Regulation of the mTORC1 growth pathway by amino acids

123 Pages·2015·13.03 MB·English

Regulation of the mTORC1 growth pathway by amino aci ds
by
MASSACHUSETTS INSTITUTE
OF rECHNOLOLGY
Zhi-Yang Tsun
B.S. Bioengineering, Molecular Biology MAY 2 7 2015
University of California, San Diego (2004)
LIBRARIES
Submitted to the Department of Biology
in Partial Fulfillment of the Requirements for the Degree of
Doctor of Philosophy
at the
Massachusetts Institute of Technology
June 2015
@2015 Zhi-Yang Tsun. All rights reserved.
The author hereby grants to MIT permission to reproduce
and to distribute publicly paper and electronic
copies of this thesis document in whole or in part
in any medium now known or hereafter created.
Signature redacted
Signature of Author.....................................
Departmqntof Biology
Marob 14, 2015
Signature redacted
C e rtifie d b y ................................................
David M. Sabatini
Professor of Biology
JI]hesis Supervisor
Signature redacted
A cce pted by ............................................
Midhael T. Hemann
Professor of Biology
Chairman, Committee for Graduate Students
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Regulation of the mTORC1 growth pathway by amino acids
by
Zhi-Yang Tsun
Submitted to the Department of Biology on May 22, 2015 in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy at the Massachusetts Institute of
Technology
Abstract:
The mTORC1 kinase is a master growth regulator that responds to numerous
environmental cues, including amino acids, to regulate many processes, such as protein,
lipid, and nucleotide synthesis, as well as autophagy. Given that mTORC1 regulates a
multitude of processes, it is not surprising that the pathway it anchors is deregulated in
various common diseases, including cancer.
The Rag GTPases interact with mTORC1 and signal amino acid sufficiency by
promoting the translocation of mTORC1 to the lysosomal surface, its site of activation.
The Rags are unusual GTPases in that they function as obligate heterodimers, which
consist of RagA or B bound to RagC or D. We show that RagC/D is a key regulator of
the interaction of mTORC1 with the Rag heterodimer and that, unexpectedly, RagC/D
must be GDP-bound for the interaction to occur. We identify FLCN and its binding
partners, FNIP1/2, as Rag-interacting proteins with GTPase activating activity for
RagC/D, but not RagA/B.
Given that many proteins known to signal amino acid sufficiency to mTORC1,
including the Rag GTPases, localize to the lysosome and that intralysosomal amino acid
accumulation is necessary for mTORC1 activation, we began our search for potential
direct amino acid sensors at the lysosomal membrane. We identify SLC38A9, an
uncharacterized protein with homology to amino acid transporters, as a lysosomal
transmembrane protein. SLC38A9 forms a supercomplex with Ragulator, the Rag
GTPases and the v-ATPase and is necessary for mTORC1 activation by amino acids,
particularly arginine. Overexpression of the full-length protein or just its Ragulator-
binding domain makes mTORC1 signaling insensitive to amino acid starvation but does
not affect its dependence on Rag activity. SLC38A9 reconstituted in proteoliposomes
transports arginine, an abundant amino acid in the lysosome and necessary for
mTORC1 pathway activity. These results place SLC38A9 between amino acids and the
Rag GTPases and are consistent with the notion that amino acids are sensed at the
lysosome. Thus, SLC38A9 is an excellent candidate for being an amino acid sensor
upstream of mTORC1.
Thesis supervisor: David M. Sabatini
Title: Member, Whitehead Institute; Professor of Biology, MIT
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Acknowledgements:
Undertaking PhD training in David Sabatini's lab has been the most
transformative years of my life. What inspires me most about David is that he leads by
example. He engenders incredible work ethic in his trainees because he works harder
than anyone in the lab. David has a piercing clarity of thought that instantly scrutinizes
new data and develops consistent models, that is matched by his scientific rigor and
fearlessness. David pursues the highest caliber of quality not just in experiments but
also in data figures, writing, and in giving seminars. Attempting to emulate these
qualities has pushed me to grow in ways I never expected. Perhaps the most valuable
lesson that I've learned working with David is to pursue important problems, and thanks
to my time in his lab, I finally have the confidence to tackle important but difficult
problems.
I would like to thank the members of my thesis committee, Steve Bell, and Bob
Sauer, for the continued guidance and support throughout my journey. I am also grateful
to Brendan Manning and Hidde Ploegh who are also part of my thesis evaluation
committee.
