signal transduction

1Molecular Biology of Molecular Biology of CancerCancer

Signal Transduction

1Reference:

Molecular Biology of the cell 4th or 5th ed.


Why is signaling important? Allows cells to respond to external

stimuli such as:a) Cytokinesb) Growth Factorsc) Hormonesd) Tissue Repair or Remodeling e) Other cellsf) Stressg) Tissue Specific Regulation h) Regulate Differentiation and Developmenti) Immune Responsej) Pathogens


Signaling is a process of cellular communication

Signals from outside to the inside result in changes to the cell:1. Gene induction/suppression2. Differentiation/Development3. Protein secretion4. Surface marker changes5. Changes in cellular distribution6. Environmental changes7. Apoptosis8. Proliferation9. Motility 10.Destruction of foreign invaders11.Destruction of aberrant cells


External signaling can be:External signaling can be:1. Molecules involved in cell-cell and cell-matrix

interactions.2. Autocrine signaling: signaling molecules released by a

cell and only affect itself (e.g. many growth factors).


External signaling can be:External signaling can be:3. Paracrine signaling: signaling molecules released by

a cell only affect target cells in close proximity to it (e.g. neurotransmitter and neurohormones).

4. Endocrine signaling: signaling molecules (hormones) synthesized by cells of the endocrine organs - act on target cells distant from their site of synthesis.


General model for signalingReceptor – Ligand mediated signaling from outside to inside

Cascade of events (2nd Messengers --involving a number of different enzymes [Ca2+, kinases, phosphatases, adapter proteins, etc.)

Cellular Changes


Transmembrane ReceptorsTransmembrane Receptors Cell-surface receptors can

be categorized into four classes:

1.Receptor with intinsic enzymatic activity Tyrosine kinase receptors

(EGF, insulin, PDGF)2. G-protein-coupled receptor e.g. receptors for epinephrine, serotonin and glucagon


Transmembrane ReceptorsTransmembrane Receptors

3. Receptor with an associated enzyme

e.g. cytokine receptors, receptors for growth hormone and interferons

Receptor guanylyl cyclases (atrial natriuretic peptide)

4. Ion-channel-linked receptor

e.g. neurotransmitter-gated ion channels


TRANSDUCTIONTRANSDUCTION

Intracellular signaling pathways typically involve phosphorylation cascades that are reversibly and tightly controlled by protein kinases and protein phosphatases.

Kinases and phosphatases can be divided into:Kinases and phosphatases can be divided into:1. transmembrane proteins or intracellular proteins.2. serine/threonine-specific or tyrosine-specific (but also a class of

dual-specific) Tyrosine phosphorylation is rare in the cell

only <0.1% of total protein phosphorylation But important in cellular regulation.

Their importance is evident from the fact that many protein tyrosine kinases (PTK) are encoded by proto-oncogenes


Receptor protein tyrosine kinase Receptor protein tyrosine kinase ((RTKs)


Receptor protein tyrosine kinase (Receptor protein tyrosine kinase (RTKs)Extracellular region

Typically several hundred aa)Ligand binds to the extracellular domain

Most known ligands are secreted soluble proteins

Membrane-bound and extracellular matrix-bound ligands can also activate receptor

Transmembrane region All have a single hydrophobic

transmembrane region followed by a few basic amino acids.)


Receptor protein tyrosine kinase (Receptor protein tyrosine kinase (RTKs) Cytoplasmic region1. Contains a protein kinase catalytic domain (PTK),

conserved in sequence of ~250 aa in length (conservation from 32-95%).

Contains a major tyrosine phosphorylation site (its phosphorylation is required for kinase activation in many cases)

2.A C-terminal region.varies from a few up to 200 aa in lengthmost of the tyrosine phosphorylation occurs here.Protein kinase activity is stimulated by binding of

ligands to the extracellular side


P

PP

Receptor protein tyrosine kinase-initiated Receptor protein tyrosine kinase-initiated signal transductionsignal transduction

Ligand bindingReceptor oligomerizationtyrosine autophosphorylation of the

receptor subunits


P

PP

Kinase activation

Binds other proteins Sign

alin

g

P

Receptor protein tyrosine kinase-initiated Receptor protein tyrosine kinase-initiated signal transductionsignal transduction

Autophosphorylation of receptors serves two purposes:1.activates catalytic activity of the PTK.2.changes the conformation of the receptor that allows it to bind

to next cytoplasmic signalling molecules in the cascade.


