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1 General Mechanisms of Cell Signaling Signaling to Cell Nucleus MUDr. Jan Pláteník, PhD. Somatic cells can be reprogrammed to pluripotent stem cells ! fibroblast iPS cardiomyocytes hepatocytes Reprogramming – what is it? Takahashi K & Yamanaka S. Cell 126, 2006, 663-676 neurones

General Mechanisms of Cell Signaling Signaling to Cell Nucleus · 2020-06-04 · 1 General Mechanisms of Cell Signaling Signaling to Cell Nucleus MUDr. Jan Pláteník, PhD. Somatic

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Page 1: General Mechanisms of Cell Signaling Signaling to Cell Nucleus · 2020-06-04 · 1 General Mechanisms of Cell Signaling Signaling to Cell Nucleus MUDr. Jan Pláteník, PhD. Somatic

1

General Mechanisms of Cell Signaling

Signaling to Cell Nucleus

MUDr. Jan Pláteník, PhD.

Somatic cells can be reprogrammed to pluripotent stem cells !

fibroblastiPS

cardiomyocytes hepatocytes

Reprogramming – what is it?

Takahashi K & Yamanaka S. Cell 126, 2006, 663-676

neurones

Page 2: General Mechanisms of Cell Signaling Signaling to Cell Nucleus · 2020-06-04 · 1 General Mechanisms of Cell Signaling Signaling to Cell Nucleus MUDr. Jan Pláteník, PhD. Somatic

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General mechanisms of cell signaling

Cell-to-cell communication:

1. Synthesis and

2. Release of the signaling molecule by the signaling cell

3. Transport of the signal to the target cell

4. Detection of signal by specific receptor protein

5. Signal-receptor complex triggers a change in cell metabolism and/or gene expression of the target cell

6. Removal/Termination of the signal

Page 3: General Mechanisms of Cell Signaling Signaling to Cell Nucleus · 2020-06-04 · 1 General Mechanisms of Cell Signaling Signaling to Cell Nucleus MUDr. Jan Pláteník, PhD. Somatic

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Signals operate over various distances :

+ AUTOCRINE

Signaling molecule (ligand): Peptides/proteins

– ACTH, insulin, glucagon, growth factors, cytokines, and many others

Small lipophilic molecules:

– steroids, thyroid hormones, prostaglandins

Small hydrophilic molecules:

– AA or derivatives of AA - epinephrine, norepinephrine, histamine, serotonine, glutamate, GABA, glycine etc.

Gases: NO, CO, H2S

Page 4: General Mechanisms of Cell Signaling Signaling to Cell Nucleus · 2020-06-04 · 1 General Mechanisms of Cell Signaling Signaling to Cell Nucleus MUDr. Jan Pláteník, PhD. Somatic

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Receptor

Always a protein

Ligand binding + Conformation change

signal transduction

Binding specificity

Effector specificity

Two classes of signaling molecules

Page 5: General Mechanisms of Cell Signaling Signaling to Cell Nucleus · 2020-06-04 · 1 General Mechanisms of Cell Signaling Signaling to Cell Nucleus MUDr. Jan Pláteník, PhD. Somatic

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Second messengers:

Cyclic adenosine monophosphate (cAMP)

Cyclic guanosine monophosphate (cGMP)

Inositol-1,4,5-tris-phosphate

1,2-diacylglycerol

Calcium

(NO, oxygen radicals)

Molecular switches:

Page 6: General Mechanisms of Cell Signaling Signaling to Cell Nucleus · 2020-06-04 · 1 General Mechanisms of Cell Signaling Signaling to Cell Nucleus MUDr. Jan Pláteník, PhD. Somatic

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Signal termination:

Receptor desensitization– endocytosis & degradation

– phosphorylation

Degradation or removal of signalling molecule– cAMP: phosphodiesterase

– Calcium: Ca2+ pumps

Dephosphorylation by protein phosphatases

Cell-surface receptors for signaling molecules:

Ion channels– for Na+, K+, Ca2+, Cl-

– e.g. receptor for acetylcholine, GABA, glutamate, glycine

Seven-spanning G protein-linked

Receptors associated with an enzymic activity

Page 7: General Mechanisms of Cell Signaling Signaling to Cell Nucleus · 2020-06-04 · 1 General Mechanisms of Cell Signaling Signaling to Cell Nucleus MUDr. Jan Pláteník, PhD. Somatic

