes of the PCK
rat. PLoS One 9, e87660.
Shiroto, T. et al. (2014) Caveolin-1 is a critical determinant
of autophagy, metabolic switching, and oxidative stress in
vascular endothelium. PLoS One, e87871.
Uetake, R. et al. (2014) Adrenomedullin-RAMP2 system suppresses
ER stress-induced tubule cell death and is involved
in kidney protection. PLoS One 9, e87667.
Chang, P.C. et al. (2014) Autophagy pathway is required for
IL-6 induced neuroendocrine differentiation and chemoresistance
of prostate cancer LNCaP cells. PLoS One 9, e88556.
Lin, L. et al. (2014) Mechanical stress triggers cardiomyocyte
autophagy through angiotensin II type 1 receptormediated
p38MAP kinase independently of angiotensin II.
PLoS One 9, e89629.
Gu, W. et al. (2014) Ambra1 is an essential regulator of
autophagy and apoptosis in SW620 cells: pro-survival role of
Ambra1. PLoS One 9, e90151.
Autophagy Signaling
AMP:
ATP
AMPK
Apoptosis
Phagophore
Atg16L1
Amino
Acids
Atg16L1
Macroautophagy
PI3K-I/Akt
Signaling
GβL
MAPK/Erk1/2
Signaling
Atg13 FIP200
ULK1
p150
PI3K
Class III
Beclin-1
Bcl-2
Atg14
Atg5
Rubicon
Atg12
Atg12
mTOR
Ambra1
Atg5
Atg10
Atg7
p53/Genotoxic
Stress
Raptor
PRAS40
ub
Cytoplasmic
Contents
Oxidative
Stress
Membrane
Nucleation
ub
Mitophagy
Mitochondrial
Damage
PARL
BNIP3
SQSTM1/p62
BNIP3L/NIX
+ NBR1 +
ALFY
Atg3
Atg7
Atg4
Sequestration
LC3-II
LC3-I
LC3
PE
PINK
SQSTM1/p62
NBR1
Ambra1
chapter 03: Cell Growth and Death
ub
Parkin
+ LC3-II
Autophagosome
Lysosome
ub -targets
Fusion
Autophagolysosome
Macroautophagy, often referred to as autophagy, is a catabolic process that results in the autophagosomic-lysosomal degradation of bulk cytoplasmic contents, abnormal
protein aggregates, and excess or damaged organelles. Autophagy is generally activated by conditions of nutrient deprivation but has also been associated with physiological
as well as pathological processes such as development, differentiation, neurodegenerative diseases, stress, infection, and cancer. The kinase mTOR is a critical regulator of
autophagy induction, with activated mTOR (Akt and MAPK signaling) suppressing autophagy, and negative regulation of mTOR (AMPK and p53 signaling) promoting it. Three
related serine/threonine kinases, UNC-51-like kinase -1, -2, and -3 (ULK1, ULK2, UKL3), which play a similar role as the yeast Atg1, act downstream of the mTOR complex.
ULK1 and ULK2 form a large complex with the mammalian homolog of an autophagy-related (Atg) gene product (mAtg13) and the scaffold protein FIP200 (an ortholog of
yeast Atg17). Class III PI3K complex, containing hVps34, Beclin-1 (a mammalian homolog of yeast Atg6), p150 (a mammalian homolog of yeast Vps15), and Atg14-like
protein (Atg14L or Barkor) or ultraviolet irradiation resistance-associated gene (UVRAG), is required for the induction of autophagy. The Atg genes control autophagosome
formation through Atg12-Atg5 and LC3-II (Atg8-II) complexes. Atg12 is conjugated to Atg5 in a ubiquitin-like reaction that requires Atg7 and Atg10 (E1 and E2-like enzymes,
respectively). The Atg12-Atg5 conjugate then interacts noncovalently with Atg16 to form a large complex. LC3/Atg8 is cleaved at its C-terminus by Atg4 protease to generate
the cytosolic LC3-I. LC3-I is conjugated to phosphatidylethanolamine (PE) also in a ubiquitin-like reaction that requires Atg7 and Atg3 (E1 and E2-like enzymes, respectively).
The lipidated form of LC3, known as LC3-II, is attached to the autophagosome membrane. Autophagy and apoptosis are connected both positively and negatively, and extensive
crosstalk exists between the two processes. During nutrient deficiency, autophagy functions as a pro-survival mechanism; however, excessive autophagy may lead to
cell death, a process morphologically distinct from apoptosis. Several pro-apoptotic signals, such as TNF, TRAIL, and FADD, also induce autophagy. Additionally, Bcl-2 inhibits
Beclin-1-dependent autophagy, thereby functioning both as a pro-survival and as an anti-autophagic regulator.
Mitophagy is a selective autophagic process specifically designed for the removal of damaged or unneeded mitochondria from a cell. Upon mitochondrial damage, the protein
PINK, which is continually degraded in the healthy state through the action of PARL, is stabilized and recruits the E3 ligase Parkin to initiate mitophagy. Polyubiquitination of
mitochondrial membrane proteins by Parkin results in the recruitment of autophagy adaptor proteins SQSTM1/p62, NBR1, and Ambra1 that bind to LC3 via their LC3-
interacting region (LIR). In addition, BNIP3 and BNIP3L/NIX, which also contain LIRs, directly recruit autophagic machinery by a ubiquitin-independent mechanism to induce
autophagosome formation in certain cell types.
Select Reviews:
Alers, S., Löffler, A.S., Wesselborg, S., and Stork, B. (2012) Mol. Cell. Biol. 32, 2–11. • Codogno, P., Mehrpour, M., and Proikas-Cezanne, T. (2012) Nat. Rev. Mol. Cell Biol.
13, 7–12. • Ding, W.X. and Yin, X.M. (2012) Biol. Chem. 393, 547–564. • Feng, D., Liu, L., Zhu, Y., and Chen, Q. (2013) Exp. Cell. Res. 319, 1697–1705. • Jin, M.
and Klionsky, D.J. (2014) FEBS Lett. 588, 2457–2463. • Papinski, D. and Kraft, C. (2014) Autophagy 10, 1338–1340. • Schneider, J.L. and Cuervo, A.M. (2014) Curr.
Opin. Genet. Dev. 26, 16–23.
© 2003–2015 Cell Signaling Technology, Inc. • We would like to thank Prof. Bingren Hu, University of Maryland School of Medicine, Baltimore, MD, for reviewing this diagram.
96 For Research Use Only. Not For Use in Diagnostic Procedures. See pages 302 & 303 for Pathway Diagrams, Application, and Reactivity keys.
www.cellsignal.com/cstpathways
97