Enzyme coupled receptors, receptor tyrosine kinases - Lecture 24 Flashcards
What is known about Tyrosine phosphorylation?
- protein phosphorylation discovered in 1906
- first evidence for tyrosine kinase in 1979
- human genome was fully sequenced in 2001, and revealed that there are ~90 tyrosine kinases
Controls the following:
-growth factor signalling (and oncogenesis) - cell adhesion, spreading, migration and shape
- cell cycle control
- gene regulation and transcription
- stimulation of glucose uptake (insulin)
- angiogenesis (sprouting of new blood vessels)
- etc.
Describe the receptor tyrosine kinase (RTK) families
- transmembrane proteins with extracellular ligand binding domains
- their carboxyl-terminal domains either have intrinsic enzymatic activity or are directly associated with cytosolic enzymes
- each subunit of an RTK has one transmembrane spanning domain
- most inactive RTKs are monomers
- ligand binding causes a conformational changes, which brings two internal kinase domains together
- exception: insulin receptor family where receptors are dimers (pre-dimerized) - have an extra level of control to maintain them in inactive state in absence of ligand
Describe the insulin receptor activation
- purified insulin receptors embedded into nanodiscs (nanoscale disc-shaped membrane patches)
- visualized by single-particle electron microscopy
- extra polypeptide loop over ATP binding pocket (block - without insulin, does not signal)
- no insulin = intracellular domains are very close together, kinase domains are very far apart
- in presence of insulin = extracellular domain moves, causes the kinase domains to come together to interact and activate
What is the point of cell surface receptors?
respond to low concentrations of ligand
signal - reception - transduction - response (need amplification)
What are RTK ligands?
receptor tyrosine kinases (RTKs) are activated by two types of ligands:
1. cell surface bounds ligands
2. secreted growth factors
What are cell surface bound ligands?
- RTK ligands
- ephrins which activate ephrin receptors
- ‘bidirectional signalling’: ligand engagement of the receptor can result in signalling from the target cell to the signalling cell
- the signal is not only sent to the target cell but is also sent back to the signalling cell (ephrins bind to other kinases and stimulate signalling)
- regulate: angiogenesis, and axon guidance
What are examples of secreted growth factors?
- epidermal growth factor (EGF)
- platelet derived growth factor (PDGF)
- insulin, insulin-like growth factor (IGF-1)
What are secreted growth factors?
- RTK ligands
- dimeric ligand: e.g. PDGF is a covalently linked dimer with two distinct receptor binding domains (sties)
- PDGF can dimerize two adjacent PDGF receptors to initiate intracellular signalling
- signal molecule binds two separate receptors to bring them together to dimerize
- monomeric ligands: e.g. EGF; dimerization orients the internal kinase domains: phosphorylation of Tyr residues in C - tail of both receptors
Describe the receptor kinase activation
- dimer formation brings the kinase domains of each cytosolic receptor tail into contact with the other (dimerization)
- transactivation: this activates the kinases to phosphorylate the adjacent tail on several tyrosines: receptor autophosphorylation (one receptor phosphorylates the tyrosines of the other and vice-versa = trans-activation/ trans-phosphorylation/ auto-phosphorylation
Describe the receptor tyrosine kinase substrate recruitment
- each phosphorylated tyrosine serves as a specific docking site for several intracellular signalling proteins, via a SH2 interaction domain - bring proteins together in a complex
What are docking sites?
Grb2 recognizes a specific phosphorylated tyrosine on the activated receptor by means of an SH2 domain and recruits Sos
(this is an example - other proteins with SH2 domains can also bind)
How do RTKs activate Ras?
- Grb-2: growth factor receptor binding protein-2 (has SH2 domain which binds the activated RTK, and also has SH3 domain - this binds Ras-GEF also called Sos)
- Sos: Son of Sevenless (Guanine nucleotide exchange factor that stimulates GDP to GTP exchange on Ras)
Where does ‘Src Homology Domain’ (SH) nomenclature come from?
