A3Trasduzione Del Segnale 07 10 2014.Ppt

8/18/2019 A3Trasduzione Del Segnale 07 10 2014.Ppt

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

General intracellular mechanisms in signal transduction

•

alteration of the activity, by conformationalchanges, of intracellular signal proteins

• Protein Kinases (MAPKs, PKA, PKC,etc.) and Phosphatases

• GTPases• Phospholipase C• Transcriptional factors

• Other enzymes

• alteration of the localization of specificadaptor proteins or enzymes

• Phosphotyrosine binding proteins

•

alteration of the concentration of smallmolecules (second messengers)

• cAMP• cGMP

• IP3• DAG• Ca2+


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Small Molecules and Ions as SecondMessengers

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

• The extracellular signal molecule that binds to the membrane is a pathway’s“first messenger”

•

Second messengers can readily spread throughout cells by diffusion

•

Second messengers participate in pathways initiated by G-protein-linkedreceptors and receptor tyrosine kinases

• Second messengers amplificate the signal


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Second messengers

Ca 2+


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

Reception

Transduction

Inactive G protein

Active G protein (102 molecules)

Inactive adenylyl cyclase

Active adenylyl cyclase (102)

ATP

Cyclic AMP (104)

Inactive protein kinase A

Inactive phosphorylase kinase

Active protein kinase A (104)

Active phosphorylase kinase (105)

Active glycogen phosphorylase (106)

Inactive glycogen phosphorylase

Glycogen

Response

Glucose-1-phosphate(108 molecules)

Note the amplification


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Protein Modules in Signal Transduction

• Signal transduction in cell occurs via protein-protein and protein-lipidinteractions based on protein modules

• Most signaling proteins consist of two or more modules

•

This permits assembly of functional signaling complexes


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Cessation of Activity in Signal TransductionPathways

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 fora 1st messenger, so messenger is released.

3. plasma membrane receptors are removed when thecombination of the 1st messenger and receptor is takeninto the cell by endocytosis.

Often a phosphorylated receptor may bind to aprotein -arrestin promoting removal of the receptorfrom the membrane by clathrin-mediated endocytosis. • -Arrestin may also bind a cytosolic Phosphodiesterase,• bringing this enzyme close to where cAMP is being produced,contributing to signal turnoff.


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Figure 15-51 Molecular Biology of the Cell (© Garland Science 2008)

Phosphorylation-dependent desentitization of a GPCR

Block the interaction between receptor and G-proteinInduce interaction between receptor and clathrin vesicles (endocitosis)


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! A protein kinase transfers the phosphate group of a donor molecule (suchas ATP) to a hydroxyl group on a protein.

! A protein phosphatase catalyzes removal of the Pi by hydrolysis.

Protein OH + ATP Protein O P

O

O

O

+ ADP

Pi H2O

Protein Kinase

Protein Phosphatase

Many enzymes are regulated by covalent attachment of phosphate, inester linkage, to the side-chain hydroxyl group of a particular amino acidresidue (serine, threonine, or tyrosine).

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

Protein Phosphorylation andDephosphorylation in Signal Transduction


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factor PDGF)

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

Protein kinases in signal transduction


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Predicted phosphorylation sites versus phosphorylated residues

Several Bioinformatic tools

allow the identification ofpotential consensus sites ofphosphorylation by aspecific protein kinase, butthey cannot take intoaccount the disposition andaccessibility of suchconsensus site in 3Dstructure


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Experimental methods for the identification of protein phosphorylation eventsinclude:

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 activityor subcellular localization of a protein or enzyme, e.g.

by promoting reversible changes in the affinity forspecific molecules, due to

inter-molecular repulsion/attraction eventsisocitrate dehydrogenase

by promoting reversible conformational changesthat modulate the enzymatic activity, due to theformation of novel hydrogen bonds orrepulsion/attraction of intra-molecular chargedregions

glycogen phosphorylaseMAPKs and RTKsSrcCdks

by promoting reversible formation of consensussites for binding of proteins that specificallyrecognize phosphorylated domains. This

mechanism involves changes in subcellularlocalizationPhosphoTyr / protein containing PTB/SH2 domainsPhosphoSer or PhosphoThr / 14-3-3 protein


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Regulation of the catalytic activity of glycogen phosphorylase


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Molecular structures of MAP kinase in itsinactive, unphosphorylated form (a) andactive, phosphorylated form (b).

Regulation of the catalytic activity of MAP Kinase

Example of cooperativephosphorylation


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Bo on and Eck Structure and re ulation of Src famil kinases Onco ene 2004 23 7918–7927

Autoinhibition of Src activity is based on phosphorylation events that modulatethe intramolecular interdomain interaction


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Other examples of regulation by phosphorylation events


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Signal molecule

Activated relaymolecule

Receptor

Inactiveprotein kinase

1 Activeproteinkinase

1



2



3

ADP

Inactiveprotein

Activeprotein

Cellularresponse

ATP

PPP i

ADP ATP

PPP

i

ADP ATP

PPP i

P

P

P

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

Protein kinases and phosphatasesare themselves substrates of otherprotein kinases and phosphatases

and their activity is often regulated byphosphorylation/dephosphorylationevents. Therefore phosphorylationevents of signal transduction proteinallow Amplification and Integration ofdifferent signals


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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.xhttp://onlinelibrary.wiley.com/doi/10.1111/j.1742-4658.2009.07027.x/full#f8


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Mechanistic aspects of multisite phosphorylation.



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Multisite protein phosphorylation –distributive versus processivemechanism



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A3Trasduzione Del Segnale 07 10 2014.Ppt