Tyrosine Kinases Flashcards Preview

ICB - Cell Signalling > Tyrosine Kinases > Flashcards

Flashcards in Tyrosine Kinases Deck (82)
Loading flashcards...
1
Q

Describe the structure of a receptor tyrosine kinase (RTK).

A

Single span TM domain, extracellular ligand binding domain , intracellular globular tyrosine kinase domain.

2
Q

How many RTKs are encoded by the human genome?

A

~80

3
Q

What is transautophosphorylation?

A

Cross phosphorylation by the tyrosine kinase domains in dimerised RTKs. Results in lots of phosphorylated tyrosines in the cytoplasmic region of the receptor.

4
Q

What varies between different RTKs?

A

Have different extracellular regions - respond to different ligands.

5
Q

What is the main purpose of transautophosphorylation?

A

To generate binding sites for effector proteins, that recognise the pTyr residues via their SH2 domains.

6
Q

What is a common outcome of RTK signalling?

A

Cell growth and proliferation

7
Q

Why have drugs been developed that target RTKs?

A

Due to their roles in cell growth and proliferation, RTK genes are often mutated or overexpressed in cancer.

8
Q

What is herceptin and why is it used?

A

Used as a cancer treatment - herceptin is a monoclonal antibody that inhibits the EGF receptor, Her2.

9
Q

How can RTK activation be studied?

A

Using western blotting of cell lysates or immunostaining with antibodies against pTyr. Techniques should compare cells treated with ligand and cells without ligand.

10
Q

How can the cellular responses to RTK signalling be studied?

A

Cellular responses can be monitored by cell counting, quantification of DNA synthesis (qPCR) or by microscopic observation (e.g. HGFR activation causes cells to migrate from an epithelium)

11
Q

What is the major difference between RTK activation and GPCR activation?

A

RTKs only have one TM helix and therefore information cannot be transmitted as a conformational change (shift in helices in GPCR) - instead most RTKs dimerise upon ligand binding.

12
Q

How can RTKs be dimerised upon ligand binding?

A

Receptors are brought together by a dimeric ligand binding - two binding sites, one for each receptor.

13
Q

If the RTK pre-exists as a dimer, what happens upon ligand binding?

A

A conformational change to activate the kinase domains.

14
Q

How does EGF binding to the EGF receptor result in dimerisation of the receptor?

A

Without ligand, the receptor forms an autoinhibitive structure - where the dimerisation arm is buried. EGF monomer binds and causes a conformational change which releases the dimerisation arm - allows receptor to form a dimer.

15
Q

Describe the structure of the insulin receptor.

A

A disulfide linked dimer

16
Q

Give other examples of how RTKs can be activated if not by RTK dimerisation upon ligand binding.

A

RTKs that exist as covalently bound or non-covalently associated dimers are activated by conformational changes upon ligand binding. Some RTKs require receptor clustering to a particular region of the membrane for there to be an output (e.g. Eph receptors). Some RTKs require coreceptors for activation (e.g. FGFRs).

17
Q

How does heparan sulfate act as a coreceptor of FGFR?

A

HS acts as a glue between two receptor molecules - needed for receptor assembly to be stable enough to transmit a signal.

18
Q

Describe the structure of heparan sulfate.

A

A polysaccharide with many sulfide groups. Length of HS can vary - different lengths bind different receptors.

19
Q

Describe the structure of the tyr kinase domain found in an RTK.

A

2 lobes - N terminal and C terminal. Active site between the two lobes.

20
Q

How is the tyr kinase domain in an RTK autoinhibited?

A

Activation loop folds over the entrance to the catalytic cleft (between the two lobes).

21
Q

Describe the active conformation of the tyr kinase domain in an RTK.

A

The activation loop moves out (receptor is dynamic) to allow access to the activation cleft.

22
Q

How does the auto-inhibition of the tyr kinase domain of an RTK affect transautophosphorylation?

A

For transphosphorylation to occur, two kinase domains must meet in the active conformation, where their activation loops do not block the catalytic cleft. Once the activation loop has been phosphorylated, it becomes locked in the active conformation.

23
Q

How do clustered receptors result in higher levels of phosphorylation?

A

Receptors are more likely to meet in the active conformation and phosphorylation spreads between receptors faster. Phosphorylation is quicker than dephosphorylation by phosphatases.

24
Q

How is the EGF receptor activated?

A

Tyr kinase domain is activated by an allosteric mechanism. EGF binding causes conformational change - one kinase pushes into the receiver kinase, causing it to open up and activate the receptor. This allows access to dimerisation arms and dimerisation of two EGF bound receptors. Allows phosphorylation of the tail regions of each receptor.

25
Q

What controls the outcome of the signalling downstream of an RTK?

A

The combination of adaptor proteins that bind to the pTyr residues in the activated RTK

26
Q

How does PLC-β contribute to downstream signalling of RTKs?

A

Produces DAG which goes on to activate PKC and IP3 which goes on to release calcium from the ER

27
Q

How does PI 3-kinase contribute to downstream signalling of RTKs?

