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BIOL2056 > Intracellular signalling > Flashcards

Intracellular signalling Flashcards

1
Q

The insulin receptor is a member of what family?

A

The tyrosine kinase receptor superfamily

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2
Q

Approximately how many tyrosine kinase receptors are there in the human genome?

A

50

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3
Q

What is the insulin receptor involved in?

A

Cell proliferation, differentiation, migrations and plays a crutial role in development and tissue homeostasis

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4
Q

What does Glucagon and insulin antagonistically regulate?

A

glucose release in the liver

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5
Q

examples of growth factors that function through tyrosine kinase receptors

A
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6
Q

Tell me some charactertistics of the tyrosine kinase receptor family

A
  • Transmembrane proteins
  • Catalytic receptors
  • Contain a ligand binding site in a portion of the receptor located outside the cell
  • An intracellular portion of the receptor contains a protein kinase active site
  • The protein kinase is specific for phosphorylating tyrosine residues
  • Ligand binding activates the protein kinase
  • This results in autophosphorylation (self-phosphorylation) of tyrosine residues in the intracellular part of the receptor
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7
Q

Tell me about GPCR ligand binding

A
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8
Q

Tell me dimerisation of ligand bound receptor tyrosine kinase as being a common mechanism for transducing signals to the interior of the cell…?

A

Cross phosphorylation of active sites when receptor is activated

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9
Q

Tell me about the dimerisation and cross-phosphorylation of the tyrosine ?

A
  • increases kinase activity and further receptor tail phosphorylation
  • Consider catalytic domains flip between Inactive and active state but will be predominantly inactive due to inhibitory action of the activation loop.
  • Dimerization enables cross phosphorylation and activation
  • Dimerization leads to the phosphorylation of other intracellular tyrosine residues
  • Essentially conversion of ligand binding into a conformational and chemical change (phosphorylation)
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10
Q

Activation loop phosphorylation stabilises what?

A

The active conformation of the insulin receptor tyrosine kinase (IRK)

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11
Q

The activation loop occludes the active site. Why can’t Tyr1162 be phosphorylated?

A

The Tyr1162, is bound in the active site but cannot be phosphorylated (in Cis) because part of the A-loop interferes with the ATP binding site and the catalytic residue Asp1150 is improperly positions to coordinate MgATP

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12
Q

What does Tyrosine phosphate of T1162 and 1163 in trans distrupt?

What does this movement facilitate?

A

existing interaction and induces new interactions with Arg1155 and Arg1164 which displaces the activation loop by about 30Å and enables ATP and substrate to bind

This movement facilitates a functional spatial arrangement of Lys1030 and Glu1047, the residues involved in MgATP coordination, and of Asp1150, which is part of the highly conserved Asp–Phe–Gly triad

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13
Q

SH2 domain binding to tyrosine phosphorylated residues generates what?

A

A simple output system describing tyrosine kinase signalling

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14
Q

What is the SH2 domain thought to interact with?

A

SH2 domain present in about 100 proteins and thought to mainly interact with phosphorylated tyrosine residues

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15
Q

Tell me about SH2 structure

A
  • approx. 100 AA
  • folds into a compact structure with a central beta sheet with two binding pockets
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16
Q

There are 27 PTB containing protein in the human genome. What do many PTB domains interact with?

A

non-phosphorylated tyrosine residues

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17
Q

Name 2 reader molecules, what they drive and what they interact with?

A

Reader molecule can drive signalling output

there are 2 types: SH2 and PTB

Both are able to interact with phosphorylated protein

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18
Q

Different SH2 domains interact with different tyrosine phosphorylated peptides, how is specificity of the SH2 determined?

A

Specificity is determined by two binding pockets

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19
Q

What are the Src-like SH2 domains specific to?