It is rare to find a lab with such talented and motivated people and yet have an
environment that is equally exceptional in collegiality. Many thanks to Rich Possemato
who mentored me during my rotation with inexhaustible patience, and Kris Wood, Yoav
Shaul, Shomit Sengupta, Peggy Hsu, Doug Wheeler, Yasemin Sancak, Maki Saitoh for
making the transition from undergraduate to graduate lab such a welcoming one. I also
appreciate the incisive conversations with Tim Peterson, who started the work on Birt-
Hogg-Dube syndrome, and whose DEPTOR paper helped convinced me to pursue my
rotation here.
Liron Bar-Peled and Lynne Chantranupong were wonderful bay-mates and
tolerated my relentless questions when I first joined the lab. Lynne-mama, as we
affectionately call her, has been an amazing bench-mate throughout my time here-so
thoughtful and showed me what 'being industrious' means. Although Rachel Wolfson
recently joined the bay, I have really appreciated her incisive feedback, vigorous
discussions, and unconditional eagerness to help. I am grateful to Shuyu Wang, with
whom I had the pleasure of sitting next to and work closely with, and who pushed me to
think deeper about science and life. And many thanks to Kuang Shen who has taught
me everything I know about experimental biochemistry. It has been a pleasure to work
with everyone who has spent time in our bay, Tony Kang, Naama Kanarek, Bobby
Saxton, and Jose Orozco. The way everyone so generously came to help during the two
instances when we had to race to submit our manuscripts is a testament to how the lab
really has become a family.
Many people in the lab have made this an inspiring and rigorous place to train:
Walter Chen for his constant encouragement and companionship as we joined the lab
together; Larry Schweitzer for incisive feedback and help with my NRSA fellowship
application; Carson Thoreen, Omer Yilmaz, Pekka Katajisto and Kris Wood for giving
inspiring advice; Roberto Zoncu, who taught me how to work with microscopes and
isolate lysosomes, and whose exemplar seminars have been inspirational; Mike Pacold,
who has been a wonderful source of necessary and unnecessary information, as well as
medical advice; Kivanc Birsoy, Bill Comb, and Do Kim for thoughtful discussions and
advice; Kathleen Ottina, who makes it so effortless to do our best science in lab, and for
sharing life advice; Amanda Hutchins, Sam Murphy, and Kevin Krupczak who ensure the
lab runs smoothly; Edie Valeri, who always brightens the day and has been so reliable
and responsive for everything non-science in the lab; Greg Wyant and Monther
Remaileh for a seamless transition as I wrapped up in lab. I have been most fortunate to
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have the opportunity to work with very talented students, Choah Kim, Tony Jones,
Elizabeth Yuan, and Alice Chen, who have certainly taught me more than I could teach
them.
I especially want to thank Tim Wang who has become a dear friend and scientific
colleague. I am grateful that we went through scientific races together, and what can
only be described as a scientific coming-of-age, together. His intellectual and scientific
prowess inspires me and his incisive feedback pushes me to grow.
I would also like to thank Mounir Koussa, who has been my partner in crime as
we embarked on this science and life journey 3 years ago. His companionship has kept
me sane while also in many ways encouraged madness. He has been unnecessarily
generous with his time in reviewing all my practice talks, grant applications, and
manuscript drafts, and I am grateful for how he has shaped my personal and scientific
development.
I am very fortunate to have friends who have not only supported me but continue
to inspire me: Nikhil Bhatla, Allen Cheng, Sasha and Masha Rayshubskiy
Anna Chambers, Jason Yamada-Hanff, Pallav Kosuri, and Dr. Sheila Nutt.
I would like to thank my parents, Maung-win Maung and Sau-Man Yang, and
sister Zhi-Fang Tsun, who continue to support me unconditionally and made possible all
the opportunities I am fortunate to have. And Charlie and Veronica Plovanich who have
been my family here on the east coast.
And of course my wife, Molly Plovanich, who not only supports and inspires me
through every step of our journey, but also provided key scientific guidance during the
most critical time. She is my better half who grounds me in life.
