Molecular and Cellular Mechanisms of Biosignaling
Prof. Yong Tae Kwon
Research interest: ubiquitin-proteasome system and autophagy in
mammals
Part I. Basics of Biosignaling (~2 lectures)
Ligand, receptor (GPCR, RTK), intracellular signaling pathways,
second messenger
Part II. Signaling, Cell Cycle, and Cancer (~3 lectures)
Growth factor-induced signaling, cell cycle, cell cycle checkpoint,
apoptosis, DNA repair, oncogenesis, cancer treatment
Part III. Molecular Cellular Physiology of Biosignaling (~3 lectures)
Intercellular and intracellular signaling in the context of cell type-
specific processes: nervous signaling, photosignaling, muscle
contraction, and coagulation
Part IV. Biosignaling in neuroendocrine system and metabolism (~2
lectures)
Topics will include metabolism, obesity, diabetes, etc.
Each Student will present 1-2 slides at the end of my
lecture.
Lehninger Principles of Biochemistry, 5th Ed
Reading materials for additional information and
examination
N-terminal amino acid as degradation signal
The N-end rule pathway
X substrate
Ub
E3 X substrate
Ubiquitin-dependent proteolysis
Is this physiologically meaningful?
Components involved in this pathway in mammals?
Physiological functions of identified components?
Substrates underlying identified functions?
Human diseases caused by mutations in the pathway?
Inhibitors to control pathophysiological conditions?
Biochemical principle in UPS?
Components and structure of N-end rule pathway
UBR1 UBR3
UBR2 UBR6
UBR4 UBR7
UBR5
Kwon, Nat Rev Mol Cell Biol 2011
Kwon, Ann Rev Biochem 2012
The Cell as a starting point to study
All Biomedical Sciences
Nucleus
Nucleolus
DNA
Gene
Precursor RNA
Mature RNA
Endoplasmic reticulum (ER)
Protein synthesis
Ribosome
mRNA
tRNA
Splicing
DNA (exon and intron)
Precursor RNA vs. mRNA: splicing
mRNA vs. cDNA
protein
Endoplasmic reticulum (ER)
Complex: ribosome, mRNA, tRNA
~35,000 genes
1 million proteins ???
Protein expression:
mRNA, ER, ribosome, tRNA, polysome
Signal Transduction
Signaling between cells is usually
mediated by interaction of a ligand
with a receptor.
The ligand-bound receptor
activates intracellular signaling
pathway to control various cellular
functions.
Basic elements of signal transduction
Intracellular vs. cell surface receptors
Hydrophobic molecules
(steroid hormones, retinoic
acids, etc) enter cells and
bind to intracellular receptors
Hydrophilic or big molecules
(proteins or polypeptide
hormones) bind to cell
surface receptors
Majority
Major types of intercellular signaling
Contact dependent signaling: gap
junction or ligand-receptor interaction
on cell surface.
Endocrine signaling: A sender cell
secretes ligands (hormones) into the
blood for long distance transport to
target receptors.
Paracrine signaling: A sender cell
secretes ligands that are diffused
(up to ~0.1 mm) to bind to target
receptors.
Synaptic signaling: An axon terminus
secretes ligands (neurotransmitters)
Into restricted environment to target
nerves or muscles.
Cell surface receptors categorized by action modes
Ligand binding to ligand-gated ion channel
receptor permits ions (e.g., Na+) to permeates
through the receptor in nervous cells.
Enzyme-linked receptors themselves are
kinases that phosphorylate effectors.
Cytokine binding to cytokine receptors
(without kinase activity) activates a kinase
effect.
G-protein-coupled receptors
(GPCR)
~50% of all known drugs
Major Features of Signal Transduction: Specificity
Numerous types of ligands
Ligand specificity of receptors: signal to noise
Major Features of Signal Transduction: Amplification
The initial ligand signal can be amplified ~10,000 times.
Through a cascade of signaling molecules.
Usually, from the plasma membrane toward the nucleus.
Major Features of Signal Transduction: Desensitization
Homeostasis in signaling
Negative feedback
Major Features of Signal Transduction: Integration
Various signals are integrated and merged into common regulatory
machinery in the cytoplasm and nucleus.
Downstream signaling pathways are usually simpler (e.g., on vs. off).
Cells can sense millions of ligands.