A

The Src-like SH2 domains (Src, Fyn,Hck and Nck) select negatively charged residues in the pTyr+1 and pTyr+2 position and use a hydrophobic pocket to select for aliphatic residues in the pTyr+3 position

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20
Q

What is the PLC-gamma1 like SH2 domains specific to?

A

The PLC-γ1-like SH2 domains (PLC-γ1 amino-terminal SH2, PLC-γ1 carboxy-terminal SH2 and SHP-2) use a long hydrophobic cleft to select for aliphatic residues from the pTyr+1 to pTyr+5 positions.

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21
Q

What are the Grb2-like SH2 domains contain?

What does this do?

A

Grb2-like SH2 domains contain a bulky tyrosine residue that protrudes from the EF loop and blocks the pTyr+3 position if the phosphopeptide is in an extended conformation.

This forces the bound peptides to make a β-turn, which is accomplished by selecting an asparagine (Asn) residue in the pTyr+2 position23,137

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22
Q
  1. Tyrosine Phosphorylation of the intracellular receptor domains drives the direct interaction and regulation of downstream signalling proteins.

What are the stages to this?

A
  1. P85 is a regulatory subunit of PI3K. It contains a SH2 domain. When bound to the receptor PI3K becomes active and generates a new signalling molecule called PIP3.
  2. PLCg is recruited by its SH2 domain to the phosphorylated receptor and in turn is activated and generates DAG and IP3.
  3. The SH2 domain of Grb2 mediates recruitment and subsequent activation of the RAS/MAPK pathway.
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23
Q
  1. Receptor mediated Tyrosine Phosphorylation of adaptor proteins also drives downstream intracellular signalling

How does it do this?

Provide an example

A

Some receptors use the recruitment of an adaptor protein to the receptor induced by by phospho-tyrosine binding.

The adaptor is then multiplied phosphorylated and post–translationally modified and initiates downstream signalling

Examples: FGFRS1 (fibroblast growth factor) receptor substrate: (FGF and NGF), GAB1 (EGF and others)

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24
Q

Tyrosine phosphorylation induces receptor ubiquination and internalisation: Coupling reader modules to drive signalling pathways

A
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25
Q

What are the 3 pathways activated by phosphorylation of tyrosine?

A
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26
Q

Middle T antigen of the polymoma transforming virus interacts with and activates a PI-kinase

Middle T transforming proteins

Induced upon infection with polyoma virus. It is required for transformation of cells.

How is this done?

A
  1. Middle T associates with a tyrosine kinase (pp60cSrc the cellular homologue of the Src oncogene)
  2. A PtdIns-Kinase activity was found associated with middle T immunoprecipitates.
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27
Q

What is the PI3K pathway regulated by?

A

RTK and GPCR receptor signalling

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28
Q

The PI3K pathway…

A

Middle T, RTK and GPCR all regulate the activity of PI3K to generate the second messenger PtdIns(3,4,5)P3

How do RTKS specifically regulate PI3K to stimulate PtdIns(3,4,5)P3 synthesis ?

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29
Q

How is the PtdIdns(3,4,5)P3 signal controlled?

A

Controlling the PtdIns(3,4,5)P3 signal

  • PTEN: human tumour suppressor often deleted or mutated in human tumours (40%)
  • PTEN removes the 3 phosphate from PtdIns(3,4,5)P3 (3-phosphatase)
  • SHIP1/2 is a 5 phosphatase which removes the 5 phosphate from PtdIns(3,4,5)P3 .
  • Although SHIP degrades PtdIns(3,4,5)P3 it also generates a new lipid PtdIns(3,4)P2 which is through to have messenger functions
  • The balance of these enzymes dictates the kinetics and extent of the generation of PtdIns(3,4,5)P3
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30
Q

What are Class 1A PI3K directly regulated by?

How?

