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Paper 4 > Cell Signalling > Flashcards

Cell Signalling Flashcards

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1
Q

Different types of signalling

A
  • Endocrine (long range)
  • Paracrine
  • Autocrine
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2
Q

Types of molecule that mediate signalling

A
  • Small e.g. hormone

- Large e.g. growth factor

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3
Q

Different types of receptor

A
  • Intracellular e.g. steroid hormone

- Transmembrane + cell surface receptor e.g. insulin

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4
Q

Responses within cells

A
  • Multiplicity of responses
  • Transcriptional, cell division
  • Strength of signal important
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5
Q

Specificity of signalling

A
  • Protein-protein interactions e.g. SH2/SH3 bromodomain

- Protein-secondary messenger interactions

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6
Q

ATP/GTP

A
  • ATP = used by kinases

- GTP = used by small GTPases

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7
Q

Laboratory methods

Studying protein kinases

A
  • 32P radiolabelling
  • Rarely use whole organisms, now cell culture
  • Culture cells → synchronise cells → replace medium w/ 32P → lyse cells → add inhibitors → run on SDS-PAGE
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8
Q

Laboratory methods

Which aa are phosphorylated

A
  • Ser/Thr/Tyr
  • Cut band out of SDS-Page
  • 2D-thin layer electrophoresis
  • N terminal sequencing
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9
Q

Laboratory methods

In vitro kinase assay

A
  • Kinase + substrate incubated w/ radiolabeled ATP
  • View w/ gel electrophoresis or autoradiography
  • OR scintillation counter
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10
Q

Laboratory methods

Modern methodology

A
  1. Phosphospecific Ab
    - Ab recognise phosphate + surrounding aa - specific
    - Phosphopeptide → inoculate rabbit → affinity purify → ELISA
  2. Phosphoproteomics
    - Iso-electrofocus → SDS PAGE
    - Proteolytic degradation → Mass spec or peptide sequencing
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11
Q

MAPK pathway

A
  • EGF binding EGFR activates tyrosine kinase
  • GRB2 binds EGFR via Grb2
  • GRB2 binds SOS via SH3
  • SOS exchanges GDP for GTP
  • Ras → Raf → MAPK → MAPKK
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12
Q

MAPK

A
  • ERK1/2
  • Structure (enzymatic cleft)
  • Activation look, TEY
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13
Q

Ds MAPK

A
  • Kinases e.g. Mnk1/2
  • TF e.g. CREB
  • Nucleosomal proteins e.g. histone H3
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14
Q

JNK

A
  • UV radiation/ stress
  • ds = mostly TF x kinases
  • TPY
  • JNKK1/2 phosphorylate T/Y
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15
Q

p38 MAPK

A
  • yeast Hog1
  • TGY phosphorylation by MKK3/6
  • ds = similar to ERK: kinases, nucleosomal protein, TF
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16
Q

Specificity of MAPK

A
  • Protein-protein
  • Kinase substrate specificity
  • Scaffold protein
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17
Q

Effects in nucleus

A
  • 2 routes to modify histone (phosphorylation or acetylation)
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18
Q

Pharmaceutical

EGF/Ras/ERK

A
  • Inhibitor found by screening library

- Anti-inflamm drug, live cell assay

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19
Q

Evolution of MAPK

A
  • Yeast pathway ↑ linear
  • Yeast = no further kinases past MAPK, x histone phosphorylation
  • Fission yeast Ras activates adenylate cyclase, budding yeast, Ras = coupled to MAPJ
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20
Q

Tumour progression

1. Io tumour

A
  • Small clump of cells
  • Run out of nutrients
  • Restricted
  • Enclosed
  • Benign + treatable
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21
Q

Tumour progression

2. Tumour growth

A
  • Hypoxia → angiogenic factors

- More aggressive

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22
Q

Tumour progression

3. Tumour progression

A
  • Blood vessels permeate

- 2o tumours

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23
Q

Tumour progression

4. Secondary tumour

A
  • Metastasis

- Rate of mutation ↑

24
Q

Time frame

A
  • Colon cancer 5-20 years
  • Cell cycle 12-18hr, 1000s of divisions
  • Cigarette consumption vs death shifted 20 years
  • ↑ rate in 60-90 yrs
  • 10 years, 90% survival skin cancer, 1 year, 20% pancreatic cancer (diff cancer)
25
Q

Genetics of cancer

A
  • Accelerator mutations/ oncogene = GoF = dominant = only 1 copy needed = x heritable (embryo die)
  • Breaks = on→off = LoF = recessive = 2 copies needed = heritable
26
Q

Virus + cancer

A
  • Retroviruses, reverse transcriptase
  • RSV
  • DNA tumour retrovirus e.g. HPV
27
Q

Focus-forming assay

A
  • Transformed cell = diff/ morphology = loss of contact inhibition = grow w/o solid support
  • Genetic library 100,000 genes = transform cells = cells pick up cancer causing genes form foci
28
Q

Tumorigenesis

A
  • To assess formation of 2o tumours, tumorigenesis needs to be anticipated in live animals
  • Tumour from focus forming assay to live animal
  • Immune suppressed mouse
29
Q

Abnormal activation of growth factor genes

A
  • Host GF gene taken up by retroviruses
  • v-sis found in virus
  • OR insertional oncogenesis (viral promoter)
30
Q

Intracellular tyrosine kinase discovery

A
  • Chicken w/ sarcoma

- Filtrate → young chicken

31
Q

Structure of RSV

A
  • Envelope = lipid bilayer
  • Core = diploid viral RNA genome + reverse transcriptase
  • Viral RNA genome = gag, pol + env in ALV
  • V-src = gag, pol, env + src
32
Q

