Investigating Intracellular Signaling

November 10th, 2010

Silverman et al, 2004

The molecular story

Intracellular signaling: the biochemical pathways that function to
regulate the cells physiology and metabolism
Function
Genetics

Biochemistry

Genes

Proteins

Molecular Biology

Why do we study Intracellular

Signaling?
Fundamentally about mechanism

To understand what takes place within a

cell as it responds and interacts with its
environment

To know what protein products are

present, how they are regulated, and in
what networks they function

To discover novel drug targets for

disease therapies

Examples of processes regulated by

intracellular signaling
1. Cell fate determination
2. Neural migration
3. Axon outgrowth
4. Axon guidance
5. Synapse formation
6. Learning and memory
7. Disease

Signaling Questions of the Day

I.

Where, when, and how much of your protein

Expression

is present?
II.

Pathway

Does your protein interact with other

proteins?

III.

Function

Do post-translational modifications occur to

your protein?

IV.

Gene
regulate gene
expression?
Regulation

Does your protein interact with DNA and

Signaling Questions of the Day

I.

Where, when, and how much of your protein

is present?
II.

Does your protein interact with other

proteins?

III.

Do post-translational modifications occur to

your protein?

IV.

Does your protein interact with DNA and

regulate gene expression?

Localizing and Quantifying Proteins

shRNA

Id2

p27
-actin

Our ability to localize and quantify the presence of a

specific protein of interest is often dependent on the
existence of GOOD antibodies

The foundation of biochemistry: Antibody

Production

Monoclonal vs. Polyclonal

Entire protein, specific

regions, or modifications

May not work for both

spatial and quantitative
purposes

Different batches may be

different

Purifying Proteins 2 major methods:

1. Column Chromatography: Proteins are separated and
purified according to their physical properties

Gel-filtration
chromatography: Size

Ion-exchange
chromatography: Charge

Hydrophobic
chromatography: Solubility

Affinity chromatography:
Binding Affinity

Purifying Proteins 2 major methods:

2. Immunoprecipitation: Precipitate protein out of
solution by using a protein specific antibody

Beads

Couple antibody
to beads

Incubate antibody
with cell lysis

Verify purified
protein with
Western

Localizing and Quantifying Proteins

SPATIAL
Cellular

Subcellular

Immunoelectron
Immunohistochemistry Microscopy
Reporter
Genes

Cell Fractionation

AMOUNT
Quantitative
Western Blot

ELISA

Immunohistochemistry

Marker
Secondary
antibody
Protein
Primary
antibody

Reporter Proteins
2. Fusion protein

1. Co-expression

Reporter

Protein promoter

Reporter

Immunoelectron Microscopy
Gold-conjugated antibodies

Cell Fractionation followed by

Western Blot

Western Blot

Samples then transferred to membrane (nitrocellulose

or PVDF) and probed with antibody

Enzyme-linked immunosorbent assay

(ELISA)
More sensitive
than Western
(nanograms vs
micrograms)
Protein in its
native conformation
rather than
denatured as in a
WB

Signaling Questions of the Day

I.

Where and how much of your protein

is present?
II.

Does your protein interact with other

proteins?

III.

Do post-translational modifications
occur to your protein?

IV.

Does your protein interact with DNA?

Co-immunoprecipitation
Protein
Complex

antibody
Sepharose
bead

Used to identify proteins that directly interact with your

protein of interest

Affinity Chromatography

1. Attach target protein to beads

2. Add soluble proteins to beads
3. Elute proteins bound to beads

4. If antibody available, run a

Western Blot to determine
presence of interacting protein

Frster Resonance Energy Transfer

(FRET)
436 nm
436 nm

480 nm

535 nm

What if we dont know the identity

of the interacting protein

Identifying Proteins
Mass Spectrometry: Used to determine the composition
of proteins in a solution based on the mass/charge ratio

Signaling Questions of the Day

I.

Where and how much of your protein

is present?
II.

