6 - Protein Kinases and Phosphatases, Calcium Signaling Flashcards
How do proteins signal to each other?
By changes in state (binding, conformational, post-translational, localization)
How do compartments aid in cell signaling?
The compartment could have different signaling molecules or conditions (ex. low pH)
What is an example of information being passed between proteins?
Phosphorylation -> conformational change -> change in catalytic activity
What are the different broad groups of kinases?
Serine/threonine kinases, or tyrosine kinases
What groups can kinases phosphorylate?
OH groups
True or false: phosphorylation events always activate a protein
False: it can also inhibit a protein
How is substrate specificity seen in kinases?
Amino acids, primary structure, shape, and location
How can amino acids lead to kinase substrate specificity?
Kinases can only phosphorylate serine, threonine, or tyrosine
How can primary structure lead to kinase substrate specificity?
The amino acids surrounding Ser/Tyr/Thr can also provide information
What is the common motif that kinases phosphorylate?
Basic-Basic-X-Ser/Thr
What amino acids are basic?
Arg and Lys
What is the perfect sequence match for PKA?
Arg-Arg-X-Ser/Thr
True or false: PKA only phosphorylates proteins that have an Arg-Arg-X-Ser/Thr motif
False: while that is the perfect match, it can also recognize other combinations
How can shape lead to kinase substrate specificity?
Secondary, tertiary, and quaternary structure all affect shape, and how the kinase can interact with the site
How can location lead to kinase substrate specificity?
Both the kinase and the substrate need to be close together to have an effect
How can the location of the kinase / substrate be controlled?
Through anchoring scaffolds (AKAPs)
What does AKAP stand for?
A-kinase anchoring proteins
What do AKAPs do?
Anchor PKAs and other proteins to a specific portion of the cell (increase specificity)
How can spacial location be observed (in an experiment) in the cell?
Through fluorescent proteins (GFP)
What does GFP stand for?
Green fluorescent protein
What does GFP do?
Shines green (allows for visualization in space)
How is GFP used to make other proteins fluorese?
Use an antibody to tag the protein with the fluorescent protein
What does modification of GFP lead to?
Different colors for visualization
What is a brainbow?
Using different fluorescent proteins and random chance to color individual neurons in the brain a different color
How does brainbow work?
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
Why do neurons in a brainbow have different colors?
Random cutting events leads to random combinations of fluorescent proteins
What is the use of brainbow?
Help study the brain and brain diseases (3D neural networks) (trace pathways)
What does a brainbow look like?
3D neural networks, with many different shades (each neuron is its own shade)
What are the subunits of PKA?
2 regulatory subunits and 2 catalytic subunits
What is the inactive state of PKA?
Regulatory subunits are bound to catalytic subunits
What is the active state of PKA?
Regulatory subunits do not bind to cayatlytic subunits (due to cAMP)
What enzymes can PKA activate?
Phosphorylase kinase, tyrosine hydroxylase, CREB
What enzymes can PKA inhibit?
Glycogen synthase
Where is CREB located?
In the nucleus
How does PKA get to CREB?
Through nuclear pores
What does activated CREB do?
Bind to DNA to activate gene transcription
How does the regulatory subunit inhibit the catalytic subunit of PKA?
Pseudo-substrate of regulatory subunit binds to and blocks catalytic subunit
What is a pseudo-substrate?
A fake sequence that blocks an enzyme’s own activity (allosteric regulation)
How does binding of cAMP activate PKA?
Remove pseudo-substrate from catalytic subunits, thus releasing them making them active
How can proteins regulate their own activity?
Through allosteric regulation (pseudo-substrates)
Where is PKC found at low calcium concentrations?
In the cytosol (not near membrane)
What prevents PKC from being active?
Pseudo-substrate blocking catalytic subunit
What removes the pseudo-substrate from PKA?
Binding of cAMP to regulatory subunits
What removes the pseudo-substrate from PKC?
Binding of DAG (after calcium binding)
What happens to PKC when calcium binds?
It moves to the cell membrane and attaches to PIP2
What happens once PKC reaches the membrane?
PS (phosphatidylserine) / PI (phosphadtidylinositol) and DAG bind to PKC to activate it
What determines subcellular specificity and localization?
Scaffolds
How is calcium regulated in the cell (4 ways)?
- Na/Ca exchanger on cell membrane
- Ca pump on ER membrane
- Ca binding molecules
- Ca pump on mitochondria
What is the purpose of calcium pumps?
Create a calcium gradient for signaling
How is the calcium cellular response stopped?
Through active pumps that move calcium back into the ER
What can calcium act on (5 molecules)?
