Introduction• Recruitment of kinases in signalling

pathways• Consequences of protein phosphorylation

RTK family:• Classification & structure/function• RTK ligands• Receptor dimerization &

autotransphosphorylation

RTK-mediated pathways:• Ras-Raf-MAP kinase pathway, use of

dominant negative mutants to map pathway

Cell surface receptors recruit activity of protein kinases in two general ways:• Non-receptor tyrosine kinasesNon-receptor tyrosine kinases: Receptors

lacking self-contained kinase function recruit activities of intracellular protein kinases to the plasma membrane

• Receptor tyrosine kinasesReceptor tyrosine kinases: Possess an intrinsic tyrosine kinase activity that is part of the receptor protein. Examples include receptors for growth factors (PDGF, EGF, insulin, etc.)

Implicated in diverse cellular responses:• Cell division• Differentiation• Motility

At least 50 RTKs identified: • Subdivided into 10 subclasses based on

differences within extracellular, ligand-binding domain of receptor

“Oncogenic” RTK mutants exist: • erbB gene encodes an N-terminal truncated,

constitutively active form of EGF receptor

Four common structural features shared among RTKs:• Extracellular ligand-binding domain• Single transmembrane domain• Cytoplasmic tyrosine kinase domain(s)• Regulatory domains

Extracellular ligand-binding domain. Cytosolic domain with tyrosine kinase (tk)

enzyme activity.

Regulatory domains

Typically small soluble proteins Work in autocrine and paracrine

manner Dimerize (may aid in receptor

dimerization) Some RTK ligands membrane-bound

Ligand-induced RTK activation induces Receptor dimerization, leading to activation of catalytic domains

Receptor autotransphosphorylation: • Further stimulates kinase activity• Leads to phosphorylation of additional proteins

involved in receptor signalling pathway• Provides “docking sites” for downstream

signalling proteins (Grb2, PI3-kinase, phospholipase C, etc.)

SH2 domains: bind P-Tyr-containing sequences

SH3 domains: bind to pro-rich (PxxP) sequences

Receptor tyrosine kinase mediated signal transduction

• does not involve G-proteins• does involve phosphorylation of proteins• … at tyrosines

ligands

inactive tyr kinase monomer

…cross-phosphorylation of tyrosines

Receptor Tyrosine Kinase Activation

ATPADP

Phosphorylated tyrosines on activated signaling proteins cell response

P-P-P- -P

-P-Pactive

tyr kinase dimer

XXX

ligands

inactive tyr kinase monomer

cross-phosphorylation

of tyrosines

MAP (mitogen-activated protein) kinase activation:

ATPADP

P-P-P- -P

-P-Pactive

tyr kinase dimer

XXX

adaptor protein

Ras-activatingprotein

GDP

GTP

Rasinactiveactive

MAP kinase-kinase-kinaseactive

MA

P k

inase-k

inase

MA

P ki

nase

nucleus

P-

P-

translocation to nucleus…

P|MAP kinase

nucleus

Transcription factors Other

nuclear proteins

P-

P-

DNA binding Changes

in protein activitychanges in

gene activity

Cell proliferation, differentiation

Ras-Raf-MAP kinase pathway R7 photoreceptor development in drosophila (fruitfly)

Tyr-P

Grb2

SH3 domains

Proline-rich regions (-PXXP-)

Sos Ras(inactive)

GDP GTP

PiRas(active)

Raf

MEK

MAP kinase

P

PP

DNA

Nucleus

MAP kinase

PPfos junPP

Increase gene expression

SH2 domain

Regulates cell growth and cell reproduction

Tyrosine kinase catalyzes transfer of phosphate group from ATP to to tyrosines

Can trigger ten or more signal transduction pathways at once

Abnormal tyrosine kinases that work even without a signal molecule may contribute to some cancers

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