BME 42-620 Engineering Molecular Cell Biology
Lecture 21:
Lecture 21:
Cell Signaling (II)
Chapter 15
1
BME42-620 Lecture 21, November 29, 2011
Final Exam Papers
1) R. Delanoue & I. Davis, Dynein anchors its mRNA cargo after apical transport in the
Drosophila blastoderm embryo,
Cell, 122:97-106, 2005.
2) D. Levy & R. Heald, Nuclear size is regulated by importin �� and Ntf2 in Xenopus,
Cell,
143:288, 2010.
3) S. Ally, A. G. Larson, et al, Opposite-polarity motors activate one another to trigger cargo
3) S. Ally, A. G. Larson, et al, Opposite polarity motors activate one another to trigger cargo
transport in live cells,
Journal of Cell Biology, 187:1071-1082, 2009.
4) Y. Shimamoto, Y. T. Maeda, et al, Insights into the micromechanical properties of the
metaphase spindle,
Cell, 145:1062-1074, 2011.
metaphase spindle,
Cell, 145:1062 1074, 2011.
5) C. A. Wilson, M. A. Tsuchida, et al, Myosin II contributes to cell-scale actin network
treadmilling through network disassembly,
Nature, 465:373-377, 2010.
6) A. Levskaya, O. D. Weiner, W. A. Lim, C. A. Voigt, Spatiotemporal control of cell signaling
using a light-switchable protein interaction,
Nature, 461:997-1001, 2009.
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Final Exam Time & Location
• Available final exam dates
Dec 9 11 (morning)
- Dec. 9, 11 (morning)
- Dec. 14, 15, 16
- Dec. 10 may be possible
• Location
- Mellon Institute 411 (in the former Lane Center)
- Other locations possible
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Final Exam Presentation Format (I)
• Each presentation should include three sections
Background
- Background
- Data presentation
- Critical review
• Time allocation
- Background section: approximately 15 minutes
- Data presentation: ~45-60 minutes
- Critical review section: approximately 10 minutes
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Final Exam Presentation Format (II)
• Organization
- For each group, approximately one student �� one section
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- Background section should be brief;
Give details but be selective
- Data presentation should include a slide summarizing main
messages
All figures in the main text must be covered
- Critical review can accompany data presentation
- Review section may include
Whether the data and methods are sound
Whether the logic development is sound
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p
Limitations, white space
Writing style
Final Exam Presentation Format (III)
• Each presentation will be graded based on
- Accuracy, clarity, logic, & completeness of presentation of all
sections
Q lit f lid ( th fi l
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- Quality of slides (as the final report); Give proper citations
• For each group, the presentation PPT file will serve as the final
group report.
• Each student should turn in a two-page report following the
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standard instructions of reading assignments.
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Outline
Overview of cell signaling
• Overview of cell signaling
• Classification of signaling related proteins
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• Receptors
• Signaling protein transducers
• Second messengers
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Overview of cell signaling
• Overview of cell signaling
• Classification of signaling related proteins
g
g
p
• Receptors
• Signaling protein transducers
• Second messengers
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Overview of Cell Signaling
• Sources of extracellular signal
- Non-cellular environment
- Cellular environment (cell-cell
Cellular environment (cell cell
communication)
- Hundreds of types of signals
C ll i li
• Cells signaling
- Stimulus sensing; communication
- Information processing;
decision making
• ��Receptors
��Signal transducers
��Effector proteins
S
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• Signaling pathways regulate
nearly all cellular functions.
Alberts MBoC 5e
Membrane & Intracellular Receptors
• Receptors bind signaling
molecules (ligands)
• Receptors are highly sensitive and
specific.
- Typical signal molecule
concentration <10-8 M
- More than 1500 human genes
encode receptors
• Most receptors are at the cell
surface.
• Some receptors are intracellular
(
li h
)
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(e.g. light, gas receptors).
Alberts MBoC 5e
General Principles of Signaling (I)
• Four forms of
intercellular signaling
• Paracrine signaling
acts locally over
different types of cells.
• Autocrine signaling
acts locally over the
same types of cells
including themselves.
• Endocrine signaling
acts over long distance.