Modular protein interaction domains were first identified in the tyrosine kinase Src. These domains are referred to as Src homology domains (SH)
- SH1 domain: tyrosine kinase domain (not called SH1 anymore)
- SH2 domain: recognizes specific phosphotyrosine motifs
- SH3 domain: binds to proline rich domains in intracellular proteins
- Some SH2 (and/or SH3) containing proteins: enzymes, adaptors, scaffold proteins, and signal regulators
What is the Ras superfamily of small GTPases
Ras, Rho, Rab, Arf, Ran
Describe the activation of the MAP kinase cascade (downstream of Ras)
- Ras activates MAP-kinase-kinase-kinase (e.g. Raf)
- Raf phosphorylates MAP-kinase-kinase (e.g MEK)
- MEK phosphorylates MAP-kinase (e.g. MAPK:ERK1/2 (MAP ERK kinase)
- MAPK phosphorylates cytoplasmic proteins or translocates to nucleus and phosphorylates transcription factors
MAPK: mitogen activated protein kinase
ERK: extracellularly regulated kinase
- cascade leads to genetic changes
Describe the compartmentalization of MAPK signalling by scaffolds
Mammalian cells utilize strategies to limit cross talk between MAP-kinase signalling pathways and to compartmentalize signalling cascade
What are scaffold proteins?
One strategy is to utilize scaffold proteins to hold the three kinases that comprise the MAP-kinase cascade in a molecular complex
- the scaffold strategy allows for the precise recruitment and regulation of MAP kinases in cellular compartments pre-determined by the both the nature of the scaffold and the receptor activating signal
Describe the EGFR signalling in carcinogenesis
Proto-oncogene:
- a normal gene which, when altered by mutation, becomes an oncogene that can contribute to cancer
- may have many different functions in the cell - can provide signals that lead to cell division - regulate programmed cell death (apoptosis)
Oncogene:
- a gene having the potential to cause a normal cell to become cancerous (EGF receptor)
- 85-90% of lung cancers are non-small cell lung cancer (NSCLC)
- EGFR over-expressed
- EGFR mutations (exons 18-21): encodes EGFR kinase domain: increase in kinase activity, hyper-activation of downstream pro-survival signalling pathways
- over expression cells express so many receptors - don’t need ligands, they bump into each other spontaneously and signal the cascade activation
MAPK
- phosphorylates cytoplasmic proteins or translocates to the nucleus and phosphorylates (regulates) transcription factors
- e.g. Ets (E-26) and Jun (from Japanese ju-nana, meaning 17, short for avian sarcoma virus 17)
STAT
- signal transducer and activator of transcription (transcription factor)
- physically associates with receptor phosphorylated, dimerized, dissociates and goes straight to the nucleus
PI3K
- phosphoinositide 3-kinase
AKT
- promotes cell survival
mTOR
- mammalian target of rapamycin (serine-threonine kinase): central regulator of cell metabolism, growth, proliferation and survival
PTEN
- phosphatase: antagonizes the PI3’ kinase/AKT pathway
Describe EGFR signalling
PIP2: phosphatidylinositol 4, 5 bisphosphate
PIP3: phosphatidylinositol 3, 4 triphosphate
PTEN: phosphatase and tensin homolog
PDK-1: Phosphoinositide-dependent kinase-1
AKT/PKB: protein kinase B
PH: pleckstrin homology domain (binds to PIP3)
Describe EGFR targeted therapy: monoclonal antibodies
FOLFOX-4:
- standard chemotherapy
- folinic acid, fluorouracil, and oxaliplatin
Cenuximab:
- monoclonal antibody
- binds to the extracellular region of the EGFR
- antagonizes ligand binding
- stimulates EGFR internalization and degradation
*antibodies to EGF receptor - not just control vs treatment - one gets gold standard treatment, other gets this + something
Describe insulin receptor signalling
Insulin receptor was activated, does not become docking site, phosphorylates IRS
IRS: insulin receptor substrate (becomes docking site, has lots of tyrosine)
aPKC: atypical protein kinase C
GSK3: glycogen synthase kinase 3
GS: glycogen synthase
AS160: Akt substrate of 160 kDa