How did src gene arise

A
  • Viral promoter overshoots provirus + uptakes c-src
  • Packed into new virus
  • MAPK pathway = packed w/ points viral oncogenes can hijack
  • Focus forming assay = just src needed
33
Q

c-src + v-src discovery

A
  • Ab against Src
  • Ab immunoprecipitated src + used gamma-labelled ATP
  • src phosphorylates IgG
  • 2D thin layer electrophoresis phosphoTyr
34
Q

c-src structure

A
  • SH2/3, myristylation
  • SH1
  • CTD = Tyr 527, interacts w/ SH2. Specific
  • SH3 interacts w/ pro-ruch region in kinase domain
35
Q

Pharmaceuticals cancer

A
  • Conventional treatment = surgery, chemotherapy, radiotherapy
  • Rational drug design
  • Kinase inhibitor issue (shared sequence similarity)
36
Q

Abelson Tyr kinase

A
  • In humans = CML, ALL, AML
  • Blood analysed in leukaemia → Philadelphia chromosome
  • Translocation btw chromosome 9 + 22 → 9q+, 22q-
  • Karyotype, 22q-
  • BRC-2qII juxtaposed w/ ABL-9q34 → new transcript
  • Diff variation of BCR/ABL
  • Treatment = block Tyr kinase, Gleevec
37
Q

Diff Tyr kinase structure/ function

A
  • EGFR = central TM domain, ectodomain
  • IGFR = tetramer, IRS1 links insulin to effectors
  • PDGF = immunoglobulin-like fold, kinase insert region
  • VEGF receptor = interrupted SH1 region
  • Eph receptor = fibronectin type II- like domain, ephori, bound on surface of adjacent cell
38
Q

EGFR + activation

A
  • 4 genes HER1-4, diff ligands
  • Monomer w/o ligand
  • Ligand binding → dimerisation → autophosph of receptors (specific → SH2 domain
39
Q

Tyr kinase + cancer

A
  • Major player
  • Overexpression e.g. breast
  • Mutation in ectodomain e.g. glioblastoma
    Erb2/HER2 overexpression = 30% breit
40
Q

EGFR mutation

A
  • Point mutation at cytoplasm
  • GTG → GAG, Val664 → Glu
  • vERB = hijacked, truncated EGFR, overexpression
  • Mutation < Overexpression
41
Q

EGFR overexpression

A
  • Genomic instability, dividing cells ↑ prone to mistakes + ↓ checks
  • Over-expression = selective advantage
42
Q

Immunotherapy

A
  • High throughput assay → inhibitor
  • Cell surface signature = ectodomain over-expressed on
  • Ab against ectodomain, humanised
  • Herceptin
43
Q

Small GTPases

A
  • Grb2 binds receptor via SH2 + Sos via 2xSH3
  • SOS gives GTP to Ras
  • OR Shc can interact w/ receptor tyr kinase
44
Q

Ras

A
  • 3 genes
  • 90% pancreatic tumour
  • Gly12 + Gln61 (block Ras hydrolysis of GTP)
  • CAAX box
  • Inhibition = farnesyl transferase inhibit (ras delocalised)
45
Q

ds from Ras

A
  • Inositol lipid signalling - PLCy or P13K
  • PDGF both, EGF = PLcy
  • PLCy = PIP2 → PIP3
  • P110 of PW3K binds Ras, p85 of P13K interacts w/ phosphotyr via SH2
  • p110 mutated
  • Akt recruited by PIP3 + phosph. by PDK1/2 on membrane
  • PTEN reverses PI3K
46
Q

Ral-GEF

A
  • ds of Ras

- Involved in cell motility + membrane morphology

47
Q

Raf

A
  • ds of Ras
  • 3 gene products A,B + C-RAF
  • 60% melanomas have mutation
  • Inhibitor against V600E in B-rAF
48
Q

TF

A
  • Ets + ELK1 oncogenes
  • TCF in nucleus
  • TCF + SRF activate immediate early genes
  • c-fos/jun/myc = IE oncogenes
49
Q

Induction of oncogenic TF

A
  • Quiesce then stimulate w/ TFs

- See transient IE gene then 2nd wave of 2o genes

50
Q

Activator protein-1

Induction

A
  • Subtractive hybridisation
  • mRNA control + stimulated cell, cDNA library, remove house keeping
  • 100-200 inducible genes
51
Q

Activator protein-1

Gene regulation

A
  • Make genomic clone + discover what us reg. elements are

- Genetic library + secret w/ probe for IE gene of interest

52
Q

Activator protein-1

us reg. element responsible for induction

A
  • TRE
  • Reporter assay, CAT
  • Induction of reporters + us reg. element
53
Q

Activator protein-1

How signal activates reg. element

A
  • EMSA
  • Protein that binds RE = retarded at top
  • Purify
54
Q

Nuclear oncogenes

A
  • Fos + Jun → AP1
  • ERK activates TCF, binds SRE + switches on For
  • Jun activates AP-1
  • Activates 2o genes
55
Q

How do Fos/Jun become oncogenic

A
  • Deletion at N-terminus in V-jun compared to c-jun, important for interaction w/ JNK
  • Deletion in CTD in v-Fos, affects phosphorylation. site, means hyperactive
  • Translocation in Burkitt’s lymphoma of c-myc (chromosome 8 → 14), myc gene under control of immunoglobulin enhancer

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