Tyrosine Kinases

Question 1

Signal Transduction

Answer 1

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

Question 2

Receptors as catalysts and amplifyers

Answer 2

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

Question 3

Types of receptors

Answer 3

1) GPCR
2) Receptor protein kinase
3) Ion channels
4) Transmembrane scaffold (adhesion receptor)
5) Enzymatic receptors

Question 4

Signalling pathway divergence/convergence

Answer 4

Divergence
- provides multiple responses to a single stimulus

Convergence
- Allows signal integration and coordination

Question 5

Regulating activation levels of signalling molecules

Answer 5

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

Question 6

Signalling pathways as Biochemical Logic Circuits

Answer 6

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

Question 7

Flexible signalling

Answer 7

Protein-protein interactions mediated by small conserved domains:
- being conserved, provides a platform for modular control systems that is more dynamic

Question 8

KINOME protein kinase superfamily

Answer 8

Different protein kinases found across different species/organisms
- varying levels of tyrosine and protein kinases with varying gene levels too

Question 9

Human KINOME

Answer 9

518 protein kinases, 90TKs, +/- 23,000 genes

• 40 atypical protein kinases

Question 10

Key signalling pathways linked to TK activity

Answer 10

1) MAPK pathway: conserved between yeast and man

- JNK and p38 MAPK pathways enable adaptive & stress-sensitive responses (impact gene expression)

Question 11

Bcr-Abl oncogene causing leukaemia

Answer 11

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

Question 12

Src proto-oncogene

Answer 12

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

Question 13

The MAPK (ERK) pathway

Answer 13

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

Question 14

Role of TKs

various cellular functions determined by their pathways…

Answer 14

• 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

Question 15

Receptor TK subfamilies and classification

Answer 15

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

Question 16

TK extracellular/intracellular domain

Answer 16

Extracellular: Responsible for ligand binding

Intracellular (kinase) domain: enables signalling pathways

Ligand binding –> signal activation mechanism unknown

Question 17

Receptor TK (RTK) Signalling Complexes

Answer 17

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)

Question 18

RTK structure (domains)

Answer 18

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

Question 19

Epidermal Growth Factor Receptor (EGFR) model system

Answer 19

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

Question 20

RTK inhibition - therapeutic use

Answer 20

Cancer therapy: Blocking EGFR useful in treating epithelial cancer

• block with specific antibodies
• block with TK inhibitors

Question 21

EGFR (ErbB1) activation and turnover

Answer 21

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

Question 22

Insulin receptor activation pathway

Answer 22

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

Question 23

The SH2 domain and RTK binding

Answer 23

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’

Question 24

The Protein Phosphotyrosine Binding (PTB) domain

Answer 24

Mediates protein binding to phosphotyrosines

• modular unit comprising 100-150 residues
• recognises phosphotyrosine motif ‘NPXpY’

Question 25

SH3 domain

Answer 25

Recognise polyproline motifs (RXXPXXP)

- proline-rich helical polyprolines help increase binding to target substrates

Question 26

SOS factor activating Ras proto-oncogene

Answer 26

Activates Ras through GTP-binding proteins

- alters cell’s behaviour and physiological response

Question 27

Protein Kinase B (c-Akt) key target of RTK pathway

Answer 27

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

Question 28

PK and disease

Answer 28

> 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)