Does your protein interact with other

proteins?

III.

Do post-translational modifications
occur to your protein?

IV.

Does your protein interact with DNA?

Post-translational Modifications
Involve a molecule that is covalently attached to one or
more amino acids of the protein, changing the structural
and functional properties of the protein

Enzyme

Post-translational Modifications
Proteins can be modified by:

ATP

Protein
kinase
P

ADP

Acetylation

Alkylation

Amidation

Biotinylation

Formylation

Glutamylation

Glycosylation

Glycylation

Hydroxylation

Isoprenylation

Lipoylation

Methylation

Phosphorylation

Sulfation

Ubiquitination

Post-translational Modifications
Modification specific antibodies: Used to localize
and quantify modified protein

Post-translational Modifications
Modification specific assays: Ex. Kinase Assay
Protein
kinase

ATP32
Substrate

Signaling Questions of the Day

I.

Where and how much of your protein

is present?
II.

Does your protein interact with other

proteins?

III.

Do post-translational modifications
occur to your protein?

IV.

Does your protein interact with DNA?

Investigating protein-DNA
interactions

Luciferase Assay

Promoter Region

Target Gene

?
Promoter Region

Luciferase

Can a protein upregulate expression of a target gene?

Chromatin Immunoprecipitation (ChIP)

Crosslink protein and DNA

Sonicate to shear DNA

Immunoprecipitate

Reverse crosslink and Purify DNA

PCR and Sequence

ChIP-Chip and ChIP-Seq

High-throughput method of determining all the gene
elements that are controlled by a transcription factor

Electrophoretic Mobility Shift Assay

(EMSA)
Assays migration of radiolabeled DNA in a gel

Lane 1: DNA alone

Lane 2: DNA + Non-binding

Protein

Lane 3: DNA + Binding

Protein

However everything is in vitro

Putting it all together in a journal

article
An example of an intracellular
signaling study

Regulation of Motor Neuron

Specification by Phosphorylation of
Neurogenin 2

Yong-Chao Ma et al.
Greenberg Lab at Harvard University
Neuron, April 2008

Background
Dopaminergic

Spinal Motor

Neural
Progenitor

Interneuron

Neural Stem Cell

Oligo

Glial
Progenitor
Astrocyte

Commitment to a neuronal fate

Establishment of cell-type identity

Background

Commitment
Neural Stem Cell

Ngn2

Neural
Progenitor

Identity
Spinal Motor

Ngn2

Neurogenin 2 (Ngn2) is a basic helix-loop-helix

transcription factor involved in spinal cord
development

Background

Neurogenin 2 (Ngn2) is a basic helix-loop-helix

transcription factor

Ngn2 promotes neurogenesis (known mechanism)

Ngn2

Ngn2

E-protein

p27KIP

E-box

NeuroD (neurogenesis)

Background
Neurogenin 2 (Ngn2) is a basic helix-loop-helix
transcription factor

Ngn2 promotes neurogenesis - Known Mechanism

Ngn2 regulates spinal motor neurons (SMN) - Unknown

Mechanism

???

Ngn2

E-protein

E-box

HB9 (SMN)

The Overarching Question:

How can a single transcription factor Ngn2 - regulate both global neurogenesis
yet restrict SMN identity?

Hypothesis:
Post-transalational modifications exist for Ngn2 that can
regulate between the ability to promote neurogenesis and
specify SMN identity.

Ngn2

E-protein

E-box

???
HB9 (SMN)

NeuroD (neurogenesis)

Hypothesis:
Post-transalational modifications exist for Ngn2 that can
regulate between the ability to promote neurogenesis and
specify SMN identity.

1. Do modifications exist?
2. What is the functional significance of the modifications
for SMN identity?
3. What is the molecular mechanism leading to SMN
identity?

Hypothesis:
Post-transalational modifications exist for Ngn2 that can
regulate between the ability to promote neurogenesis and
specify SMN identity.

