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.2011 Nov 15;21(5):888-95.
doi: 10.1016/j.devcel.2011.08.028.

Tao-1 phosphorylates Hippo/MST kinases to regulate the Hippo-Salvador-Warts tumor suppressor pathway

Affiliations

Tao-1 phosphorylates Hippo/MST kinases to regulate the Hippo-Salvador-Warts tumor suppressor pathway

Julian C Boggiano et al. Dev Cell..

Abstract

Recent studies have shown that the Hippo-Salvador-Warts (HSW) pathway restrains tissue growth by phosphorylating and inactivating the oncoprotein Yorkie. How growth-suppressive signals are transduced upstream of Hippo remains unclear. We show that the Sterile 20 family kinase, Tao-1, directly phosphorylates T195 in the Hippo activation loop and that, like other HSW pathway genes, Tao-1 functions to restrict cell proliferation in developing imaginal epithelia. This relationship appears to be evolutionarily conserved, because mammalian Tao-1 similarly affects MST kinases. In S2 cells, Tao-1 mediates the effects of the upstream HSW components Merlin and Expanded, consistent with the idea that Tao-1 functions in tissues to regulate Hippo phosphorylation. These results demonstrate that one family of Ste20 kinases can activate another and identify Tao-1 as a component of the regulatory network controlling HSW pathway signaling, and therefore tissue growth, during development.

Copyright © 2011 Elsevier Inc. All rights reserved.

