Midterm 2 Flashcards
(41 cards)
1
Q
Receptor Tyrosine Kinases
A
Phosphorylate specific tyrosines
2
Q
Tyrosine Kinase Associated Receptors
A
Associate with intracellular proteins that have tyrosine kinase activity
3
Q
Receptor-like Tyrosine Phosphatases
A
Remove phosphate group
4
Q
Receptor Serine/Threonine Kinases
A
Phosphorylate specific serine/threonine
5
Q
Receptor Guanylyl Cyclases
A
Directly catalyzes the production of cGMP
6
Q
Histidine Kinase Associated Receptors
A
Kinase phosphorylates itself on histidine and then transfers the phosphate to a second IC signalling protein
7
Q
Cell-Cell Communication
A
Informs the cell of WHAT they are (or what to become), WHERE they are, and WHEN they should do something
8
Q
Roles of Growth Factors: Epidermal Growth Factor
A
Proliferation of several epithelial cells including skin repair after wound
9
Q
Roles of Growth Factors: Platelet Derived Growth Factor
A
Fibroblast (connective tissue) proliferation and motility
10
Q
Roles of Growth Factors: Insulin
A
Stimulate carbohydrate utilization and protein synthesis
11
Q
Roles of Growth Factors: Nerve Growth Factor
A
Stimulate survival and growth of some neurons
12
Q
Roles of Growth Factors: Fibroblast Growth Factor
A
Muscle/cartilage development
13
Q
Roles of Growth Factors: Colony Stimulating Factor
A
Macrophage stimulation
14
Q
Roles of Growth Factors: Vascular Endothelial Growth Factor
A
Stimulate angiogenesis
15
Q
Growth Factors
A
Small secreted proteins capable of stimulating cell proliferation, differentiation or motility
16
Q
Identification of EGF
A
- Stanley Cohen in 1950s
- Identified as active substance in extract of salivary gland that increased epidermal cell proliferation
- Rationalized that any substance that altered the timing of developmental processes would be biologically significant
- EGF enhanced epidermal growth and led to the observed precocious eyelid opening
17
Q
EGF-Like Growth Factors
A
- Expressed as transmembrane pro-forms
- Ectodomain portion sheds and goes on to communicate throughout the cell
18
Q
Receptor Tyrosine Kinases
A
- Family of membrane receptors that bind extracellular peptide growth factors and transduce signals from their intracellular tyrosine kinase domains to a series of intracellular cascades to regulate cellular growth/proliferation and/or metabolic activity
- All have homology in their sequences, leading to the identification of the family
19
Q
EGF and 32P Incorporation
A
-Stimulates the incorporation of 32P from [gamma-32P]-ATP into cell membranes
-Took membranes that expressed a lot of EGFR and incubated them with radioactive ATP
-Found that the EGF stimulated something along the lines of phosphorylation of something, leading to the significant increase in 32P in the ones with EGF
-
20
Q
RTKs: Auto-Phosphorylation
A
- Receptors are auto-phosporylated on tyrosine residues
- Ran gels and found that the protein that was being phosphorylated corresponded with almost exactly the predicted size of EGFR. Increased levels of phosphorylation were found on the receptor itself, therefore it was postulated that the first substrate of the kinase was the receptor itself.
21
Q
RTKs: Activation
A
- Activated through receptor dimerization and transphosphorylation
1. GF binds
2. Dimerization occurs
3. One kinase moiety on one receptor will transphosphorylate tyrosines on the other receptor strain and kick-start EC signalling cascade
22
Q
RTKs: Crystal Structure
A
- Crystal structures show that dimerization is entirely receptor mediated
- Although EGF ligand does not span the dimerization interface, it simultaneously engages two separate surfaces on the same monomer that allow un-masking of dimerization interface
23
Q
EGFR Activation
A
- Ectodomain is composed of 4 domains
- During the activation process, 2 EGFs are required (1 per receptor)
- EGF binds between domains I and III –> 130 degree rotation to expose domain II (which has the dimerization arm)
- Direct intramolecular interactions between domains II and IV restrain the domain II/III relationship that characterize the inactivated configuration
- The II/IV tether buries the dimerization arm of domain II against domain IV and prevents dimerization (when inactive)
24
Q
EGF/Her/ERBB Receptor Family
A
- EGF can bind different combinations of the 4 HER receptors, which are all very similar but are expressed on different cells and tissues
- 11+ ligands including EGF, TGFalpha, amphiregulin, heregulins, neuregulins
- All receptors must form dimers to be active
1 + 1,2,3,4
2 + 1,3
3 + 1,2
4 + 1,2,4
25
ERBB1
Her1/EGFR
- Forms homo or hetero dimers
- Bind a wide variety of ligands
- KO mice die mid-gestation/birth, so this receptor is extremely important in development (esp neurological)
26
ERBB2
Her2
- Does not bind GF because it is always constitutively active (dimerization arm always exposed)
- Does not form homodimers
- Heterodimers containing ERBB2 are more potent mitogens, internalize slowly and bind more ligands
27
Signalling Pathway
1. Signal is recognized
2. Effector is activated, which can lead to changes in
- metabolism of the cell
- gene regulation
- cytoskeletal protein (leading to the cell migrating and possible binding changes)
Signal --> Receptor sensor) --> Transduction Cascade --> Targets --> Response
28
Transduction Cascade Elucidation
-Via studying the drosophila eye
-Sevenless receptor is required for response to Boss ligand, which is secreted by R8 in order for the cell to become R7
-Each eye contains 7 photoreceptors and the 7th one expressed the sevenless receptor (which is similar to EGFR). The R8 cell secretes Boss. If there is a mutant that does not express sevenless, then there is no 7th photoreceptor cell.