A

Phosphorylates lipids in the plasma membrane which then become docking sites for other effector proteins in the signalling pathway.

28
Q

How do non-enzymatic adaptor proteins contribute to the downstream signalling of RTKs?

A

Act as mediators to result in the activation of Ras GTPase and the MAP kinase pathway.

29
Q

What is the outcome of the MAP kinase pathway?

A

The mitogen activated protein kinase (MAPK) pathway causes cell proliferation.

30
Q

How can Src kinase cause cancer?

A

An avian virus picked up the Src gene from a mammalian host, produces a truncated Src that is constitutively active - this can then cause sarcomas in muscle tissue if humans catch the virus from birds.

31
Q

Describe the structure of Src kinase.

A

Has a kinase domain, an SH2 domain and an SH3 domain. Anchored to the cell membrane by a lipid anchor.

32
Q

Describe the inactive state of Src.

A

SH2 binds pTyr in the tail region of the Src kinase, constraining the polypeptide to its inactive conformation.

33
Q

How is Src activated?

A

By removal of the phosphate group in the tail region - this causes a conformational change (SH2 no longer bound to pTyr) and the SH3 domain becomes accessible to ligand. Src can then auto-phosphorylate to become active.

34
Q

Describe the structure of an SH2 domain.

A

Has two binding sites - one with a positive residue at the bottom to bind the negative phosphate group in pTyr, and a smaller binding pocket to bind a hydrophobic residue found two aas along from pTyr.

35
Q

How many different SH2 domains are encoded by the human genome?

A

More than 100

36
Q

What is recognised by SH3 domains and how do SH3 domains bind their target?

A

Recognise proline-rich sequences, can have different recognition sequences. A groove on the surface of the SH3 domain binds polyproline.

37
Q

What responses are signalled by insulin binding to the insulin receptor?

A

Stimulates glycogen breakdown and protein synthesis

38
Q

Where is the phosphorylation found following activation of the insulin receptor?

A

On the receptor associated protein, IRS1 (rather than being on the receptor itself).

39
Q

Describe the structure of IRS1.

A

Has a PTB domain to bind pTyr on the activated insulin receptor. Has a PH (plescktrin homology) domain to anchor it to phosphorylated lipids in the membrane.

40
Q

What is Grb2?

A

A cytosolic adaptor protein with SH2 domains to bind pTyr and an SH3 domain to bind effector proteins.

41
Q

Describe the structure and function of Sos.

A

Has a PH domain to anchor it to phosphorylated lipids in the membrane and goes on to activate Ras GTPase.

42
Q

Describe the signalling downstream of the insulin receptor.

A

IRS1 phosphorylated by activated insulin receptor. Grb2 binds IRS1 via its SH2 domains. Sos binds Grb2 via its SH3 domains and can now activate Ras GTPase.

43
Q

How can adaptor proteins allow complexity in signalling pathways?

A

Adaptor proteins, e.g. Grb2, can bind scaffold proteins - which can then be used to form multiprotein complexes.

44
Q

How is the recruitment of Sos doubly regulated?

A

Needs both PH domain binding to PIP3 in the membrane and Grb2 binding to the RTK or RTK associated protein.

45
Q

Give an overview of the RasMAP kinase pathway?

A
  1. Ras GTPase activates Raf kinase
  2. Raf kinases activates Mek kinase
  3. Mek kinase activates Erk kinase
  4. Erk kinase activates different effector proteins, which into the nucleus to activate transcription of the target gene.
46
Q

How are the kinases in the RasMAP kinase pathway activated?

A

By phosphorylation, catalysed by the previous kinase - with the exception of Raf kinase which is activated by Ras GTPase.

47
Q

How does Ras GTPase activate the first kinase in the pathway (MAPKKK)?

A

Ras GTPase has a binding site for GDP - when this is phosphorylated to give GTP, there is a shift in the switch helix. This conformational change is detected by the first kinase in the pathway. ALLOSTERIC MECHANISM

48
Q

How is Ras GTPase activated?

A

Activated by Sos which is a Ras GEF. Sos binds activated RTK via binding to Grb2 adaptor protein.

49
Q

How many MAPKKKs, MAPKKs and MAPKs are encoded in the human genome?

A

16 MAPKKKs, 9 MAPKKs and 14 MAPKs

50
Q

How are different responses in the Ras MAP kinase pathway generated?

A

By using different combinations of kinases - Raf, Mek and Erk are only one possible combination.

51
Q

What happens to active Erk that remains in the cytosol?

A

Erk phosphorylates p90(RSK) which can then enter the nucleus and bind to the SRE (serum response element) upstream of the c-fos gene, with TCF. Activates transcription.

52
Q

What happens to active Erk that goes into the nucleus?

A

Erk phosphorylates TCF which binds to the SRE upstream of the c-fos gene with p90(RSK). Activates transcription.

53
Q

What is c-fos?

A

An early response gene - one of the first genes to be switched on during proliferation. Acts as a transcription factor to activate genes involved in the cell cycle.

