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BIO 317 > 6 - Protein Kinases and Phosphatases, Calcium Signaling > Flashcards

6 - Protein Kinases and Phosphatases, Calcium Signaling Flashcards

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1
Q

How do proteins signal to each other?

A

By changes in state (binding, conformational, post-translational, localization)

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2
Q

How do compartments aid in cell signaling?

A

The compartment could have different signaling molecules or conditions (ex. low pH)

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3
Q

What is an example of information being passed between proteins?

A

Phosphorylation -> conformational change -> change in catalytic activity

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4
Q

What are the different broad groups of kinases?

A

Serine/threonine kinases, or tyrosine kinases

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5
Q

What groups can kinases phosphorylate?

A

OH groups

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6
Q

True or false: phosphorylation events always activate a protein

A

False: it can also inhibit a protein

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7
Q

How is substrate specificity seen in kinases?

A

Amino acids, primary structure, shape, and location

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8
Q

How can amino acids lead to kinase substrate specificity?

A

Kinases can only phosphorylate serine, threonine, or tyrosine

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9
Q

How can primary structure lead to kinase substrate specificity?

A

The amino acids surrounding Ser/Tyr/Thr can also provide information

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10
Q

What is the common motif that kinases phosphorylate?

A

Basic-Basic-X-Ser/Thr

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11
Q

What amino acids are basic?

A

Arg and Lys

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12
Q

What is the perfect sequence match for PKA?

A

Arg-Arg-X-Ser/Thr

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13
Q

True or false: PKA only phosphorylates proteins that have an Arg-Arg-X-Ser/Thr motif

A

False: while that is the perfect match, it can also recognize other combinations

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14
Q

How can shape lead to kinase substrate specificity?

A

Secondary, tertiary, and quaternary structure all affect shape, and how the kinase can interact with the site

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15
Q

How can location lead to kinase substrate specificity?

A

Both the kinase and the substrate need to be close together to have an effect

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16
Q

How can the location of the kinase / substrate be controlled?

A

Through anchoring scaffolds (AKAPs)

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17
Q

What does AKAP stand for?

A

A-kinase anchoring proteins

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18
Q

What do AKAPs do?

A

Anchor PKAs and other proteins to a specific portion of the cell (increase specificity)

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19
Q

How can spacial location be observed (in an experiment) in the cell?

A

Through fluorescent proteins (GFP)

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20
Q

What does GFP stand for?

A

Green fluorescent protein

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21
Q

What does GFP do?

A

Shines green (allows for visualization in space)

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22
Q

How is GFP used to make other proteins fluorese?

A

Use an antibody to tag the protein with the fluorescent protein

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23
Q

What does modification of GFP lead to?

A

Different colors for visualization

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24
Q

What is a brainbow?

A

Using different fluorescent proteins and random chance to color individual neurons in the brain a different color

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25
Q

How does brainbow work?

A

LoxP sites allow for random cutting of the DNA strand with the fluorescent proteins. The mixing of these different proteins leads to different colors that can be visualized

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26
Q

Why do neurons in a brainbow have different colors?

A

Random cutting events leads to random combinations of fluorescent proteins

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27
Q

What is the use of brainbow?

A

Help study the brain and brain diseases (3D neural networks) (trace pathways)

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28
Q

What does a brainbow look like?

A

3D neural networks, with many different shades (each neuron is its own shade)

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29
Q

What are the subunits of PKA?

A

2 regulatory subunits and 2 catalytic subunits

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30
Q

What is the inactive state of PKA?

A

Regulatory subunits are bound to catalytic subunits

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31
Q

What is the active state of PKA?

A

Regulatory subunits do not bind to cayatlytic subunits (due to cAMP)

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32
Q

What enzymes can PKA activate?

A

Phosphorylase kinase, tyrosine hydroxylase, CREB

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33
Q

What enzymes can PKA inhibit?

A

Glycogen synthase

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34
Q

Where is CREB located?

A

In the nucleus

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35
Q

How does PKA get to CREB?

A

Through nuclear pores

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36
Q

What does activated CREB do?

A

Bind to DNA to activate gene transcription

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37
Q

How does the regulatory subunit inhibit the catalytic subunit of PKA?

A

Pseudo-substrate of regulatory subunit binds to and blocks catalytic subunit

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38
Q

What is a pseudo-substrate?

A

A fake sequence that blocks an enzyme’s own activity (allosteric regulation)

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39
Q

How does binding of cAMP activate PKA?

A

Remove pseudo-substrate from catalytic subunits, thus releasing them making them active

40
Q

How can proteins regulate their own activity?

A

Through allosteric regulation (pseudo-substrates)

41
Q

Where is PKC found at low calcium concentrations?

A

In the cytosol (not near membrane)

42
Q

What prevents PKC from being active?

A

Pseudo-substrate blocking catalytic subunit

43
Q

What removes the pseudo-substrate from PKA?

A

Binding of cAMP to regulatory subunits

44
Q

What removes the pseudo-substrate from PKC?

A

Binding of DAG (after calcium binding)

45
Q

What happens to PKC when calcium binds?

A

It moves to the cell membrane and attaches to PIP2

46
Q

What happens once PKC reaches the membrane?

A

PS (phosphatidylserine) / PI (phosphadtidylinositol) and DAG bind to PKC to activate it

47
Q

What determines subcellular specificity and localization?

A

Scaffolds

48
Q

How is calcium regulated in the cell (4 ways)?

A
  1. Na/Ca exchanger on cell membrane
  2. Ca pump on ER membrane
  3. Ca binding molecules
  4. Ca pump on mitochondria
49
Q

What is the purpose of calcium pumps?

