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CHE 541 > 16 - Phosphorylation > Flashcards

16 - Phosphorylation Flashcards

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1
Q

What is the consensus sequence for PKA?

A

Arg-Arg-X-Ser (phosphorylate Ser)

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2
Q

Besides primary sequence, what plays a role in kinase recognition?

A

3D structure

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3
Q

What is the specificity of phosphatases?

A

Non-specific

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4
Q

How is specificity regulated in vivo?

A

Through localization and substrate targeting

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5
Q

How is a sequence logo read?

A

The bigger the letter, the more preferred it is

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6
Q

What are some methods (3) to find a consensus sequence)?

A
  1. Comparison of sequences around known phosphorylation sites
  2. Vary one amino acid near phosphoacceptor site, measure Km and vmax
  3. Synthetic peptide libraries to screen for kinase specificity
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7
Q

What are two mechanisms to regulate kinase specificity?

A

A protein domain on the enzyme or substrate, or an associated targeting subunit

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8
Q

What are some examples of targeting via domains?

A

Transmembrane region to anchor to membrane (RTKs), PTP1B directs to cytoplasmic face of ER, PTPMEG1 anchors to cytoskeleton, SH2/SH3 domains for protein/protein interactions

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9
Q

How big are signaling domains?

A

50-100 amino acids

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10
Q

What are some general features of signaling domains?pT

A

Fold into distinct structures, found in unrelated proteins, sequence conservation, modular in structure and function

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11
Q

What does SH2 bind to?

A

pTyr

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12
Q

What does SH3 bind to?

A

Pro rich sequences

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13
Q

How does the EGFR pathways with PLC-g work?

A

The pTyr on EGFR becomes a docking site for PLC-g based on SH2 domains, which targets it for the signaling complex

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14
Q

How does a separate polypeptide increase specificity?

A

It can bridge the kinase and substrate to target it to a particular organelle

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15
Q

What does AKAP stand for?

A

A-kinase anchoring proteins

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16
Q

How does PKA become activated?

A

cAMP binding to regulatory subunits releases the catalytic subunits

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17
Q

What do AKAPs do?

A

Localize PKA and other signaling molecules

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18
Q

What do Cdks do?

A

Regulate cell division cycle

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19
Q

How are Cdks targeted?

A

Different cyclins enable catalytic domains to phosphorylate different substrates at different phases of the cell cycle

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20
Q

How can phosphatases be targeted?

A

They can be complexed with different targeting subunits to limit specificity to certain proteins

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21
Q

What is the general structure of a kinase?

A

N-lobe, C-lobe, and substrate binding pocket

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22
Q

What does the N-lobe of a kinase do?

A

Makes contacts with ATP

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23
Q

What does the C-lobe of a kinase do?

A

Makes contact with protein substrate

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24
Q

What residue is critical for kinase activity and why?