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Muscle receptor for acetylcholine

Cell-surface receptors for signaling molecules:

Ion channels

Seven-spanning G protein-linked

Receptors associated with an enzymic activity

Page 8: General Mechanisms of Cell Signaling Signaling to Cell Nucleus · 2020-06-04 · 1 General Mechanisms of Cell Signaling Signaling to Cell Nucleus MUDr. Jan Pláteník, PhD. Somatic

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G-protein

targets:

- membrane

enzymes

- ion channels

Page 9: General Mechanisms of Cell Signaling Signaling to Cell Nucleus · 2020-06-04 · 1 General Mechanisms of Cell Signaling Signaling to Cell Nucleus MUDr. Jan Pláteník, PhD. Somatic

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Cyclic AMP& protein kinase A

R R

CC

+ 4 cAMP

R R

C C

cAMP

cAMP

cAMP

cAMP

inactive PKA

Active PKA

PKA controls synthesis/breakdown of glycogen in liver and muscle

Page 10: General Mechanisms of Cell Signaling Signaling to Cell Nucleus · 2020-06-04 · 1 General Mechanisms of Cell Signaling Signaling to Cell Nucleus MUDr. Jan Pláteník, PhD. Somatic

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NO signaling through guanylate cyclase:

Phospholipase C pathway

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Calcium in the cell: In cytosol only 0.1-0.2 μM, about 1 μM

is a signal

Source of the signal is:

– outside: ligand-operated Ca2+ channels

voltage-operated Ca2+ channels

– ER stores: PI3 receptor/channel

ryanodine receptor/channel– cell membrane potential-dependent (striated muscle)

– Ca2+ -dependent (heart, CNS)

Calmodulin (148 AMK)

Page 12: General Mechanisms of Cell Signaling Signaling to Cell Nucleus · 2020-06-04 · 1 General Mechanisms of Cell Signaling Signaling to Cell Nucleus MUDr. Jan Pláteník, PhD. Somatic

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Information in Ca2+ signal is encoded by its

– LOCALISATION

– FREQUENCY

– AMPLITUDE

(Berridge et al., Nature 1998, 395: 645-648)

Cell-surface receptors for signaling molecules:

Ion channels

Seven-spanning G protein-linked

Receptors associated with an enzymic activity– intrinsic catalytic activity: receptor tyrosine

kinases (RTKs, e.g. rec. for EGF, insulin)

– associated with soluble tyrosine kinases (e.g. rec. for cytokines, interferons)

Page 13: General Mechanisms of Cell Signaling Signaling to Cell Nucleus · 2020-06-04 · 1 General Mechanisms of Cell Signaling Signaling to Cell Nucleus MUDr. Jan Pláteník, PhD. Somatic

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Receptor tyrosine kinases

e.g.: Ras/MAPK

Monomer receptor-TK

Inactive RasGDP

Growth factor

Page 14: General Mechanisms of Cell Signaling Signaling to Cell Nucleus · 2020-06-04 · 1 General Mechanisms of Cell Signaling Signaling to Cell Nucleus MUDr. Jan Pláteník, PhD. Somatic

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P

P

P P

P

P

Ligand binding, receptor dimerisation

and autophosphorylation

P

P

P P

P

PGRB2

SH2

SH3

Page 15: General Mechanisms of Cell Signaling Signaling to Cell Nucleus · 2020-06-04 · 1 General Mechanisms of Cell Signaling Signaling to Cell Nucleus MUDr. Jan Pláteník, PhD. Somatic

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P

P

P P

P

PGRB2

SH2

SH3

Sos (Guanine Nucleotide Exchange factor)

P

P

P P

P

PGRB2

SH2

SH3

Sos (Guanine Nucleotide Exchange factor)

GTP

Page 16: General Mechanisms of Cell Signaling Signaling to Cell Nucleus · 2020-06-04 · 1 General Mechanisms of Cell Signaling Signaling to Cell Nucleus MUDr. Jan Pláteník, PhD. Somatic

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P

P

P P

P

PGRB2

SH2

SH3

Sos (Guanine Nucleotide Exchange factor)

GTP

Ras activated

P

P

P P

P

PGRB2GTP

Ras

H2N

COOH

Raf (Ser/Thr kinase)

