Lecture 7; Cell signalling four

Question 1

What are secondary messangers?

Answer 1

Second Messengers are freely diffusable signaling molecules (generated in the cell)
Examples include

Ca++ 
cAMP
cGMP
IP3 
NO

Question 2

What residues are most often phosphorylated in the cell and what are the effects of this?

Answer 2

99% of phosporylation on cellular proteins is at serines or threonines

• changing protein function/enzyme activity
• changing location e.g. excluding transcription factors from cell nucleus
• altering proteins binding partners binding partner.

Question 3

What Phosphorylates Ser Thr residues?

Answer 3

Ser/Thr kinases, regulated by being phosphorylated.

These kinases lead to phosphorylation cascades and amplification as each kinase can p/ph more than one molecule

i.e low conc signal can have very large IC effects

Each kinase in the cascade has a specific target (specificity) and thus determines the end points that are phosphorylated

Question 4

What do AA sequences either side of the target residue create?

Answer 4

AA sequences give the protein very specific biochemical properties and therefore determines which specific enzyme can bind

SPECIFCITY

Question 5

Do g proteins exist beyond GPCR?

Answer 5

Yes, small G-proteins exist which ultilise GTP and GDP to regulate their function.

I.e

• Rac/Rho/cdc42 (Family)
• Ras family
• Rab family involved in vescle trafficking
• ARF (family) involved in vesicular trafficking

Question 6

What are g proteins and how are they regulated?

Answer 6

They are GTP dependant and are regulated by signalling pathways that control GTP addition and removal

i. e GAPs = removal
i. e GEFs = addition

Question 7

What does the Rac family do?

Answer 7

Even though they are closely related sequence wise they have different functions
e.g. Rac, Rho and cdc42 all affect actin cytoskeleton but in different ways

Question 8

What does the g protein RAS do?

Answer 8

involved in regulating growth pathways

Question 9

What can mutations in RAS result in?

Answer 9

Mutations in Ras are quite common in cancer and these are capable of inducing uncontrolled cell growth (i.e Ras is an important oncogene)

Question 10

What regulates RAS?

Answer 10

A GTP exchange factor (GEF) called SOS that is specific for Ras can promote the swapping of GTP for GDP on Ras.

Question 11

Describe step one in RAS signalling?

Answer 11

SOS is activated by Growth factor signaling pathways so facilitates growth factor activation of Ras. (adds GTP)

Question 12

What is step 2 in RAS signalling?

Answer 12

GTP Loaded Ras interacts with and activates the protein kinase Raf

Question 13

What is step 3 in RAS signalling?

Answer 13

which in turn activates the Map Kinase pathways via a phosphorylation cascade

(signal amplification)

Question 14

What is step 4 in RAS signalling?

Answer 14

which activates expression of genes involved in growth

Question 15

What regulates RAS post signalling?

Answer 15

GTPase Activating protein specific for Ras activates the GTPase activity in Ras catalysing the hydrolysis if GTP back to GDP. Now it can no longer activate RAF.

Question 16

What provides a link between two components in a signalling pathway?

Answer 16

Adapter Proteins Provide a Link Between Two Components in Signaling Pathway e.g. Grb2

Question 17

How is Grb2 an adapter protein?

Answer 17

Upon activation of PDGF Receptor a complex can form at the plasma membrane where Grb2 links the PDGF receptor to SOS and brings about activation of Ras

PDGF (growth factor receptor) adaptor protein activates SOS which regulates RAS

Question 18

Give another example of an adapter protein that links to the RAS pathway;

Answer 18

This pathway can also involve another adaptor protein called SHC which itself becomes tyrosine phosphorylated e.g in the case of insulin mediated activation of Ras

Question 19

What are scaffold proteins?

Answer 19

• Protein binding molecules

- Bigger than adapter molecules

Question 20

What is the function of scaffold proteins?

Answer 20

They can bind a number of signalling molecules and localise them to a particular part of the cell, concentrating them for a purpose

Question 21

Give some examples of scaffold protiens;

Answer 21

• AKAPs (A-Kinase anchoring proteins) - localises PKA with related signalling molecules
• RACKs (Receptors for A and C-Kinase) -localises PKC signalling
• JIP - anchors several components of the JNK cascade to help the targeting
• IRS proteins - act as scaffolds for insulin signaling complexes

Question 22

What are MAP kinases a good example of?

Answer 22

• Ser/Thr Kinase Amplification Cascades

- Where a Scaffold Can Direct Specificity

Question 23

Why do scaffolds need to direct specificity in MAP kinases?