-Genetic screens revealed that there were more components to the cascade than just EGF --> EGFR --> Ras --> STUFF
+They took the flies and treated them with shit to introduce mutations at random and then screened for defective eyes. Of those who had defective eyes, they screened for the 7th photoreceptor and then did gene sequencing to map the mutations.
29
Ras
- G-proteins that act as molecular switches and act downstream of EGF
- GTP-bound ACTIVE form
- Catalyzed by guanine exchange factors
30
Sos
Son of Sevenless
- Guanine exchange factor for Ras
- Promotes the exchange of GDP for GTP on GTP binding proteins in order to activate them
31
Grb2 and Sos
-Missing link between receptor and Ras
-Ras binds GTP/GDP and then Sos catalyzes the change
-Grb2 is an adaptor domain which links the two proteins together
-Grb2 is composed of
+SH2 - binds phosphorylated tyrosine
+SH3 - binds hydrophobic, proline-rich sequence
32
Activation of EGFR
1. Binding of hormone causes receptor dimerization kinase activation and phosphorylation of cytosolic receptor tyrosine residues
2. Binding of GRB2 and Sos couples receptor to inactive Ras
- Binding of GRB2 recruits SH3 domain to bind proline rich region of Sos
3. Sos promotes dissociation of GDP from Ras; GTP binds and active Ras dissociates from Sos
4. MAP Kinase Cascade
33
SH2
- Conserved regions of about 100 amino acids that are not required for catalytic activity
- Bind tightly to tyrosine-phosphorylated peptides
- Mediate protein-protein interactions of effectors with growth factor receptors
- Specificity resides in the sequence context of the pTyr residue (the 3 aa's next to the tyrosine are important)
- The cytoplasmic tails of EGFR have a number of tyrosines that are phosphorylated, and all have SH2 and are recruited to specific residues (ie. GRB2 is recruited to 1068 and 1086 Tyr) depending on the context
34
SH3
- Compact region of about 60 amino acids
- Signalling complex assembly and regulatory functions
- Bind proline-rich target sequences
- Composed of two beta-sheets placed at right angles
- Binding surface lined with hydrophobic residues that contract proline residues in target
- Hydrophobic pocket is flanked by variable loops that determine binding specificity
35
MAP Kinase Cascade
1. Ras activated by exchange of GDP for GTP
2. Active Ras recruits, binds and activates Raf (MAPKKK)
3. GTP hydrolysis leads to dissociation of Ras from Raf
4. Raf activates MEK (MAPKK)
5. MEK activates MAPK
6. Dimeric form of active MAPK translocates to nucleus and activates transcription factors
36
Early-Response Genes
- Addition of growth factors to quiescent cells cause rapid increase in expression of about 100 early-response genes
- These genes encode proteins necessary to propel the cell through the cell cycle
37
Control of Cell Proliferation
- Most cells will not proliferate unless prompted to do so by mitogenic growth factors
- EC signals are processed and distilled to a binary decision - proliferation or quiescence
38
Cell Cycle
1. G1 - Cell senses the presence of all the signals
2. S - DNA replication; cell has committed to divide
3. G2 - Pre-mitosis/division; all the protein and apparatus that will divide get into place
4. M - mitosis
- At the exit of mitosis phase, the two daughter cells re-enter G1 and are going to re-asses to see if they have the signals to proliferate until they reach the R point
- At the R point, they will decide if they are going to divide or enter the G0 phase (quiescent)
39
Hallmarks of Cancer Cells
- Self-sufficiency in growth signals
- Insensitivity to anti-growth signals
- Evading apoptosis
- Limitless replicative potential
- Sustained angiogenesis (for sustained nutrients)
- Tissue invasion and metastasis
- Genetic instability
40
Oncogenes
- Any gene that encodes a protein able to transform cells in culture or induce cancer in animals
- Most are derived from normal cellular genes whose products participate in cellular growth-controlling pathways
- First were found in tumour causing viruses and were found to be related to cellular genes
- Activation of a proto-oncogene generally involves a gain-of-function mutation, which is a point mutation leading to constitutively active protein product, gene amplification, or chromosomal translocation that causes inappropriate expression or activity of a growth-regulatory gene
41
Activation Mechanisms of Proto-Oncogenes
1. Translocation or Transposition - Gene moved to new locus, under new controls --> Normal growth-stimulating protein in excess
2. Gene Amplification - Multiple copies of the gene --> Normal growth-stimulating protein in excess
3. Point Mutation within the gene --> Hyperactive or degradation resistant protein
