Ser/Thr kinases and phosphatases

Question 1

Advantages to phosphorylation as regulatory mechanism (3)

Answer 1

It’s fast, can be amplified, and is easily reversible

Question 2

Phosphorylated amino acid residues (2 common, 1 rare)

Answer 2

Serine/Thr most common, tyrosine more rare

Question 3

What does phosphorylation change?

Answer 3

Hormone response, cell shape, protein synthesis, gene expression, hormone release, muscle contraction, cell metabolism

Question 4

T/F. 3/4 all proteins are reversibly phosphorylated.

Answer 4

False, closer to 1/3 +

Question 5

T/F. Phosphorylation can happen on any amino acid

Answer 5

False, ser/thr/tyr

Question 6

PKA structure

Answer 6

PKA is a dimer of 1 catalytic subunit and 1 regulatory subunit, held together by AKAP (Together, this forms the R2C2 complex.)

Question 7

PKA activation

Answer 7

PKA is activated by binding of 4 cAMP molecules (or the binding of 2 cAMP molecules to each regulatory unit)

Question 8

cAMP is produced by the -

Answer 8

activity of adenylyl cyclase

Question 9

Why do we care about cAMP production?

Answer 9

cAMP binding of PKA releases catalytic subunits, now free to phosphorylate ser/thr residues of proteins

Question 10

Function of AKAPs?

Answer 10

anchoring proteins for PKA subunitys

Question 11

Two ways to turn off signaling?

Answer 11

1j. Desensitization (i.e. removing receptor)
2. Turn off downstream signaling (i.e. cAMP removed by phosphodiesterases or removal of phosphate groups by phosphatases)

Question 12

Example of PKA physiological relevance

Answer 12

PKA regulates water reabsorption to concentrate urine; without this→diabetes insipidus

Question 13

Where does PKC receive priming phosphorylation from, and what happens after?

Answer 13

PKC receives priming phosphorylation from PDK-1, and then it autophosphorylate its own C-terminus

Question 14

Conventional PKC activation requires what?

Answer 14

DAG and Ca2+, products of phospholipase C cleavage of PIP2

Question 15

What are three types of PKCs

Answer 15

Conventional, Novel and atypical, require different factors for activation

Question 16

Mechanism of PKC phosphorylation?

Answer 16

The binding of second messengers allow psuedosubstrate domain to separate from the substrate binding pocket of the enzyme, allowing PKC to phosphorylate the ser/thr residue of proteins with a PKC consensus

Question 17

Example of physiological relevance of PKC

Answer 17

PKC dysregulation involved in many cancers, PKC helps prime mitochondria after minor ischemic event which can survive major infarct much better

Question 18

cAMP removed by

Answer 18

phosphodiesterases

Question 19

how is PKA implicated in cystic fibrosis?

Answer 19

Hormone binds to receptor, stimulates Gs, Ad cyclase making cAMP, cofactor for PKA, which then signals to CFTR channel to shuttle Cl out of cell – channel dysfunctional in cystic fibrosis

Question 20

Diffs between PKA and PKG (2)

Answer 20

No G protein, guanylate receptor protein directly coupled. Two diff types of guanylate cyclase, including cytosolic. Activated by NO. Dimer, instead of dimer of dimers

Question 21

PKG physiological relevance

Answer 21

Atrial natriuretic peptide/factor (ANP/ANF). Produced by cardiac distension, goes to kidney, increases sodium and water excretion, which decreases blood volume. ANP hormone binds to guanylyl cyclase receptor, creates cGMP and produces PKG, which blocks absorbtion of sodium from kidney, so more Na goes into blood

Question 22

Viagra mechanism

Answer 22

Inhibits phosphodiesterase, drastically decrease half life of cGMP in cell, smooth muscle relaxation and increased blood flow

Question 23

T/F. Free Ca2+ is kept in moderately high intracellular concentrations

Answer 23

False, kept in very small concentrations

Question 24

Enzyme involved in sensing cellular Ca2+

Answer 24

calmodulin, when bound activates CaM kinase

Question 25

what does CaM kinase need in order to be fully functional?

Answer 25

Ca2+ bound to Ca2+, autophosphorylation

Question 26

T/F. Nitric oxide signals via membrane-bound GC

Answer 26

False, signals via intracellular GC

Question 27

T/F. Calmodulin binds to Ca2+ to keep free cytosolic Ca2+ low

Answer 27

False, to sense calcium levels

Question 28

T/F. PKG pathway signaling involves activation of a G-protein coupled receptor

Answer 28

False, no G-proteins involved, it’s guanylyl cyclase dependent

Question 29

T/F. Conventional PKC activation requires phosphatidyl serine, Ca2+ and DAG

Answer 29

True!

Question 30

T/F. CaM kinase binds directly to Ca2+

Answer 30

False, binds to calmodulin which is bound to Ca2+

Question 31

What are first two steps of all MAPK pathways?

Answer 31

Stimulus, then activator (can be receptor tyrosine kinase, etc)

Question 32

Where is a GEF involved in MAPK?

Answer 32

In conversion of stimulatus to activator, you have exchange of GDP for GTP

Question 33

Ca2+/calmodulin physiological relevance

Answer 33

phosphorylates myosin, leads to muscle contraction

Question 34

Generic MAPK pathway

Answer 34

Stimulus → activator → Map Kinase Kinase Kinase (MKKK) → Map Kinase Kinase (MKK) → Map Kinase (MAPK) → substrate

Question 35

Example of GEF

Answer 35

SOS (Son of sevenless)

Question 36

Example of G-protein

Answer 36

Ras-GTP

Question 37

phosphatase function

Answer 37

They catalyze the hydrolysis of a phosphate on a protein

Question 38

MAPK pathway more specific example

Answer 38

1. Three successive kinases are activated: MKKK phosphorylates MKK, which phosphorylates MAPK
   - i.g. Raf phosphorylates MEK, which phosphorylates ERK
2. The MAPK then translocates to the nucleus and acts on the final targets, usually a transcription factor (i.e. ERK goes to the nucleus to phosphorylate Elk, which works with SRF as a transcription factor)