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Figures

Figure 1
Figure 1. Loss of Tao-1 function causes overproliferation
(A–C)ap>Tao-1 RNAi causes massive overgrowth of 3rd instar wing imaginal discs compared to wild-type discs. GFP marks theapterous domain, and Notch or E-Cadherin is in blue. Ventral is down. (D–F)Tao-1 knockdown causes overproliferation in the eye imaginal disc. Larval eye imaginal discs expressingTao-1 RNAi under the control of thedorsal enhancer driver (de>Tao-1 RNAi) are overgrown dorsally compared to wild-type eye discs. Anterior is to the left and ventral down in all eye disc images. (G–I) E-cadherin staining of pupal eyes 43 hr APF showing increased numbers of interommatidial cells inGMR>Tao-1 RNAi eyes compared toGMR-Gal4/+ eyes. Yellow arrows point out regions where interommatidial cell layer is doubled. (J–L) Representative images ofGMR-Gal4/+ andGMR>Tao-1 RNAi third instar eye discs labeled with BrdU to indicate mitotically active cells. Note increased incorporation of BrdU posterior to the second mitotic wave (yellow arrow) inGMR>Tao-1 RNAi discs. The morphogenetic furrow is marked by the red arrowhead.Tao-1 RNAi transgenes on the second chromosome (images in B, E, H, K) and third chromosome (images in C, F, I, L) give similar phenotypes. Unless otherwise noted, all subsequent experiments usingTao-1 RNAi were conducted with the transgene on the third chromosome, which was used primarily for convenience in building strains.
Figure 2
Figure 2.Tao-1 depletion causes upregulation of many known targets of the HSW pathway
(A–D)ap>Tao-1 RNAi results in upregulation of known HSW targets within the dorsal compartment of the wing. (A) Notch, (B) Crumbs, (C) aPKC, and (D) Expanded are apically enriched inTao-1 RNAi wing discs. Arrows mark the D–V boundary. (E–E’) In the eye imaginal disc,four-jointed-LacZ, a transcription reporter for HSW signaling, is upregulated in heat-shock flip-out cell clones expressingTao-1 RNAi (marked by GFP). (F–G) In the wing,Tao-1 knockdown under theen-Gal4 driver results in increased activity of thebantam miRNA (as assayed by the bantam-GFP sensor, Brennecke et al., 2003), ayki target gene, suggesting increasedbantam transcription. Arrow indicates A–P boundary. (H–H’) Expression of Flag-taggedTao-1 under theengrailed promoter (en>Tao-1-Flag) results in lower Ex levels in the posterior compartment of wing discs. In all images ventral is down and posterior to the right.
Figure 3
Figure 3.Tao-1 functionally interacts with HSW pathway components
(A) Lethality caused by loss ofTao-1 is suppressed by expressing awts transgene. Surviving adults were scored 1–2 days after eclosion and the percentage of expected offspring was plotted for each genotype. The number within each bar represents the expected number of adult progeny for each genotype. (B–D)Tao-1 RNAi suppresses the effect of Wts overexpression in the wing. (B–C) Maleen-Gal4/+ control wings appear normal, whereasen>wts wings have a smaller posterior compartment. (D)en>wts; Tao-1 RNAi animals survive to adults and have an intermediate wing phenotype. (E) Tao-1 promotes Wts phosphorylation inDrosophila S2 cells. Tao-1 and Wts were transiently expressed in S2 cells and Wts phosphorylation was measured using a phospho-specific antibody (Yu et al., 2010). Levels of Wts phosphorylation (lane 1) were increased in the presence of Hpo and Tao-1 (lanes 2 and 3). In contrast, Wts phosphorylation was severely diminished in the presence of a kinase-dead form of Tao-1 (lane 4). (F–H’) HSW pathway target gene upregulation inTao-1 RNAi knockdown requiresyki. (F–G’) Reduction ofyki using theen-Gal4 driver leads to a reduction in Ex staining, whileTao-1 RNAi results in increased Ex expression. (H–H’) Double knockdown ofTao-1 andyki causes decreased levels of Ex, similar toyki RNAi alone.
Figure 4
Figure 4. Tao-1 phosphorylates Hpo to activate the HSW pathway
(A) Hpo is required for Tao-1 to promote Wts phosphorylation in S2 cells. Normally, Wts is phopshorylated in the presence of Tao-1 (lane 2); however, transfectinghpo dsRNA prevents Wts phosphorylation (lane 3). (B) Tao-1 promotes Hpo phosphorylation at a critical regulatory residue (T195). Cells were transfected with a kinase-dead form of Hpo (HpoKD) to prevent auto-phosphorylation. A low level of Hpo phosphorylation was detected in these cells using a commercially available phospho-MST1/2 antibody (lane 1), which specifically recognizes phosphorylation at T195. This level of phosphorylation can be diminished by depleting endogenous Tao-1 (lane 2), or by expressing a kinase-dead form of Tao-1 (Tao-1KD; lane 3). Hpo phosphorylation is greatly increased in the presence of wild-type Tao-1 (lane 4). (C–E’)Tao-1 functions throughhpo to regulate HSW pathway target genes. (C–C’) As shown previously, loss ofTao-1 results in increased Ex levels. (D–D’)UAS-hpo expressed using adpp-Gal4 driver (dpp>hpo) results in decreased Ex expression. (E–E’)dpp>hpo; Tao-1 RNAi also results in decreased Ex staining, suggesting thathpo functions downstream ofTao-1. All flies raised at 18°C. (F) Tao-1 directly phosphorylates T195 within the activation loop of Hpo. Flag-tagged Tao-1 was immunoprecipitated from S2 cells and incubated with recombinant Hpo kinase domain (aa 1–367; GST-Hpokin dom). Immunoprecipitated Tao-1 displayed a mobility shift (lanes 2 and 4) relative to Tao-1KD (lanes 1 and 3), indicating that Tao-1 was catalytically active. Staining with phospho-MST1/2 antibody revealed that Tao-1 phosphorylates the kinase dead mutant of Hpo at T195 (lane 2). The T195A mutation confirmed that the phospho-MST1/2 antibody specifically recognizes phosphorylation at T195 (lane 4).
Figure 5
Figure 5. Mer and Ex require Tao-1 to activate the HSW pathway
(A) Mer and Ex require Tao-1 to induce increased Wts phosphorylation. Normally Wts phosphorylation increases in the presence of Mer and Ex (lane 2). However, depleting Tao-1 with dsRNA eliminates this effect (lane 3). (B) Tao-1 induced Wts phosphorylation is not dependent on Mer and Ex. Depletion of Mer and Ex by dsRNA (lane 3) does not affect Tao-1 induced p-Wts staining (lane 2). (C) Tao-1 promotes Hpo phosphorylation independently of Mer, Ex and Kibra. S2 cells were transfected with dsRNA targetingMer (lane 3),ex (lane 4) orkibra (lane 5). Knockdown of these genes had no effect on Tao-1’s ability to promote Hpo phosphorylation (compare lane 2 to lanes 3–5).
Figure 6
Figure 6. A model for Tao-1’s function in the HSW pathway
Tao-1 directly phosphorylates Hpo at T195 in the kinase activation loop, leading to the activation of Wts and inhibition of Yki, which remains cytoplasmic when phosphorylated. In the absence of HSW pathway activity, Yki translocates to the nucleus to promote transcription of target genes that promote growth and inhibit cell death. Genetic and biochemical experiments position Tao-1 upstream of Hpo and suggest that Mer and Ex function through Tao-1 to activate HSW signaling. Kibra, Mer and Ex can form a protein complex and promote HSW activation, but it remains unclear if Kibra functions upstream or in parallel to Tao-1.
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