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g
Alberts MBoC 5e
General Principles of Signaling (II)
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g ( )
• Many signaling proteins act as
molecular switches
• Two ways to activate/deactivate
signaling proteins
• Human genomes encodes ~520
kinases and ~150 phosphatases
Alberts MBoC 5e
• Two main types of kinases
- tyrosine kinase
- serine/threonine kinase
Alberts MBoC 5e
• Two types of GTP-binding proteins
- Trimeric G proteins
Monomeric GTPases
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- Monomeric GTPases
General Principles of Signaling (III)
• Different pathways
have different rates of
have different rates of
response.
• Pathways involve
• Pathways involve
gene expression
regulation are usually
slow.
Alberts MBoC 5e
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General Principles of Signaling (IV)
• Cascade of signaling events
��Receptors
��Signal transducers
��Effector proteins
• Relay, integration, and distribution
of signals require transducers.
• Signaling pathways regulate nearly
all cellular functions
all cellular functions.
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Alberts MBoC 5e
Specific Reponses of Cells to Signaling
p
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g
g
• A cell in a multicellular organism may be
exposed to hundreds of signals.
exposed to hundreds of signals.
• Different types of cells respond differently
to the same type of signals
to the same type of signals.
• A major challenge is to understand how
the cells process such information and
the cells process such information and
make decisions.
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Feedback Loops in Signaling Networks
• Two types of feedback loops
Positive feedback
- Positive feedback
- Negative feedback
• Positive feedback loop
- Bistability
• Negative feedback loop
- Robustness to noise
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Adaptation of Sensitivity to Signaling
• Cells can adapt to external stimuli through sensitivity adjustment.
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Overview of cell signaling
• Overview of cell signaling
• Classification of signaling related proteins
g
g
p
• Receptors
• Signaling protein transducers
• Second messengers
18
Overview of Cell Signaling
• Cascade of signaling events
��Receptors
��Signal transducers
��Effector proteins
• Relay, integration, and distribution
of signals require transducers.
• Signaling pathways regulate nearly
all cellular functions
all cellular functions.
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Alberts MBoC 5e
Transducers in Signaling
• Signaling proteins
Kinases
Phosphatases
GTPases
Adapters
• Second messengers
cAMP cGMP
, cGMP
Lipids
Calcium
NO (nitrogen monoxide)
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Overview of cell signaling
• Overview of cell signaling
• Classification of signaling related proteins
g
g
p
• Receptors
• Signaling protein transducers
• Second messengers
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Membrane Receptors
• Most extracellular signal
molecules bind to specific
membrane receptors.
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f
• Three largest classes of
receptors, defining three
transduction mechanisms.
• Two common strategies
used to transfer signals
- conformation changes
- clustering
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G-Protein Coupled Receptors (I)
G Protein Coupled Receptors (I)
• Signal molecules of GPCR
include
photons
- photons
- molecules of taste and smell
- hormones, neurotransmitters, ��
- proteins, small peptides, etc��
• Function
- Nearly all human senses: sight, smell,
taste
- Behavior and mood regulation
- Regulation of immune system and
inflammation
- Nervous system regulation
• Half of known drugs work
through GPCR directly or
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through GPCR directly or
indirectly
Different Trimeric G-Protein Families
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Example: Regulation of cAMP by G Proteins
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y
• Cyclic AMP is synthesized
from ATP by adenylyl
cyclase.
• Cyclic AMP is degraded by
• Cyclic AMP is degraded by
cAMP phosphodiesterases
through hydrolysis.
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Enzyme Coupled Receptors
• Enzyme coupled receptors:
receptor serine/threonine kinases
receptor tyrosine kinase
cytokine receptors
guanylyl cyclase receptors
• Latent gene regulatory pathway receptors
Notch receptors
Hedgehog receptors
TNF receptors
Toll-like receptors
Toll like receptors
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Protein Kinase & Phosphatase (I)
Kinase
Phosphorylated
Phosphatase
Protein
Phosphorylated
protein
Presence/absence of a single phosphate group turns
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on/off a signaling protein
Protein Kinase & Phosphatase (II)
• Normally part of a signaling cascade
• Often serve as signal amplifiers
• Human genomes encodes ~520 kinases and ~150 phosphatases
• Two main types of kinases
- serine/threonine kinase (>99%)
- tyrosine kinase
C
C
R
V
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Common Common
Rare
Very rare
Abundance in eukaryotes
Receptor Serine/Threonine Kinases
p
• Binds to about 40 human proteins, e.g.