Answer 23

Scaffold proteins are important in directing the specificity in the map kinase cascades because there are a lot of similarities in these pathways and cross reaction might occur without the scaffold proteins

Question 24

How else can protein kinases regulate signalling cascades?

Answer 24

Protein Kinase Cascades Can Regulate Signaling Pathway by Regulating Protein Stability via Ubiquitination

Question 25

How does ubiquination promote stability?

Answer 25

Ser phosphorylation of some proteins can stimulate them to be ubiquitinated Following the addition of an initial ubiquitin molecule further can be added = (polyubiquitination) leading to Poly-Ubiquitination = rapid breakdown via the proteasome complex

Question 26

What is the function of phospholipases?

Answer 26

Phospholipases cleave phospholipids to produce lipid second messengers

Question 27

What is a major phospholipase?

Answer 27

Phospholipase C is a major signaling molecule causing cleavage of PIP2 to release IP3 (stimulating Ca++ influx) and diacylglycerol (which activates Protein Kinase C)

Question 28

What are the two major types of PLC?

Answer 28

- PLCb activated by GPCR via Gq | - PLCg contains an SH2 domain and is activated for example by the PDGF receptor

Question 29

Give an example of signalling through PLC;

Answer 29

PLC can cleave a phospholipid - The diacylglycerol can Recruit and activate C1 domain containing proteins e.g.Protein Kinase C - The Inositol tris-phosphate can Activate calcium channels in ER and so increase cytoplasmicCa2+

Question 30

What produces PIP3?

Answer 30

``` Phosphoinositide Kinases (PIKinases) - 8 isoforms, 3 classes, ``` i.e Growth factor stimulation rapidly activates class I PI 3-kinase which stimulates production of PIP3

Question 31

What is the function of PIP3?

Answer 31

PIP3 has a very potent effect in stimulating cell functions such as growth, migration and metabolism

Question 32

Describe in general how closely phosphoinositides are related;

Answer 32

Imagine the different phosphoinositides as keys with same overall conformation but with different prongs allowing each to open a different lock

Question 33

Describe the synthesis of PIP3;

Answer 33

PI 3-kinase is main form regulated (i.e it phosphorylates the 3 position of inositol ring to produce PIP3) Phosphoinositide Kinases (lipid kinase)

Question 34

Describe the mechanism of action of PIP3;

Answer 34

Molecules at a very defined patch on the plasma membrane provides a mechanism to colocalises the enzyme with the substrate at a very high concentration (e.g PDK1 with PKB) so a reaction that was unlikely to occur in the cytosol is now very likely to occur The interaction of the PIP3 with the PH domain induces conformational changes that to produce an increase in catalytic activity and expose sites that are targets for phosphorylation

Question 35

Describe a molecule with a PH domain that relies on PI 3 kinase;

Answer 35

Membrane Localisation of pREX (PH domain) Relies of Activation of PI 3-kinase and Results in Activation of Rac Results in increased cell movement

Question 36

Describe a cascade that relies on PI3 kinase;

Answer 36

Activation of PKB Serine Kinase Cascade Relies of Activation of PI 3-kinase PIP3 activates PDK-1, which phosphorylates PKB that is also bound to PIP 3 (formed by PI 3 kinase) PKB in turn phoshprylates a range of proteins resulting in Results in increased cell growth, cell survival and cell metabolism Message; Activation of PKB Serine Kinase Cascade Relies of Activation of PI 3-kinase

Question 37

Describe the class 1a PI 3 kinase structure;

Answer 37

Class-Ia PI 3-kinase Contains Two Subunits (p85 ADAPTER +p110) With Multiple Regulatory Domains Which are Mainly Held Together by p85 Inter SH2 Domain Interactions In this state the PI 3-kinase is inactive because 1. It is not at the membrane where substrate is 2. The N-SH2 domain is blocking kinase activity

Question 38

What facilitates P110 recruitment to the growth factor receptor?

Answer 38

(p85) Adapter Subunit SH2 Domains Facilitate Recruitment of p110 to pTYR Proteins via phosphorylation

Question 39

How does PI 3 Kinase subunit p110 become activated?

Answer 39

Interaction of SH2 Domains with pTYR Proteins Induces Conformational Change Allowing Activation of p110 In this state the PI 3-kinase is active because 1. It is at the membrane where substrate is 2. The N-SH2 domain isn’t blocking kinase activity

Question 40

How can the catalytic activity of PI 3 kinase be regulated?

Answer 40

Ras and Gβγ Further Strengthen Localisation to PM and Can Also Regulate Catalytic Activity