Protein Kinases Flashcards
What three amino acid residues are preferred for phosphorylation in eukaryotes and why?
Through what type of reaction does a protein kinase catalyse?
What is the opposite reaction & what enzyme mediate this?
What helps to stabilise the purine ring of ATP in phosphoryl transfer?
What 3 structural parameters of protein kinase helps assist the gamma phosphate in the correct orientation for transfer?
What 2 structural parameters stabilises the gamma phosphate in transfer?
Serine, threonine, tyrosine - OH group
Condensation reaction
Hydrolysis - phosphatases
Purine-binding pocket
P-loop/glycine-rich loop, Mg2+ and a second divalent cation
Positively charged amino acid (lysine) residues & positive end of alpha-helix dipole
How do Mg2+ and a second divalent cation aid gamma phosphate transfer?
How does a water molecule act as a structural parameter in the transfer of phosphate in ATP?
Why is there a bias for phosphotyrosine residues in literature?
What kind of structure does PKA have?
What does it consist of?
How does cAMP activate PKA?
In what mechanism does cAMP bind?
Coordinates beta & gamma phosphate in high energy eclipsed conformation - increases electrostatic repulsion & electrophilicity of gamma phosphate
Completes coordination with metal cations & helps anchor protein
Better tools & easier to find due to 0.01% of all phosphoresidues - larger signal against noise
Heterotetramer - 4 subunits
Mixed alpha-beta
Binds to 2 binding sites on each regulatory subunit - once 4 cAMP binds the protein dissociates into a dimer of regulatory subunits and 2x monomers of catalytic subunits
Cooperative allosterically - 1 cAMP binds increasing the affinity for the other binding sites
What are the two lobes in PKA involved in and where are they located?
What are the 2 other sites?
What is between the 2 lobes?
What other feature of PKA is there?
What are the 5 helices/loops involved in PKA?
What is the rate dependent step of the PKA cycle?
Small lobe (top) in ATP-binding
Large lobe (bottom) in peptide binding
Phosphorylation site - for own activation
Peptide-binding site - to phosphorylate
Linker
C-terminal tail
Glycine-rich loop, catalytic loop, activation loop, magnesium loop, C-helix
ADP dissociation
Motifs of the small lobe (ATP-binding):
What does the glycine-rich loop do?
What are its properties?
What does it dictate?
What does the beta strand 2 do?
How does this alter phosphate transfer?
What two things does the beta strand 3 do?
What are the 2 other features of the small lobe?
Glycine & phenylalanine residues hydrogen bond to beta phosphate in ATP
Flexible/mobile due to glycine residues
Whether enzyme is open/closed
Valine forms hydrophobic interactions with purine ring of ATP
Anchors purine ring for efficient gamma phosphate transfer
- Lysine forms electrostatic interactions with alpha & beta phosphates
- Lysine forms electrostatic interactions with glutamate on the C-helix
C-helix & C-tail
Motifs of the large lobe (peptide binding):
What does the catalytic loop do?
Mg2+ loop?
Activation loop?
F-helix?
Lysine forms electrostatic interactions with gamma phosphate for transfer
Aspartate of loop binds to Mg2+ on ATP
Role for interacting with correct substrate
Forms stable core for other helices to pack around - contributes to catalytic competence & compact structure
What happens to each lobe interactions after phosphate transfer?
What happens to the conformational change of the lobes after phosphate transfer?
How is the hydrophobic core/catalytic spine of the kinase stabilised?
How does this effect the PKA?
Small lobe interactions remain same (alpha & beta) but large lobe interactions change & structure changes
Conformational change due to lack of ATP so lobes not held together
When the purine ring of ATP binds - causes hydrophobic residues to stack
Conformational change of kinase domain for activation
What are the 3 ways consensus sequences arise?
What other 2 ways promotes phosphorylation?
How can you produce a pattern coding for specificity of amino acid residues?
What can the pattern be used for? 2 ways
What is the bias with this?
What can remove this bias?
Physical accessibility of amino acids (e.g at loops/turns)
Cryptic sites (accessible after protein-protein interactions)
Consensus sequence - flanking amino acids
Adaptor proteins & protein-protein interactions
Multiple sequence alignment of optimal consensus sequences
- identify new substrates
- engineer a substrate & choose P
doesn’t take into account unknown substrates as uses data from known ones
Producing degenerative peptide libraries
What are the 4 steps to producing a degenerative peptide library?
