Lecture 10

Question 1

What triggers the phosphorylation of RyR2 (ryanodine receptor) by PKA

Answer 1

The initial cause is a stressor (adrenaline, epinephrine)

The signalling events activate adenylyl cyclase (a membrane protein)

Adenylyl cyclase converts ATP to cAMP

Question 2

What does cAMP do

Answer 2

It activates PKA by binding to its regulatory subunit that stops it from being active

After binding to it, the PKA is active and phosphorylates RYR

Question 3

What does activating the RYR do

Answer 3

In the SR lumen and it triggers heart rate to increase

Question 4

What does CaMKII do

Answer 4

Same as PKA, phosphorylates ryr2 but needs ca-can to activate it first

Question 5

What is the phosphorylation target site on the RYR receptor for camKII

Answer 5

A serine residue

Question 6

What is a phospomimetic mutation

Give an example of how this applied to RYR receptor

Answer 6

Converting a residue to another residue that mimics a phosphate (has a negative charge)

The serine target in the receptor can be replaced by aspartate to mic is being phosphorylated

Question 7

What is the result of a phosphomimetic mutation in RYR and what does this tell us

Answer 7

The activity of PKA increases once’s the seine looks phosphorylated

And the disordered region of the RYR becomes alpha helical

This tells us that kinases have cross talk where they indirectly influence each other (the phosphorylation of RYR made PKA increase)

Slide 3

Question 8

How is the phosophomimetic mutation of ser to asp not perfect

Answer 8

Because phosphate has -2 charge

Asp has -1

Question 9

Can proteins be targeted by more than one kinase

Answer 9

Yes and the different kiansss have their own recognition sites for the protein

Question 10

Give an example of something that’s targeted by many kinases and what kinases target it

Answer 10

Caldesmon

By PKA, PKC, cam k1/II, casien kinase II

Question 11

What is caldesmon

Answer 11

A smooth muscle contraction protein

But not targeted by myosin kinases

Question 12

What do protein phosphotase do

Answer 12

Remove phosphate groups from ser, thr, or tyr side chains

Question 13

What do protein phosphotase generally have in them

Answer 13

One type Dependent on metal ions (like calcineurin) so have two metal ions (fe/mn/zn/mg)

But the mg is less common

One types have A cysteine residue in the active site

Question 14

What are the two types of protein tyrosine kinases?

Answer 14

Receptor (RTK)

Non receptor (TK)

Question 15

What do Receptor tyrosine kinases need to be active

What is their structure

Answer 15

They need to dimerize to be active

The kinase domain is in the cytosol and very similar across all types of RTK

the head is extracellular and different across all types of RTK (interacts with diff proteins)

Question 16

What do Non receptor tyrosine kinases have

Answer 16

They’re attached to the membrane via palmitoyl or myristoyl

Intercellular

(lipid groups that get added to the proteins and insert into the membrane)

Question 17

Different rtk’s respond to

Answer 17

Different stimuli (which is why the extracellular domains are all different)

Question 18

Explain the dimerization process of RTK

Answer 18

Before it’s signal binds, it’s two domains are separate and inactive

Dimerizes once it ligand binds and gets activated

The kinase domain (in cytosol) phosphorylates itself

The Diff proteins with sh2 domains dock and bind to these phosphorylated sites

Question 19

What is the kinase activation cascade

Answer 19

After the sh2 protiens dock at the kinase domain of RTK, signalling MAPK pathway happens

Question 20

Does the RTK add a phosphoryl or phosphate group

Answer 20

Phosphoryl

Question 21

What does the kinase domain of RTK look like

Answer 21

Has a beta sheet heavy region

Question 22

How does the src family non receptor tyrosine protein kinase work

Answer 22

N-SH3-SH2-kinase-C

The sh3 domain binds to two prolines (pxxp) motif on the inactive kinase

The sh2 binds to a phosphotyrosine on the c terminus

A phosphotase removes the tyrosines phosphate, sh2 domain detaches

The kinase auto phosphorylates itself and becomes active

Question 23

Slide 12

Answer 23

What do we need to know

Question 24

What amino acids do sh2 domains recingnize

Answer 24

Phospotyrosine residues

Question 25

What amino acids do sh2 domains recingnize

Answer 25

Phospotyrosine residues

Question 26

How does the sh2 binding pocket form

Answer 26

So it’s bind phosphorylated tyr

Inside the pocket is postively charged to bind the negative phosphate on the phosphotyrosine

The pocket is also hydrophobic since the tyr is hydrophobic because of its aromatic ring

Question 27

How many negative charges on phosphotyrosine at physiological ph

Answer 27

Two

Question 28

What are scaffold protiens and give examples

Answer 28

They organize the positions of other proteins and create functional protein networks

They act as scaffolds for other proteins to bind

Ex. Shank, PSD-95, Grip

Question 29

What domains are in the shank1 protein

Answer 29

Ankyrin

SH3

PDZ

Proline rich region (recognized by sh3 domains)

SAM

Question 30

What are the two types of sequences that SH3 domains bind to

Answer 30

Class 1 from the C-N of the sh3 domain:
Pxxp

Class 2 From the N-C of the sh3 domain: xpxxp

Question 31

What sites of proteins does the pdz domain in shank 1 bind

Answer 31

Binds the c teminal domain of other proteins

Question 32

How many pdz domains does shank have

What about grip

Answer 32

1

Multiple

Question 33

What are ankyrin repeats

Answer 33

2-3 or more repeats if an anchor to bind other proteins

By multiple of the repeats together, they form loops that bind to protiens

Question 34

How many ankyrin repeats does shank 1 have

Answer 34

7

Question 35

What does a single ankyrin domain look like

Answer 35

1 domain without a loop

Question 36

What is the SAM domain in shank 1

Answer 36

Interacts with itself to form dimers and oligomers

This means it can build a network of shank proteins

Question 37

What are all of the proteins interaction domains

Answer 37

SH3
SH2
C1
C2
PDZ
SAM
ANK repeat