Lecture 10 Flashcards
What triggers the phosphorylation of RyR2 (ryanodine receptor) by PKA
The initial cause is a stressor (adrenaline, epinephrine)
The signalling events activate adenylyl cyclase (a membrane protein)
Adenylyl cyclase converts ATP to cAMP
What does cAMP do
It activates PKA by binding to its regulatory subunit that stops it from being active
After binding to it, the PKA is active and phosphorylates RYR
What does activating the RYR do
In the SR lumen and it triggers heart rate to increase
What does CaMKII do
Same as PKA, phosphorylates ryr2 but needs ca-can to activate it first
What is the phosphorylation target site on the RYR receptor for camKII
A serine residue
What is a phospomimetic mutation
Give an example of how this applied to RYR receptor
Converting a residue to another residue that mimics a phosphate (has a negative charge)
The serine target in the receptor can be replaced by aspartate to mic is being phosphorylated
What is the result of a phosphomimetic mutation in RYR and what does this tell us
The activity of PKA increases once’s the seine looks phosphorylated
And the disordered region of the RYR becomes alpha helical
This tells us that kinases have cross talk where they indirectly influence each other (the phosphorylation of RYR made PKA increase)
Slide 3
How is the phosophomimetic mutation of ser to asp not perfect
Because phosphate has -2 charge
Asp has -1
Can proteins be targeted by more than one kinase
Yes and the different kiansss have their own recognition sites for the protein
Give an example of something that’s targeted by many kinases and what kinases target it
Caldesmon
By PKA, PKC, cam k1/II, casien kinase II
What is caldesmon
A smooth muscle contraction protein
But not targeted by myosin kinases
What do protein phosphotase do
Remove phosphate groups from ser, thr, or tyr side chains
What do protein phosphotase generally have in them
One type Dependent on metal ions (like calcineurin) so have two metal ions (fe/mn/zn/mg)
But the mg is less common
One types have A cysteine residue in the active site
What are the two types of protein tyrosine kinases?
Receptor (RTK)
Non receptor (TK)
What do Receptor tyrosine kinases need to be active
What is their structure
They need to dimerize to be active
The kinase domain is in the cytosol and very similar across all types of RTK
the head is extracellular and different across all types of RTK (interacts with diff proteins)
What do Non receptor tyrosine kinases have
They’re attached to the membrane via palmitoyl or myristoyl
Intercellular
(lipid groups that get added to the proteins and insert into the membrane)
Different rtk’s respond to
Different stimuli (which is why the extracellular domains are all different)
Explain the dimerization process of RTK
Before it’s signal binds, it’s two domains are separate and inactive
Dimerizes once it ligand binds and gets activated
The kinase domain (in cytosol) phosphorylates itself
The Diff proteins with sh2 domains dock and bind to these phosphorylated sites
What is the kinase activation cascade
After the sh2 protiens dock at the kinase domain of RTK, signalling MAPK pathway happens
Does the RTK add a phosphoryl or phosphate group
Phosphoryl
What does the kinase domain of RTK look like
Has a beta sheet heavy region
How does the src family non receptor tyrosine protein kinase work
N-SH3-SH2-kinase-C
The sh3 domain binds to two prolines (pxxp) motif on the inactive kinase
The sh2 binds to a phosphotyrosine on the c terminus
A phosphotase removes the tyrosines phosphate, sh2 domain detaches
The kinase auto phosphorylates itself and becomes active
Slide 12
What do we need to know
What amino acids do sh2 domains recingnize
Phospotyrosine residues
What amino acids do sh2 domains recingnize
Phospotyrosine residues
How does the sh2 binding pocket form
So it’s bind phosphorylated tyr
Inside the pocket is postively charged to bind the negative phosphate on the phosphotyrosine
The pocket is also hydrophobic since the tyr is hydrophobic because of its aromatic ring
How many negative charges on phosphotyrosine at physiological ph
Two
What are scaffold protiens and give examples
They organize the positions of other proteins and create functional protein networks
They act as scaffolds for other proteins to bind
Ex. Shank, PSD-95, Grip
What domains are in the shank1 protein
Ankyrin
SH3
PDZ
Proline rich region (recognized by sh3 domains)
SAM
What are the two types of sequences that SH3 domains bind to
Class 1 from the C-N of the sh3 domain:
Pxxp
Class 2 From the N-C of the sh3 domain: xpxxp
What sites of proteins does the pdz domain in shank 1 bind
Binds the c teminal domain of other proteins
How many pdz domains does shank have
What about grip
1
Multiple
What are ankyrin repeats
2-3 or more repeats of an anchor to bind other proteins
By multiple of the repeats together, they form loops that bind to protiens
How many ankyrin repeats does shank 1 have
7
What does a single ankyrin domain look like
1 domain without a loop
What is the SAM domain in shank 1
Interacts with itself to form dimers and oligomers
This means it can build a network of shank proteins
What are all of the proteins interaction domains
SH3
SH2
C1
C2
PDZ
SAM
ANK repeat
