JC3 paper Flashcards
what enzyme is shown to control endosomal to lysosomal sorting of EGFR?
type I gamma PIP5-kinase (PIPKIγi5)
synthesises PtdIns4,5P2
What does EGFR stand for?
epidermal growth factor receptor
what plays a role in controlling the signaling of EGFR?
-trafficking and degradation of EGFR
what does PIPKγi5 interact with?
- interacts with SNX5 (sorting nexin 5)
- SNX5 binds to PtdIns4,5P2 and other phosphoinositides
where do SNX5 and PIPKIγi5 localise to?
-localise to endosomes
What happens when there is loss of either SNX5 or PIPKIγi5?
- EGFR trafficking form the early endosome into the ILVs of a MVB is BLOCKED
- loss of ILV sorting prolongs EGFR signalling
How do SNX5 and PIPKIγi5 facilitate ILV sorting?
- they prevent ubiquitination of hrs
- this allows hrs to bind to EGFR which is required for ILV sorting
what does EGFR govern/what is its function?
Governs growth and differentiation of cells during development and adult homeostasis
what can happen to EGFR once it has been internalised?
IT undergoes trafficking to decide whether it:
- gets recycled back to PM
- is degraded by lysosome
- translocation to the nucleus
what does PtdIns4,5p2 do?
regulates endosomal trafficking
phosphoinositide
How long does EGFR continue to signal in the cell once being endocytosed?
- signals until agonist is separated from receptor
- signals until trafficked into ILVs of the MVB
give principles of immunoprecipitation
-precipitating a particular protein (or complex)out of a solution using antibodies for that protein
- sepharose beads are used which can be sedimented by centrifugation
- Antibody for protein (e.g.SNX5) attached to bead.
- add bead to solution - will bind to protein/SNX5 if it present and pull down SNX5 complex when centrifuged
- wash and remove unbound proteins
- separate proteins and run on gel/immunoblot for SNX5 and PIPKIγi5
- shows if there is an interaction between SNX5 and PIP5 (there is)
Give the principles of a GST pull down
- Gluthionine bound to sepharose bead
- protein of interest has a fusion tag (e.g. GST)
- GST tag on protien of interest will bind to gluthionine (affinity ligand) which is attached to the sepharose bead.
- when centrifuged the beads with centrifuge with protein of interest attached
what happens when PIPKIγi5 siRNA is used?
- no PIPKIγi5 produced
- there is no trafficking to the lysosome of EGFR (stays in EE)
- EGFR is more abundant and activated
give an early endosome marker
EEA1
give a lysosomal marker
LAMP1
how can you tell that PIPKIγi5 siRNA prevents EGFR trafficking to the lysosome?
- Use immuno-fluorescence
- Stain cells with EGFR and LAMP1 antibodies- see overlap between the 2 (colocalization)
-when PIPKIγi5 siRNA is used there is less/no colocalising between LAMP1 and EGFR
what is actin used for in blot?
- a loading control
- shows all lanes have equal loaded amount of protein/ cells
Why was EGFR labelled with gold?
as heavy metal so shows up when using electron microscopy
what is BSA and why is it used?
bovine serum albumin
- used as it prevents non-specific binding
why was ubiquitin modified to have histadine molecules?
- histadine has a high affinity to nickel ions
- has high affinity to nickel matrix (nickel agarose)
- using the nickel argarose and histadine labelled ubiquitin allows us to identify ubiquitinated proteins
(non-ubiquitinated proteins will be washed off of the gel)
