学会レポ II

• Interaction of paxillin with poly(A)-binding protein 1 regulates cell migration via translational control of Raf synthesis. (Norman of Beatson Institute)

Paxillin, vinculin, talin などの focal adhesion complex の構成タンパク質と相互作用するタンパク質をプロテオミクスで調べた研究。ノートを日本語訳する元気がないので、そのまま。
Anti-paxillin antibody immunoprecipitiaon -> Mass spectrophy. This identified poly(A)-binding protein 1 (PABP1) as a paxillin binding protein.
Paxillin has 4 LD domains in its amino-terminus and interacts with actopaxin, actin, vinculin, FAK and MT.
PABP, along with eIF4G and eIF4E, generates pseudocircularisation of mRNA for efficient translation, but paxillin is NOT a component of this protein-RNA complex. Suggesting a different role of paxillin in PABP binding.
They checked the localization of mRNA bound paxillin by anti-paxillin staining and membrane fractionation. This showed paxillin to be in the ER fraction and perinuclear zone.
Next, they shut out the nuclear export of protein by treating cells with lactomycin B, nuclear export inhibitor. This treatment induced nuclear accmumilation of paxillin and PABP1. RNAi knock down of paxillin also resulted in PABP1 nuclear accumilation. These results indicate the possilbity of shuttling of PABP1 and paxillin complex between cytosol and nucleus. This possibility was further comfirmed by the fact that nuclear export signal exists in paxillin but NOT in PABP1.
Constructing PABP1 mutant that has mutation in paxillin binding domain was successful and over expression of the mutant paxillin caused migration defect and focal adhesion complex was more stable.
Focal adhesion turn over is mediated by phosphorylation of focal adhesion component. They searched for the candidate kinase, and identified Raf1 to be involved in this process.
So, taking the results so far into account, they hypothesised that activity of cell motility depends on Raf1 activity and PABP1-paxillin interaction is involved in this process.
Translational efficiency of a certain mRNA can be monitored by measuring the amount of ribosomal complexs loaded onto the mRNA. This can be inferred from the fraction from sucrose gradient fractionation and subseaquent detection of mRNA.
By this analysis, Raf1 mRNA was revealed to be heavilly translated, having more than five ribosomal complexes on and this translational efficiency was declined upon RNAi knock down of paxillin.
By recruitment of specific mRNA, in this case Raf1 and also beta-actin, to the site of focal adhesion complex, localized translation of Raf1 and beta-actin occurs. Local increase in the protein concentration of Raf1 induces dissasembly of focal adhesion complex, and increase in beta-actin concentration at the same place elogates the actin fibre to promote filopodial cell protrusion.
この過程で、細胞運動というのは細胞質内での分子の相互作用にととまらず、核から細胞膜へ、そしてその逆の過程の繰り返しによって制御されている非常に複雑な過程であることが描き出されているように思われる。果たして、このような状況を解析できるのだろうか？システムバイオロジーならばなんとかなるのかなぁ？
More over, paxillin is thought to function as a scaffold for MAPK cascade. It is possible that Raf1 is regulated transcriptionally together with the post transcriptional control shown in this talk.