G-Protein-Coupled Receptors (GPCRs)
Represent ~5% of all
proteins
Targets of ~50% of all known
drugs
Hormones,
neurotransmitters,
Local mediators
Heterotrimeric GTP-binding
proteins (G proteins)
Heterotrimeric G proteins coupled with GPCR
s: stimulates
adenylate cyclase,
cAMP up.
i: inhibits adenylate
cyclase, cAMP down
q: phospholipase C
IP3, DAG, Ca++
12: guanine-nucleotide
exchange factors (GEFs)
Extracellular ligands
Epinephrine (adrenaline) and its synthetic analogs
Transduction of the epinephrine signal:
the -adrenergic pathway
GTP and cAMP
Molecular switches that turn on and off proteins
Phosphorylation and GTP-binding as major molecular switches
Phosphorylation: the most widely used protein modification by kinases
GTP-binding: switch on and off by the status of GTP-binding of G-proteins
Inactivation of G-proteins by GAP
GTP-bound: active
Bound GTP hydrolyzed by GTPase
activities (GAP) of Ras and its GAP
Switch I and switch II relaxed
into an inactive conformation
Not interact with downstream targets
such as Raf.
Activation and inactivation of G proteins
-GTP (active) + (active)
-GDT (inactive)
1 GPCR vs. 10-100 G-proteins
G proteins activate downstream
effectors
GTPase activator proteins
(GAPs) and regulators of G protein
signaling (RGSs) inactivate G proteins
By modulating the GTPase activity
Constitutive activation of Gs
by cholera toxin
Cholera toxin
The bacterium Vibrio cholerae
Responsible for the harmful effects
of cholera infection
s: constitutive activation
Adenylate cyclase/cAMP
ADP-ribosylation of Gs
Chloride channel in intestine
Diarrhea
Adenylate cyclases regulated by Gs and Gi
Second Messengers
Ligand-bound receptors may mediate signaling via second messengers,
small intracellular molecules.
Fast signaling for the entire population of proteins that are present in the cell.
cAMP as a second
messenger for GPCR
The GPCR effector adenylate
cyclase
Breakdown by cyclic nucleotide
phosphodiesterases (PDE)
Activated by Gs and inhibited by
Gi
Basal conc.: 10-7
Activated: 2-100 fold
Synthesis and degradation of cAMP
Activation of cAMP-dependent protein kinase (PKA)
When [cAMP] is low, two regulatory
(R) subunits associate with catalytic
subunits. The complex is
catalytically inactive.
When [cAMP] rises in response to a
hormonal signal, each R subunit
binds two cAMPs.
Dramatic reorganization that pulls its
inhibitory sequence away from the C
subunit.
Open up the substrate-binding cleft
and releasing each C subunit in its
catalytically active form.
Activation of cAMP-dependent protein kinase (PKA)
cAMP function is mediated by
cAMP-dependent protein kinase
(PKA)
PKA phosphorylates many other
proteins
cAMP-regulated gene regulatory
proteins (CREBs)
cAMP-sensitive regulatory
elements (CRE)
PKA controlled by cAMP
Amplification in epinephrine cascade
Epinephrine triggers a series of
reactions in hepatocytes.
Catalysts activate catalysts, resulting
in great amplification of the signal.
Unbound / Inactive Bound / Active Bound / Inactive
Fig. 13.23. Phosphorylation to desensitize GPCR
Protein kinase A (PKA)
GPCR-specific protein kinases (GRKs)
fast slow
Phosphorylation
Fig. 13.10. Termination of receptor-dependent signal transduction is essential
to return to basal (pre-activation) state or to prevent overactivation
Desensitization of the -adrenergic receptor in the
continued presence of epinephrine1
-adrenergic protein
kinase (ARK)
-arrestin (arr).
Gq
phospholipase C and IP3
Two intracellular second messengers
are produced in the hormone-sensitive
phosphatidylinositol system: inositol
1,4,5-trisphosphate (IP3) and
diacylglycerol.
Both contribute to the activation of
protein kinase C.
By raising cytosolic [Ca2+], IP3 also
activates other Ca2+-dependent
enzymes.
Ca++ as a 2nd messenger
In response to many hormones
and neurotransmitters
By binding to Ca++-dependent
regulators
10-3 M (out, ER, SR) vs. 10-7 M
(in)
Na+/Ca++ exchanger
Ca++ pump: ATP
Ca++ signaling
Fig. 13.29. Ca++ signaling and its action
Triggering of oscillations in intracellular [Ca2+] by
extracellular signals
A dye (fura) that undergoes
fluorescence changes when it
binds Ca2+.
Fluorescence intensity represented
by color.
The cells are heterogeneous in
their responses.
Some have high intracellular [Ca2+]
(red), others much lower (blue).