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Table of Contents
A b stra ct .................................................................................................... . .3
A cknow ledgem ents ..................................................................................... 4
Chapter 1: Introduction
I. Intro d u ctio n .............................................................................................. . .8
II. T he m T O R P athw ay................................................................................... 8
A. Rapamycin: a discovery tool and therapeutic........................................ 8
B. mTORC1 and mTORC2.................................................................... 9
C. Amino acid signaling machinery....................................................... 11
D. Downstream effectors of mTORC1.....................................................12
Ill. mTOR signaling in cancer..........................................................................13
A .U pstream of m T O R C 1........................................................................13
B. Birt-Hogg-Dub6 Syndrome...................................................................15
C. Outputs of mTORC1 Altered in Cancer.............................................. 16
D. Rapalogues in cancer therapy...........................................................17
IV .A m ino acid transport............................................................................... 19
A. History: digestion, protein absorption, and AA uptake in tissues.........19
B. SLC38 AA transporter family.............................................................21
C. Arginine transport at the plasma and lysosomal membranes.................. 23
V. Preface for work presented in this thesis.......................................................25
R e fe re n ce s ............................................................................................... ...2 8
Chapter 2: The Folliculin tumor suppressor is a GAP for RagC/D GTPases that
signal amino acid levels to mTORC1
S u m m a ry ...................................................................................................... 3 9
In tro d u ct io n ................................................................................................... 4 0
R e sults ..................................................................... ................ . . 4 1
The RagC Nucleotide State Determines mTORC1 Binding to the Rag
H e te ro d im e r...................................................................................... . 4 1
FLCN Interacts with the Rags in an Amino Acid-Sensitive Fashion................43
FLCN is Necessary for mTORC1 Activation and Localization to the Lysosomal
M e m b ra ne s ....................................................... . . ...................... . . 4 5
FLCN Co-Localizes with the Rag GTPases on the Lysosomal Surface in an
Amino Acid-Sensitive Fashion....................................................................46
FLCN-FNIP2 is a GAP for RagC and RagD............................................ 47
D is c u s s io n ..................................................................................................... 5 0
F ig u re s ..................................................................................................... . .5 2
F ig u re Leg e nd s ........................................................................................ . . 56
Experimental Procedures............................................................................. 60
R e fe re n ce s ............................................................................................... ...6 6
S up ple m e nta l Info ........................................................................................ . 72
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Chapter 3: Lysosomal amino acid transporter SLC38A9 signals arginine
sufficiency to mTORC1
S u m m a ry ...................................................................................................... 7 9
Intro d u ctio n ............................................................................................... . 8 0
R e s u lts ..................................................................................................... . .8 1
SLC38A9 Interacts with Ragulator and the Rag GTPases......................... 81
SLC38A9, a Lysosomal Membrane Protein Required for mTORC1 Activation... 82
SLC38A9.1 Overexpression Makes mTORC1 Signaling Insensitive to Amino
Acids..................... ................................. 83
Modulation of the SLC38A9-Rag-Ragulator Interactions by Amino Acids..........84
SLC38A9.1 is an Amino Acid Transporter...................................................85
D isc u s s io n ............................................................................................... . .. 8 6
F ig u re s ..................................................................................................... . .8 8
F ig u re Le g e nd s ........................................................................................ . . 9 3
R e fe re n ce s ............ ................................................................................... . .9 6
Experimental Procedures............................................................................. 99
S u p p le m e nta l Info .......................................................................................... 10 7
Chapter 4: Future directions and discussions
I. T he R ag H ete ro d im e r..................................................................................119
11. Lysosomal amino acid concentrations in human cells........................................120
111. Nutrient sensing in the compartmentalized cell...............................................121
R e fe re n c e s .................................................................................................. 1 2 3
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CHAPTER 1
I. INTRODUCTION
Cell growth, or the accumulation of mass, is a resource-intensive process. As such, cells
have evolved sophisticated systems to ensure that the rate of growth is controlled not
only by the availability of nutrients, but also by signaling pathways that report growth
factors and cellular stresses. One such system is the mechanistic target of rapamycin
complex 1 (mTORC1) pathway. mTORC1 integrates a diverse set of signals, such as
growth factors, nutrient and energy levels, to regulate many anabolic and catabolic
processes, including protein, lipid, and nucleotide synthesis, as well as autophagy.
Given that mTORC1 regulates a multitude of processes, it is not surprising that the
pathway it anchors is deregulated in various common diseases, including cancer,
diabetes, and aging. Therefore, understanding the molecular underpinnings of this
pathway will be essential for therapeutic intervention.
II. THE MTOR PATHWAY
A. Rapamycin: a discovery tool and therapeutic
In the 1970s, Rapamycin was isolated from Streptomyces hygroscopicus in a soil
sample from Easter Island, also called Rapa Nui. Identified from an antibiotic screen
performed in Ayerst Research Labs, rapamycin lacked antibacterial activity but had
potent growth-inhibitory effects on yeast, causing G1 arrest (Robert and Gregory, 1996).
These anti-proliferative effects also applied to human cancer cells, extending these
effects from yeast to human (Eng et al., 1984). Rapamycin was also shown to have
immunosuppressive effects in two mouse models of auto-immune disease. Once FDA
approved rapamycin for use as an immunosuppressant in kidney transplant patients,
several pharmaceutical companies developed derivatives, called rapalogues, with better
bioavailability (Robert and Gregory, 1996). Rapamycin and its chemical derivatives,
rapalogues, are also used as anti-restenosis agents in drug-eluting stents and in
chemotherapy for cancer. Rapamycin extends lifespan in every model organism tested,
and rapalogues are in pre-clinical trials for aging-related morbidities.
