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 Gs
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 Gs

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)