MEF were transfected in suspension with 1.6 g DNA using DreamFect Gold transfection reagent.
This article demonstrates the high efficiency of DreamFect Gold from OZ Biosciences for DNA transfection into MEF cells cultivated in suspension.article reference: J Biol Chem. 2014 Jan 10;289(2):1128-41. doi: 10.1074/jbc.M113.498444.
β-arrestin promotes Wnt-induced low density lipoprotein receptor-related protein 6 (Lrp6) phosphorylation via increased membrane recruitment of Amer1 protein.
Kríz V, Pospíchalová V, Masek J, Kilander MB, Slavík J, Tanneberger K, Schulte G, Machala M, Kozubík A, Behrens J, Bryja V.
Abstract
β-Arrestin is a scaffold protein
that regulates signal transduction by seven transmembrane-spanning
receptors. Among other functions it is also critically required for
Wnt/β-catenin signal transduction. In the present study we provide for
the first time a mechanistic basis for the β-arrestin function in
Wnt/β-catenin signaling. We demonstrate that β-arrestin is required for
efficient Wnt3a-induced Lrp6 phosphorylation, a key event in downstream
signaling. β-Arrestin regulates Lrp6 phosphorylation via a novel
interaction with phosphatidylinositol 4,5-bisphosphate
(PtdIns(4,5)P2)-binding protein Amer1/WTX/Fam123b. Amer1 has been shown very recently to bridge Wnt-induced
and Dishevelled-associated PtdIns(4,5)P2 production to the
phosphorylation of Lrp6. Using fluorescence recovery after
photobleaching we show here that β-arrestin is required for the
Wnt3a-induced Amer1 membrane dynamics and downstream signaling. Finally,
we show that β-arrestin interacts with PtdIns kinases PI4KIIα and
PIP5KIβ. Importantly, cells lacking β-arrestin showed higher
steady-state levels of the relevant PtdInsP and were unable to increase
levels of these PtdInsP in response to Wnt3a. In summary, our data show
that β-arrestins regulate Wnt3a-induced Lrp6 phosphorylation by the
regulation of the membrane dynamics of Amer1. We propose that
β-arrestins via their scaffolding function facilitate Amer1 interaction
with PtdIns(4,5)P2, which is produced locally upon Wnt3a stimulation by
β-arrestin- and Dishevelled-associated kinases.
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