Mediators of Cell signaling

ICG601
Dhiraj Kumar
 Mediators of Cell signaling
• Cell surface receptors (RTKs, non-RTKs)

• Nuclear receptors

• Signaling intermediates (Kinases, phosphatases)

• Second messengers (Ca2+, IP3, DAG, ROS etc.)

• Protein domains in Signaling

• Signaling pathways

• Crosstalk among signaling pathways

• Graphs and Networks to understand cell signaling

Cellular tools for information
transmission
Signaling networks are composed of highly
specialized proteins with devolved functions

•Receptors

•Transducers

•Adapters

•Scaffolds

•Effectors

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 Intercellular communication

Generation of signal in one cell

Protein phosphorylation

Transmission of signal Serine

Threonine
Tyrosine

Recognition and response to

the signal in the target cell
Protein phosphorylation during cell signaling
Why phosphorylation evolved as preferred way of cellular signal transduction?
Protein phosphorylation during cell signaling

Phosphate group: pKa is 6.7, at neutral pH contains 2 negative charge

Phosphorylated residues: Serine/Threonine/Tyrosine

4 oxygen atoms present on the phosphate group, allows for additional H-bonding

The phosphate ester of Serine/Threonine/Tyrosine are quite stable at RT and neutral pH

No spontaneous hydrolysis, requires specific enzymes called phosphatases

Flexible and Reversible means of regulating protein activity
 Principal mechanism of
intercellular communication
 Nature of external signals

Physical Chemical Electrical Optical

Touch (adhesion) Growth factors

Temperature Hormones
pH
Osmolarity
 Cell-to-cell communication:
Hormones and Growth factors

Hormones: Signaling molecules for communication between the cells

Growth factors: Hormones that are protein and regulate cell proliferation
Endocrine, Paracrine and autocrine signaling
Hormones: Ligands for nuclear receptors
Steroid
Hormones
Hormones: Ligands for trans-membrane
receptors
Recognition of hormones by receptors
Signaling through nuclear receptors
Ligand and structure of few nuclear receptors
Orphan receptors
G-protein coupled receptors
GTPase Superfamily
Inhibition of GPCR signaling
Receptor tyrosine kinases

Examples??

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Antigen Receptors (BCR and
TCR), Pattern receptors

Downstream signaling process

delegated to molecules that
interact with the receptors

Eg. Antigen receptors (B and T

cells, TLRs, cytokine receptors
etc.

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 Signal Transducers

Kinases: Phosphatases:

Tyrosine kinase Tyrosine Phosphatases

Serine/Threonine Kinase Serine/Threonine Phosphatases
Dual specificity kinase Dual specificity Phosphatases

Human genome contains >1000 kinases and phosphatases!!

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 Interactions for information
transmission
•The ability of a protein to deliver a signal depends on its activity
and location

Interactions between proteins or proteins-lipids can control:

1) proximity to substrate
2) proximity to an upstream regulator
3) conformation and hence activity of a signaling protein

These regulatory interactions are mediated by specific protein domains

action

SH3 SH2 Kinase domain

Myr-

site 1: Yact site 2: Yinhib

interaction Advance Cell and Molecular Biology
 Kinases
SH3 SH2 Kinase domain
Myr-

Site 1: Yactivating Site 2: Yinhib

csk Y2
CD45 SH2
SH3
Active
KD
Y1 P

Y2 P SH2
SH3 2 mechanisms P
P Pro
Pro
KD Y2 SH2 Pro
Y1 SH3

KD
Inactive Y1 P
Active
Closed conformation

Phosphorylation may change activity, affinity orAdvance

location
Cellof
andaMolecular
proteinBiology
in the cell
 Second Messengers
MESSENGERS examples

metal ions Calcium

Second messengers Zinc, also Mn 2+

•Diffusible (?) messengers interact Lipids DAG

with and cause changes in activity + inositol lipids PIPs, IPs
or location of a protein

•Messengers must be mobile, stable cAMP, cGMP

Cyclic nucleotides
•Require own regulatory networks ADR-ribose,
+sugar nucleosides
cADP-ribose

dissolved gas NO, H2S

H2O2 and other reactive Oxygen species

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 Adaptors and Scaffolds

No enzymatic activity, provide platform for molecular interaction, alter effective

substrate concentration at the site of reactions
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Protein modules and their targets - example 1 - SH2