Page 17: General Mechanisms of Cell Signaling Signaling to Cell Nucleus · 2020-06-04 · 1 General Mechanisms of Cell Signaling Signaling to Cell Nucleus MUDr. Jan Pláteník, PhD. Somatic

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P

P

P P

P

PGRB2GTP

Ras

H2N

COOH

Raf (Ser/Thr kinase)

MEK (Ser/Thr & Tyr kinase)P

P

P

P P

P

PGRB2GTP

Ras

H2N

COOH

Raf (Ser/Thr kinase)

MAPKKK: MAP Kinase Kinase Kinase

MEK (Ser/Thr & Tyr kinase)

MAPKK: MAP Kinase Kinase

P

PPP

ERK 1/2 (MAPK: Mitogen Activated

Protein Kinase)

Page 18: General Mechanisms of Cell Signaling Signaling to Cell Nucleus · 2020-06-04 · 1 General Mechanisms of Cell Signaling Signaling to Cell Nucleus MUDr. Jan Pláteník, PhD. Somatic

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Source of figure:

Cell Signaling Technology Inc.

Page 19: General Mechanisms of Cell Signaling Signaling to Cell Nucleus · 2020-06-04 · 1 General Mechanisms of Cell Signaling Signaling to Cell Nucleus MUDr. Jan Pláteník, PhD. Somatic

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Signaling to the Cell Nucleus

How signal affects protein function

Modification of structure/ function of proteins present in the cell

Change in spectrum /amount of proteins in the cell

– .... regulation of gene expression

Page 20: General Mechanisms of Cell Signaling Signaling to Cell Nucleus · 2020-06-04 · 1 General Mechanisms of Cell Signaling Signaling to Cell Nucleus MUDr. Jan Pláteník, PhD. Somatic

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Regulation of eukaryotic gene transcription

Model of interaction of several transcription activators with the mediator complex

Lodish et al.: Molecular Cell Biology, W.H.Freeman & Co, 5th ed., 2004

Page 21: General Mechanisms of Cell Signaling Signaling to Cell Nucleus · 2020-06-04 · 1 General Mechanisms of Cell Signaling Signaling to Cell Nucleus MUDr. Jan Pláteník, PhD. Somatic

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Eukaryotic transcription factors:

Classification according to structural motifs:

– homeodomains

– „zinc fingers“

– „leucine zippers“

– bHLH (basic Helix-Loop-Helix) proteins

Eukaryotic transcription factors:

Classification according to way of expression/regulation:

– inducible

– constitutive

– ligand-activated

Page 22: General Mechanisms of Cell Signaling Signaling to Cell Nucleus · 2020-06-04 · 1 General Mechanisms of Cell Signaling Signaling to Cell Nucleus MUDr. Jan Pláteník, PhD. Somatic

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Inducible TFs: e.g. AP1 (Activator Protein 1)

Jun family: c-Jun, JunB, JunD

Fos family: c-Fos, FosB, Fra-1, Fra-2

TGACTCA

TRE [TPA (phorbol ester) response element]

Jun Fos

Jun Jun

Jun ATF

Constitutive TFs: e.g. CREB

(cAMP/Ca2+ response element binding protein)

TGACGTCA

CRE [cAMP/Ca2+ response element]

CREB CREB

P

Ser133

Page 23: General Mechanisms of Cell Signaling Signaling to Cell Nucleus · 2020-06-04 · 1 General Mechanisms of Cell Signaling Signaling to Cell Nucleus MUDr. Jan Pláteník, PhD. Somatic

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Ligand-activated TFs:Superfamily of nuclear receptors

Lodish et al.: Molecular Cell Biology, W.H.Freeman & Co, 5th ed., 2004

Signaling to the cell nucleus

what goes into nucleus:

– ligand

– receptor

– transcription factor

– another signalling molecule (kinase, second messenger)

Page 24: General Mechanisms of Cell Signaling Signaling to Cell Nucleus · 2020-06-04 · 1 General Mechanisms of Cell Signaling Signaling to Cell Nucleus MUDr. Jan Pláteník, PhD. Somatic

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Ex.1: Ligand for nuclear receptor/ligand-activated transcription factor translocates

to the nucleus

Receptors for vitamin D3, thyroxin, retinoic acid

Ex.2: Nuclear receptor/ligand-activated transcription factor translocates to the nucleus

