Phosphoinositide Signalling Flashcards
How does membrane localisation drive signalling processes?
Increases the effective concentration of components in signalling pathways 700 fold. More likely to interact and activate downstream signalling.
3 mechanisms of localising proteins to the plasma membrane.
- Protein-protein interactions - phosphobinding motifs (SH2, PTB), ubiquitin motifs
- Lipid tethers - Myristoylation, prenylation.
- Lipid interacting motifs - (PH, FYVE, PX, PHD), DAG binding domain (c2) or membrane interaction domain (c1)
Example of membrane localisation - Ras
Farnesylation of Ras binds the membrane to act and induces tumourigenesis when oncogenic. If not localised, it cannot act as a small Mw G-protein.
Golgi localised Ras activates ERK and PI3K, ER localised was activates JNK.
How can PKB become oncogenic?
PKB activated by membrane localisation, binding to PIP3.
Mutated E17K, fixing it to the membrane and becoming oncogenic.
Structure of PtdIns
Draw and name 7
Explain the experiment that proved the turnover cycle and how the cycle functions.
Incubated pancreatic tissue with 32P, isolated PIs. Radiolabelling of PIs can see how PIP2 is replenished at the plasma membrane - Turnover Cycle.
PIP2 -> DAG + IP3 (by PLC)
DAG -> Phosphatidic Acid
CMP-synthase adds CMP group
PI synthase recreates PI with no phosphates.
PI4K adds 1st, PI5K adds 2nd.
What is the function of PLC?
What are the downstream targets of its products?
PLC converts PIP2 to DAG and IP3.
DAG downstream targets = PKC (proliferation), Ras, Munc-13 (exocytosis)
IP3 downstream targets = Ca2+ release
How are PIs transferred back to the plasma membrane to replenish PIP2?
PITP mediated bulk transport.( PI-transport protein NA)
PITP picks up PI from ER, exchanges with PC at plasma membrane and takes PC back to ER. Until equilibrium is reached.
Sec proteins have a structurally similar domain that can perform this function.
Explain the Bankaitis review.
Individual responses in the cell are dependent on specific Sec/PITP proteins.
Loss of PI4K = loss of all downstream signalling
Loss of 1 PITP = loss of specific pathway
Could be involved in cancer therapeutics.
How were PI interacting proteins found?
Examples
Bead created containing specific PIs. Add cell lysate and wash unbound. Analyse bound proteins by mass spec.
PH, PX, FYVE, domains - 400 proteins
How is PIP2 signalling involved in actin polymerisation?
Polymerisation at the cell leading edge to cause cell migration, absence of retraction = elongation at the trailing edge.
Overexpression of PIP5K increased PIP2 and actin polymerisation / cytoskeletal dynamics - measured by GFP-Actin.
Causes Nucleation, Severing and Uncapping
How is PIP2 involved in regulation of actin nucleation?
ARP2/3 COMPLEX and WASP
ARP2/3 attaches to actin filaments and creates new nucleation sites for growth of branched filaments.
ARP2/3 activity is stimulated by WASP proteins.
WASP activation caused by AND logic gate - PIP2 binding AND either Cdc42 or Nck binding.
WASP (VCA domain) kept inactive by VCA-CRIB binding domain. NWASP/PIP2/Cdc42 = same activity as VCA alone.
How is PIP2 involved in regulation of actin severing?
Cofilin binds PIP2 through Lys-rich patches. PIP2 interaction sequesters cofilin to the membrane, leading to an inactivation of its severing ability.
EGF activating PLC = PIP2 hydrolysis, cofilin translocates away from the membrane, bind F-actin and induce severing to increase polymerisation (new barbed ends)
How is PIP2 involved in regulation of actin uncapping?
Profilin binds PIP2 through Lys-rich patches. This sequesters profilin and stops the actin-profilin capping, leading to an increase in polymerisation.
Data in vivo shows positive and negative impact on actin polymerisation.
What are focal adhesions?
- Link the ECM to the cytoskeleton through transmembrane integrins.
- Help the migrating edge of the cell move further
- Integrins are receptors for the ECM and organise intracellular platforms that act on motility and proliferation.