• Modules of about ~100 amino acids

•Binds specifically to pY containing peptide motifs
•Conserved and variable pocket (for pY and 3-6 residue C-
terminal of pY respectively)
•Affinity in micromolar range

Protein modules and their targets - example 2 - SH3

• Binds to proline rich peptide (left handed poly-pro

type II.
• Minimum consensus—P-X-X-P-
• Each Pro- preceded by an aliphatic residue, together
recognized by hydrophobic pocket of SH3 domain.
• Affinity in micromolar range
• Can be enhanced by additional 3D structure acquired.

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Protein modules and their targets - example 3 - Bromo domain

• About ~100 Amino acids

• Found in chromatin associated proteins
• Binds with acetylated lysine containing peptides
• Binds to acetyl-Lysine

Protein modules and their targets - example 4 - EF domain

• ~ 40 residues
• binds with intracellular Calcium ions
• Leads to large or subtle change in conformation eg regulatory or
structural categories.

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 Protein modules and their targets - example 5 - PH domain

• Binds with specific phosphoinositides with high

affinity( micro to nano molar)
• makes signaling intermediates responsive to lipid
messengers
• PI-4,5-bisphosphate, PI-3,4-P2 or PI-3,4,5-P3

• Specifically used for targeting cytoplasmic proteins to

membranes.

Some multi-domain proteins

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Complex pathways of signal
transduction

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Advance Cell and Molecular Biology
… is regulated by subcellular compartmentalisation
Mitogen stimulation
Ras. Ras.
Resting GDP GTP
RTK
Kinases
cytosol
Raf-1
Ras.GDP

Grb2-SOS P
MEK MEK P
(GEF)

ERK P Cytoplasmic
ERK P
Raf-1 substrates

TF P P
Nuclear ERK P
substrates
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Regulation of biological
function by signaling

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Why have a 3 component kinase cascade ?

1) Signal amplification 2) Signal fine tuning

three knobs are better than one

On / Off Volume Balance

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… regulated by protein:protein interactions

Mitogens
Growth factors
Receptor
receptor
e
nas
Ras ki
P P

Raf
P 14-3-3
SUR-8
Hsp90 P
P
Adaptors

Ksr
MEK
&
MP1 P P Scaffolds
ERK

P P

ERK
P
P
Elk
SAP Gene
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… regulated by protein:protein interactions

Mitogens
Growth factors
Receptor
receptor
e
nas
Ras ki
P P
P
Raf
RKIP Disruptors
P P

MEK

P P

ERK

P P

ERK
P
P
Elk
SAP Gene
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… regulated by protein:protein interactions

Mitogens
Growth factors
Receptor
receptor
e
nas
Ras ki
P P
P
Raf

RKIP
MEK

ERK

Elk
SAP Gene
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… and is regulated by posttranslational
modifications
Raf-1 domain structure & phosphorylation sites

“Activation
RBD CRD 375 loop”
K

ATP
CR1 CR2 CR3
S S SY SS S
43 259 338 341 491 494 621
P P P P P P P

Activating
phosphorylations

Inhibitory phosphorylations

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 Receptor
e.g. 7-TMR
cell membrane
g g
a tyrosine
Ras
AdCyc a b b kinase shc SOS Ras
cAMP heterotrimeric
grb2 Akt
Rap1 GEF G-protein Rac
cAMP ATP cAMP
cAMP Raf-1 PI-3
PKA cAMP K PAK
B-Raf cAMP AMP
PKA
PDE MEK
MEK1,2 ERK1,2
ERK1,2
cytosol
MKP

transcription
factors

nucleus
How can we
understand this
activation inhibition phosphorylation
complexity?
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 Receptor
e.g. 7-TMR
cell membrane
g g
a tyrosine
Ras
AdCyc a b b kinase shc SOS Ras
cAMP heterotrimeric
grb2 Akt
Rap1 GEF G-protein Rac
cAMP ATP cAMP
cAMP Raf-1 PI-3
PKA cAMP K PAK
B-Raf cAMP AMP
PKA
PDE MEK
MEK1,2 ERK1,2
ERK1,2
cytosol
MKP

transcription
factors

nucleus
The biochemical view
activation inhibition phosphorylation

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 Reductionism: Failure to integrate the available knowledge
together

