Final Review Flashcards
What are EGFs?
53 amino acid long (relatively small), when purified works in many cell types, EGF binds to EGFR
What are EGFRs?
EGF receptor consists of 2 SEPARATED monomers (same chemical composition) when unbound; each monomer consists of
- extracellular binding site for EGF
2.intracellular tyrosine kinase domain
How were EGFs discovered
- Accidentally discovered as contaminant in salivary gland of male mice when studying NGF (nerve growth factor); lead to premature eyelid opening and tooth eruptions in mice
- V-Src was 1st identified tyrosine kinase, accidentally discovered using Rous Sarcoma Virus probe
C-SRC has 5 parts:
- N-terminal myristoylation site (M)
- SH3 domain
- SH2 domain
- SH1 kinase domain
- C terminal Tail (inhibitory domain)
*V-Src missing part of C-terminal tail leading to constitutive active kinase activity (always on)
*Tyrosine phosphorylation correlates with ability of V-SRC to transform cells
** EGFR cytoplasmic domain has region of homology with SRC SH1 (kinase domain)
- V-Sis discovered to be an autocrine platelet-derived growth factor (PDGF); therefore the cell makes its own receptor and growth factor leading to autonomous cell proliferation
What happens when EGF binds to EGFR, what happens when EGF is NOT bound to EGFR?
When EGF is NOT PRESENT, EGFR remains monomeric (2 subunits)
When 2 EGF is PRESENT it binds to EGFR, inducing dimerization of receptor leading to the phosphorylation of the carboxyl-terminal tails on the intracellular domain
*cross-phosphorylation creates attachment point for OTHER proteins part of EGF signal transduction pathaway
What is the transduction pathway? (8)
- 2 EGF’s binds to 2 monomer -extracellular binding domains of EGFR; leads to dimerization and phosphorylation of EGFR
- Phosphorylation of C-terminal tail on intracellular side serves as anchor for Grb-2 protein
- Grb-2 recruits Sos protein
- Sos recruits Ras, a small g protein, which expels GDP and recruits GTP activating it
- Activated Ras then activates Raf protein kinase
- Raf activates MEKs protein kinase
- MEKs activate ERKs (extracellular signal-regulated kinases
- Activated/Phosphorylated ERKs move into nucleus from cytosol and stimulate transcription factors to increase gene expression, this increases rate of protein synthesis leading to enlargement of cytoskeleton and thus cell growth
*Erk phoshorylate ETs (erythroblast transformation specific) and other Transcription Factors proto-oncoprroteins to cause immediate early gene induction t cause cell proliferation
What does Viral-ErbB have missing?
V-ErbB is missing segment in ectodomain/extracellular EGF binding domain therefore leading to it being constitutively active + activating growth singals without need for ligand binding
What are the different mechanisms for constitutive activation of EGFR in cancer?
- Over-expression
- Mutations that change structure of receptor
*both lead to ligand-independent firing, no need for ligand to bind to activate growth factor
What are the EGFR family members and how are they targeted by anti-cancer drugs
- EGFR=HER1=ErbB1
- HER2=ErbB2
- HER3=ErbB3
- HER4=ErbB4
*EGFR and HER2 are most commonly mutated receptors, don’t see as much HER3 or HER4
*ligands (what binds to receptors) can be shared among RTKs
*RTKs can form heterodimers with one another
What are the subdomains of EGFR and how do they specifically bind in presence of a ligand (EGF)?
There are 4 subdomains of EGF, ligand domain on I and III but unable to be reached by ligand in ABSENCE of EGF since II and IV are folded; when EGF is PRESENT II and IV unfold revealing ligand domain I and III
What are the 4 EGFR activating alterations in human cancer and what do they lead to?
- Amplification
- Insertion
- Deletion
- Point Mutations
*these alterations lead to either 1) destabilization of inactive conformation or 2) stabilization of active conformation or
3) both
what is -nib?
kinase inhibitor
What is -tinib?
tyrosine kinase inhibitor
What is -mab
monoclonal antibody
How are growth factors hijacked in cancer?
constitutive activation of RTKs and production of autocrine growth factors are common in human malignancies
What is an autocrine growth factor?
same cell makes own receptor and growth factor leading to autonomous cell proliferation; an example would be V-sis and how it makes autocrine platelet derived growth factor (PDGF)
What are tyrosine kinases?
tyrosine kinases are enzymes that selectively phosphorylate tyrosine residues in different substrates
What is Grb2? What does it do? How does it lead to Ras activation?
