Contribution of Orb2A Stability in Regulated Amyloid-Like Oligomerization of Drosophila Orb2
Figure 6
Tob promotes LimK-Orb2 association and Orb2 phosphorylation.
(A) Tob is a substrate for Lim kinase. An in vitro kinase assay was performed using recombinant maltose binding protein (MBP) and MBP-tagged mammalian Btg, Tob1, Tob2, and Drosophila Tob (dTob) as substrates (left panel). Drosophila Tob was phosphorylated, as were Tob1 and Tob2, while the more distantly related Btg exhibited background levels of phosphorylation similar to MBP (right panel). The calculated molecular weight of MBP tag is 42.5 KDa and that of MBP-tagged Drosophila Tob is ∼101 KDa. (B) Lim kinase interacts with Tob. (Right panel) Tob was immunoprecipitated from adult head extracts expressing HA-tagged LimK under Elav-GeneSwitch inducible driver line and blotted with anti-HA antibodies for LimK. (Left panel) The +RU486 lane contains 5% of the lysate used for immunoprecipitation in the right panel. (C) Lim kinase phosphorylates Tob-associated Orb2A and Orb2B. Orb2 with and without Tob was immunopurified and dephosphorylated prior to use as substrates in the in vitro kinase assay. Phosphorylation was assessed in the presence (+LimK) or absence (−LimK) of LimK. Orb2 by itself shows a low level of phosphorylation, suggesting either endogenous LimK or some other kinase is copurified. Phosphorylation significantly increases with the addition of LimK. Western blots on the right show expression levels of the individual components as loading controls. (D) LimKinase phosphorylates recombinant Orb2B in the presence of recombinant MBP-tagged Tob. Due to insolubility, it was not possible to test recombinant Orb2A in this assay. The LimK used in this study is also autophosphorylated. (E) LimKinase (tagged with V5-epitope) forms a complex with Orb2 only in the presence of Tob that interacts with Orb2. In presence of TobΔ28, which interacts weakly with Orb2, only background level of LimK was detected in the Orb2 complex. Also see Figure S6.