Tyrosine Kinase Receptors Flashcards
What are the types of signal transduction mechanisms?
- Conformational-coupling - preformed complex
- Conformational-coupling - diffusion dependent complex formation
- Posttranslational modification (glycosylation/phosphorylation etc…)
- Protein degradation
Mechanism: soluble ligand -> receptor-based recognition/interaction -> cell-based information processing -> cellular responses
What are the roles of receptors in intracellular signalling?
A receptor can convert physical signals into chemical signals
Receptors can act as catalysts and molecular amplifiers
Receptors sense diverse stimuli but initiate a limited repertoire of cellular signals
Receptors act by increasing the rates of key regulatory reactions (catalytic activity)
Most signalling events involves changes in reaction rates (kinetic) rather than their equilibria (thermodynamic)
What are some types of membrane receptors?
GPCR, receptor protein kinase, ion channel, transmembrane scaffold, guanylyl cyclase
What are the types of signalling pathways?
Ligand binding changes receptor conformation (conversion of inactive to activated state) leading to initiation of signalling
Types:
Linear/parallel
Divergence (multiple pathway activation) allows multiple responses to a single signal
Convergence allows signal integration and coordination
Multiply branched
What is involved in regulation of signalling?
Activating and deactivating reactions are usually executed by different regulatory proteins
Separating activation and inactivation enables fine-tuning regulation of signal amplitude (size) and timing
Allostery is the ability of a molecule to alter the conformation of a target protein when it binds non-covalently to that protein
Covalent modification of a protein’s chemical structure is also frequently used to regulate its activity e.g. phosphorylation or dephosphorylation
Describe flexible signalling control?
This is mediated by protein-protein interactions
They can be mediated by small, conserved domains
Modular interaction domains are essential for signal transmission
Adaptors consist exclusively of binding domains or motifs
Distinct species (isoforms) of similar signalling proteins expand the regulatory mechanisms possible in signalling pathways Cells may express one or several protein isoforms to fulfil signalling needs
Describe the tyrosine kinase family in humans?
Human genome encodes ~518 protein kinases
90 human tyrosine kinases (<20% of kinases)
58 human receptor tyrosine kinases (integral membrane proteins) divided into 20 subfamilies
32 cytoplasmic (non-receptor) human tyrosine kinases
Describe the soluble tyrosine kinase family types?
Src family, Abl family and Tec family
They all have a kinase domain
Some have lipid tails
Give an example from the Src family?
C-Src is a cellular proto-oncogene that is highly similar to the chicken v-Src oncogene
C-Src is activated by release of intrasteric inhibition
Activation of c-Src involves liberation of modular binding domains for activation-dependent interactions and phosphorylation of target substrates
C-Src often associates with receptors, including receptor tyrosine kinases
Give an example from the AbI family?
Bcr gene and Abl tyrosine kinase fuse in Philadelphia translocation that causes chronic myelogenous leukaemia (CML)
The TK is constitutively active in leukocytes causing them to proliferated = leukaemia
New drug - Imatinib (Gleevec) targets the active site of the tyrosine kinase and blocks its activity – causing a block in cancer
Give an overview of tyrosine kinases?
Many receptor protein tyrosine kinases (RTKs) are activated by growth factors
They are phosphorylated to initiate either dimerisation or activation
They initiate downstream pathways
Mutations in RTKs can be oncogenic
What are some subfamilies of receptor tyrosine kinases?
Human genome encodes 58 different receptor tyrosine kinase genes
Divided into 20 different sub-families
Highly studied ones include:
Platelet-derived growth factor receptor (PDGFR)
Epidermal growth factor receptor (EGFR or ErbB1)
Insulin receptor (IR)
Vascular endothelial growth factor receptor 2 (VEGFR2)
They are important targets in disease therapy
What is the structure of receptor tyrosine kinases?
