6 - Protein Kinases and Phosphatases, Calcium Signaling

Question 1

How do proteins signal to each other?

Answer 1

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

Question 2

How do compartments aid in cell signaling?

Answer 2

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

Question 3

What is an example of information being passed between proteins?

Answer 3

Phosphorylation -> conformational change -> change in catalytic activity

Question 4

What are the different broad groups of kinases?

Answer 4

Serine/threonine kinases, or tyrosine kinases

Question 5

What groups can kinases phosphorylate?

Answer 5

OH groups

Question 6

True or false: phosphorylation events always activate a protein

Answer 6

False: it can also inhibit a protein

Question 7

How is substrate specificity seen in kinases?

Answer 7

Amino acids, primary structure, shape, and location

Question 8

How can amino acids lead to kinase substrate specificity?

Answer 8

Kinases can only phosphorylate serine, threonine, or tyrosine

Question 9

How can primary structure lead to kinase substrate specificity?

Answer 9

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

Question 10

What is the common motif that kinases phosphorylate?

Answer 10

Basic-Basic-X-Ser/Thr

Question 11

What amino acids are basic?

Answer 11

Arg and Lys

Question 12

What is the perfect sequence match for PKA?

Answer 12

Arg-Arg-X-Ser/Thr

Question 13

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

Answer 13

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

Question 14

How can shape lead to kinase substrate specificity?

Answer 14

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

Question 15

How can location lead to kinase substrate specificity?

Answer 15

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

Question 16

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

Answer 16

Through anchoring scaffolds (AKAPs)

Question 17

What does AKAP stand for?

Answer 17

A-kinase anchoring proteins

Question 18

What do AKAPs do?

Answer 18

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

Question 19

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

Answer 19

Through fluorescent proteins (GFP)

Question 20

What does GFP stand for?

Answer 20

Green fluorescent protein

Question 21

What does GFP do?

Answer 21

Shines green (allows for visualization in space)

Question 22

How is GFP used to make other proteins fluorese?

Answer 22

Use an antibody to tag the protein with the fluorescent protein

Question 23

What does modification of GFP lead to?

Answer 23

Different colors for visualization

Question 24

What is a brainbow?

Answer 24

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

Question 25

How does brainbow work?

Answer 25

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

Question 26

Why do neurons in a brainbow have different colors?

Answer 26

Random cutting events leads to random combinations of fluorescent proteins

Question 27

What is the use of brainbow?

Answer 27

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

Question 28

What does a brainbow look like?

Answer 28

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

Question 29

What are the subunits of PKA?

Answer 29

2 regulatory subunits and 2 catalytic subunits

Question 30

What is the inactive state of PKA?

Answer 30

Regulatory subunits are bound to catalytic subunits

Question 31

What is the active state of PKA?

Answer 31

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

Question 32

What enzymes can PKA activate?

Answer 32

Phosphorylase kinase, tyrosine hydroxylase, CREB

Question 33

What enzymes can PKA inhibit?

Answer 33

Glycogen synthase

Question 34

Where is CREB located?

Answer 34

In the nucleus

Question 35

How does PKA get to CREB?

Answer 35

Through nuclear pores

Question 36

What does activated CREB do?

Answer 36

Bind to DNA to activate gene transcription

Question 37

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

Answer 37

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

Question 38

What is a pseudo-substrate?

Answer 38

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

Question 39

How does binding of cAMP activate PKA?

Answer 39

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

Question 40

How can proteins regulate their own activity?

Answer 40

Through allosteric regulation (pseudo-substrates)

Question 41

Where is PKC found at low calcium concentrations?

Answer 41

In the cytosol (not near membrane)

Question 42

What prevents PKC from being active?

Answer 42

Pseudo-substrate blocking catalytic subunit

Question 43

What removes the pseudo-substrate from PKA?

Answer 43

Binding of cAMP to regulatory subunits

Question 44

What removes the pseudo-substrate from PKC?

Answer 44

Binding of DAG (after calcium binding)

Question 45

What happens to PKC when calcium binds?

Answer 45

It moves to the cell membrane and attaches to PIP2

Question 46

What happens once PKC reaches the membrane?

Answer 46

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

Question 47

What determines subcellular specificity and localization?