-Growth Factor Receptor Bound protein 2
Grb2 is an adaptor protein that serves as the “glue” for other proteins but does not have any enzymatic activity
-Grb2 has SH2 domain (+ some SH3) which is necessary and sufficient to bind pY within EGFR C-terminal, as it brings Grb2 to receptor C-terminal to cause downstream signal to activate proteins
What is SH2 domain function?
-different SH2 domains bind to different pY residues (tyrosine phosphorylated residue) and is dictated by the 3 amino acid residues following the pY sites which dictate the specificity of SH2 domain binding
-multiple SH2-containing proteins associate with specific pY residues of activated EGFR terminus (form of signal amplification) some are enzymes like kinases, PCC (phospholipase C), phosphates while others are adaptors like Grb2
What is the significance of Ras in cancer and how is it mutated?
3 Ras proteins (H, K, N Ras) which are frequently mutated at:
- Glycine 12 (G12)
- Glycine 13 (G13)
- Glutamine 61 (Q61)
*therefore when turned into different amino acid=oncogene
*Grb2 recruits SOS/GEF to activate Ras (small G protein) via stimulating GTP binding + expelling GDP
*Gap inactivates Ras by promoting GTP hydrolysis, i.e. expel GTP and GDP binds to Ras
*Therefore activating Ras point mutations keep Ras proteins in bound, active form thus going through transduction pathway indefinitely
What is the Ras cycle?
-GTP binding induced by SOS/GEF activates Ras
-GTPase activity stimulated by GAP inactivates Ras via hydrolysis reaction
What is the structure of Ras and how does it bind to GTP? GDP?
-H-Ras structure has guanine nucleotide binding domain
-G1, G2, G3… contacts GTP/GFP and binding affinity very high so once bound Ras does not let go
-Ras cannot distinguish between GTP/GDP
What do mutant Ras do?
-K-Ras activation mutants have 1) reduced intrinsic and 2) GAP stimulated GTPase activites
-therefore, change into an other amino acid reesults in partial loss of GDP activity and decrease of both basal rate and GAP
-similar findings found in H-Ras
How does Ras hot-spot mutations affect intrinsic GTPase activity?
-Q61 (glutamine 61) is involved in catalysis and mutation BLOCKS this
-mutation of G12 sterically hinders Q61-mediated catalysis
How does Ras hot-spot mutations affect GAP-induced activity?
-G12 and Q61 interacts with GAPs “Arg Finger” which greatly augments GTP hydrolysis
-mutations in G12 and G13 (lies next to G12) cause steric hindrance for GAP and Q61 mediated catalysis resulting in glutamine being unable to function
How does Ras induce RTK signaling downstream?
-Raf:a Ser/Thr kinase acts DOWNSTREAM of Ras
-evolutionary conserved “RTK-Grb2-Sos/GEF-Ras-Raf-MEK-ERK” pathaway
How does Ras-GTP bind to Raf and activate it?
-Ras GTP binds Raf and activates it via autophosphorylation
switch 1 mediates Ras-GTP interaction with downstream effector proteins
-switch 2 critical for interactions w/ GEF (For Ras-GTP) and GAP (for Ras-GDP)
Why can kinase cascade cause signal amplification?
because a single active enzyme can phosphorylate multiple downstream kinase or substrates
ex:ERK which is final kinase transcription factor goes to nucleus and binds DNA turning it ON or OFF
How does ERK cause proliferation?
ERKs are the final kinase transcription factor which go directly to the nucleus and activate transcription factors and ETs (erythroblast transformation specific)” a transcription factor that acts downstream of ERK via phosphorylation
What do oncoprotein do?
oncoprotein constitutively activate downstream signaling pathways and ignore upstream thereby contributing to uncontrolled cell proliferation
What are pathways and oncoprotein importance in human cancer?
- many ways which cancer arises
- oncoprotein aberrantly induce signaling pathways
-specificity of signaling pathways often depend on conditional (or regulated) protein-protein interaction
-oncoprotein can cause multiple downstream effects including deregulation of cell prolif, cell death, etc
-critical steps in oncogenic signaling pathways are targeted by precision cancer therapies
If you activate Raf, MEK, or ERK (part of MAPK cascade) is it sufficient to induce proliferation?
Yes, rescue proliferation defect, but other known Ras downstream effectors like P13K could not be rescued
What are immediate early genes (IEGs)?