Feedback mechanism
Triggering of oscillations in intracellular [Ca2+] by
extracellular signals
Fura is used in a single
hepatocyte.
Norepinephrine (added
at the arrow) causes
oscillations of [Ca2+] from
200 to 500 nM.
Similar oscillations are
induced in other cell
types by other
extracellular signals.
Transient and highly
localized increases
in [Ca2+]
A weak [IP3]-producing stimulus
may cause a single [Ca2+]
channel to open briefly.
A somewhat stronger stimulus
may cause all the Ca2+ channels
in a cluster to open.
A sufficiently large puff produces
elevated [Ca2+] over an area
great enough to include
neighboring clusters of Ca2+
channels.
Opening of the channels in
neighboring clusters propagates
this effect, resulting in a wave of
elevated [Ca2+] moving along the
ER.
Ca++ and calmodulin (CaM)
Calmodulin-dependent kinases
Auto-phosphorylation: sustained activity
Receptor tyrosine kinase
Growth factors: EGF, PDGF etc
Receptor tyrosine kinases
Tyr kinase domain vs. Ligand-binding domain
Growth factor receptors for insulin (INS-R), vascular epidermal growth factor (VEGFR),
platelet-derived growth factor (PDGF-R), epidermal growth factor (EGF-R), nerve
growth factor (NGF-R), and fibroblast growth factor (FGF-R).
Assembly of signaling molecules
Many signaling processes
are mediated by an assembly
of multiple signaling
molecules.
SH2 domain interacting with
P-Tyr + 3 residues of
substrate
Some binding modules of signaling proteins
RTS signaling and Ras GTPase
~30% of all cancers involve Ras mutations.
RTK-RAS-MAPK cascade
RTK: kinase receptor
Ras: GTPase
MAPKKK: kinase
MAPKK: kinase
MAPK: kinase
Transcription factors
(e.g., c-Fos)
Why multi-step in signaling?
Signal amplified in number and duration
Signal fine regulated
Signal cross talks
Regulation of gene expression by
insulin through a MAP kinase
cascade
Insulin receptor: INS-R
INS-R autophosphorylation
Insulin receptor substrate -1 (IRS-1)
Ras
Raf-1
MEK: MAPKK
ERK: MAPK
Elk1
Serum response factor (SRF)
Receptor Serine/Threonine Kinases
TGF and BMPs
Tissue development and
differentiation
Mutations in these pathways
found in cancers
SMAD-dependent transcription
Fig. 13.18. TGF-activated receptor serine/threonine
kinase cascade
The JAK-STAT transduction
mechanism for the
erythropoietin receptor
Erythropoietin (EPO)
Dimerization of EPO receptor.
JAK phosphorylates EPO-R.
(a)STAT5 binds to P–EPO-R
JAK - p-STAT
STAT5 dimer
Exposing a nuclear localization sequence
(NLS).
(b) Grb2 binds P–EPO-R and triggers the
MAPK cascade.
Cytokines: paracrine/autocrine
polypeptides
Interleukins: immune response
in leukocyte
Interferons: immune response
against viruses or bacteria
G proteins
s: stimulates
adenylate cyclase,
cAMP up.
i: inhibits adenylate
cyclase
q: phospholipase C
IP3, DAG, Ca++
12: GEFs
Fig. 13.21. Heterotrimeric G proteins coupled with GPCR
Cross talk between GPCR and RTK
Insulin - INS-R
Phosphorylates -adrenergic
receptor.
PKB
Internalization of adrenergic
receptor.
Alternatively, INS-R–catalyzed
phosphorylation of a GPCR
INS-R uses GPCR to enhance
its own signaling.
cGMP as a second messenger
Two isozymes of guanylyl cyclase that participate in
signal transduction
cGMP synthesized by
guanylate cyclase
Membrane-spanning forms
that are activated by their
extracellular ligands: atrial
natriuretic factor (ANF) and
guanylin.
A soluble heme-containing
enzyme.
Activated by intracellular nitric
oxide (NO).
NO as a signaling molecule
1998 Nobel Prize
Guanylate cyclases
cGMP synthesis activated by NO
Phosphodiesterase (PDE)
cGMP degradation
NO, Vasodilation, and Drug
Endothelial cells: NO
Vascular smooth muscle cells
guanylate cyclase
cGMP
cGMP-sensitive kinase
Viagra
Erectile dysfunction
PDE5 inhibitor
[NO] increase
80-4000-fold less potent than PDE3 (cardiac
muscle)
15-fold less potent than PDE6 (retina)
Each student is required to present a slide
shown below at the end of class
Please use propagated materials, websites, or slides
themselves
Intracellular vs. cell surface receptors
Hydrophobic molecules
(steroid hormones, retinoic
acids, etc) enter cells and
bind to intracellular receptors
Hydrophilic or big molecules
(proteins or polypeptide
hormones) bind to cell
surface receptors
Majority
Major types of intercellular signaling
Contact dependent signaling: gap
junction or ligand-receptor interaction
on cell surface.
