Test 3: 44-45 Flashcards
Your patient has a single amino acid change in the Ras gene. This mutation leads to a reduction in the GTPase function of Ras. Is this a problem? What disease does your patient most likely suffer from?
Cancer: Ras is mutated in 20-30% of all cancers
Your canine patient has prostatic carcinoma. From the deep recesses of your vet school memory, you decide to sequence the B-RAF gene exon 15. What are you looking for?
A point mutation causing a change from valine to
glutamic acid at amino acid 450
Your patient has pulmonary valve stenosis and atrial septal defects and hypertrophic cardiomyopathy, short stature, learning problems, impaired blood clotting, and a characteristic configuration of facial features including a webbed neck and a flat nose bridge.
Noonan Disease: Mutation in the Sos gene
Your patient is an 11-year-old spayed female mongrel dog weighing 14.1 kg and with bilateral mandibular lymph node metastases from a mast cell tumor. The primary tumor on the muzzle had been completely resected by the referring veterinarian 1 month previously. Initial examination revealed bilateral enlarged mandibular lymph nodes measuring 2.2 × 1.8 × 1.5 cm on the left and approximately 1 cm on the right. No relapse was detected on the muzzle. Cytological examination of the enlarged mandibular lymph nodes by aspiration biopsy showed metastatic tumor cells. Although chemotherapy was initiated with concurrent administration of intravenous vinblastine at 2 mg/m2 weekly and oral prednisolone at 10–15 mg/day, the lymph node lesions progressed rapidly. The tumor sample was subjected to DNA sequence analysis. Mutations in c-kit Exon 11 were found. What will you do?
Tr eat with imatinib
What are some growth factors?
Small Peptides (EGF, TGFa, TGFb, FGF)
Insulin (Insulin-like growth factors)
Steroids (dexamethasone, hydrocortisone)
what are some functions of growth factors
Cell Proliferation
Cellular Differentiation
Extracellular Matrix Formation
Cell Secretion
Cell Motility
Morphogens during Development
when do growth factors function
will trigger a cell in G0 or G1 phase and trigger cell to go through one round of cell cycle (proliferation and division)
How do growth factors work in general?
GF binds to type of receptor with an intercellular and extracellular component
these receptors live on cell surface and will convert extracellular contact to intracellular signal
The receptor-growth factor complex does not need to come into the cell to work
3 types of growth factor receptors
tyrosine kinase (sometimes serine)
7 alpha helical (7TM, GPCR)
steroid receptors
RTK pathway overview
RTK: tyrosine kinase
adapter protein
monomeric G proteins
serine/threonine kinases
transcription factors
gene regulation
7 alpha helical pathway overview
7 alpha helical (GPCR)
trimeric G proteins
cyclic nucleotides
membrane channels
membrane signals
gene regulation
Binding of growth factor to receptor causes
Binding to its Receptor
Often results in receptor dimerization
Causes conformational change in the receptor
This change leads to kinase activation
Receptor cytoplasmic domain is phosphorylated
Now the internal cytoplasmic domain must interact with other proteins to send the signal
what protein interacts with the activated RTK receptor
RTK binds with growth factor, dimerizes, changes shape and becomes phosphorylated
GRB2 binds (type of adapter protein)
protein domains
SH2, PTB
interact with phosphotyrosine (p-Tyr)
which protein domain interacts with p-Tyr
p-Tyr= phosphotyrosine
SH2, PTB
protein domains
SH3, WW
interact with certain configurations of proline
what type of protein domains interact with interact with certain configurations of proline
SH3 and WW
protein domains
PDZ