Answer 47

Scaffolds

Question 48

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

Answer 48

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

Question 49

What is the purpose of calcium pumps?

Answer 49

Create a calcium gradient for signaling

Question 50

How is the calcium cellular response stopped?

Answer 50

Through active pumps that move calcium back into the ER

Question 51

What can calcium act on (5 molecules)?

Answer 51

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

Question 52

What does CaM stand for?

Answer 52

Calmodulin

Question 53

What is the structure of calmodulin?

Answer 53

4 EF hands (binding sites for calcium)

Question 54

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

Answer 54

False: calmodulin has no enzymatic activity

Question 55

What does calmodulin do?

Answer 55

Act as a calcium sensor to modulate activity of other enzymes

Question 56

What does calmodulin couple?

Answer 56

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

Question 57

How does active calmodulin activate other proteins?

Answer 57

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

Question 58

What is the range of calcium signaling?

Answer 58

Local, but very sustained and widespread signaling

Question 59

What is CaMKII important for?

Answer 59

Long term memory in the brain

Question 60

What does CaMKII stand for?

Answer 60

Cam Kinase II (calcium/calmodulin dependent kinase II)

Question 61

What is the structure of CaMKII in its inactive form?

Answer 61

Inhibitory domain blocks catalytic domain

Question 62

How does calmodulin interact with CaMKII?

Answer 62

Wraps around inhibitory domain to release catalytic domain

Question 63

What is autophosphorylation?

Answer 63

Phosphorylation of itself

Question 64

How is autophosphorylation used in CaMKII?

Answer 64

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

Question 65

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

Answer 65

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

Question 66

What is meant by "calcium-independent" CaMKII?

Answer 66

Phosphorylation keeps CaMKII active, even without calcium and calmodulin

Question 67

What eventually inactivates CaMKII?

Answer 67

Phosphatases (remove phosphate groups)

Question 68

Which amino acid is phosphorylated in CaMKII?

Answer 68

T286 (Threonine286)

Question 69

What amino acid is bound to calmodulin in CaMKII?

Answer 69

F (phenylalanine)

Question 70

What is the compact state of CaMKII?

Answer 70

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

Question 71

What is the extended state of CaMKII?

Answer 71

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

Question 72

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

Answer 72

Equilibrium

Question 73

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

Answer 73

Length of linker regions

Question 74

What does the linker length of CaMKII determine?

Answer 74

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

Question 75

What happens when calmodulin binds to CaMKII?

Answer 75

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

Question 76

True or false: CaMKII is active when it is extended

Answer 76

False: it is only active when calmodulin binds to CaMKII

Question 77

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

Answer 77

CaMKII will rapidly become inactive

Question 78

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

Answer 78

CaMKII will remain active (autonomous)

Question 79

What is "trapping" (in terms of CaMKII)?

Answer 79

T286 phosphorylation strengthens the binding of calmodulin to CaMKII

Question 80

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

Answer 80

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

Question 81

What is transphosphorylation?

Answer 81

Phosphorylation of adjacent subunits

Question 82

How is transphosphorylation seen in CaMKII?

Answer 82

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

Question 83

What determines the overall activity of CaMKII?

Answer 83

The number of subunits that are activated

Question 84

True or false: CaMKII has a molecular memory for calcium

Answer 84

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

Question 85

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

Answer 85

Equal spikes spaced out

Question 86

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

Answer 86

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

Question 87

What explains the CaMKII pattern with low frequency of calcium?

Answer 87

Not enough time of phosphorylate, so it keeps becoming inactive

Question 88

What explains the CaMKII pattern with high frequency of calcium?

Answer 88

Has time to phosphorylate, so it remains highly active

Question 89

How is specificity maintained if many receptors use cAMP?

Answer 89

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

Question 90

What is meant by a "local pool" of cAMP?

Answer 90

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

Question 91

How are these "pools of cAMP" specific?

Answer 91

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

Question 92

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

Answer 92

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

Question 93

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

Answer 93

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

Question 94

What does calmodulin sense (spatially and temporally)?

Answer 94

A brief flood of calcium in a local region

Question 95

True or false: calcium can only signal through calmodulin

Answer 95

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

Question 96

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

Answer 96

The frequency of calcium oscillations