(I) Ligand binding induces receptor (I) Ligand binding induces receptor oligomerizationoligomerization

1. Ligand is a dimer2. Ligands cluster on scaffolding3. Ligands cluster in signaling cell4. Ligands induce receptor-receptor interaction


Examples: PDGF (platelet-derived growth factor)PDGF are dimeric - homodimers or

heterodimers of A and B chains.PDGF A chain binds only a PDGF receptorB chain binds both a and b receptors

Different composition of the PDGF appears to have different cellular responses.

a ba

AA

a a

A B

a a a ba b b

BBBB BBA B


Apart from the ligands, the extracellular domains of the receptors are also involved in the dimer formationExamples: EGF (epidermal growth

factor)Ligands are monomeric.Ligands induce both homo- and heterodimers of

their receptors.


(II) Tyrosine phosphorylation of receptors(II) Tyrosine phosphorylation of receptors Ligand binding Receptor oligomerization

Juxtapositioning of the cytoplasmic domains of the receptors Conformational changes


PP

The conformational changes allow Mg2+-ATP to bind the major autophosphorylation site in PTK (normally buried in the active site)initial trans-phosphorylation occurs on a tyrosine

residue in the other monomer of the receptor complex (Tyr857 in PDGF receptor)


P

P

P

P

PP PP

Other Tyr residues in the receptors can now be phosphorylated by the activated receptor PTK

These phosphorylations serve as molecular switches to specifically bind cytoplasmic signaling molecules


P

P

P

P SH2 SH3

Grb2

(III) Interaction of receptors with (III) Interaction of receptors with cytoplasmic proteinscytoplasmic proteins

The next step in PTK-mediated signaling involves interaction with cytoplasmic proteins that contain protein-protein interaction modules.

CONCEPTProtein modules direct specific

interactions in signal transduction pathways.

Various modules are frequently found in the same proteins


(III) Interaction of receptors with (III) Interaction of receptors with cytoplasmic proteinscytoplasmic proteins

RTKs signal via their phosphorylated tyrosine residuesThe phosphotyrosines

form binding sites to which subsequent signaling and scaffolding proteins bind to propagate the signal


Signal Transduction by the SRC Signal Transduction by the SRC FamilyFamily

SRC family of Protein tyrosine kinases: 8 known members of the family:

(SRC, LCK, BLK, HCK, FGR, YES, LYN, FYN )

60-75% amino acid identity between them (outside the unique region)

Sequences:myristylation sequenceunique regionSH3 domainSH2 domaincatalytic domainregulatory region

Myristy

lation

SH3 SH2 Protein kinaseUnique

Membrane

Y Y


SH2 (SRC Homology 2) domainSH2 (SRC Homology 2) domain

SH2 binds to phosphotyrosine and the immediate C-terminal residues (3-5) in a sequence-specific fashionthe autophosphorylated tyrosine residue in a

receptor PTK binds specifically to one or more SH2-containing proteins, but may not bind to other SH2-containing proteins


Variations in the nature of the hydrophobic socket in different SH2 domains allow them to bind to phosphotyrosine adjacent to different sequences

The other side of the pocket is more variable and allows specific recognition of the residues at the C-terminal of the phosphotyrosine.

One side of the pocket is lined with conserved basic aa and binds the phosphotyrosine

SH2 (SRC Homology 2) domainSH2 (SRC Homology 2) domain


Functions of SRC familyFunctions of SRC familyPotential substrates of SRC1.Signal transducing proteins

many potential substrates are identified2.Cytoskeletal proteins

on activation, a portion of SRC become associated with cytoskeleton.

nonactivated SRC and nontransforming mutants of v-SRC are not associated with cytoskeleton.

transformation is associated with large changes in cytoskeleton organization


Turning off or quenching of receptor PTK Turning off or quenching of receptor PTK signalingsignaling

1. Dephosphorylation Tyrosine phosphatases

reverse the effects of Tyr phosphorylation

They are both soluble and receptor-like

Some are constitutive, most are regulated


Turning off or quenching of receptor PTK Turning off or quenching of receptor PTK signalingsignaling

2. Receptor internalization

endocytosis, may be autophosphorylation-mediated

3. Negative feedback loop by phosphorylation

PP

PP

PP

PPP

signal transduction

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