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Signal

transduc.on 3

General intracellular mechanisms in signal transduction

alteration of the activity, by conformational


changes, of intracellular signal proteins
Protein Kinases (MAPKs, PKA, PKC,
etc.) and Phosphatases
GTPases
Phospholipase C
Transcriptional factors
Other enzymes

alteration of the localization of specific


adaptor proteins or enzymes
Phosphotyrosine binding proteins

alteration of the concentration of small


molecules (second messengers)

Figure 15-17 Molecular Biology of the Cell ( Garland Science 2008)

cAMP
cGMP
IP3
DAG
Ca2+

Small Molecules and Ions as Second


Messengers

Second messengers are small, nonprotein, water-soluble molecules or ions

The extracellular signal molecule that binds to the membrane is a pathways


first messenger

Second messengers can readily spread throughout cells by diffusion

Second messengers participate in pathways initiated by G-protein-linked


receptors and receptor tyrosine kinases

Second messengers amplificate the signal

Second messengers

Ca 2+

Reception
Binding of epinephrine to G-protein-linked receptor (1 molecule)

Transduction

Note the amplification

Inactive G protein
Active G protein (102 molecules)

Inactive adenylyl cyclase


Active adenylyl cyclase (102)

ATP
Cyclic AMP (104)

Inactive protein kinase A


Active protein kinase A (104)

Inactive phosphorylase kinase


Active phosphorylase kinase (105)
Inactive glycogen phosphorylase
Active glycogen phosphorylase (106)
Response
Glycogen
Glucose-1-phosphate
(108 molecules)

Protein Modules in Signal Transduction

Signal transduction in cell occurs via protein-protein and protein-lipid


interactions based on protein modules

Most signaling proteins consist of two or more modules

This permits assembly of functional signaling complexes

Cessation of Activity in Signal Transduction


Pathways
A major way to cease receptor activity is to decrease the concentration of the 1st
messenger in the region of the receptor.
Methods for decreasing 1st messenger concentration include:
1. enzymes metabolize 1st messenger
2. receptor becomes chemically altered
(usually by phosphorylation, which lowers affinity for
a 1st messenger, so messenger is released.
3. plasma membrane receptors are removed when the
combination of the 1st messenger and receptor is taken
into the cell by endocytosis.
Often a phosphorylated receptor may bind to a
protein b-arrestin promoting removal of the receptor
from the membrane by clathrin-mediated endocytosis.
b-Arrestin may also bind a cytosolic Phosphodiesterase,
bringing this enzyme close to where cAMP is being produced,
contributing to signal turnoff.

Phosphorylation-dependent desentitization of a GPCR

Block the interaction between receptor and G-protein


Induce interaction between receptor and clathrin vesicles (endocitosis)
Figure 15-51 Molecular Biology of the Cell ( Garland Science 2008)

Protein Phosphorylation and


Dephosphorylation in Signal Transduction
Many enzymes are regulated by covalent attachment of phosphate, in
ester linkage, to the side-chain hydroxyl group of a particular amino acid
residue (serine, threonine, or tyrosine).
In many pathways, the signal is transmitted by a cascade of protein
phosphorylations

Protein Kinase
OH + ATP

Protein

Protein

+ ADP

O
Pi

H2O

Protein Phosphatase
w A protein kinase transfers the phosphate group of a donor molecule (such
as ATP) to a hydroxyl group on a protein.
w A protein phosphatase catalyzes removal of the Pi by hydrolysis.

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30% of all proteins undergoes to phosphorylation events, even on multi-sites.


Protein kinases in signal transduction

factor (PDGF)

Predicted phosphorylation sites versus phosphorylated residues

Several Bioinformatic tools


allow the identification of
potential consensus sites of
phosphorylation by a
specific protein kinase, but
they cannot take into
account the disposition and
accessibility of such
consensus site in 3D
structure

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Experimental methods for the identification of protein phosphorylation events


include:
Kinase Activity Assays on peptides carrying the potential phosphorylation sites
Band shift assay

Western blotting with Antibodies directed against


protein phosphorylated in that specific residue

Mass spectrometry analysis

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Phosphorylation may directly alter with a switch mechanism the biological activity
or subcellular localization of a protein or enzyme, e.g.
by promoting reversible changes in the affinity for
specific molecules, due to
inter-molecular repulsion/attraction events
isocitrate dehydrogenase
by promoting reversible conformational changes
that modulate the enzymatic activity, due to the
formation of novel hydrogen bonds or
repulsion/attraction of intra-molecular charged
regions
glycogen phosphorylase
MAPKs and RTKs
Src
Cdks
by promoting reversible formation of consensus
sites for binding of proteins that specifically
recognize phosphorylated domains. This
mechanism involves changes in subcellular
localization
PhosphoTyr / protein containing PTB/SH2 domains
PhosphoSer or PhosphoThr / 14-3-3 protein
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Regulation of the catalytic activity of glycogen phosphorylase

Regulation of the catalytic activity of MAP Kinase

Molecular structures of MAP kinase in its


inactive, unphosphorylated form (a) and
active, phosphorylated form (b).

Example of cooperative
phosphorylation
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Autoinhibition of Src activity is based on phosphorylation events that modulate


the intramolecular interdomain interaction

Boggon and Eck Structure and regulation of Src family kinases Oncogene (2004) 23, 79187927

Other examples of regulation by phosphorylation events

In many pathways, the signal is transmitted by a cascade of protein phosphorylations


Signal molecule

Receptor

Protein kinases and phosphatases


are themselves substrates of other
protein kinases and phosphatases
and their activity is often regulated by
phosphorylation/dephosphorylation
events. Therefore phosphorylation
events of signal transduction protein
allow Amplification and Integration of
different signals

Activated relay
molecule

Inactive
protein kinase
1

Active
protein
kinase
1
Inactive
protein kinase
2

ATP

Pi

ADP

Active
protein
kinase
2

PP

Inactive
protein kinase
3

ATP

Pi

ADP

Active
protein
kinase
3

PP

Inactive
protein

ATP

Pi

PP

ADP

Active
protein

Cellular
response

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Integration of signalling events by multisite protein phosphorylation

FEBS Journal
Volume 276, Issue 12, pages 3177-3198, 29 APR 2009 DOI: 10.1111/j.1742-4658.2009.07027.x
http://onlinelibrary.wiley.com/doi/10.1111/j.1742-4658.2009.07027.x/full#f8

Mechanistic aspects of multisite phosphorylation.

FEBS Journal
Volume 276, Issue 12, pages 3177-3198, 29 APR 2009 DOI: 10.1111/j.1742-4658.2009.07027.x
http://onlinelibrary.wiley.com/doi/10.1111/j.1742-4658.2009.07027.x/full#f7

Multisite protein phosphorylation distributive versus processive


mechanism

FEBS Journal
Volume 276, Issue 12, pages 3177-3198, 29 APR 2009 DOI: 10.1111/j.1742-4658.2009.07027.x
http://onlinelibrary.wiley.com/doi/10.1111/j.1742-4658.2009.07027.x/full#f7

FEBS Journal
Volume 276, Issue 12, pages 3177-3198, 29 APR 2009 DOI: 10.1111/j.1742-4658.2009.07027.x
http://onlinelibrary.wiley.com/doi/10.1111/j.1742-4658.2009.07027.x/full#f7

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