W10L3 Flashcards
Tyrosine phosphorylation
Protein phosphorylation discovered in 1906
99.9% of all phosphorylation in the cell was serine and threonine
First evidence for tyrosine kinase in 1979
Human genome was fully sequenced in 2001, and revealed that there are ~ 90 tyrosine kinases
* Growth factor signaling (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.
Receptor Tyrosine Kinase (RTK) Families
Usually monomers, single TM domain proteins, has extracellular and intracellular domain
Transmembrane proteins with extracellular ligand binding domains
Their carboxyl-terminal domains either have intrinsic enzymatic activity or are directly associated with cytosolic enzymes
- either have tyrosine kinase activity or are associated with a molecule that can do tyrosine kinase activity
Each subunit of an RTK has one transmembrane spanning domain
- exception is insulin receptor. Insulin receptor is pre-dimerized
Most inactive RTKs are monomers
- Ligand binding causes a conformational change, which brings two internal kinase domains together. Causes the receptors to dimerize or oligomerize (come together in bunches so they can transphosphorylate each other and activate signalling cascade)
- Exception: Insulin receptor family where receptors are dimers (have an extra level of control to maintain them in the inactive state in the absence of ligand). They are pre-dimerized
Insulin Receptor activation
Purified insulin receptors embedded into nanodiscs (nanoscale disc-shaped membrane patches)
Visualized by single-particle electron microscopy
In inactive state, where there is no insulin, the extracellular domains are close to each other. The intracellular kinase loops are far away.
In active state with insulin, there is conformation change. The kinase domains come close to each other and interact
There is also extra polypeptide loop over ATP binding pocket. All kinases need ATP to grab a phosphate from the ATP and put it onto the substrate. ATP needs to bind to every kinase in our body. If there is no ATP in the pocket, then the kinase is silent and it cannot do its job
- insulin receptor has an extra polypeptide loop to block the ATP from coming in
What is the point of cell surface receptors?
If ligands cannot cross membrane, then kinases need to be at cell surface, and need to respond to low concentrations of ligand (10^-9 to 10^-12 M)
Because there is very little ligand, there needs to be amplification
Signal –> reception –> Transduction with amplification –> responses
Receptor tyrosine kinases (RTKs) are activated by two types of ligands
Receptor tyrosine kinases (RTKs) are activated by two types of ligands:
i) Cell Surface Bound Ligands
ii) Secreted Growth Factors
RTK - Cell Surface Bound Ligands
Cell Surface Bound Ligands:
Example is ephrins (Ephrins which activate ephrin receptors)
- ephrins (the ligand) on cell 1 (signalling cell), ephrin receptors on cell 2 (target cell)
- ephrin has intracellular portion, TM portion, and extracellular portion
- extracellular domain binds to a receptor on the target cell to activate it
- the ephrin receptors are RTK, they transphosphorylate, dimerize to become activated
‘Bidirectional signaling’: ligand engagement of the receptor can result in signaling from the target cell to the signaling cell
- the signal is not only sent to the target cell but is also sent back to the signaling cell (ephrins bind to other kinases within the signalling cell and stimulate signaling in the signalling cell)
- so both cells get signalling
Ephrins regulate:
* Angiogenesis
* Axon guidance
RTK - Secreted Growth Factors examples
- epidermal growth factor (EGF)
- platelet derived growth factor (PDGF)
- insulin, insulin-like growth factor (IGF-1)
Secreted Growth Factors
- Dimeric Ligand
e.g., PDGF is a covalently linked dimer with two distinct receptor binding domains (or sites)
- PDGF can dimerize two adjacent PDGF receptors to initiate intracellular signaling
- PDGF is the ligand, it binds to 2 different receptors and brings them together - Monomeric Ligands
- e.g., EGF is a monomer and can cause things to dimerize
- EGF goes to ligand binding site. Then, it brings 2 receptors together in close proximity.
- Dimerization orients the internal kinase domains: phosphorylation of Tyr (tyrosine) residues of the intracellular C–tail (carboxy tail) of both receptors
Receptor Tyrosine Kinase Activation
Step 1. Dimerization
- Dimer formation brings the kinase domains of each cytosolic receptor tail into contact with the other
Step 2. Transactivation
- this activates the kinases to phosphorylate the adjacent tail on several tyrosines: receptor autophosphorylation
Transactivation = transphosphorylation = autophosphorylation
^^^ these 3 terms are the same thing
The tyrosine on the left kinase domain will phosphorylate the kinase on the right kinase, and vice versa.
The job of the tyrosine kinase is to take phosphate from ATP and transfer it onto tyrosine
Receptor Tyrosine Kinase substrate recruitment
Each phosphorylated tyrosine serves as a specific docking site for several intracellular signaling proteins, via a SH2 interaction domain
Steps:
1. Ligand binds to receptor
2. The receptor transphosphorylates, creating phosphotyrosines
3. Phosphotyrosines are docking sites for other intracellular proteins now, so you can add more complexity to the system. Occurs via SH2 interaction domain
- each tyrosine can recruit a specific protein
Example:
- Grb2 recognizes a specific phosphorylated tyrosine on the activated receptor by means of an SH2 domain and recruits Sos
– Grb-2: growth factor receptor binding protein-2
– Sos: Son of Sevenless (Guanine nucleotide exchange factor that stimulates GDP to GTP exchange on Ras)
- Sos changes the GDP on inactive Ras to GTP, this results in active Ras protein.
- leads to downstream signals
Where does ‘Src Homology Domain’ 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
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
Signal regulators
Ras superfamily of small GTPases
- Ras family
- function: cell proliferation, differentiation, survival, apoptosis, gene expression
- Ras is mutated in tumours. Mutated Ras is always on; always in cell cycle - Rho family
- function: cytoskeletal dynamics, cell shape, polarity, adhesion, and movement; cell-cycle progression; gene expression - Rab family
- function: membrane and protein traffic in the endocytic and secretory pathways - Arf family
- function: vesicular trafficking, endocytosis, and exocytosis - Ran family
- function: nucleocytoplasmic transport; mitotic spindle organization
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)
MAPK phosphorylates cytoplasmic proteins or translocates to nucleus and phosphorylates transcription factors
- changes in protein activity or changes in gene transcription
MAPK: mitogen activated protein kinase ERK: Extracellularly Regulated Kinase
Compartmentalization of MAPK signaling by scaffolds
Mammalian cells utilize strategies to limit cross talk between MAP-kinase signaling pathways and to compartmentalize signaling cascade.
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 the signal.