TGF-�� and bone morphogenetic
t i
protein.
• TGF-�� acts through receptor
��
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p
serine/threonine kinase and Smads.
TGF
• TGF-��
- Embryonic development signaling.
- Inhibits proliferation of most adult cells.
Stimulate extracellular matrix production
- Stimulate extracellular matrix production
- Regulate cell death in development.
- Regulate tissue repair and immune
response in adults.
Smad: Sma in C. elegans & Mad in Drosophila
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p
Receptor Tyrosine Kinase (I)
• Phosphorylate tyrosines on themselves and a small set of
intracellular signaling proteins.
• Receptor tyrosine kinase
- extracellular ligand-binding domain
- cytoplasmic tyrosine kinase domain
- single transmembrane helix
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Receptor Tyrosine Kinase (II)
Alberts MBoC 5e
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Cytokine Receptors
• Cytokines are polypeptide hormones
or growth factors that act as a local
mediator in cell-cell communication
mediator in cell-cell communication.
• Immune cells secrete cytokines when
pathogens are encountered
pathogens are encountered.
• Cytokines recruit immune cells in
response to pathogens.
response to pathogens.
• Cytokine receptors activate the JAK-
STAT signaling pathway.
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y
• JAK-STAT pathway provides a fast
track to the nucleus.
JAK: Janus kinases
STAT: signal transducer and
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activators of transcription
Intracellular Receptor: Guanylyl Cyclase Receptors
• Soluble guanylyl cyclase is a
mammalian NO/CO sensor
mammalian NO/CO sensor.
• NO signaling is critical to many
physiological processes
involving cardiovascular and
neuronal systems.
• Related drugs work by blocking
the breakdown of cGMP
the breakdown of cGMP.
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Notch Receptors (I)
p
( )
• Latent gene regulatory proteins are activated
by protein degradation.
• Protein ligand: Delta (fly), LAG-2 (worm);
Receptors: Notch, Lin-12 (worm)
• Most widely used in
- cell fate regulation (development)
Lai,
Development, 131:965, 2004
cell fate regulation (development)
- pattern formation (development)
- tissue renewal (post-development)
• Main function: lateral inhibition
- Amplify and consolidate molecular
differences between adjacent cells during
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differences between adjacent cells during
embryonic development
Notch Receptors (II)
p
( )
• Binding of Delta triggers
cleavage of Notch.
• Released Notch tail migrates
into the nucleus to convert
Rbpsuh protein from a
transcriptional repressor into
a transcriptional activator.
• Activation of Notch is
irreversible.
• The simplest known pathway
from cell surface to nucleus.
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Lai,
Development, 131:965, 2004
Hedgehog Receptors
• Protein ligand: Hh; Receptors: Ptc & Smo
• Hh binds and inactivate Ptc, which activates
Smo and gene transcription.
M i f ti
• Main functions
- Regulates cellular differentiation in
embryonic development
- Maintaining stem cells in postembryonic
Maintaining stem cells in postembryonic
tissues (tissue renewal)
• Mutation of Hh causes developmental
defects
Lum & Beachy,
Science, 304:1755, 2004
defects.
• Mutation of Ptc and Smo causes skin
cancer
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cancer.
Toll-like Receptors (TLRs)
• Mammals have TLRs
that recognize specific
foreign molecules.
Main function
• Main function
- To sense and
respond to infection
• At the core of our
i h it d
i t
t
B tl
N t
430 257 2004
inherited resistance to
disease
Beutler,
Nature, 430:257, 2004
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Tumor Necrosis Factor (TNF) Receptors
• Binding of TNF with its receptors
triggers mutiple signaling
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pathways.
• Function
-Triggering apoptosis of tumor cells
-Mediate inflammatory response
-Regulate immune system function
• Inappropriate TNF signaling has
Inappropriate TNF signaling has
been implicated in many human
diseases.
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Chen et al, Science, 296:1634, 2002.
