Lecture 7; Cell signalling four Flashcards
What are secondary messangers?
Second Messengers are freely diffusable signaling molecules (generated in the cell)
Examples include
Ca++ cAMP cGMP IP3 NO
What residues are most often phosphorylated in the cell and what are the effects of this?
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.
What Phosphorylates Ser Thr residues?
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
What do AA sequences either side of the target residue create?
AA sequences give the protein very specific biochemical properties and therefore determines which specific enzyme can bind
SPECIFCITY
Do g proteins exist beyond GPCR?
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
What are g proteins and how are they regulated?
They are GTP dependant and are regulated by signalling pathways that control GTP addition and removal
i. e GAPs = removal
i. e GEFs = addition
What does the Rac family do?
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
What does the g protein RAS do?
involved in regulating growth pathways
What can mutations in RAS result in?
Mutations in Ras are quite common in cancer and these are capable of inducing uncontrolled cell growth (i.e Ras is an important oncogene)
What regulates RAS?
A GTP exchange factor (GEF) called SOS that is specific for Ras can promote the swapping of GTP for GDP on Ras.
Describe step one in RAS signalling?
SOS is activated by Growth factor signaling pathways so facilitates growth factor activation of Ras. (adds GTP)
What is step 2 in RAS signalling?
GTP Loaded Ras interacts with and activates the protein kinase Raf
What is step 3 in RAS signalling?
which in turn activates the Map Kinase pathways via a phosphorylation cascade
(signal amplification)
What is step 4 in RAS signalling?
which activates expression of genes involved in growth
What regulates RAS post signalling?
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.
What provides a link between two components in a signalling pathway?
Adapter Proteins Provide a Link Between Two Components in Signaling Pathway e.g. Grb2
How is Grb2 an adapter protein?
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
Give another example of an adapter protein that links to the RAS pathway;
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
What are scaffold proteins?
- Protein binding molecules
- Bigger than adapter molecules
What is the function of scaffold proteins?
They can bind a number of signalling molecules and localise them to a particular part of the cell, concentrating them for a purpose
Give some examples of scaffold protiens;
- 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
What are MAP kinases a good example of?
- Ser/Thr Kinase Amplification Cascades
- Where a Scaffold Can Direct Specificity
Why do scaffolds need to direct specificity in MAP kinases?
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
How else can protein kinases regulate signalling cascades?
Protein Kinase Cascades Can Regulate Signaling Pathway by Regulating Protein Stability via Ubiquitination
How does ubiquination promote stability?
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
What is the function of phospholipases?
Phospholipases cleave phospholipids to produce lipid second messengers
What is a major phospholipase?
Phospholipase C is a major signaling molecule causing cleavage of PIP2 to release IP3 (stimulating Ca++ influx) and diacylglycerol (which activates Protein Kinase C)
What are the two major types of PLC?
- PLCb activated by GPCR via Gq
- PLCg contains an SH2 domain and is activated for example by the PDGF receptor
Give an example of signalling through PLC;
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+
What produces PIP3?
Phosphoinositide Kinases (PIKinases) - 8 isoforms, 3 classes,
i.e Growth factor stimulation rapidly activates class I PI 3-kinase which stimulates production of PIP3
What is the function of PIP3?
PIP3 has a very potent effect in stimulating cell functions such as growth, migration and metabolism
Describe in general how closely phosphoinositides are related;
Imagine the different phosphoinositides as keys with same overall conformation but with different prongs allowing each to open a different lock
Describe the synthesis of PIP3;
PI 3-kinase is main form regulated (i.e it phosphorylates the 3 position of inositol ring to produce PIP3)
Phosphoinositide Kinases (lipid kinase)
Describe the mechanism of action of PIP3;
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
Describe a molecule with a PH domain that relies on PI 3 kinase;
Membrane Localisation of pREX (PH domain) Relies of Activation of PI 3-kinase and Results in Activation of Rac
Results in increased cell movement
Describe a cascade that relies on PI3 kinase;
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
Describe the class 1a PI 3 kinase structure;
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
- It is not at the membrane where substrate is
- The N-SH2 domain is blocking kinase activity
What facilitates P110 recruitment to the growth factor receptor?
(p85) Adapter Subunit SH2 Domains Facilitate Recruitment of p110 to pTYR Proteins via phosphorylation
How does PI 3 Kinase subunit p110 become activated?
Interaction of SH2 Domains with pTYR Proteins Induces Conformational Change Allowing Activation of p110
In this state the PI 3-kinase is active because
- It is at the membrane where substrate is
- The N-SH2 domain isn’t blocking kinase activity
How can the catalytic activity of PI 3 kinase be regulated?
Ras and Gβγ Further Strengthen Localisation to PM and Can Also Regulate Catalytic Activity