Domains and function: Extracellular - ligand binding Transmembrane - Membrane anchor Juxtamembrane - negative regulation Tyrosine kinase - catalytic C-terminal tail - signal regulation
The cytoplasmic domain can be phosphorylated on different tyrosine residues
Two RTK form a homo/heterodimer and cross phosphorylate each = phosphotyrosine
Give an overview of receptor tyrosine kinase signalling?
Their complexes increase signal efficiency
RTKs lay ‘flat’ freely within the membrane bilayer
Targeted recruitment of RTKs into signalling complexes
Increase in efficiency of phosphorylation and switching ‘on’
What are some types of RTK signalling pathways?
MAPK pathway - stimulus activates transducers = cellular responses
ERK pathway - uses growth factors = proliferation
JNK pathway - stimulated by osmotic/redox stress or radiation = proliferation, apoptosis and embryonic development
P38 pathway - Osmotic stress and radiation = apoptosis, cytokine release and cell cycle arrest
Describe the MAPK (ERK) pathway?
Growth factors stimulate receptor tyrosine kinase activity
This transmits signals to the mitogen-activated protein kinase pathway
This signals to other parts of the cell - including the nucleus
One of the main targets is Ras (a GTP-associated protein) - this signals to other protein kinases within the pathway
What can the ligands be in initiating the MAPK pathway?
Growth and survival factors - they have a tyrosine kinase domain
Transforming growth factor B superfamily - they have a serine/threonine kinase domain
Both work via ligands and conformational change activating the kinase domains
What can occur from tyrosine phosphorylation?
Growth factor signalling and oncogenesis
Cell adhesion, spreading, migration and shape
Cell differentiation in development
Cell cycle control
Gene regulation and transcription
Endocytosis and exocytosis
Insulin stimulation of glucose uptake
Angiogenesis (formation of new blood vessels)
Regulation of ion channels in nerve transmission
Give an overview of tyrosine kinase activation?
This leads to protein recruitment and signalling
RTK ligand binding promotes - receptor oligomerisation and autophosphorylation
Signalling proteins and adaptors bind to phosphotyrosine residues in the cytoplasmic domain of the activated receptor
e.g. ERK activation can phosphorylate transcription factors to activate gene expression
Activation causes signalling platform assembly
After dimerisation and tyrosine autophosphorylation:
Signalling factors and enzymes are recruited to the RTK-ligand complex
This is now viewed as a signalling platform
What are some RTK interacting factors?
There are tonnes - some categories are:
Enzymes/transcription factors
Adaptors/regulators
Adaptors/docking proteins
How do interacting factors bind to RTKs?
This involves the recognition of pY motifs
The OH group on tyrosine kinase is phosphorylated
This is now a negative pocket which can bind in the conserved pocket
What are the different mechanisms by which RTKs activate?
- A monomer recruited into a dimer that then becomes phosphorylated
- Assembled into a pre-formed complex and the binding of the ligand makes them switch between an active/inactive state
They can use PDGF, EGF, FGF+HSPG, Insulin, membrane ligand and collagen/ECM
Example of activation: Describe EGFR (ErB1) activation and turnover?
Steps in EGFR signalling and trafficking:
- EGF ligand binding
- Tyrosine phosphorylation (trans-autophosphorylation)
- Phospho-EGFR binding to cytosolic proteins
- Endocytosis
- Recycling or degradation
The rate/ratio between degradation or recycling regulates function
What can RTK activation lead to?
Complex protein-protein interactions
Activation of PI3K and Akt
Activation of Ras and MAPK
Insulin receptor (InsR) is also an RTK
Describe insulin receptor activation?
This leads to IRS recruitment
Pre-existing disulphide-linked heterotetramers of 2a2b subunits
The a-subunit inhibits b-subunit kinase activity in the absence of insulin
This allows phosphorylation to occur and switch between active/inactive
Describe small GTP-binding proteins?
