Rae1 feeds back to stabilize Warts, Hippo, and Merlin.
(A) FLAG-Rae1 over-expression shows a dose-dependent increase in myc-Wts levels in S2 cells. (B) Rae1 over-expression increases myc-Wts levels (lane 2) compared to control (lane 1) in Drosophila salivary glands. (C) Reducing Rae1 levels slightly by removing one copy (lane 2) reduces myc-Wts levels relative to control (lane 1) in Drosophila wing discs. (D) Rae1 reduction by siRNA reduces pLats levels in HeLa cells. (E) FLAG-Rae1 over-expression shows a dose-dependent increase in myc-Hpo levels in S2 cells. (F) Rae1 over-expression increases pHpo levels (as monitored by pMST/2 antibodies that crossreact with Drosophila Hpo, ) (lane 3) compared to control (lane 2) in Drosophila salivary glands. (G) FLAG-Rae1 over-expression increases HA-Mer levels in S2 cells. (H) Constitutive, low level Rae1-GFP over-expression shows association with the nuclear periphery in salivary glands. (I-J) Increasing Rae1-GFP levels using alleles that impair Hippo signaling (hpoMGH1/+, I) or ubiquitination (Uba1B1/+, J) show increased pools of Rae1-GFP and some association with the cell membrane (arrowheads in I, J) strongest at higher levels of Rae1 (J). All images (I-J) were taken with identical exposure times and settings. Scale bars in H-J indicate 20 μm. (K-K”) Immunofluorescence of Wts over-expression in the salivary glands (ptc>myc-wts) shows Wts association with plasma membranes and diffuse staining in the cytoplasm. Arrowheads point to cells with higher levels of cytoplasmic Wts staining. (L-L”‘) Co-over-expression of Rae1 and Wts in the salivary glands (ptc>myc-wts, Rae1GFP) increases Wts levels and association with the plasma membrane including overlapping domains with Rae1. Cells with higher cytoplasmic Wts staining are no longer seen. Membrane recruitment of Wts has been reported to promote its activity [64–65]. Scale bars in K-L indicate 50 μm. (M) Rae1 over-expression increases acetylated tubulin levels in Drosophila salivary glands. (N) Reducing Rae1 levels decreases acetylated tubulin levels in Drosophila wing discs. Relative levels of Wts in A-C, pLats in D, Hpo in E, pHpo in F, Mer in G, and acetylated tubulin in M, N (normalized by Tubulin) are indicated.