-Include transcription factor (Tf oncoprotein)
-immediate early genes include Fos, Myc, Jun
What is multistep tumorigenesis? (Overview 4)
tumorigenesis is driven by series of mutations and epigenetic changes in DNA that affect genes controlling cell proliferation, cell survival, genome stability, and other processes
1. Process of tumor formation is complex involving multiple molecular changes
2. Molecular changes involve the inactivation of tumor suppressor genes and activation of oncogenes
3. # of molecular changes that occur in genome human cancer cells far exceeds the number REQUIRED for tumorigenesis to reach completion, complicating identifying the critical alterations
4. Tumor heterogeneity arises as consequence of hi mutation rates coupled with selection
Why do most cancers take decades to form?
age of diagnosis most prevalent in late 60s and early 80s; environmental factors play role, for example incident of lung cancer increased decades AFTER cigarette smoking became popular
What is the number of rate-limiting steps needed to drive tumorigenesis?
number of rate-limiting alterations required for cancer to form-6 rate limiting steps
What is the crypt in colon cells look like and how does it function?
colon cancer is 2nd most common cancer
-role of colon is to reabsorb water lined with crypts, cells produced in crypt around 200-300 cells
-every 3 to 5 days epithelial lining replaced as protective measure
-composed of 4 cell types + myofibroblast which surround crypt
1. Enterocytes
2. Globlet cells
3. TA cells (transient-amplifying cell)
4. Stem cells
-Stem cells divide asymetrically, some move up some don’t
What is loss of APC and significance initiating tumorigenesis?
-sequences of molecular changes vary but loss of APC/mutation usually early event, leading to tumor progression
-APC is a gatekeeper gene, highly conserved in Mice
-once lost often see formation of adenomas
-loss of APC and activation of KRas leads to metastatic colon cancer= alterations in gatekeepers (APC) and caretakers (Kras) needed for cancers to form
*some molecular changes mutually exclusive but highly variable with number and order of mutations
What are the two models for tumorigenesis, and what is the main theory now?
- Clonal Sweep: accumulation of all mutations had advantage versus some just some mutations; would see sequential mutation; mutations that confer selective advantage overtake bulk of tumor
ex: APC and KRas mutations have advantage over just tumors with APC mutant - Big Bang (main theory): lots of mutations occur early on, then coevolve meaning mutations do NOT compete with each other; so final tumor composed of mixture of distinct clonal populations
-see cancers that are heterozygous meaning not composed of just 1 cancer cell but multiple with different mutations/genetic makeup–experimentally validated
How was the big bang model studied?
-to determine tumorigenesis process can preform hi-resolution sequencing
-do left and right part of tumor have same or different mutations?
-found tumors OFTEN highly heterogenous meaning big bang (mutations occurred early on) so public mutations (present in entire tumor) and private mutations (happen on other side)
What is Wnt signaling and what is the significance?
-Wnt signaling leads to the accumulation of beta-catinin, but if APC present, B-catenin gets phosphorylated and degraded; localized to the cytoplasm
-APC present, normal Wnt is OFF=target gene suppressed
-APC absent/mutated, cancer Wnt ON= target gene activated; localizes to the nucleus
What is the difference between drivers (gatekeepers) vs passengers (caretakers)?
driver mutations are mutations present in MOST tumor cells, while passenger mutations are in SOME but not ALL tumor cells
What evidence suggests that cancer is a multi-step process?
-Histopathology (from normal tissue to carcinoma)
- Late onset of lung cancer in smokers
- Tumors can be found in mixtures of adenomas and carcinomas
- Molecular changes coupled to tumor progression (Loss of APC and activation of KRAS lead to metastatic colon cancer together, but not alone.)
- *Mutations in RAS and p53 Drive Skin Carcinogenesis (from painting example)
-Mutations in PIK3CA or KRAS alone were shown to be not sufficient to cause cancer
Multi-step process doesn’t always occur in the same sequence. What are the changes?
-sequences of molecular changes vary, however loss of APC/mutation USUALLY early event leading to tumor progression, but tumors can be initiated by mechanisms other than loss of APC
-some molecular changes mutually exclusive
-molecular changes are numerous
What is an initiator? Examples?
mutagenic-like, example:BP, DMBA
What is a promoter? Examples?
non-mutagenic, when coupled with an initiation can contribute to tumorigenesis ex:TPA, known skin irritant