- Ca dependent enzymes
- Ca dependent kinases
- Structural proteins
- Nuclear proteins
- Calmodulin
What does CaM stand for?
Calmodulin
What is the structure of calmodulin?
4 EF hands (binding sites for calcium)
True or false: calmodulin is an enzyme that senses calcium levels
False: calmodulin has no enzymatic activity
What does calmodulin do?
Act as a calcium sensor to modulate activity of other enzymes
What does calmodulin couple?
Single input (calcium signal) to a wide range of cellular responses
How does active calmodulin activate other proteins?
Globular hands can wrap around peptides to open active sites or ion channels
What is the range of calcium signaling?
Local, but very sustained and widespread signaling
What is CaMKII important for?
Long term memory in the brain
What does CaMKII stand for?
Cam Kinase II (calcium/calmodulin dependent kinase II)
What is the structure of CaMKII in its inactive form?
Inhibitory domain blocks catalytic domain
How does calmodulin interact with CaMKII?
Wraps around inhibitory domain to release catalytic domain
What is autophosphorylation?
Phosphorylation of itself
How is autophosphorylation used in CaMKII?
Keeps the subunit active (stops inhibitory domain from binding to it, even without calmodulin)
True or false: when there is little calcium / calmodulin present, there is no activity of CaMKII
False: if there is phosphorylation, it remains active (autonomous)
What is meant by “calcium-independent” CaMKII?
Phosphorylation keeps CaMKII active, even without calcium and calmodulin
What eventually inactivates CaMKII?
Phosphatases (remove phosphate groups)
Which amino acid is phosphorylated in CaMKII?
T286 (Threonine286)
What amino acid is bound to calmodulin in CaMKII?
F (phenylalanine)
What is the compact state of CaMKII?
All subunits are inactive, and compact (no binding to calmodulin possible)
What is the extended state of CaMKII?
Subunit is extended, which makes it possible to bind to calmodulin
What converts the compact state to the extended state of CaMKII?
Equilibrium
What determines the equilibrium of the compact and extended state in CaMKII?
Length of linker regions
What does the linker length of CaMKII determine?
Equilibrium point (how often the subunits are extended, and how much calcium is needed to generate a response)
What happens when calmodulin binds to CaMKII?
Removes pseudo-substrate, enzyme is active, and can autophosphorylate
True or false: CaMKII is active when it is extended
False: it is only active when calmodulin binds to CaMKII
What happens to CaMKII if calcium levels drop, and there is no phosphorylation?
CaMKII will rapidly become inactive
What happens to CaMKII if calcium levels drop, and there is phosphorylation?
CaMKII will remain active (autonomous)
What is “trapping” (in terms of CaMKII)?
T286 phosphorylation strengthens the binding of calmodulin to CaMKII
True or false: when CaMKII is autonomous, its activity is constant (regardless of calcium)
False: while it has activity without calcium, it’s activity is strengthened (x5) when calmodulin is bound
What is transphosphorylation?
Phosphorylation of adjacent subunits
How is transphosphorylation seen in CaMKII?
One active subunit can phosphorylate other subunits to keep them active (autonomous)
What determines the overall activity of CaMKII?
The number of subunits that are activated
True or false: CaMKII has a molecular memory for calcium
True: based on the frequency and the phosphorylation, CaMKII can “remember” how much calcium was present
What is the activity of CaMKII with low frequency of calcium?
Equal spikes spaced out
What is the activity of CaMKII with high frequency of calcium?
Each steps adds (sigmoidal curve) until it reaches max activity
What explains the CaMKII pattern with low frequency of calcium?
Not enough time of phosphorylate, so it keeps becoming inactive
What explains the CaMKII pattern with high frequency of calcium?
Has time to phosphorylate, so it remains highly active
How is specificity maintained if many receptors use cAMP?
Highly regulated in space and time (scaffolds, local pools, etc.)
What is meant by a “local pool” of cAMP?
cAMP produced can only affect a certain area (perfume in a warehouse)
How are these “pools of cAMP” specific?
Specific in what receptor makes them, and what proteins are in the local area (scaffolds) to react to them
True or false: when IP3 binds to the ER, the entire ER releases calcium
False: only the portion of the ER close to the IP3 signaling is going to release calcium (localized area)
True or false: calmodulin has a range of activity, depending on how much calcium binds to it
False: it is only activated when all 4 binding sites are filled with calcium
What does calmodulin sense (spatially and temporally)?
A brief flood of calcium in a local region
True or false: calcium can only signal through calmodulin
False: it can also bind directly to ion channels to open them, for example
What is the molecular memory of CaMKII for calcium based on?
The frequency of calcium oscillations