A

Create a calcium gradient for signaling

50
Q

How is the calcium cellular response stopped?

A

Through active pumps that move calcium back into the ER

51
Q

What can calcium act on (5 molecules)?

A
  1. Ca dependent enzymes
  2. Ca dependent kinases
  3. Structural proteins
  4. Nuclear proteins
  5. Calmodulin
52
Q

What does CaM stand for?

A

Calmodulin

53
Q

What is the structure of calmodulin?

A

4 EF hands (binding sites for calcium)

54
Q

True or false: calmodulin is an enzyme that senses calcium levels

A

False: calmodulin has no enzymatic activity

55
Q

What does calmodulin do?

A

Act as a calcium sensor to modulate activity of other enzymes

56
Q

What does calmodulin couple?

A

Single input (calcium signal) to a wide range of cellular responses

57
Q

How does active calmodulin activate other proteins?

A

Globular hands can wrap around peptides to open active sites or ion channels

58
Q

What is the range of calcium signaling?

A

Local, but very sustained and widespread signaling

59
Q

What is CaMKII important for?

A

Long term memory in the brain

60
Q

What does CaMKII stand for?

A

Cam Kinase II (calcium/calmodulin dependent kinase II)

61
Q

What is the structure of CaMKII in its inactive form?

A

Inhibitory domain blocks catalytic domain

62
Q

How does calmodulin interact with CaMKII?

A

Wraps around inhibitory domain to release catalytic domain

63
Q

What is autophosphorylation?

A

Phosphorylation of itself

64
Q

How is autophosphorylation used in CaMKII?

A

Keeps the subunit active (stops inhibitory domain from binding to it, even without calmodulin)

65
Q

True or false: when there is little calcium / calmodulin present, there is no activity of CaMKII

A

False: if there is phosphorylation, it remains active (autonomous)

66
Q

What is meant by “calcium-independent” CaMKII?

A

Phosphorylation keeps CaMKII active, even without calcium and calmodulin

67
Q

What eventually inactivates CaMKII?

A

Phosphatases (remove phosphate groups)

68
Q

Which amino acid is phosphorylated in CaMKII?

A

T286 (Threonine286)

69
Q

What amino acid is bound to calmodulin in CaMKII?

A

F (phenylalanine)

70
Q

What is the compact state of CaMKII?

A

All subunits are inactive, and compact (no binding to calmodulin possible)

71
Q

What is the extended state of CaMKII?

A

Subunit is extended, which makes it possible to bind to calmodulin

72
Q

What converts the compact state to the extended state of CaMKII?

A

Equilibrium

73
Q

What determines the equilibrium of the compact and extended state in CaMKII?

A

Length of linker regions

74
Q

What does the linker length of CaMKII determine?

A

Equilibrium point (how often the subunits are extended, and how much calcium is needed to generate a response)

75
Q

What happens when calmodulin binds to CaMKII?

A

Removes pseudo-substrate, enzyme is active, and can autophosphorylate

76
Q

True or false: CaMKII is active when it is extended

A

False: it is only active when calmodulin binds to CaMKII

77
Q

What happens to CaMKII if calcium levels drop, and there is no phosphorylation?

A

CaMKII will rapidly become inactive

78
Q

What happens to CaMKII if calcium levels drop, and there is phosphorylation?

A

CaMKII will remain active (autonomous)

79
Q

What is “trapping” (in terms of CaMKII)?

A

T286 phosphorylation strengthens the binding of calmodulin to CaMKII

80
Q

True or false: when CaMKII is autonomous, its activity is constant (regardless of calcium)

A

False: while it has activity without calcium, it’s activity is strengthened (x5) when calmodulin is bound

81
Q

What is transphosphorylation?

A

Phosphorylation of adjacent subunits

82
Q

How is transphosphorylation seen in CaMKII?

A

One active subunit can phosphorylate other subunits to keep them active (autonomous)

83
Q

What determines the overall activity of CaMKII?

A

The number of subunits that are activated

84
Q

True or false: CaMKII has a molecular memory for calcium

A

True: based on the frequency and the phosphorylation, CaMKII can “remember” how much calcium was present

85
Q

What is the activity of CaMKII with low frequency of calcium?

A

Equal spikes spaced out

86
Q

What is the activity of CaMKII with high frequency of calcium?

A

Each steps adds (sigmoidal curve) until it reaches max activity

87
Q

What explains the CaMKII pattern with low frequency of calcium?

A

Not enough time of phosphorylate, so it keeps becoming inactive

88
Q

What explains the CaMKII pattern with high frequency of calcium?

A

Has time to phosphorylate, so it remains highly active

89
Q

How is specificity maintained if many receptors use cAMP?

A

Highly regulated in space and time (scaffolds, local pools, etc.)

90
Q

What is meant by a “local pool” of cAMP?

A

cAMP produced can only affect a certain area (perfume in a warehouse)

91
Q

How are these “pools of cAMP” specific?

A

Specific in what receptor makes them, and what proteins are in the local area (scaffolds) to react to them

92
Q

True or false: when IP3 binds to the ER, the entire ER releases calcium

A

False: only the portion of the ER close to the IP3 signaling is going to release calcium (localized area)

93
Q

True or false: calmodulin has a range of activity, depending on how much calcium binds to it

A

False: it is only activated when all 4 binding sites are filled with calcium

94
Q

What does calmodulin sense (spatially and temporally)?

A

A brief flood of calcium in a local region

95
Q

True or false: calcium can only signal through calmodulin

A

False: it can also bind directly to ion channels to open them, for example

96
Q

What is the molecular memory of CaMKII for calcium based on?

A

The frequency of calcium oscillations

BIO 317 (27 decks)

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