What is a dwell time?
How can the dwell time be decreased assuming that phosphorylation is a stochastic process?
What is also key in phosphorylation?
- Make library of randomised peptides - lock in central residue for phosphorylated and randomise the other amino acids
- Add purified kinase & ATP-Mg2+ to peptides for phosphorylation
- Extract phosphorylated peptides with affinity binding step with Fe ions & produce sub-library of phosphopeptides
- Identify enrichment of particular amino acids in particular positions - use amino acid sequencer
Amount of time given in this case for two proteins to undergo interactions
- Consensus sequence at the active site
- Interactions through other binding domains & optimal consensus sequences for those
Longer contact means higher likelihood of successful reaction - longer dwell time
What 3 elements is the catalytic subunit made up of and on which lobes are they?
What begins the ‘cascade’/activation of kinase activation in PKA?
What is the conformational requirement for the C-helix?
Catalytic loop?
Activation loop?
C-helix (small), activation loop (large), catalytic loop (large)
Phosphorylation at the activation loop
Such its glutamate coordinates to the lysine of the beta 3 strand which coordinates ATP
Coordinates breaking bonds of 1 side of gamma phosphate to reform on other side on transfer
To dictate choosing the correct substrate consensus sequence once it’s in the active form
What does phosphorylation of the activation loop achieve conformationally? 2 things
How is the C-helix put into its correct conformation after activation (phosphorylation of the activation loop)?
How is the catalytic loop put into its correct conformation after activation?
How does the activation loop change its own conformation?
Why is GSK3 (glycogen synthase kinase) an exception?
How else can you maximise inhibition of PKA?
On what basis does this work?
- Stabilises & induces correct orientation of the hydrophobic regulatory spine
- Correct conformational change of the activation loop, C-helix & catalytic loop
+ve histidine of C-helix is attracted to phosphate which pulls C-helix in orientation such that its glutamate interacts with the lysine of the beta 3 strand
Arginine +ve interacts with phosphate
changes so can interact with consensus sequence - has a +ve lysine & attracted to -ve phosphate
Doesn’t require autophosphorylation in trans - as substrate has been ‘primed’/phosphorylated prior so can be targeted for a 2nd & 3rd processive phosphorylation event
Couple with pseudosubstrate peptide in the regulatory subunits
Regulatory subunits hold catalytic subunits in place when no cAMP present
How is CDK2 activated?
How would you describe CDK2’s activation/T-loop?
How can you make a psuedosubstrate peptide sequence?
How can a pseudosubstrate act as a trans inhibitor?
What are PKA’s two methods of trans inhibition?
How can a pseudosubstrate act as a cis inhibitor?
When cyclin binds - positions PSTAIRE to interact with lysine of T-loop such that it is extruded from the catalytic site for phosphorylation
Cryptic phosphorylation site
Replace phosphorylated residue of a peptide to an amino acid without a hydroxyl group
Binds to the kinase active site
- Regulatory domains bind to kinase domain until cAMP binds
- Regulatory domains contain pseudosubstrate sequence that inserts into active site
N or C-terminus of kinase has a pseudopeptide sequence & inserts into active site
What are the 2 equations for Kd?
What are the units of Kd and Ka?
What is the K on for proteins binding small ligands?
What is the K on for proteins binding other proteins?
What is the equation for half life?
What is the relationship between Kd, half life & affinity?
What happens to the dimensionality of the system & degree of freedom when proteins go from the cytoplasm to the membrane?
What does this mean for Kon and complex formation?
[AB]/[A][B] = Koff/Kon
Ka = M-1, Kd = M
10^8M-1S-1
10^6M-1S-1
0.7/kd
Smaller the Kd, the longer the half life & higher affinity
3D -> 2D so dimensionality decreases & degree of freedom decreases
Kon increases so rate of [AB] formation increases as likelihood of successful collisions increases
What are the 3 steps to Src’s inhibitory mechanism?
What is the optimal consensus sequence for binding to the SH2 domain?