A

Lys72, because it binds ATP in the binding pocket

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25
How can a dead kinase for studying a signaling pathway be created?
By mutating Lys72, making the kinase inactive
26
What is autophosphorylation?
Kinase phosphorylates itself
27
What are some examples of autophosphorylation?
Receptor tyrosine kinases
28
What is transautophosphorylation?
Intermolecular (one dimer phosphorylates the other, cross)
29
True or false: an RTK can only dimerize in 1:1 stoichiometry
False: it can also dimerize in 1:2 stoichiometry
30
What happens when an RTK is activated?
It dimerizes, and autophosphorylates the other dimer
31
What does phosphorylation of an RTK do?
Increases activity and recruitment
32
How does insulin receptor kinase work?
In the inactive state, the activation loop blocks the activate site. When phosphorylated, (Tyr 1158, 1162, 1163), the activation loop moves, allowing ATP and substrate binding
33
What is Abl?
A nonreceptor Tyr kinase that has increased activity in disease conditions
34
What is Gleevec?
A small molecule that inhibits Abl
35
How does Gleevec work?
It binds to unphosphorylated Abl, and locks it in its inactive confirmation
36
What disease is caused by overactive Abl?
CML (chronic myelogenous leukemia)
37
What is the structure of a phosphatase?
Shallow cleft where protein binds, with a cleft for a phosphorylated amino acid
38
How does the structure of a phosphatase suggest its nonspecificity?
It has a shallow cleft, so almost any protein with a phospho-amino acid can bind to the cleft
39
What is the catalytic residue in a phosphatase?
Cys (acts as a nucleophile at the base of the cleft)
40
How can the specificity of a phosphatase be increased?
Through associated protein domains
41
How come many kinases have low basal activity?
Autoinhibition
42
How does autoinhibition work?
A secondary domain binds to catalytic domain and keep activity low
43
How does autoinhibition of CamKII work?
The catalytic and inhibitory domains are part of the same molecule, and the peptide folds in on itself when inactivated
44
How does autoinhibition of PKC work?
The catalytic and inhibitory domains are part of the same molecule, and the peptide folds in on itself when inactivated
45
How does autoinhibition of PKA work?
The catalytic and inhibitory domains are separate polypeptides, that interact with each other when inactivated
46
What is a pseudosubstrate?
A sequence that resembles consensus sequence, but cannot be phosphorylated
47
What is an example of a pseudosubstrate?
Arg-Arg-X-Ala (Ser --> Ala, cannot be phosphorylated)
48
What methods (4) can be used to show autoinhibition?
1. Release autoinhibitory domain by limited proteolysis to generate active kinase 2. Mutagenesis of pseudosubstrate region to generate active kinase 3. Create synthetic peptides that inhibit kinase (based on autoinhibitory site) 4. X-ray crystallography (like Src and twitchin)
49
What type of kinase is Src kinase?
Tyr kinase
50
What is the structure of Src kinase?
4 subunits: SH2, SH3, unique domain, and catalytic domain
51
What Tyr is activative in Src?
Y416
52
What Tyr is inhibiting in Src?
Y527
53
What does Y527 do in Src?
When phosphorylated, it interacts with SH2 domain to inhibit the enzyme
54
How does the SH3 domain interact in Src?
It binds to Pro rich regions to inhibit kinase activity
55
How is Src usually activated?
Through the SH3 pathway
56
How can Src be activated?
Either dephosphorylate Y527, or have another protein bind to SH2/SH3 domains
57
What is twitchin?
A protein involved in C. elegans motion
58
How does twitchin inactivation work?
Autoinhibited by separate regulatory domain that blocks the substrate binding celft
59
How is titin kinase activated?
Through mechanical strain
60
How may phosphorylation change the activity of an enzyme (5 ways)?
1. Alter Km 2. Alter vmax 3. Alter affinity for allosteric activator or inhibitor 4. Promote or disrupt protein-protein interactions 5. Alter membrane association or change localization
61
What is the most important aspect of phosphorylation?
The introduction of a negative charge
62
What does glycogen phosphorylase do?
Controls metabolism of glycogen by catalyzing first step in reaction
63
What is the activate state of glycogen phosphorylase?
Phosphorylation at Ser14
64
What is the glycogen phosphorylase pathway?
Epinephrine --> cAMP --> PKA --> phosphorylase kinase --> glycogen phosphorylase
65
What changes when glycogen phosphorylase is phosphorylated?
Small subtle shift (35 A away from Ser14)
66
How does phosphorylation of Ser14 activate glycogen phosphorylase?
Binds to Arg69 and Arg43 (electrostatics) to being dimer subunits together, and causes a 10 degree rotation to open catalytic site (global change)
67
What type of effect (direct or indirect) is phosphorylation on glycogen phosphorylase?
Indirect: change in Ser14 induces a subtle change on the overall structure
68
What does ICDH stand for?
Isocitrate dehydrogenase
69
What does ICDH do?
Converts isocitrate to alpha-ketoglutarate in the Krebs cycle
70
What happens when ICDH is phosphorylated?
It is inactivated
71
What is glyoxylate bypass, and how is it activated?
A bypass to conserve carbon, caused by inactivation of ICDH (phosphorylation)
72
How does ICDH coordinate with its substrate?
The negative charges on citrate coordinate with the positive enzyme
73
What happens when Ser113 of ICDH is phosphorylated?
It blocks isocitrate bonding
74
What does Ser113 do in ICDH?
Part of active site (helps with coordination), and site of phosphorylation
75
What type of effect (direct or indirect) does phosphorylation have on ICDH?
Direct: phosphorylation directly blocks substrate binding (does not affect tertiary structure)
76
How was it shown that the negative charge was important for ICDH?
Mutagenesis of Asp and Glu deletions residues decreased enzymatic activity (no negative charge)
77
How can phosphorylation be mimicked in a protein?
Mutagenesis to an Asp or Glu (simulate negative charge)
78
How were the mutants of ICDH analyzed?
By measuring changes in vmax and Km

CHE 541 (20 decks)

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