Tyrosine Kinases Flashcards
Signal Transduction
Conveying messages into the cell interior (based on external signals)
Various mechanisms:
1) Conformational coupling
2) Diffusion dependent conformational coupling
3) Post-translational modification
4) Protein degradation
Receptors as catalysts and amplifyers
Converting signals into chemical signals, sense diverse range of stimuli but activate a limited repertoire of signals:
- act to increase rate of regulatory proteins
- amplify a single signal into multiple responses
Types of receptors
1) GPCR
2) Receptor protein kinase
3) Ion channels
4) Transmembrane scaffold (adhesion receptor)
5) Enzymatic receptors
Signalling pathway divergence/convergence
Divergence
- provides multiple responses to a single stimulus
Convergence
- Allows signal integration and coordination
Regulating activation levels of signalling molecules
Allostery and modification can occur independently and separately
Allostery:
- a molecule binds non-covalently to target protein to alter its conformation (activation or deactivation)
Modification:
- phosphorylation or dephosphorylation of protein’s structure can regulate activity my altering its binding affinity
Signalling pathways as Biochemical Logic Circuits
Various signalling pathways integrate to process more complex information
Positive feedback loop: Irreversible ON switch
Positive feed-forward loop: responds to prolonged input
Conformational lock: dual control switch
Provides a flexible/adaptive system
Flexible signalling
Protein-protein interactions mediated by small conserved domains:
- being conserved, provides a platform for modular control systems that is more dynamic
KINOME protein kinase superfamily
Different protein kinases found across different species/organisms
- varying levels of tyrosine and protein kinases with varying gene levels too
Human KINOME
518 protein kinases, 90TKs, +/- 23,000 genes
- 40 atypical protein kinases
Key signalling pathways linked to TK activity
1) MAPK pathway: conserved between yeast and man
- JNK and p38 MAPK pathways enable adaptive & stress-sensitive responses (impact gene expression)
Bcr-Abl oncogene causing leukaemia
Fusion of Bcr & Abl TKs in Philadelphia translocation causes chronic myelogenous leukaemia:
- Causes constitutively activation of TK in leukocytes promoting constant proliferation
- treated with Imatinib (Gleevec) that targets the TK active site & blocks its activity
Src proto-oncogene
Cellular proto-oncogene very similar to chicken v-Src oncogene
- c-Src activated by release of intrasteric inhibition freeing up modular binding domains for activation and thus activating target substrates
The MAPK (ERK) pathway
ERK signalling molecule can travel into cell’s nucleus and activate various transcription factors
- the MKP-1 molecule mediates a strict regulatory feedback loop to limit levels of ERK import into nucleus
Role of TKs
various cellular functions determined by their pathways…
- growth factor signalling
- cell adhesion, spreading, migration and shape
- cell differentiation during development
- cell cycle control
- gene regulation/transcription
- endo/exocytosis
- insulin stimulation
- angiogenesis
- regulating nerve ion channels
Receptor TK subfamilies and classification
20 different subfamilies including Ephrins, FGFRs, ErbBs, PDGFRs (platelet-derived), VEGFRs
- important targets in disease therapy
Similar architecture:
- N terminus facing out towards extracellular media
- C terminus inward
Classified based on ability to bind different ligands:
- different structures on extracellular domain
- embedded TK domain of ~300 residues
TK extracellular/intracellular domain
Extracellular: Responsible for ligand binding
Intracellular (kinase) domain: enables signalling pathways
Ligand binding –> signal activation mechanism unknown
Receptor TK (RTK) Signalling Complexes
Free floating RTKs in membrane are recruited to cluster into signalling complexes - increasing efficiency of phosphorylation and switching ON
- RTKs cause different responses depending on tissue/cell type (different signalling molecules present)
RTK structure (domains)
N-terminal, extracellular domain
- Ligand binding function
TM domain
- membrane anchor
Juxtamembrane domain
- negative regulation function
Tyrosine Kinase
- catalytic, activation of signal pathways
C-terminal tain
- signal regulation
Epidermal Growth Factor Receptor (EGFR) model system
1) Extracellular domain binds EGF causing signal transduction
2) TK domain activated (phosphotyrosine)
3) Signalling proteins like Grb2 bind to phosphotyrosine residues in the cytoplasm to become activated
4) Active Grb2 activates Ras pathway, recruiting Raf and eventually activating the ERK pathway
RTK inhibition - therapeutic use
Cancer therapy: Blocking EGFR useful in treating epithelial cancer
- block with specific antibodies
- block with TK inhibitors
EGFR (ErbB1) activation and turnover
Upon activation (ligand binding), receptor is trafficked to cell interior by endocytosis
The EGFR can be recycled or degraded to help regulate activation levels:
- Recycling = more signalling & proliferation
- Degradation = shorter lifespan & less signalling
Insulin receptor activation pathway
Receptor activation (insulin binding) causes recruitment of Insulin Receptor Substrate 1 (IRS1) which promotes PI3K activity
Insulin Receptor (INSR) made of 2alpha-2beta subunits - alpha subunit inhibits beta subunit's kinase activity in absence of insulin
The SH2 domain and RTK binding
Present in various different signalling proteins and other kinases (modular unit comprising ~100 residues)
This domain binds specifically with the phosphotyrosine found on RTKs
- specificity comes as a result of surrounding residues limiting engangement
- Recognises specific phosphotyrosine motif ‘pYXXXX’
The Protein Phosphotyrosine Binding (PTB) domain
Mediates protein binding to phosphotyrosines
- modular unit comprising 100-150 residues
- recognises phosphotyrosine motif ‘NPXpY’
SH3 domain
Recognise polyproline motifs (RXXPXXP)
- proline-rich helical polyprolines help increase binding to target substrates
SOS factor activating Ras proto-oncogene
Activates Ras through GTP-binding proteins
- alters cell’s behaviour and physiological response
Protein Kinase B (c-Akt) key target of RTK pathway
Activated by PIP3, phosphorylates target substrates:
- promotes protein synthesis, prevents cell death, promotes differentiation, stimulates GLUT4 translocation to muscle cell surface
important in VEGF system
PK and disease
> 30% of PKs implicated in disease hence is a key drug target (as are non-kinase proteins)
- developing drugs to target mutated forms of TKs (somatic/germline mutation)