Endocrine signaling: A sender cell
secretes ligands (hormones) into the
blood for long distance transport to
target receptors.
Paracrine signaling: A sender cell
secretes ligands that are diffused
(up to ~0.1 mm) to bind to target
receptors.
Synaptic signaling: An axon terminus
secretes ligands (neurotransmitters)
Into restricted environment to target
nerves or muscles.
Cell surface receptors categorized by action modes
Ligand binding to ligand-gated ion channel
receptor permits ions (e.g., Na+) to permeates
through the receptor in nervous cells.
Enzyme-linked receptors themselves are
kinases that phosphorylate effectors.
Cytokine binding to cytokine receptors
(without kinase activity) activates a kinase
effect.
G-protein-coupled receptors
(GPCR)
~50% of all known drugs
G-Protein-Coupled Receptors (GPCRs)
Represent ~5% of all
proteins
Targets of ~50% of all known
drugs
Hormones,
neurotransmitters,
Local mediators
Heterotrimeric GTP-binding
proteins (G proteins)
Heterotrimeric G proteins coupled with GPCR
s: stimulates
adenylate cyclase,
cAMP up.
i: inhibits adenylate
cyclase, cAMP down
q: phospholipase C
IP3, DAG, Ca++
12: guanine-nucleotide
exchange factors (GEFs)
Activation and inactivation of G proteins
-GTP (active) + (active)
-GDT (inactive)
1 GPCR vs. 10-100 G-proteins
G proteins activate downstream
effectors
GTPase activator proteins
(GAPs) and regulators of G protein
signaling (RGSs) inactivate G proteins
By modulating the GTPase activity
Adenylate cyclases regulated by Gs and Gi
cAMP function is mediated by
cAMP-dependent protein kinase
(PKA)
PKA phosphorylates many other
proteins
cAMP-regulated gene regulatory
proteins (CREBs)
cAMP-sensitive regulatory
elements (CRE)
PKA controlled by cAMP
Gq
phospholipase C and IP3
Two intracellular second messengers
are produced in the hormone-sensitive
phosphatidylinositol system: inositol
1,4,5-trisphosphate (IP3) and
diacylglycerol.
Both contribute to the activation of
protein kinase C.
By raising cytosolic [Ca2+], IP3 also
activates other Ca2+-dependent
enzymes.
Fig. 13.29. Ca++ signaling and its action
Ca++ and calmodulin (CaM)
Calmodulin-dependent kinases
Auto-phosphorylation: sustained activity
Receptor tyrosine kinase
Growth factors: EGF, PDGF etc
RTS signaling and Ras GTPase
~30% of all cancers involve Ras mutations.
RTK-RAS-MAPK cascade
RTK: kinase receptor
Ras: GTPase
MAPKKK: kinase
MAPKK: kinase
MAPK: kinase
Transcription factors
(e.g., c-Fos)
Why multi-step in signaling?
Signal amplified in number and duration
Signal fine regulated
Signal cross talks
Regulation of gene expression by
insulin through a MAP kinase
cascade
Insulin receptor: INS-R
INS-R autophosphorylation
Insulin receptor substrate -1 (IRS-1)
Ras
Raf-1
MEK: MAPKK
ERK: MAPK
Elk1
Serum response factor (SRF)
Receptor Serine/Threonine Kinases
TGF and BMPs
Tissue development and
differentiation
Mutations in these pathways
found in cancers
SMAD-dependent transcription
Fig. 13.18. TGF-activated receptor serine/threonine
kinase cascade
NO as a signaling molecule
1998 Nobel Prize
Guanylate cyclases
cGMP synthesis activated by NO
Phosphodiesterase (PDE)
cGMP degradation
NO, Vasodilation, and Drug
Endothelial cells: NO
Vascular smooth muscle cells
guanylate cyclase
cGMP
cGMP-sensitive kinase
Viagra
Erectile dysfunction
PDE5 inhibitor
[NO] increase
80-4000-fold less potent than PDE3 (cardiac
muscle)
15-fold less potent than PDE6 (retina)