What feature of the SH2-cSrc binding association means that SH3 binding can change?
- C-terminal tyrosine phosphorylated by Csk
- C-terminal binds to SH2 domain
- This conformation changes conformation of SH3 such that it can bind to the type II polyproline helix linker
pYEEI
High Koff - meaning that SH3 can unbind from linker & have time to reorganise residues & motif conformation for activation loop to be phosphorylated & activated
What are the 3 steps for Src’s inhibitory mechanism?
What is the optimal sequence for the phosphorylated peptide to bind to SH2?
How would you describe c-Src & SH2’s binding dissociation & how does this affect the kinase domain?
When Src is activated/phosphorylated, what are the 3 things that happens?
- C-terminal Src is phosphorylated by c-Src kinase (Csk) at its tyrosine residue
- Phosphorylated c-Src binds to SH2 domain
- Conformational change of SH2 domain results in SH3 binding to type II polyproline helix of the linker region
pY E E I
High Kd (Koff) - so allows for dissociation & also of the SH3-linker dissociation so kinase has opportunity to be activated in this time if it is phosphorylated when both autoinhibition mechanisms are off
- C-helix rotates & positions such that its glutamate interacts with beta strand 3 lysine
- Catalytic loop & c-helix reorganisation allows for active kinase domain
- Trans autophosphorylation (Src can activate activation loop of another) or phosphorylation of substrate
What happens when Csk is knocked out? What does this mean about the c-terminal tail of src?
What happens when the Y at C-tail is a F?
Why does the c-tail & SH2 interaction need to be effective?
As well as a high Koff rate, what is the other feature between c-Src-SH2?
What does this allow for?
C-terminal tail is not phosphorylated so Src kinase is active - means that the phosphorylated tail is a suppressive signal
No phosphorylation as no Oxygen - can’t enter autoinhibited state
Activation of Src leads to activated oncogenes
High Kon (association)
Can re-associate with proteins with a more optimal consensus sequence preventing autoinhibition
What are the 2 ways you could introduce adaptor proteins to prevent autoinhibition (activate) Src?
How does this prevent autoinhibition?
What other 3 ways could you activate Src?
- Introduce protein with optimal sequence for tyrosine phosphorylation
- Protein with a stable polyproline helix structure
Proteins bind to either SH2 domain or SH3 domain respectively but this doesn’t result in inhibition of kinase as the C-tail & linker region are not directly bound to the domains
- Dephosphorylate tyrosine at c-terminal
- Physically displace SH3
- Phosphorylate the activation loop with a Src kinase (auto)
What are the 3 consequences of protein phosphorylation?
What is unique about the consensus sequence reading by tyrosine kinases?
What 3 things does this ensure about this particular pathway?
How large is an SH2 domain?
What does the SH2 domain fold look like?
What are the main features of the Src SH2 domain?
- Change in catalytic activity of an enzyme
- Change in its location for protein-protein interactions
- Both
Read twice - once by the tyrosine kinase & then phosphopeptide sequence read by a docking protein
- Longer protein-protein interactions
- Longer protein half-life
- Fidelity of transduction
100-110 amino acids / 12kDa
Anti-parallel beta sheet in the middle made up of 4ß strands and 2 alpha helices on each side
pY (tyrosine) and pY+3 positively charged binding pockets
What determines pY in the Src SH2 domain?
What determines pY+3 in the Src SH2 domain?
If the optimal consensus sequence for a substrate of Src SH2 domain is pYEEI, what does this tell you about ßD3, ßD5 and ßD’1? (pY+1 and pY+3)
What is selectivity dependent on?
What happens if you want to change the selectivity/amino acid at pY+3?
If ßD5 is F or Y (aromatic hydrophobic) then what is the general consensus sequence?
If ßD5 is I or C (non-aromatic hydrophobic) then what is the consensus sequence?
What does this say about each pY+3?