hydrophobic interactions
what type of protein domains interact with hydrophobic interactions
PDZ
protein domain
PH
FYVE
interact with phospholipid interactions
what type of protein domains interact with interact with phospholipid interactions
PH FYVE
explain how GRB2 works
adapter protein with 2 SH3 and 1 SH2 domains, loosely binded to Sos
SH2 domain will attach to p-Tyr= phosphotyrosine
SH3 domains bind to proline
once SH2 binds to pTyr of receptor GRB2 will strongly bind with Sos
what is Sos
guanine nucleotide releasing protein
Loosely attached to GRB2 in RTK (tyrosine kinase receptor)
becomes strongly attached to GRB2 when GRB2 SH2 protein domain binds to the pTyr on the RTK, this changes GRB2 and now strongly binds to Sos at GRB2 SH3 protein domain for prolines
Sos will make GDP → GTP (will let go of one guanine)
Ras will bind to GTP and becomes active
this activation of Ras will stimulate kinase cascade which will make cell divide
RAS activates what
RAF→ MEK→ MAPK/ERK
MAPK/ ERK leads to :
nucleotide synthesis, gene expression, protein synthesis, cell growth→ cell growth
MAPK leads to :
nucleotide synthesis, gene expression, protein synthesis, cell growth
MAPK is created by
active RAS→ RAF→ MEK→ MAPK
Growth factor also stimulate phospholipids by
PI3K
Phosphoinositide 3-kinases
used to make phospholipids which are used to activate kinase by unmasking kinase domain or change confirmation of kinase domain
full pathway of RTK
receptor tyrosine kinase
growth factor attaches to receptor, receptor dimerizes and phosphorylated
GRB2 binds to p-Tyr of receptor, which stimulated GRB2 to bind strongly to prolines of Sos, which will kick a guanine off GDP to create GTP which will active Ras
Ras will trigger kinase cascade
Ras→ Raf→ Mek→ MAPK = cell growth
how to deactivate MAPK
MKP-1 (MAP Kinase Phosphatase)
how to deactivate RAF → MEK
RKIP (Raf Kinase Inhibitor Protein: disrupts Raf-MEK interaction)
how to deactivate RAS
Ras GTPase Activity
how to stop RTK
MKP-1 (MAP Kinase Phosphatase)
RKIP (Raf Kinase Inhibitor Protein: disrupts Raf-MEK interaction)
Ras GTPase Activity
GAP (GTPase Activating Protein)
Receptor Phosphatases
Receptor Degradation or Recycling
mutation in GAP can create ___
neurofibromatosis
Ras can deactivate, always on, always sending signal to divide
mutations in Ras lead to ___ % of cancers
20-30%
Ras stays active, cell will continuously grow and divide
Your patient has pulmonary valve stenosis and atrial septal defects and hypertrophic cardiomyopathy, short stature, learning problems, impaired blood clotting, and a characteristic configuration of facial features including a webbed neck and a flat nose bridge.
Noonan Disease: Mutation in the Sos or Raf genes. Proteins stay active longer than normal disrupting normal development.
Noonan Disease:
Mutation in the Sos or Raf genes. Proteins stay active longer than normal disrupting normal development.
Your patient has pulmonary valve stenosis and atrial septal defects and hypertrophic cardiomyopathy, short stature, learning problems, impaired blood clotting, and a characteristic configuration of facial features including a webbed neck and a flat nose bridge.
Your patient has a single amino acid change in the Ras gene. This mutation leads to a reduction in the GTPase function of Ras. Is this a problem? What disease does you patient most likely suffer from?
Cancer: Ras is mutated in 20-30% of all cancers
Your canine patient has prostatic carcinoma. From the deep recesses of your vet school memory, you decide to sequence the BRAF gene exon 15. What are you looking for?
A point mutation causing a change from valine to glutamic acid at amino acid 450
80% of patients will have this mutation
Can treat with imatinib?