NF-kB Pathway
• Activation of Toll-like receptors or TNF receptors triggers
a signaling cascade that releases NFkB.
g
g
• NFkB proteins regulate transcriptions of hundreds of
ti i t i i
genes participate in immune responses.
• Excessive or inappropriate inflammation response can
pp p
p
cause tissue damage and severe pain.
Chronic inflammation can lead to cancer
• Chronic inflammation can lead to cancer.
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Challenges in Analyzing Signaling Pathways
• Hundreds of signaling pathways.
Human cancer pathways
• Pathways frequently branch and converge.
• Positive and negative feedback loops are
common.
• Outcomes of signaling pathways can be
spatial and temporal dependent.
• Analysis typically uses graph models.
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References
• J. Hancock, Cell Signaling, 3rd ed., Oxford University Press, 2010.
• F. Marks et al, Cell Signal Processing, Garland Science, 2008.
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Overview of cell signaling
• Overview of cell signaling
• Classification of signaling related proteins
g
g
p
• Receptors
• Signaling protein transducers
• Second messengers
42
GTP-Binding Proteins
• Trimeric G-protein & Monomeric small
GTPase
• Large family of related proteins
• Evolved from a common ancestor by
gene duplication and divergence
• Use GTP binding and hydrolysis to
switch between two states of activity
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Copyright 2008 by Saunders/Elsevier. All rights reserved.
Monomeric GTPases
Participate in many cellular activities:
• Membrane traffic
Arf Rab Sar
Membrane traffic
Arf, Rab, Sar
• Nuclear transport
Ran
• Signal transduction
Ras
Reg lation of the c toskeleton
Rho
• Regulation of the cytoskeleton
Rho
• Protein synthesis
EF-Tu
• Protein translocation into ER
SRP
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Actin Regulation
• GTPase: Molecule switch;
Family of proteins that are
Family of proteins that are
activated by GTP binding
and inactivated by GTP
hydrolysis and phosphate
dissociation.
• Rho GTPase:
Rho GTPase:
cdc42: its activation triggers
actin polymerization and
bundling at filopodia.
Rho: its activation promotes
actin bundling.
Rac: its activation promotes
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Rac: its activation promotes
polymerization at the cell
periphery.
Adaptor Domains (I)
• Adaptor domains mediate
interactions of proteins with each
other and with membrane.
• These domains are compactly folded
p
y
and incorporated into a variety of
proteins.
• Adaptors facilitate the formation of
protein complexes and make signal
transduction more reliable.
SH1 : tyrosine kinase domain
SH2 S h
l
2 bi d h
h t
i
tid
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SH2: Src homology 2, binds phosphotyrosine peptides
SH3: Src homology 3 binds polyproline type II helices
Adaptor Domains (II)
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Overview of cell signaling
• Overview of cell signaling
• Classification of signaling related proteins
g
g
p
• Receptors
• Signaling protein transducers
• Second messengers
48
Overview of Second Messengers (I)
• Types of second messengers
- Cyclic nucleotides: cAMP, cGMP
C l i
- Calcium
- Lipids
- Nitric oxide
• Small molecules.
• Information encoded by local
Information encoded by local
concentrations.
• Advantages
- Range (e.g. broadcasting)
- Response speed (up to ms)
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• Second messengers are interrelated.
Overview of Second Messengers (II)
• Production (source)
• Localization
• Target
• Degradation (sink)
Degradation (sink)
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Cyclic Nucleotide (I)
• Producer:
cAMP �� adenylyl cyclase
cGMP �� guanylyl cyclase
• Degrader:
cAMP phosphodiesterase
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p
p
cGMP phospoodiesterase
cAMP (I)
• Diffuse rapidly through cytoplasm as in free solution
• May be modulated locally (through upstream G-
proteins)
• Concentration in resting cell ~10-8M
• Can amplify signal by 100-fold on time scale of ms.
T
t
• Targets:
- kinase
- cyclic nucleotide-gated ion channels
Exchange factors for small GTPases (Rap1 Rap2)
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- Exchange factors for small GTPases (Rap1, Rap2)
cAMP (II)
• cAMP regulates PKA
• PKA targets metabolic
enzymes, transcription
factors and ion
channels
• Guanylyl cyclase
(cGMP producer) is
activated by NO and
CO
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Questions ?
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