Cell Signalling Flashcards
Different types of signalling
- Endocrine (long range)
- Paracrine
- Autocrine
Types of molecule that mediate signalling
- Small e.g. hormone
- Large e.g. growth factor
Different types of receptor
- Intracellular e.g. steroid hormone
- Transmembrane + cell surface receptor e.g. insulin
Responses within cells
- Multiplicity of responses
- Transcriptional, cell division
- Strength of signal important
Specificity of signalling
- Protein-protein interactions e.g. SH2/SH3 bromodomain
- Protein-secondary messenger interactions
ATP/GTP
- ATP = used by kinases
- GTP = used by small GTPases
Laboratory methods
Studying protein kinases
- 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
Laboratory methods
Which aa are phosphorylated
- Ser/Thr/Tyr
- Cut band out of SDS-Page
- 2D-thin layer electrophoresis
- N terminal sequencing
Laboratory methods
In vitro kinase assay
- Kinase + substrate incubated w/ radiolabeled ATP
- View w/ gel electrophoresis or autoradiography
- OR scintillation counter
Laboratory methods
Modern methodology
- Phosphospecific Ab
- Ab recognise phosphate + surrounding aa - specific
- Phosphopeptide → inoculate rabbit → affinity purify → ELISA - Phosphoproteomics
- Iso-electrofocus → SDS PAGE
- Proteolytic degradation → Mass spec or peptide sequencing
MAPK pathway
- 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
MAPK
- ERK1/2
- Structure (enzymatic cleft)
- Activation look, TEY
Ds MAPK
- Kinases e.g. Mnk1/2
- TF e.g. CREB
- Nucleosomal proteins e.g. histone H3
JNK
- UV radiation/ stress
- ds = mostly TF x kinases
- TPY
- JNKK1/2 phosphorylate T/Y
p38 MAPK
- yeast Hog1
- TGY phosphorylation by MKK3/6
- ds = similar to ERK: kinases, nucleosomal protein, TF
Specificity of MAPK
- Protein-protein
- Kinase substrate specificity
- Scaffold protein
Effects in nucleus
- 2 routes to modify histone (phosphorylation or acetylation)
Pharmaceutical
EGF/Ras/ERK
- Inhibitor found by screening library
- Anti-inflamm drug, live cell assay
Evolution of MAPK
- Yeast pathway ↑ linear
- Yeast = no further kinases past MAPK, x histone phosphorylation
- Fission yeast Ras activates adenylate cyclase, budding yeast, Ras = coupled to MAPJ
Tumour progression
1. Io tumour
- Small clump of cells
- Run out of nutrients
- Restricted
- Enclosed
- Benign + treatable
Tumour progression
2. Tumour growth
- Hypoxia → angiogenic factors
- More aggressive
Tumour progression
3. Tumour progression
- Blood vessels permeate
- 2o tumours
Tumour progression
4. Secondary tumour
- Metastasis
- Rate of mutation ↑
Time frame
- 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)
Genetics of cancer
- Accelerator mutations/ oncogene = GoF = dominant = only 1 copy needed = x heritable (embryo die)
- Breaks = on→off = LoF = recessive = 2 copies needed = heritable
Virus + cancer
- Retroviruses, reverse transcriptase
- RSV
- DNA tumour retrovirus e.g. HPV
Focus-forming assay
- 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
Tumorigenesis
- 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
Abnormal activation of growth factor genes
- Host GF gene taken up by retroviruses
- v-sis found in virus
- OR insertional oncogenesis (viral promoter)
Intracellular tyrosine kinase discovery
- Chicken w/ sarcoma
- Filtrate → young chicken
Structure of RSV
- Envelope = lipid bilayer
- Core = diploid viral RNA genome + reverse transcriptase
- Viral RNA genome = gag, pol + env in ALV
- V-src = gag, pol, env + src
How did src gene arise
- 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
c-src + v-src discovery
- Ab against Src
- Ab immunoprecipitated src + used gamma-labelled ATP
- src phosphorylates IgG
- 2D thin layer electrophoresis phosphoTyr
c-src structure
- SH2/3, myristylation
- SH1
- CTD = Tyr 527, interacts w/ SH2. Specific
- SH3 interacts w/ pro-ruch region in kinase domain
Pharmaceuticals cancer
- Conventional treatment = surgery, chemotherapy, radiotherapy
- Rational drug design
- Kinase inhibitor issue (shared sequence similarity)
Abelson Tyr kinase
- 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
Diff Tyr kinase structure/ function
- 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
EGFR + activation
- 4 genes HER1-4, diff ligands
- Monomer w/o ligand
- Ligand binding → dimerisation → autophosph of receptors (specific → SH2 domain
Tyr kinase + cancer
- Major player
- Overexpression e.g. breast
- Mutation in ectodomain e.g. glioblastoma
Erb2/HER2 overexpression = 30% breit
EGFR mutation
- Point mutation at cytoplasm
- GTG → GAG, Val664 → Glu
- vERB = hijacked, truncated EGFR, overexpression
- Mutation < Overexpression
EGFR overexpression
- Genomic instability, dividing cells ↑ prone to mistakes + ↓ checks
- Over-expression = selective advantage
Immunotherapy
- High throughput assay → inhibitor
- Cell surface signature = ectodomain over-expressed on
- Ab against ectodomain, humanised
- Herceptin
Small GTPases
- Grb2 binds receptor via SH2 + Sos via 2xSH3
- SOS gives GTP to Ras
- OR Shc can interact w/ receptor tyr kinase
Ras
- 3 genes
- 90% pancreatic tumour
- Gly12 + Gln61 (block Ras hydrolysis of GTP)
- CAAX box
- Inhibition = farnesyl transferase inhibit (ras delocalised)
ds from Ras
- 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
Ral-GEF
- ds of Ras
- Involved in cell motility + membrane morphology
Raf
- ds of Ras
- 3 gene products A,B + C-RAF
- 60% melanomas have mutation
- Inhibitor against V600E in B-rAF
TF
- Ets + ELK1 oncogenes
- TCF in nucleus
- TCF + SRF activate immediate early genes
- c-fos/jun/myc = IE oncogenes
Induction of oncogenic TF
- Quiesce then stimulate w/ TFs
- See transient IE gene then 2nd wave of 2o genes
Activator protein-1
Induction
- Subtractive hybridisation
- mRNA control + stimulated cell, cDNA library, remove house keeping
- 100-200 inducible genes
Activator protein-1
Gene regulation
- Make genomic clone + discover what us reg. elements are
- Genetic library + secret w/ probe for IE gene of interest
Activator protein-1
us reg. element responsible for induction
- TRE
- Reporter assay, CAT
- Induction of reporters + us reg. element
Activator protein-1
How signal activates reg. element
- EMSA
- Protein that binds RE = retarded at top
- Purify
Nuclear oncogenes
- Fos + Jun → AP1
- ERK activates TCF, binds SRE + switches on For
- Jun activates AP-1
- Activates 2o genes
How do Fos/Jun become oncogenic
- 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