Ras

Raf-1
MEK1,2

ERK1,2

transcription
factors

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The digital view
Signal

Processing
Unit

Death Survival Growth

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 Stress Signal

Death Survival Growth

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 Growth Signal

Death Survival Growth

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Death Survival Growth

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 Types of biological network
Gene regulatory network

Metabolic network

Signal transduction network

Protein-protein interaction network

At different scale
• Immunological network
• Ecological network
• Intracellular vesicular network

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 challenges
Identifying complex macromolecular interaction between genes, proteins and metabolites

Once identified, Network models can be used to:

• Simulate the process it represents
• Predict the features of its dynamic behavior
• Extrapolate cellular phenotypes

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 Features of graphs
Biological networks are best described and visualized using graphs

• Node degree (in- and out- degree for directed graphs)

• Average degree distribution (k), k=E/N (N=total number of nodes in a graph, E=edges)

• Shortest and mean path length

• Clustering coefficient (interactions among the adjacent nodes)

• Betweenness and stress (centrality parameters)

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 Node degree
Undirected network Directed network

G=(V, E) G=(8, 10)

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 degree distribution P(k)
Degree distribution P(k): probability that a given node n has exactly k links in the network

P(k) = N (total no. of links)/K (total no. of nodes)

Degree distribution: used to classify different kinds of networks

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Shortest path length and mean
path length
How many links to pass through in order to connect two
nodes
Connect node A to E

All possible paths Steps Shortest path

A-G, G-F, F-E 3
A-H, H-E 2
A-B, B-F, F-E 3
A-C, C-B, B-F, F-E 4
H

Mean Path Length???

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 Clustering coefficient C(k)
CI=2nI /k(k- nI =links between first neighbors
k(k-1)/2 = Total possible links between first neighbors
1)

nI =3, k(k-1)/2 =3 nI =1, k(k-1)/2 =3 nI =0, k(k-1)/2 =3

Hence Ci=1 Hence Ci=1/3 Hence Ci=0/3

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Clustering coefficient of entire
network
nI =links between first neighbors
k(k-1)/2 = Total possible links between first neighbors

CI=2nI /k(k-
1)

21/36 0.583333333

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 Types of graphs (networks)

Random graphs (Erdos-Renyi Network)

Scale-free Networks

Hierarchical Networks

Degree distribution and clustering together defines network types

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 Random networks

Equal probability for each pair of nodes to be connected

Most of the nodes have degree distribution very similar to

the average

Degree distribution
Clustering coefficient independent of nodes

Clustering coefficient

Random networks have no preferential

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interactions
And hence contain hardly any information
Scale free network
Power-law degree distribution

Frequency of event varies with power of some attribute

Degree distribution???

Clustering coefficient is independent of k

No modularity

Preferential attachment of new incoming node to

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The highly connected node (hub) in the network
Hierarchical Network

Modularity, local clustering and scale free organization!!

Sparsely connected nodes are part of highly clustered areas

Communication between different clustered area maintained by

Interactions between few hubs!!!

Modular architecture, C(k) dependent on k

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 Most real-world networks are
modular
They exhibit scale-free property

Sub-modules of the larger network show similar property as the parent network

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 Centrality parameters of
networks

Mean path
Degree Clustering
length

Stress Betweenness

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 Stress and betweenness

Number of shortest paths passing through a node

• Calculate shortest paths between each pair of nodes in the network

• Nodes present in higher number of shortest path compared to the average are stressed nodes

• Betweenness refers to the no. of shortest path passing through a given node between two
different regions in the network.

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Stress and betweenness

Stress algorithm Betweenness algorithm

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Nodes with lethal phenotype
showed relatively higher
betweenness centrality value

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 Lecture 3

• Complexity of cell signaling

• The circuitry of cell signaling machinery
• Signaling motifs (FFLs, Feedbacks etc.)
• Studying a conserved signaling pathway
(MAP Kinase Pathway)
• Cell signaling during chemotaxis
• Cell signaling during inflammation