1. Do modifications exist?
2. What is the functional significance of the modifications
for SMN identity?
3. What is the molecular mechanism leading to SMN
identity?

Ngn2 is Phosphorylated

Phosphorylated Ngn2 expressed in SMN

Working Model

Ngn2 is phosphorylated at S231 and S234

Phosphorylated Ngn2 expressed in differentiating SMN

Ngn2

E-protein

E-box

???
HB9 (SMN)

NeuroD (neurogenesis)

Hypothesis:
Post-transalational modifications exist for Ngn2 that can
regulate between the ability to promote neurogenesis and
specify SMN identity.

1. Do modifications exist?
2. What is the functional significance of the modifications
for SMN identity?
3. What is the molecular mechanism leading to SMN
identity?

Lack of phosphorylation results in reduction in

SMN number

Mutate: S231A S234A (lose phosphorylation)

Lack of phosphorylation results in reduction of

SMN number

Phosphorylation has no effect on Neurogenesis

related factors

Working Model

Lack of Ngn2 phosphorylation leads to SMN loss

Phosphorylation not necessary for neurogenesis factors

Ngn2

E-protein

E-box

???
HB9 (SMN)

NeuroD (neurogenesis)

Hypothesis:
Post-transalational modifications exist for Ngn2 that can
regulate between the ability to promote neurogenesis and
specify SMN identity.

1. Do modifications exist?
2. What is the functional significance of the modifications
for SMN identity?
3. What is the molecular mechanism leading to SMN
identity?

Does Ngn2 cooperate with known SMN

signaling pathways?

SMN express a unique combination of proteins involved

in transcription (Lhx3, Isl1, NL1)

SMN factors work together in a complex

Ngn2 and SMN factors expressed together in

differentiating SMN

Ngn2

E-protein

E-box

???
HB9 (SMN)

SMN
Factors

Ngn2 physically interacts with SMN factors

in the cell nucleus

Immunoprecipitation after nuclear fractionation

Ngn2 interaction with SMN factors dependent

on phosphorylation

Working Model

Ngn2 physically interacts with Lhx3, Isl1 and NL1 (SMN factors)

Ngn2 and SMN factor interaction dependent on

phosphorylation

Ngn2

E-protein

E-box

P
Lhx3
Isl1 and NL1

???
HB9 (SMN)

How is Ngn2 Phosphorylated?

In silico screen: used available programs to identify consensus

sequence on Ngn2 with known kinases involved in motor
neuron development

Key Player: GSK3

Known to be activated by Sonic
Hedgehog (Shh)

GSK3 phosphorylates Ngn2

Shh signaling induces phophorylated Ngn2

expression in SMN

Working Model
Ngn2 is phosphorylated by GSK3 at S231 and S234

Ngn2

P
E-protein

E-box

HB9 (SMN)

Putting It All Together: The role of Ngn2 in

neurogenesis and SMN identity specification

Ngn2

E-protein

E-box

???
HB9 (SMN)

NeuroD (neurogenesis)

Working Model
Ngn2 regulates neurogenesis in a nonphosphorylated state

Ngn2

E-protein

E-box

NeuroD (neurogenesis)

Working Model
Shh activates GSK3 during SMN differentiation

P
Ngn2

E-protein

E-box

HB9 (SMN)

Working Model
GSK3 phosphorylates Ngn2 at S231 and S234

Ngn2

E-protein

E-box

HB9 (SMN)

Working Model
Phosphorylated Ngn2 and Lhx3, Isl1 and NL1 expressed in
differentiating motor neurons

Lhx3
Isl1 and NL1

Ngn2

E-protein

E-box

HB9 (SMN)

Working Model
Once Ngn2 is phosphorylated it interacts with by Lhx3, Isl1
and NL1 to transcribe SMN specific genes

Ngn2

P
E-protein

E-box

Lhx3
Isl1 and NL1

HB9 (SMN)

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Cell Culture and In Vitro Assays