A

RTK activation (one activated by receptor tyrosine kinases)

  • Class1 A p110 catalytic domains interact with the p85 regulatory domains.
  • Interaction of p110 and p85:
  • suppresses the activity of P110 subunit;
  • prevents proteolytic degradation of P110 and
  • enable recruitment of the holo enzyme to an activated RTK receptor.
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31
Q

How do tyrosine kinase receptors couple to the Class 1A PI3K?

A

Tyrosine kinase receptors couple to the class 1A PI3K through the interaction of the SH2 domain of p85 with a specific tyrosine phosphorylated receptor peptide.

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32
Q

Why are Class 1A PI3K recruited?

A

To activate tyrosine kinase receptors

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33
Q

What does the SH2 domain of the p85 subunit mediate in tyrosine kinase receptors?

A

The SH2 domain of the p85 subunit mediates the interaction with the YxxM motif on the receptor or an adaptor protein

34
Q

Tell me the stages to the binding of the SH2 domain to the receptor tyrosine phosphorylated peptide?

A
  1. Brings the enzyme to the membrane where its substrate PtdIns(4,5)P2 resides
  2. Relieves the inhibition of the p85 subunit upon the catalytic activity of p110
  3. Stimulates interaction of the kinase with the membrane.
35
Q

How does PtdIns(3,4,5)P3 control down stream signalling?

A

PtdIns(3,4,5)P3 controls downstream signalling by recruiting proteins with specific PtdIns(3,4,5)P3 binding domains

  1. Dramatic shift in phosphoinositide signalling paradigm when it was realised that some PH domains interact with phosphoinositides.
  2. The chemical synthesis of specific phosphoinositides enabled the generation of tool compounds to be able to be used to rapidly purify proteins that interact with specific PI species.
36
Q

Over 400 proteins have been identified that interact with phosphoinositides. Of these about 50 appear to interact specifically with PtdIns(3,4,5)P3. How are these proteins implicated?

A
  1. Cytoskeletal dynamics
  2. Vesicle trafficking
  3. Nuclear functions
37
Q

What was the PH domain first found in?

What does the PH domain mediate?

A

PH domain:

First found in the platelet protein pleckstrin and shown to be a module that binds PtdIns(4,5)P2.

PH domain also mediate protein/protein interactions e.g bg subunit of heterotrimeric G proteins . Also involved in generating dimerization interfaces.

38
Q

What are PH domains present in?

A

PH domains are present in many proteins including protein kinases, guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) for small GTP-binding proteins, lipid transport proteins and phospholipases

39
Q

Tell me about the structure of the PH domain

A
  • It is a domain of approximately 120 amino acids.
  • A common structural feature is that they contain two nearly orthogonal β sheets formed by seven β strands arranged in groups of four and three
  • flanked by a C-terminal helix that `holds’ the structure together.
40
Q

How is the inositide-binding site almost always formed in the PH domain?

A

The inositide-binding site is almost always formed by the β1-β2 and β3-β4 strands and the variable loops connecting them

41
Q

SH2 domain mediated activation of the PI3-kinase drives what?

A

SH2 domain mediated activation of PI3-Kinase drives the synthesis of PIP3 which induces new output signalling through PtdIns(3,4,5)P3 “readers”

42
Q

How does PtdIns(3,4,5)P3 initiates downstream signalling ?

A

by interacting and recruiting proteins to the membrane that contain specific PIP3 interaction motifs such as the Pleckstrin homology (PH) domain

NB. PKB is not the only downstream domain for PIP3 but one of the good examples

43
Q

Activation of the PtdIns(3,4,5)P3 reader PKB/AKT protein kinase couples insulin protein phosphorylation and downstream target pathway regulation