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The Target Of Rapamycin:
As a small molecule, rapamycin has an unusual mechanism of action. It binds to a small
intracellular protein called FKBP12, and in the form of this complex, inhibits its target. To
identify the target of rapamycin, screens in yeast were performed to isolate mutants that
are resistant to its growth-inhibitory effects. Mutant alleles of three genes were
identified-recessive mutations in FKBP12, and dominant mutations in TOR1, and
TOR2 (Heitman et al., 1991; Koltin et al., 1991). The FKBP12 null cells did not
recapitulate the growth arrest, whereas the TOR1/TOR2 double mutants mimicked the
growth arrest imposed by rapamycin, suggesting that rapamycin inhibits the TOR
proteins (Helliwell et al., 1994). Direct evidence that rapamycin inhibited TOR came from
biochemical purification from mammalian sources. Sequence analysis of the proteins
revealed homology to the yeast TORs (Brown et al., 1994; Chen et al., 1994; Sabatini et
al., 1994; Sabers et al., 1995). We now refer to this protein as the mechanistic target of
rapamycin, or mTOR.
B. mTORC1 and mTORC2
We now know that mTOR is the catalytic kinase domain of two distinct complexes,
mTOR complex 1 (mTORC1), and mTOR complex 2 (mTORC2). In response to growth
factors and nutrients, mTORC1 regulates key cell growth processes, such as protein,
lipid, and nucleotide biosynthesis, and autophagy. Considered part of the P13K-Akt
pathway, mTORC2 is less well-understood but was found to be the elusive kinase for
one of the two phosphorylation sites for Akt activation in response to growth factor
signaling. Thus, as part of two distinct complexes, mTOR is both upstream and
downstream of itself.
mTORC2
Yeast TOR2, but not TOR1, had rapamycin-insensitive functions (Xiao-Feng et al.,
1995). This issue was clarified with the discovery of two distinct complexes containing
TOR2. TOR1 or TOR2 can associate with the rapamycin-sensitive TOR complex 1
(TORC1), which included KOG1 (homologous to human raptor) and LST8 (homologous
to human GbL/mLST8). On the other hand, only TOR2 was found in TOR complex 2
(TORC2), which comprised of AVO1 (homologous to human SIN1), AVO2, AVO3
(homologous to human rictor) (Loewith et al., 2002). In mammals, there is one mTOR
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protein and it is found in both complexes. mTORC2 is defined by its rictor component
and also contains mLST8, SIN1 (Sarbassov et al., 2004). Deletion of TORC2
components in yeast and knockdown in mammalian cells showed actin cytoskeleton
defects (Jacinto et al., 2004).
Interest in the mTOR pathway escalated to a new high when mTORC2 was found to be
the elusive kinase for full Akt activation. The PI3K/PTEN/Akt pathway mediates insulin
signaling and is one of the most commonly mutated pathways in cancer. Full activation
of Akt requires phosphorylation at two sites. PDK1 executes the first round of
phosphorylation on T308, and mTORC2 phosphorylates S473 for full Akt activation
(Sarbassov et al., 2005b). In turn, Akt drives cell growth, cell cycle progression, glucose
metabolism, and survival by signaling to its targets TSC2, Cyclin1, GSK3, and BAD
(Greer and Brunet, 2005). How mTORC2 is activated by P13K is still poorly understood.
mTORC1
Concurrent with yeast studies, raptor was found to be a defining component of the
nutrient-responsive, rapamycin-sensitive, mammalian mTORC1 (Hara et al., 2002; Kim
et al., 2002). Raptor is not required for mTOR kinase activity in vitro, but is thought to
help in substrate recruitment (Sabatini, 2006). As described above, GbL/mLST8 is also a
component of mTORC1, but its function has been debated. PRAS40 is the final
component of mTORC1 and is an insulin-sensitive inhibitor of mTORC1 (Sancak et al.,
2007b).
The mechanisms through which mTORC1 senses and integrates stimuli are of great
interest. One key upstream factor is the Tuberous Sclerosis Complex, TSC1-TSC2
tumor suppressor, which suppresses mTORC1 activity in response to deprivation of
growth factor or energy (Brugarolas et al., 2004; Castro et al., 2003; Corradetti et al.,
2005; Garami et al., 2003; Inoki et al., 2003a; Inoki et al., 2003b; Ma et al., 2005; Reiling
and Hafen, 2004; Roux et al., 2004; Saucedo et al., 2003; Stocker et al., 2003; Tee et
al., 2003a; Tee et al., 2002; Tee et al., 2003b; Zhang et al., 2003). The TSC complex
does so by inhibiting Rheb, a GTP-binding protein that is an essential activator of the
mTORC1 kinase activity (Long et al., 2005; Sancak et al., 2007a).
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