Lecture 2: Signal Transduction Pathways Flashcards
How would you define “Signal Transduction”?
= a series of processes, by which a (chemical or physical) signal is transmitted through a cell by a series of molecular events
NOTE:
- in the extracellular side we have receptors triggering a response (e.g. hormones, light..) -> starting the signal -> changes in gene transcription
What are the common features of signal transduction pathways?
- chemical or physical signal is transmitter through the cell
- result = cellular response
- crosstalking signalling pathways integrate various signals and coordinate/modulate response
-there need to be integratory signals - signal gets amplified
- signalling has to be switched off at some point (negative feedback)
Whatare the general functions of MAP-Kinase pathway? Explain the steps of this pathway.
- Functions: proliferation, growth, survival
1) Growth Factor (GF) induces dimerization
2) Autophosphorylation of tyrosine -> passing on signal
3) Conformational change allow binding of Grb2 = adaptor protein (to tyrosine) and Sos (to Grb2)
4) Conformational change in Grb2 and Sos
5) Sos acts on Ras GDP -> kick out GDP and reload with GTP
6) Conformation change in Ras -> Interaction with Raf Kinase
7) Raf phosphorylates Kinase MEK
8) MEK phosphorylates Kinase ERK -> translocation to nucleus
9) Changes in transcription of genes
What types do we have under “receptor tyrosine kinase”?
- Specificity for a certain signal
- Linked to enzymatic domain -> can pass the signal to the next effector molecules
Types:
1) Receptor tyrosine kinase
2) Tyrosine kinase-associated receptors
3) Receptor tyrosine phosphatases
4) Transmembrane serine/threonine kinases
5) Transmembrane guanyl cyclase - involved in production of cGMP
NOTE - picture
- Signal detection domain
- Transmembrane domain
- Kinase/enzyme domain
How do RTKs look like from structural stand of point -> what happens if growth hormone comes in?
1) and 2) are composed of 2 beta sheets, the gap/loops in between interacts with the growth hormone
Two RTKs are binding the same hormone -> brings them close in proximity -> dimerization
How does phosphorylation work?
Kinase has ATP bound as a cofactor -> converts ATP to ADP -> the phosphate group gets bound to the tyrosine -> becomes negatively charged (in comparison to regular tyrosine which is more hydrophobic)
- helps molecules to recognize the site
So how does the Kinase basic cycle looks like?
Kinase binds ATP -> substrate binds to phosphorylated Kinase -> phosphoryl transfer to the protein substrate -> releases substrate -> releases ADP
=> cycle repeats
What can you say about the structure of RTKs?
- Active side of a kinase is in between two lobes N-lobe (smaller) and C-lobe (bigger) connected by a flexible Hinge-region
- H-r allows relative lobe orientation and dynamic cycle of nucleotide binding, hydrolysis and release
- Activation-loop is located in C-lobe and contains DFG-motif (Asp-Phe-Gly) plus one or more activating phosphorylation sites
NOTE: picture also shows ATP bound, stabilized by Mg
What are the structural changes between “active” and “inactive” states?
Active (phosphorylated) state = extended activation loop -> stabilizes kinase substrate binding
- F points outwards
- D (Asp) points inwards to coordinate Mg that can hold the ATP
Inactive state = D and F switch positions e.g. D facing out -> DFG-out state = prevents nucleotide binding
- Together with these conformational changes, we also see changes in alpha C helix, helps stabilize inactive Kinase (blue)
How can we inhibit Tyrosine Kinase? How come it doesn’t work in some patients?
Imatinib (yellow) = tyrosine kinase inhibitor, used for certain kinase involved in leukemia
- close to the ATP site in a specific kinase
-> keeps it in semi-activated state, but no substrate can bind => inactive, cannot act
HOWEVER some patients can have a “gatekeeper mutation” in Thr315lle (blue) -> kicks imatinib out -> inhibition no longer works -> tumor can continue growing
Just look at the following picture:
The Ras - has many different subfamilies
Why is Ras called “the heart of signal transduction”?
- 3D conformation approximates the shape of heart
- The whole sequence is displayed in the picture above
Ras comes in different isomers - how are they similar? How do they differ?
All Ras family members the G-domain (also have Caax motive) is the same -> difference comes from C-terminal region consisting of Linker and Anchor
- Differs in a way that these proteins are linked to the plasma region => H-ras, N- ras, K0ras
How can the different Ras isomers be anchored?
Isomers can have anchors stuck to cysteibe:
- Farnesylated C-term cysteine (F)
- Palmotoylated C-term cysteine (P)
- And in one case (K-RasB) also positively charged Lys-stretch (the sequence of K)(K)
=> Based on the number and composition we can infer how stable it is within the membrane
- H-Ras seems more stable than N-Ras
Why is it incorrect to call Ras a GTPases?
Because it is not really an enzyme - it doesn’t catalyze the conversion of GDP into GTP
- It is pretty lausy, there is low frequency of hydrolysis happening (thus if put into a solution with GDP and left alone -> not much would change over time)
What 2 states of Ras do we have? What in general happens to swuths between them
Two completely different states = molecular switch
- OFF state = no signalling, GDP bound
- ON state = signalling, GTP bound
In OFF state we cannot actually convert GDP into GTP by adding phosphate -> we have to kick GDP out and replace it with new GTP = ON state -> downstream effects a.f. Raf Kinase -> cellular response
-NOTE: works as an enzyme when hydrolyzin GTP to remove Pi, but it cannot by itself switch from OFF to ON
- Sos helps with that
Look at the differences in the states:
Why do we call Ras lousy? How does it compensate for it
Because the intrinsic GTPase activity is very, very low -> it needs help with conducting the chemical reactions
- Auxillary protein GEF (Guanine Nucleotide Exchange Factor) helpts with GDP-> GTP
- Auxillary proteins GAP (GTPase Activating Proteins) help with GTP -> GDP
How does GAP work?
- Auxillary proteins GAP (GTPase Activating Proteins) help with GTP -> GDP
- brings in argenine to the active sites of Ras (Arg finger positions itself against triphosphate) -> GTP is stretched in a way that makes it vulnerable to be “attached” by water (attacks gamma phosphate)
-> Switch regions get released to the sides
How does GEF work?
- GEF binds -> hinders the binding site for Mg2+ -> P-loop is displaced, cannot properly hold phosphate groups -> new GTP comes in and displayes the older GDP -> kicks out GDP, GTP binds (high amount in the cytosol)
What is next?
Downstream Effectors
-> Raf Kinase - close to plasma membrane -> gets phosphorylated -> can go and phosphorylate all kinds of other Kinases
-> MEK
-> ERK
-> nucleus
What can you say about MAP-ERK Kinases?
= mitogen activating pathway (MAP) that ends with ERK
- initially found when studying mitosis
- All are Ser-Thr Kinases activating themselves in a subsequent manner
=> building multiplayer regulation network itergrating several signals (intereference with other pathways can occur) of crosstalking signal pathways
So what is the last step of this whole pathway?
ERK enters nucleus -> acts on a huge machinery of gene transcription -> determines what proteins will get made
- Important: specificity for target genes and their transcription complexes, tissue specificity (which cells are we looking at?)
How do we switch this off? Can you think of a mutation that involves this pathway?
Switching off - connecting to Ras hydrolyzing GTP to GDP
-> BUT mutation can occur
- G12V = most common mutation
- shown in 70% of cancers
- Glycine 12 mutates to Valine -> there wouldn’t be enough space for catalytic water, plus valine = hydrophobic -> catalytic water has no way of coming in -> GTP won’t be converted into GDP => Ras is constantly ON