A
44
Q

Abbreviations to know

A
  • PDE: phosphodiesterase (remember cAMP0
  • GSK: glycogen synthase kinase (cAMP switches this pathway off)
  • SREBP1: Sterol regulatory element-binding proteins. Transcription factor that regulates fatty acid production.
  • TCS1/2: tuberous sclerosis complex. TSC1–TSC2 (hamartin–tuberin). This complex controls the RHEB small molecular weight G protein which controls MTORC1 complex protein kinase activity
  • mTORC1: mechanistic target of rapamycin complex 1. this is a protein kinase complex that essentially controls protein synthesis. It has multiple inputs into it that enable it to act as an integrator of signalling.
  • mTORC2: mechanistic target of rapamycin complex 2. this is a protein kinase complex that shares the same kinase subunit as mTORC1 but it is involved in the selective regulation of substrates. Its main substrate is the AKT/PKB kinase.
  • PDK1: phosphoinositide dependent kinase 1. a protein kinase that regulates PKB. It is also an important kinase that regulates the maturation of a number of other protein kinases
  • AS180: ATK substrate 160. this is a rab gap that control the GTP loading of the smg RAB which in turn controls vesicle trafficking.
45
Q

Tell me about activation of the P13K pathway in the liver, adipose tissue and fibroblasts…

A

Receptor coupled PtdIns(3,4,5)P3 synthesis drives specific downstream signalling pathways in different tissues

46
Q

Many different types of receptors drive PIP3 synthesis and downstream signalling

A
47
Q

Deregulation of the PI3K/PtdIns(3,4,5)P3 pathway is frequent event in ?

A

Tumour development RTK mediated PLC activation

48
Q

What are the core enzymes of the phospholipase C family composed of?

A

Core enzyme composed of the PH domain, four tandem EF domains, a split tim barrel (X Y) and a C2 domain.

49
Q

Where is the active site for the phospholipase C family?

A

The active site is in the TIM, which is interupted by an auto-inhibitory loop insert. In PLCzeta the loop is activatory and its removal inhibits the enzyme.

50
Q

What can the PH domain interact with?

A

The PH domain can interact with phosphoinositides, rac, bg (betagamma)

51
Q

What can the C2 domain interact with?

A

The C2 domain interacts with calcium and with the membrane surface and contributes to Gaq binding together with the C-terminal.

52
Q

PLC are very active enzymes that are autoinibited and activation mechanisms essentially move the loop out of the cleft

A
53
Q

Phospholipase C family

A
54
Q

SH2 mediated recruitment of PLCgamma induces what?

A

tyrosine phosphorylation of PLCg required for its activation

55
Q

SH2 domain fo the PLCgamma enables its recruitment to what?

A

The RTK receptor

56
Q

Tyrosine Phosphorylation at Y783 and y1253 are important for what?

A

PLC activation

However, this is not all that is required for PLCg activation. Stimulation of cells with either PDGF or EGF induces strong interaction with each receptor and comparable tyrosine phosphorylation of PLCg. However, PDGF induces strong PLC activation compared to the weak activation induced by EGF
What else is required?

57
Q

Cross talk with the PI3K pathway drives full activation of PLCg and PtdIns(4,5)P2 hydrolysis

A
58
Q

The small molecular weight (monomeric) G proteins of the Ras family are molecular switches that control what?

A

diverse cellular outputs

59
Q

What is GRB2?

A

GRB2 is an adaptor protein that contains SH2 and SH3 domains. SH3 specific for proline rich motifs. SH2 specific for tyrosine phosphorylated regions

60
Q

What do the SH2 domains of the adaptor protein GRB2 interact with ?

A

The SH2 domains of the adaptor protein GRB2 interact with specific tyrosine phosphorylated residues on an RTK.

61
Q

What does GRB2 recruit?

A

GRB2 recruits the SOS (son of seven less) protein through the interaction of its SH3 domain with a proline rich motif on SOS

62
Q

Whats SOS?

A

SOS is a guanine nucleotide exchange factor for RAS

Its activity is enhanced by the interaction of its PH domain with PtdIns(4,5)P2

63
Q

RTK mediated RAS activation

A
64
Q

RAS regulates two important pathways that coordinate proliferative responses downstream of many different types of receptors

A
65
Q

What does GTP exchange lead to?

