Many of these pages are rough notes and protocols that I'm working on. I'm trying to publish as many of my ideas on oww to promote an open type of science, for discussion and collaboration. Please feel free to browse and read them. If you find any errors or anything of interest then please comment in the discussion page, or e-mail me your thoughts.
- FOXO regulates lifespan in the fly
- Acts as a transcription factor when dephosphorylated
- (SS) Does it only act as a transcription factor when dephosphorylated? In other words, if there were DNA floating around in the cytoplasm, would FOXO transcribe that DNA when it is phosphorylated? **JC, It's Eukaryote, so only DNA in nucleus.
- Phosphorylated by AKT
- (SS) Within the NLS, right?
- Move FOXO to the nuclues on exogenous signal (yeast pheromone), with Samantha Sutton's phospholocator.
Look for FOXO in yeast and use the existing system if it exists in yeastDoesn't look like it exists, only other FORKHEADS. anyhow, better to try it in s2 cells.
- Suggestion to use fly S2 cells instead of yeast. using the existing constructs and seeing if they work.
BS says: Acts as a transcription factor when dephosphorylated
- Examples where phosphorylation of Foxo can lead to increased activity
*MST1 phosphorylates Foxo at the forkhead-domain (Ser212 in Foxo1). Which results in disruption of the 14-3-3 Foxo complex. *JNK phosphorylates Foxo within the transactivation domain (Thr447 and Thr451 in Foxo4), promoting Foxo nuclear translocation
Jnk homologous kinase in Drosophila (homologous to Hog1).
(SS) Idea #1. So if you want to use the Phospholocator in S2 cells, we need to find a way to phosphorylate the Phospholocator NLS with an endogenous drosophila kinase. This kinase should:
- Be activatable by an external stimulus
- Have well-defined PO4 motif (the site that is phosphorylated) and a well-defined docking site, if applicable. We will swap in the new motifs in place of the Fus3 motifs in the phospholocator.
A few ideas on kinases:
- Erk2 homologous kinase in Drosophila (homologous to Fus3).
- p38 homologous kinase in Drosophila (homologous to Hog1).
- Jnk homologous kinase in Drosophila (homologous to Hog1).
- The good thing about these three kinases is that they are well studied and do have defined motifs.
Are these kinases at all implicated in the starvation, longevity, or mating responses in Drosophila? Are there papers showing how to activate these kinases with an extracellular stimulus?
(SS) Idea #2. Build a constitutively active Fus3 kinase (it's been done in the past), and transfect into Drosophila cells. Inhibit the kinase until you are ready for it to be active... then wash away inhibitor, and you'll have active Fus3.
- Inhibitor is expensive, and hard to get.
- Who knows what Fus3 will do inside Drosophila cells? For all we know, it will phosphorylate some really important starvation proteins. Hard to control for.
Plan of action
- Identify the NLS in FOXO
- Use Samantha's phospholocator (NLS + Fus3) or (Cdc28/Clb2)
- But these might not work in S2 cells, so might need to rethink.
- Reengineer the AKT phosphorylation site to use the existing FOXO NLS
- much more difficult at this stage.
- (SS) So I'm not quite sure what this refers to, but if it's what we discussed while walking to the bus, here's what it entails:
- Use the kinase you find from idea #1 above.
- Identify the FOXO NLS, and the residue at which FOXO NLS is phosphorylated by AKT.
- Mutate the residues next to the phosphorylation site, so that now the phosphorylated residue is flanked by the consensus site for your new kinase.
- Add a docking site for your new kinase.
- Test if the FOXO NLS still works
- This part is uncertain.
- Test if your new kinase can phosphorylate the NLS, and if so, if it causes a change in translocation
- This part is uncertain.
- Activity of daf-16 (FOXO) is inhibited by a conserved phosphatidylinsitol-3-OH kinase (PI(3)K)/protein kinase D (PDK)/Akt pathway in response to DAF-2 (Insulin receptor) activity. see Tatar, Science 2003