Why does ßD5 not interact with pY+2?
pY+3
ßD residues 3 & 5, ßD’1 and residues surrounding pY+6
pY+1 = E (+ve)
so ßD3 and ßD5 = K (+ve) and Y respectively
pY+3 = I (hydrophobic)
so ßD’1 = Y (hydrophobic)
Size & charge/polarity of amino acid
Need to consider 5 amino acids in loop around pY+3
pY - charged - charged - hydrophobic
pY - hydrophobic - X - hydrophobic
If it is large, aromatic & hydrophobic then the pY+3 will be large & hydrophobic
If it is small, non-aromatic & hydrophobic then pY+3 is deeper to accommodate pY+6 residues
pY and pY+3 are only accessible due to their position in binding pockets
What domains does GRB2 adaptor protein have?
How does GRB2 activate Ras when growth factor receptors are activated? 3 steps
What two other enzymes do GFR regulate?
2 x SH2 and 1 x SH3
- SH3 domain interacts with polyproline helix structure on Sos
- SH2 domains of GRB2-Sos recognises phosphorylated tyrosine residues on GF receptors
- Sos catalyses Ras-GDP to induce conformational change such that GDP dissociates & GTP associates
PLC-gamma & P13K
What is the structure of the PTB domain?
How are PTB domains similar to SH2?
How is it different? (2)
How are PTB domains similar to PH domains?
Different?
Where do phosphopeptides bind onto PTB domains?
In what proteins are PTB domains found?
ß-sandwich: beta barrel with alpha helix at the bottom acting as a ‘cap’ and loops on the top of the barrel
Binds to phosphotyrosines
21kDa/180 amino acids - larger
Have selectivity for residues proximal to the pY (-1, 1) not pY+3,6 etc
Have a similar structure
PH binds phospholipids not phosphopeptides
At the base of the domain to make antiparallel contacts with beta strand residues
Shc
What kind of proteins are 14-3-3?
What residues do these proteins bind to?
What is each monomer made of?
How is the dimer formed and what structure is key for its function?
How is the channel optimised for phosphopeptide binding?
What causes bidentate binding of 14-3-3’s substrate?
Ubiquitous homo/hetero dimers
Phosphoserine & phosphothreonine
9 alpha helices
N-terminal helices A B C of both monomers interact to form a wide channel for phosphoser/thr binding
Lined with K & R residues as well as Y of helices C & E to maintain positively charged, hydrophobic channel
Proline residue at p+1 produces a kink in the phosphopeptide
What is bidentate binding?
How does it allow for high affinity binding?
What happens if one binding interaction is released?
Why is this significant?
How can Raf be inactivated by 14-3-3?
A phosphopeptide can be bound to 14-3-3 at two phosphoresidues
Two weak associations are multiplied such that it reduces Kd
Peptide remains anchored at other site long enough for it to re-associate
Allows to control proliferative effects
Raf has 2 phosphoserine sites - so can both be bound by 14-3-3 into its low activity form
What 2 things to WW domains bind to?
What is its structure & size?
How does Pin1’s WW domain allow for the dephosphorylation of Cdc25C? 2 steps
Why is this significant?
In the yeast homologous of Pin1 Ess1, what 3 residues/molecules of Ess1 interact with the phosphoserine of the peptide?
What does this tell us about the specificity of binding of WW domains?
- Short proline rich domains containing polyproline helices
- Phosphopeptides where proline res is p+1
3 anti-parallel ß strands - 40 amino acids
- WW domain binds to the Pro-pSer/Thr bond in Cdc25C & catalyses its isomerisation from cis to trans
- PP2A phosphatase then dephosphorylated Cdc25C
Controls the cell cycle
- Arginine coordinates 2 phosphate oxygen
- Serine coordinates 1 phosphate oxygen
- Water forms hydrogen bond between tyrosine & 1 phosphate oxygen
Arginine & serine residues in WW are required for phosphoserine/phosphothreonine binding
What do FHA domains bind?
What do they regulate? (3)
What consensus sequences do they read?
What is its structure?
What is conserved in its structure?
What is variable in its structure?
What could suggest that the FHA domain folds independently from the rest of the protein?
What residue is strongly selected in the consensus sequence?
Phosphothreonines
Cell cycle checkpoints, phosphatases, transcriptional control
Entire phosphopeptide from pT-4 to pT+3
11-stranded ß-sandwich (mixed ß/a)
6 ß-strands on one face of the sandwich
N & C-terminus ß-strands on the other face of the sandwich
Structural proximity between N & C-terminus
pT+3
