Flashcards in Lecture 10 - ras-Dependent Signaling Deck (59):
What is the connection between ras-dependent signaling and oncogenes?
A lot of retrovirus oncogenes have downstream ras-dependent signaling pathways
What are the 2 types of oncogenes?
1. v-src: oncogene in a retrovirus
2. c-src: native proto-oncogene in cell
What is a proto-oncogene? What does it code for?
Normal gene that could become an oncogene due to mutations or increased expression. Proto-oncogenes code for proteins that help to regulate cell growth and differentiation.
What is a defective oncogenic virus?
Virus that has lost viral functions and takes advantage of an infection by helper viruses by infecting at the same time and inserting their genome into the helper virus package to propagate
What is Ras?
Monomeric super family of GTPases
What is the function of Ras?
Regulate cell growth through Ser/Thre protein kinases
What are the 5 subfamilies of Ras?
What is the function of Rho?
Reorganizes cytoskeleton through Ser/Thr protein kinases
2 other names for GTPase?
GTP binding protein = G protein
Compare the rate of the ras GTPase activity to that of the G alpha GTPase activity
Compare the rate of the ras GDP dissociation to that of the G alpha GDP dissociation
What % of cancers have activating mutations of c-ras?
How do ras GTPase make up for their slower rate of GTPase activity?
They recruit accessory proteins = GTPase activating proteins (GAPs)
What is the function of GAPs? How do they do it?
They markedly accelerate the intrinsic activity of c-ras by 10^4-10^5 by either activating the GTPase activity or accelerating GDP dissociation
What is the connection between GAPs and trimeric G proteins? What can we call this?
Trimer G proteins' substrates (eg: PLPC-beta) have GAP like activity resident on the G alpha subunit binding site so that when it binds, the substrate is activated but also it triggers the subunit to hydrolyze GTP
ANOTHER built-in OFF switch mechanism
What is the purpose of receptor tyrosine kinases recruiting GAPs to bind on phosphotyrosines?
These receptors are capable of inactivating ras by stimulating GTP hydrolysis
How is ras activated?
GDP dissociation + GTP binding
How is ras inactivated?
What proteins are responsible for activating ras? 3 names
How do they do this?
Guanine nucleotide exchange factor(GEF) = guanine nucleotide releasing protein (GNRP) = guanine nucleotide exchange protein (GNEP)
They markedly accelerate GDP dissociation
What are guanine nucleotide dissociation inhibitors (GDIs)?
They stabilize the c-ras-GDP state, stimulating c-ras inactivation
Inhibition of GAP: ras activation or inactivation?
Activation of GAP: ras activation or inactivation?
Inhibition of GEF: ras activation or inactivation?
Activation of GEF: ras activation or inactivation?
Inhibition of GDI: ras activation or inactivation?
Activation of GDI: ras activation or inactivation?
How is Rho activated/inactivated?
Same mechanisms as for Ras
Where are GEFs found? 2 places
1. Bound to receptor tyrosine kinases via a Grb-2 adaptor protein
2. In the nucleus
What controls if ras is activated or inactivated by receptor tyrosine kinases?
Whether GEFs or GAPs are recruited to bind to receptor tyrosine kinases
Once activated, what does ras do?
Induces the MAP-kinase cascade
Describe the 5 steps of the MAP-kinase cascade.
1. Ras binds MAP-kinase-kinase-kinase and activates PKC to phosphorylate it = activation
2. MAP-kinase-kinase-kinase phosphorylates MAP-kinase-kinase
3. MAP-kinase-kinase phosphorylates MAP-kinase
4. MAP-kinase dissociates from the scaffold to go into the nucleus
5. MAP-kinase phosphorylates substrates
What does MAP stand for?
Mitogen Activated Protein
When is MAP active?
Under conditions of cell division or growth
What are the 2 types of substrates that MAP-kinase phosphorylates?
2. Gene regulatory proteins
What kinds of kinases are MAP kinases?
Ser/Thr EXCEPT for MEK, which is a Tyr kinase
What is another name for MAP kinase?
ERK = Extracellular Regulated Kinase
What is another name for MAP kinase kinase?
What are 2 other names for MAP kinase kinase kinase?
How do the MAP kinases recognize their substrates?
They do not recognize specific sequence but recognize specific conformations
What is PAK kinase?
Does each type of receptor/category of activity have its own MAPK cascade within a cell? Examples? What is a possible reason for this?
Examples: cell cycle arrest, glycerol synthesis, filamentous growth, cell wall remodeling, meiosis, sporulation (all examples in yeast)
Reason: substrates can enter the MAP kinase pathways at different points, so it would be hard to only have one cascade per cell
If one of the MAP kinases in one activity pathway fails, can a MAP kinase from another cascade come save this activity? Why/Why not?
NOPE because these kinases are held together by scaffolds and are functioning like a single unit
What are 4 different types of scaffolds holding MAP kinases cascades together?
What is sevenless?
Mutated membrane protein receptor tyrosine kinase that is activated by "bride of sevenless" and can then be bound by "son of sevenless", a GEF (with a Grb-2 adaptor, of course) which activates ras
What is another name for Grb-2?
Drk = downstream of receptor kinase
What are DUSPs? Function?
Dual-Specificity Protein Phosphatases that dephosphorylate MAP kinases (Ser/Thr OR Tyr)
What are the 2 types of genes activated through ras signaling? 2 names for each
1. Immediate early genes = primary response genes
2. Late response genes = secondary response genes
Timeframe of transcription induction for primary response genes?
Is transcription induction of primary response genes dependent on protein synthesis? What does this mean?
The proteins that are activating these genes already exist and do not need to be synthesized
Is the shutoff of transcription of primary response genes dependent on protein synthesis?
What is the mRNA half-life of primary response genes?
Short: 5-15 min
Over what time frame are secondary response genes expressed?
Is transcription induction of secondary response genes dependent on protein synthesis?
Describe the expression of primary response genes in non-active cells?
Describe the cascade of events that happens once the MAP kinase enters the nucleus.
1. Phosphorylates proteins, which are now activated
2. Activated proteins bind to primary response gene sequences to induce expression (protein synthesis independent)
3. mRNA is produced and then translated into primary response proteins
4a. Proteins are transcription factors that induce the expression of secondary response genes (protein synthesis dependent)
4b. Proteins destabilize the mRNA or turn off expression of the primary response gene (shut off is protein synthesis dependent)
When does the concentration of primary response mRNA drop?
When the concentration of secondary response mRNA rise
What are the 5 kinases that phosphorylate gene regulatory proteins?
How does MAP kinase phosphorylation allow it to dissociate from the scaffold to go into the nucleus?
Phosphorylation reveals its nuclear localization signals