They are multiuse switches that are:
Active when bound to GTP
Inactive when bound to GDP
GDP/GTP exchange catalysts such as Sos are known as GEFs (guanine nucleotide exchange factors) & promote activation
GAPs accelerate hydrolysis and deactivation
GDP dissociation inhibitors (GDIs) slow spontaneous nucleotide exchange
What are the growth factors that small GTP-binding proteins activate?
The growth factors they activate are - Ras, Rac, Rho, Arf and Rab proteins
They lead to proliferation, differentiation, membrane ruffling, actin stress fibres and membrane trafficking
Give some examples of interaction modules/domains?
SH2 – binds pY
PTB – binds pY
FHA – binds pT (phospho-threonine)
SH3 – binds proline-rich sequences
WW – binds proline-rich motifs
PH – binds phosphoinositide (and protein?)
FYVE – binds PI(3)P
EVH1 – binds proline-based motifs
PDZ – binds carboxy-terminal valine-based motifs e.g. F/W-F/W-D-T/S-W-V
Death Domain (DD) – interacts with other DD modules
Describe the SH2 domain?
This mediates binding to phosphotyrosine (pY)
SH2 domain = modular unit comprising ~100 residues that recognises a phosphotyrosine motif within signalling proteins
Binds pYXXXX type of sequences
Viral v-Src tyrosine kinase is an oncogene
Describe the PTB domain?
This mediates binding to phosphotyrosine
PTB domain = modular unit comprising ~100-150 residues that recognises a phosphotyrosine motif
Binds NPXpY type of sequences
Found in many adaptor proteins
Describe the SH3 domains?
They recognise polyproline motifs
The SH3 domain is a modular unit comprising ~50 residues that recognises a proline-rich helical structure in signalling factors
Describe protein kinase b (PKB or c-AKT) signalling?
Generation of PIP3 leads to the recruitment of c-AKT to the membrane and bind in its PH domain
This causes a conformational change in c-AKT to an active state
This takes place as PIP3 allows the binding of PDK1, which phosphorylates the c-AKT
Active c-AKT will then go on to downstream signals and phosphorylate multiple targets: Cell survival (suppression of apoptosis), cell differentiation, glucose uptake, protein synthesis and glycolysis
How is vascular physiology regulated?
It is regulated by VEGF-VEGFR
This is the building of blood vessels - angiogenesis
It enables the endothelial cells to engage in repair and maintenance of blood pressure
What are some other types of RTKs?
Unimolecular receptor tyrosine kinase - EGFR, PDGFR, IR and VEGFR
Bimolecular receptor tyrosine kinase - IFN-R, TCR, cytokine receptors, Src, FAK and JAK
Describe the kinome and disease?
> 30% of protein kinases implicated in various diseases
More kinases likely to be implicated in other disease states
Kinases are tractable drug targets; several commercially approved drugs that target kinases used for therapy
Proteins (non-kinase) that regulate kinases or functionally linked in a pathway are also increasingly viewed as drug targets that can subtly modify kinase activity but lessen toxic side effects
Describe the kinome and cancer?
The protein kinase catalytic domain is one of the most common modules found in cancer genes; DNA-binding and transcriptional regulation domains are also common
Describe inhibition of human protein kinases in therapy?
This can produce therapeutic benefits
A small-molecule inhibitor causes inhibition of tyrosine phosphorylation of the receptor = inhibition of downstream signalling
Or therapeutic antibodies cause activation of immune responses - also inhibiting tyrosine phosphorylation of the receptor and downstream signalling
What is the phosphatome?
Protein phosphatase family
~140 protein phosphatases
38 protein tyrosine phosphatases
38 serine/threonine phosphatases – 18 are PP1/PP2A-like, 20 are PP2C-like
62 dual specificity phosphatases (DSPs) (e.g. MKPs, PTEN)
8 haloacid dehalogenase (HAD) dual-specificity phosphatases
~2% of human genome encodes kinase/phosphatase proteins
~5% of human genome encodes kinase, phosphatase, regulatory, scaffolding and anchoring proteins all concerned with intracellular signalling