A

GTP exchange leads to conformational changes in RAS that enable reader interaction

66
Q

What does RAS bound to GTP show?

A

RAS bound to GTP shows conformational changes in switch1 (SW1) and switch 2 (SW2) loops.

67
Q

RAS bound to GTP shows conformational changes in switch1 (SW1) and switch 2 (SW2) loops. What does the change in these loops enable?

A

The changes in the conformation of these loops enables the interaction with downstream reader proteins.

68
Q

Mutations in the SW region of the RAS can what?

A

Mutations in the SW regions can ablate specific interaction with reader proteins.

69
Q

Tell me about mutations in the RAS switch 1 residues

A

Mutations in Ras switch I residues T35S, E37G and D38E eliminate PI3K binding However, T35S and D38E do not affect Raf binding. The E37G mutation also abolishes binding to Raf but not to RalGEF. The Y40C mutation does not affect PI3K binding, but it abrogates Raf and RalGEF binding.

70
Q

Tell me about the mutations in the switch II region of RAS

A

In the switch II region, the Y64G mutation eliminates PI3K and NF1 binding but has no effect on Raf binding

71
Q

Activation of downstream pathways by RAS switch loop mutants

The binary switch action of RAS converts GTP binding into the regulation of protein phosphorylation driven by the RAF/MAP kinase pathway

A
72
Q

The binary switch action of RAS converts GTP binding into the activation of PI3K and PtdIns(3,4,5)P3 signalling

A
73
Q

What are the thee isoforms of RAS, what are each ?

A

There are Three isoforms of RAS: H-RAS. N-RAS and K-RAS.

  • HRAS transcripts are highly expressed in brain, muscle and skin and lowest in liver
  • KRAS transcripts are readily detected in gut, lung and thymus.
  • NRAS transcripts are primarily expressed in the testis and thymus
74
Q

What do the isoforms of RAS show and how are they modified?

A

Show high degree of homology with main differences in the C terminal hypervariable region.

They are post-translationally modified with lipid chains that target them to membranes.

75
Q

When are the RAS isoforms often mutated?

What do they show and give an example for each isoform

A

They are often mutated in cancer (95% of pancreatic tumour have mutated K-RAS) with different isoforms showing tumour specificity:

  1. KRAS primarily in colon and pancreatic cancers
  2. NRAS in myeloid leukemia and
  3. HRAS in bladder carcinomas
76
Q

What mutations stop RAS from hydrolysing GTP?

A

The G12, G13 and Q61 mutations essentially stop RAS from hydrolysing GTP. This locks RAS in the active state inducing downstream signalling (mutations of this type occur at these 3 locations)

77
Q

Deregulation of RAS signalling in human tumours drives cell proliferation

A
78
Q

What is Glycine-12 of the P-loop (binds the GDP/GTP) is critical for?

A

Glycine-12 of the P-loop (binds the GDP/GTP) is critical for oncogenic activation of Ras because any mutation of this residue [except Gly12Pro (G12P)] activates the oncogenic potential of Ras

79
Q

What would any mutation including Gly12Ala would lead to?

A

Any mutation including Gly12Ala would lead to a clash with Arg789 and with Gln61.

80
Q

What is GIn61 essential for?

A

Gln61 is essential for GTP hydrolysis and forms contacts with the ARG789 as well as with the gamma phosphate of GTP.

81
Q

What does the mutation of Gln61 lead to?

A

Mutation of Gln61 to any other residue beside glutamate leads to a loss of GTP hydrolysis and oncogenic activation

82
Q

Examples of SH2 domain containing proteins

A
  • 120 proteins with SH2 domains
  • Contact many aspects of cell regulation.
  • Many appear to be adaptor proteins
  • RTK signalling is dependent on the repertoire of SH2 binding proteins present in a cell and on the specific tyrosine phosphorylation site.

BIOL2056 (7 decks)

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