Receptors for glucocorticoids and estrogens

Lodish et al.: Molecular Cell Biology, W.H.Freeman & Co, 5th ed., 2004

Page 25: General Mechanisms of Cell Signaling Signaling to Cell Nucleus · 2020-06-04 · 1 General Mechanisms of Cell Signaling Signaling to Cell Nucleus MUDr. Jan Pláteník, PhD. Somatic

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Ex.3: Cytoplasmatic receptor activates transcription factor, which translocates to

nucleus

Signaling from cytokine receptors: JAK-STAT system

STAT:

Signal Transducer &

Activator of Transcription

JAK:

Just Another Kinase

Lodish et al.: Molecular Cell Biology, W.H.Freeman & Co, 5th ed., 2004

Ex.4: Constitutive TF CREB in the nucleus activated by various kinases, nuclear translocation of:

-complex MAPK/RSK

-Ca/calmodulin

-PKA

Page 26: General Mechanisms of Cell Signaling Signaling to Cell Nucleus · 2020-06-04 · 1 General Mechanisms of Cell Signaling Signaling to Cell Nucleus MUDr. Jan Pláteník, PhD. Somatic

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Ex.5: Hypoxia-inducible factor:

Joseph C. LaManna et al. J Exp Biol 2004;207:3163-3169

Cascade of transcription response

Second

messengers

Kinase

activation &

Nuclear

translocation

Constitutive

transcription

factors

Inducible

transcription

factors

(immediate-

early genes)

Target genes

(delayed-response

genes)

Fig.: Pláteník et al., Life Sci. 2000: 67, 335-364.

Page 27: General Mechanisms of Cell Signaling Signaling to Cell Nucleus · 2020-06-04 · 1 General Mechanisms of Cell Signaling Signaling to Cell Nucleus MUDr. Jan Pláteník, PhD. Somatic

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Signal amplification

© 1998 GARLAND PUBLISHING

Signal integration

Page 28: General Mechanisms of Cell Signaling Signaling to Cell Nucleus · 2020-06-04 · 1 General Mechanisms of Cell Signaling Signaling to Cell Nucleus MUDr. Jan Pláteník, PhD. Somatic

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Cell allways integrates and responds to many signals:

Page 29: General Mechanisms of Cell Signaling Signaling to Cell Nucleus · 2020-06-04 · 1 General Mechanisms of Cell Signaling Signaling to Cell Nucleus MUDr. Jan Pláteník, PhD. Somatic

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Cross-talk of signaling pathways

Cross-talk of signaling pathways

Page 30: General Mechanisms of Cell Signaling Signaling to Cell Nucleus · 2020-06-04 · 1 General Mechanisms of Cell Signaling Signaling to Cell Nucleus MUDr. Jan Pláteník, PhD. Somatic

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Human genome analyses (Science 291, 2001):

The human

kinome:

518 kinases

(Science 298, 2002)

Source of figure:

Cell Signaling Technology Inc.

Page 31: General Mechanisms of Cell Signaling Signaling to Cell Nucleus · 2020-06-04 · 1 General Mechanisms of Cell Signaling Signaling to Cell Nucleus MUDr. Jan Pláteník, PhD. Somatic

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“MASTER SWITCH”:One gene/protein controls all ...

Protein A Protein B Protein C Protein D

Horb, M.E., et al.: Experimental conversion of liver to pancreas.

Current Biology, 13, 105 - 115, (2003).

Transient expression of single gene construct, coding modified key TF Pdx1, causes permanent change of liver cell to pancreatic cell, producing insulin, glucagon and amylase...

Page 32: General Mechanisms of Cell Signaling Signaling to Cell Nucleus · 2020-06-04 · 1 General Mechanisms of Cell Signaling Signaling to Cell Nucleus MUDr. Jan Pláteník, PhD. Somatic

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Somatic cells can be reprogrammed to pluripotent stem cells !

fibroblastiPS

cardiomyocytes hepatocytes

Oct3/4, Sox2, Klf4, c-Myc

Takahashi K & Yamanaka S. Cell 126, 2006, 663-676

neurones

Fig from: Yamanaka S. Cell 137, 2009, 13-17.