54
Q

How is Ras activation by RTKs switched off?

A

Phoshatases remove phosphate groups from the RTK. Ras is inactivated by Ras-GAP.

55
Q

Describe signal amplification in the Ras-MAP kinase pathway.

A

One Raf kinase can phosphorylate many Meks, which can each phosphorylate many Erks.

56
Q

How is the strength and duration of the signal moderated in the Ras-MAP kinase pathway?

A

By positive and negative feedback loops

57
Q

How is cross communication between MAP kinases prevented?

A

By scaffold proteins - localise the kinases involved in the response to the receptor in the correct order.

58
Q

Give examples of scaffold proteins used in the MAP kinase pathway and when they are used.

A

KSR - used during cell proliferation

JIP - used during the stress response

59
Q

What is the clinical relevance of the genes involved in the MAPK pathway?

A

Ras and B-RAF are protooncogenes - cause cancer when mutated. Ras mutations are seen in 15% of cancers and B-RAF mutations are seen in 60% of melanomas.

60
Q

Why is B-RAF inhibition ineffective at prolonging the lives of melanoma patients?

A

The tumour develops by switching on a different signalling pathway, independent of Raf and the cancer cells become resistant to the treatment.

61
Q

What family of GTPases are activated by the Eph receptors?

A

Rho GTPases

62
Q

Describe the signalling performed by Eph receptors and Rho GTPases.

A

Signalling between the cytoskeleton and the cell surface receptors. Important for growth cone formation - allows neurons to match up to form synapses.

63
Q

What is the result of Rho activation?

A

Generation of stress fibres - actin structures that fan out from focal adhesions.

64
Q

What is the result of Rac activation?

A

Cell edge begins to move forward as a lamina (sheet)

65
Q

What is the result of cdc42 activation?

A

Generation of filopodia - spiky protrusions from the cell.

66
Q

Describe the roles of phosphoinositides.

A

Act as signal transducers. Regulate the actin cytoskeleton, Golgi, PKC, Akt and other effectors.

67
Q

Describe the structure of phosphoinositides.

A

2 FA chains linked to glycerol, linked to a phosphoinositol by a phosphodiester bond. Found on cytoplasmic side of the membrane.

68
Q

How are extra phosphate groups added to phosphoinositide to give PIP2 and PIP3?

A

Phosphorylation by inositol kinases.

69
Q

What is the main role of PIP2?

A

Stimulates calcium release from the ER

70
Q

What is the main role of PIP3?

A

Recruits proteins to the membrane via their SH3 domains.

71
Q

What is the output of the PI3K-Akt pathway?

A

Cell growth via the inhibition of apoptosis - prosurvival signal.

72
Q

How are PDK1 and Akt recruited to the cell membrane?

A

Their SH3 domains binding to PIP3

73
Q

How are high levels of PIP3 generated in order to recruit PI3K and Akt?

A

Activated RTK stimulates PI3K to produce high levels of PIP3 - by phosphorylation of phosphoinositides.

74
Q

How is Akt activated?

A

By phoshorylation - catalysed by PDK1 and mTOR

75
Q

Describe the PI3K-Akt pathway.

A
  1. RTK activation activates PI3K to generate PIP3
  2. Recruitment of Akt and PDK1 to the membrane - via SH3 domains
  3. Akt is activated by PDK1 when the two kinases meet at the membrane
  4. Akt dissociates from the membrane and inhibits Bad - by phosphorylation
  5. Bad can no longer inhibit the apoptosis inhibitory protein
  6. Inhibition of apoptosis
76
Q

What is the purpose of mTOR?

A

Monitors whether there are enough nutrients present during PI3K-Akt signalling - responds to growth factors.

77
Q

What is PTEN?

A

Phosphatase that removes phosphate group from PI3K to switch off the PI3K-Akt pathway - acts as a tumour suppressor.

78
Q

Name the 3 ways in which RTK signalling is terminated.

A

Dephosphorylation, endocytosis and ubiquitinylation.

79
Q

How are RTKs switched off by ubiquitinylation?

A

Cbl ubiquitin ligase is recruited to the membrane via its SH2 domains. Results in ubiquitin addition to the receptor - targets the receptor for degradation.

80
Q

Describe the difference between Raf-Mek-Erk signalling following RTK activation with EGF and NGF?

A

Downstream of EGFR - negative feedback quickly switches off the pathway, resulting in cell proliferation.
Downstream of NGFR (TrkA) - positive feedback loop creates a bistable system, results in differentiation and neurite outgrowth as Ras-MAP pathway remains switched on.

81
Q

What is Shc1 and what is its function?

A

A scaffold protein similar to Grb2. Binds to activated EGFR via an SH2 domain. Approximately 30 different proteins can then bind to Shc1 in different combinations to regulate the outcome of the Ras-MAPK pathway. This allows temporal regulation of the pathway - different proteins bind at different times.

82
Q

How were the proteins that bind Shc1 identified?

A

Using pull down assays and then MS/MS sequencing.