Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

CHE 541 > 16 - Phosphorylation > Flashcards

16 - Phosphorylation Flashcards

1
Q

What is the consensus sequence for PKA?

A

Arg-Arg-X-Ser (phosphorylate Ser)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

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

A

3D structure

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What is the specificity of phosphatases?

A

Non-specific

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

How is specificity regulated in vivo?

A

Through localization and substrate targeting

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

How is a sequence logo read?

A

The bigger the letter, the more preferred it is

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

What are two mechanisms to regulate kinase specificity?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

How big are signaling domains?

A

50-100 amino acids

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

What does SH2 bind to?

A

pTyr

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

What does SH3 bind to?

A

Pro rich sequences

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

How does a separate polypeptide increase specificity?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

What does AKAP stand for?

A

A-kinase anchoring proteins

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

How does PKA become activated?

A

cAMP binding to regulatory subunits releases the catalytic subunits

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

What do AKAPs do?

A

Localize PKA and other signaling molecules

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

What do Cdks do?

A

Regulate cell division cycle

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

How are Cdks targeted?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

How can phosphatases be targeted?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

What is the general structure of a kinase?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

What does the N-lobe of a kinase do?

A

Makes contacts with ATP

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

What does the C-lobe of a kinase do?

A

Makes contact with protein substrate

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

What residue is critical for kinase activity and why?

A

Lys72, because it binds ATP in the binding pocket

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
Q

How can a dead kinase for studying a signaling pathway be created?

A

By mutating Lys72, making the kinase inactive

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
26
Q

What is autophosphorylation?

A

Kinase phosphorylates itself

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
27
Q

What are some examples of autophosphorylation?

A

Receptor tyrosine kinases

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
28
Q

What is transautophosphorylation?

A

Intermolecular (one dimer phosphorylates the other, cross)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
29
Q

True or false: an RTK can only dimerize in 1:1 stoichiometry

A

False: it can also dimerize in 1:2 stoichiometry

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
30
Q

What happens when an RTK is activated?

A

It dimerizes, and autophosphorylates the other dimer

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
31
Q

What does phosphorylation of an RTK do?

A

Increases activity and recruitment

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
32
Q

How does insulin receptor kinase work?

A

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
Q

What is Abl?

A

A nonreceptor Tyr kinase that has increased activity in disease conditions

34
Q

What is Gleevec?

A

A small molecule that inhibits Abl

35
Q

How does Gleevec work?

A

It binds to unphosphorylated Abl, and locks it in its inactive confirmation

36
Q

What disease is caused by overactive Abl?

A

CML (chronic myelogenous leukemia)

37
Q

What is the structure of a phosphatase?

A

Shallow cleft where protein binds, with a cleft for a phosphorylated amino acid

38
Q

How does the structure of a phosphatase suggest its nonspecificity?

A

It has a shallow cleft, so almost any protein with a phospho-amino acid can bind to the cleft

39
Q

What is the catalytic residue in a phosphatase?

A

Cys (acts as a nucleophile at the base of the cleft)

40
Q

How can the specificity of a phosphatase be increased?

A

Through associated protein domains

41
Q

How come many kinases have low basal activity?

A

Autoinhibition

42
Q

How does autoinhibition work?

A

A secondary domain binds to catalytic domain and keep activity low

43
Q

How does autoinhibition of CamKII work?

A

The catalytic and inhibitory domains are part of the same molecule, and the peptide folds in on itself when inactivated

44
Q

How does autoinhibition of PKC work?

A

The catalytic and inhibitory domains are part of the same molecule, and the peptide folds in on itself when inactivated

45
Q

How does autoinhibition of PKA work?

A

The catalytic and inhibitory domains are separate polypeptides, that interact with each other when inactivated

46
Q

What is a pseudosubstrate?

A

A sequence that resembles consensus sequence, but cannot be phosphorylated

47
Q

What is an example of a pseudosubstrate?

A

Arg-Arg-X-Ala (Ser –> Ala, cannot be phosphorylated)

48
Q

What methods (4) can be used to show autoinhibition?

A
  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
Q

What type of kinase is Src kinase?

A

Tyr kinase

50
Q

What is the structure of Src kinase?

A

4 subunits: SH2, SH3, unique domain, and catalytic domain

51
Q

What Tyr is activative in Src?

A

Y416

52
Q

What Tyr is inhibiting in Src?

A

Y527

53
Q

What does Y527 do in Src?

A

When phosphorylated, it interacts with SH2 domain to inhibit the enzyme

54
Q

How does the SH3 domain interact in Src?

A

It binds to Pro rich regions to inhibit kinase activity

55
Q

How is Src usually activated?

A

Through the SH3 pathway

56
Q

How can Src be activated?

A

Either dephosphorylate Y527, or have another protein bind to SH2/SH3 domains

57
Q

What is twitchin?

A

A protein involved in C. elegans motion

58
Q

How does twitchin inactivation work?

A

Autoinhibited by separate regulatory domain that blocks the substrate binding celft

59
Q

How is titin kinase activated?

A

Through mechanical strain

60
Q

How may phosphorylation change the activity of an enzyme (5 ways)?

A
  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
Q

What is the most important aspect of phosphorylation?

A

The introduction of a negative charge

62
Q

What does glycogen phosphorylase do?

A

Controls metabolism of glycogen by catalyzing first step in reaction

63
Q

What is the activate state of glycogen phosphorylase?

A

Phosphorylation at Ser14

64
Q

What is the glycogen phosphorylase pathway?

A

Epinephrine –> cAMP –> PKA –> phosphorylase kinase –> glycogen phosphorylase

65
Q

What changes when glycogen phosphorylase is phosphorylated?

A

Small subtle shift (35 A away from Ser14)

66
Q

How does phosphorylation of Ser14 activate glycogen phosphorylase?

A

Binds to Arg69 and Arg43 (electrostatics) to being dimer subunits together, and causes a 10 degree rotation to open catalytic site (global change)

67
Q

What type of effect (direct or indirect) is phosphorylation on glycogen phosphorylase?

A

Indirect: change in Ser14 induces a subtle change on the overall structure

68
Q

What does ICDH stand for?

A

Isocitrate dehydrogenase

69
Q

What does ICDH do?

A

Converts isocitrate to alpha-ketoglutarate in the Krebs cycle

70
Q

What happens when ICDH is phosphorylated?

A

It is inactivated

71
Q

What is glyoxylate bypass, and how is it activated?

A

A bypass to conserve carbon, caused by inactivation of ICDH (phosphorylation)

72
Q

How does ICDH coordinate with its substrate?

A

The negative charges on citrate coordinate with the positive enzyme

73
Q

What happens when Ser113 of ICDH is phosphorylated?

A

It blocks isocitrate bonding

74
Q

What does Ser113 do in ICDH?

A

Part of active site (helps with coordination), and site of phosphorylation

75
Q

What type of effect (direct or indirect) does phosphorylation have on ICDH?

A

Direct: phosphorylation directly blocks substrate binding (does not affect tertiary structure)

76
Q

How was it shown that the negative charge was important for ICDH?

A

Mutagenesis of Asp and Glu deletions residues decreased enzymatic activity (no negative charge)

77
Q

How can phosphorylation be mimicked in a protein?

A

Mutagenesis to an Asp or Glu (simulate negative charge)

78
Q

How were the mutants of ICDH analyzed?

A

By measuring changes in vmax and Km

CHE 541 (20 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions