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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
See next slide