Ras→ RAF
A point mutation causing a change from valine to glutamic acid at amino acid 450 causes what? and why
mutation in BRAF which leads to prostatic carcinoma
what is an alternate way to activate RAS pathway instead of RTK
integrins interact with fibronectin
Jak STAT pathway
cytokines bind to Jak, Jak phosphorylates transcription factors(STAT) that sit in the cytoplasm, these will dimerize and go into nucleus and turn on genes needed for proliferation
Jak: Janus Kinase (Tyrosine kinase)
STAT: Signal Transducer of Activated Transcription
The Jak-STAT pathway is disrupted in many hemapotoietic cancers
The ___ pathway is disrupted in many hemapotoietic cancers
Jak-STAT
mutation in ___ which leads to prostatic carcinoma
BRAF
TGF beta SMAD pathway
TGFb: Transforming Growth Factor Beta
SMAD: Mothers Against dpp
TGF beta binds to TGFbeta receptors, this causes kinase activity which will phosphorylate SMAD proteins, which will hetrodimerize and enter nucleus
___ hold kinase in place/ together
scaffold proteins help with amplification and efficiency
receptor tyrosine kinase has three parts
outside of cell-where growth factors bind
transmembrane
intracellular- conformational change which activates tyrosine kinase protein which leads to phosphorylation of tyrosine
explain RTK
growth factors binds
transmembrane
conformational change which activates tyrosine kinase protein which leads to phosphorylation of tyrosine
adapter protein GRB2 sees this pTyr and binds
GRB2 is weakly bound to SOS until it binds to pTyr then strongly binds
SOS forces GDP to GTP which activates Ras
Ras→ Raf→ Mek→ MAPK→ (will phosphorylate proteins that lead to gene expression)
Raf→ Mek→ MAPK are all ___
serine/threonine kinase
will give phosphate to eachother
RAS binds to cell membrane with the help of ___
Farnesyl
and Farnesyl Transferase
Farnesyl is ___ and likes to be where in the cell
hydrophobic, inside the cell membrane
will bind to RAS and drag it to cell membrane where it can be activated and trigger
RAS→ RAF→ MEK→MAPK
how to stop Ras from binding to Farnesyl
CAAX
4 amino acid inhibitor protein
cistine - 2 hydrophobic amino acids and any amino acid
how to kill mutated RAS
trigger RAS into inactive RAS
then inhibit SOS from activating iRAS back into aRAS
what drug to to stop kinase
Gleevec (Imatinib)
The tumor sample was subjected to DNA sequence analysis. Mutations in c-kit Exon 11 were found. What will you do?
Treat with imatinib (gleevac)
stops kinase activity
explain angiogenesis therapy for cancer
kill healthy endothelial cells that provide blood supply to cancer
no blood supply cancer will shrink, if it regrows can treat again
effective but doesn’t help long term lifespan. leads to increase in metastasis to area with healthy blood supply
3 steps of wound healing
Inflammation
Proliferative Granulation tissue formation
Matrix formation and remodeling
Thrombin causes release of ___ from platelet alpha granules.
TGF-b, PDGF, FGF
chemofactors that attract fibroblasts, macrophages, neutrophils, and keratinocytes
Thrombin causes the release of chemofactors such as TGF-b, PDGF, FGF that attract ___
fibroblasts, macrophages, neutrophils, and keratinocytes
Cell migration during wound healing cause the release of ___ from the extracellular matrix
growth factors
___ begin to remove debris.
Macrophages
granulation of tissue during wound healing :
- Dense population of fibroblasts, macrophages, and neovasculature in loose matrix of collagen, fibronectin and hyaluronic acid.
- TGF-b increases expression of genes needed for extracellular matrix formation.
- TGF-b and PDGF attract fibroblasts to wound site. They become myofibroblasts which aid in wound closure.
TGF-b and PDGF attract fibroblasts to wound site. They become ___ which aid in wound closure.
myofibroblasts (will squeeze wound shut)
Matrix formation and remodeling during wound healing:
- Gradual dissolution of granulation tissue.
- Formation of scar tissue.
- Scar not as good as normal tissue.
if you add ___ to surface wounds and burns, would healing will increase 2 fold
EGF
if you add ___ and ___ to incisions wound healing will improve
TGFB and PDGF
why don’t fetuses scar
decrease in TFG beta = slower healing, normal skin forms instead of scar
___ can be added to help with impaired wound healing such as chemotherapy
TGF beta, PDGF, EGF
__ or __ can be used to improve angiogenesis
TGF beta and FGF
VEGF
when stimulated a 7 alpha helical transmembrane receptor will most likely interact with ___
trimeric G protein
You patient has a defect in the GTPase activating protein (GAP). What will be the most likely affect?
Ras will remain in the GTP bound state
Why does RAS associated with the cell membrane
post translational farnesylation
When a growth factor binds to its receptor, what usually occurs on its internal cytoplasmic domain?
specific tyrosines are phosphorylated
As a clinician, you have drugs that specifically inhibit the function of GRB2, RAS, growth factor receptor, SOS and MAP kinase. You patient has a defect in Raf that causes it to constitutively active. What drug do you use?
MAP kinase inhibitor
Why is gleevac a useful drug for treatment of some malignancies?
it blocks the ATP binding sit on the Abl kinase
