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. 2007 Apr 4;26(7):1772-81.
doi: 10.1038/sj.emboj.7601630. Epub 2007 Mar 8.

Mob as tumor suppressor is activated by Hippo kinase for growth inhibition in Drosophila

Xiaomu Wei  1 , Takeshi Shimizu, Zhi-Chun Lai
Affiliations

Mob as tumor suppressor is activated by Hippo kinase for growth inhibition in Drosophila

Xiaomu Wei et al. EMBO J. .

Abstract

Tissue growth and organ size are determined by coordinated cell proliferation and apoptosis in development. Recent studies have demonstrated that Hippo (Hpo) signaling plays a crucial role in coordinating these processes by restricting cell proliferation and promoting apoptosis. Here we provide evidence that the Mob as tumor suppressor protein, Mats, functions as a key component of the Hpo signaling pathway. We found that Mats associates with Hpo in a protein complex and is a target of the Hpo serine/threonine protein kinase. Mats phosphorylation by Hpo increases its affinity with Warts (Wts)/large tumor suppressor (Lats) serine/threonine protein kinase and ability to upregulate Wts catalytic activity to target downstream molecules such as Yorkie (Yki). Consistently, our epistatic analysis suggests that mats acts downstream of hpo. Coexpression analysis indicated that Mats can indeed potentiate Hpo-mediated growth inhibition in vivo. Our results support a model in which Mats is activated by Hpo through phosphorylation for growth inhibition, and this regulatory mechanism is conserved from flies to mammals.

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Figures

Figure 1
Figure 1
Genetic interactions between Hpo pathway components hpo, sav, wts and mats. Scanning electron microscopy (SEM) images of adult compound eyes are shown. (A) Wild type. (B) In ey-Gal4/UAS-hpo flies, overexpression of Hpo in the developing eye reduced eye size. (C) An N-terminal fragment of Hpo (HpoN, a constitutively active form) in ey-Gal4/UAS-hpoN flies can also reduce eye size. (D) Overexpression of a C-terminal fragment of Hpo (HpoC, a putative dominant-negative form) in ey-Gal4/UAS-hpoC flies had no obvious effect on eye morphology. (E) The eye of ey-Gal4/UAS-mats flies was morphologically normal. Coexpression of Mats with Hpo in the eye of ey-Gal4/UAS-hpo; +/UAS-mats flies (F) or HpoN in ey-Gal4/UAS-hpoN; +/UAS-mats flies (G) markedly enhanced the small eye phenotype so that the eye became much smaller or abolished. (H) Coexpression of Mats with HpoC in ey-Gal4/UAS-hpoC; +/UAS-mats flies did not cause any obvious defect in the eye. (I) Overexpression of Wts in ey-Gal4/UAS-Myc-wts flies by using a strong transgenic line 6R reduced eye size and caused the eye to have a cone-like shape. Coexpression of wts with hpo in ey-Gal4/UAS-Myc-wts UAS-hpo flies (J) or hpoN in ey-Gal4/UAS-Myc-wts UAS-hpoN flies (K) completely abolished eye development. In contrast, the small and cone-like eye phenotypes induced by Wts were suppressed by HpoC coexpression in ey-Gal4/UAS-Myc-wts UAS-hpoC flies (L) or by reduction of endogenous activity of hpo in ey-Gal4/UAS-Myc-wts; +/hpoBF33 flies or sav in ey-Gal4/UAS-Myc-wts; +/sav3 flies (M, N, respectively). (O) Overexpression of Sav in ey-Gal4/UAS-HA-sav flies did not disrupt eye formation. However, Sav was able to enhance the small eye phenotype caused by Wts in ey-Gal4/UAS-Myc-wts UAS-HA-sav flies (P). Anterior is to the left in all panels.
Figure 2
Figure 2
mats is epistatic to hpo. Third instar larval eye imaginal discs are shown. Clones in mosaic eye discs were positively marked by GFP (green) in (A–D) using the MARCM method (Lee and Luo, 1999), and mats mutant clones were marked by the absence of β-galactosidase (green) in (F–H). (A) An eye disc that contains GFP-labeled wild-type MARCM clones is shown as a control. The genotype is w ey-FLP UAS-GFP/+; Tub-Gal4 FRT82B Tub-Gal80/FRT82B P[w+]90E. (B) An eye disc that contains GFP-labeled mats mutant MARCM clones exhibited an overgrowth phenotype. The genotype is w ey-FLP UAS-GFP/+; Tub-Gal4 FRT82B Tub-Gal80/FRT82B matse235. (C) Overexpression of Hpo in wild-type MARCM clones markedly inhibited tissue growth. The genotype is w ey-FLP UAS-GFP/+; UAS-hpo/+; Tub-Gal4 FRT82B Tub-Gal80/FRT82B P[w+]90E. (D) Loss of mats function effectively suppressed the growth inhibitory effect of Hpo. The genotype is w ey-FLP UAS-GFP/+; +/UAS-hpo; Tub-Gal4 FRT82B Tub-Gal80/FRT82B matse235. (E) GMR-Gal4 UAS-hpoN/+ and (F–H) w ey-FLP/+; +/GMR-Gal4 UAS-hpoN; FRT82B arm-lacZ/FRT82B matse235 eye discs were used for TUNEL staining of apoptotic cells (red). When driven by GMR-Gal4, HpoN was sufficient to cause apoptosis in the region posterior to the morphogenetic furrow (MF) in eye discs (E), and this HpoN-induced apoptosis occurred mostly in cells that were wild type for mats (F–H). Anterior is to the left in (A–D) and up-left in (E–H). Yellow arrows identify the morphogenetic furrow.
Figure 3
Figure 3
Mats associates with Hpo and is phosphorylated by Hpo protein kinase. (A) Hpo specifically phosphorylates Mats in vitro. pAc-hpo-Flag, pAc-hpoKD-Flag and pAc-sav-HA were transfected into S2 cells as indicated. Lysates of transfected cells were used for Western blot (WB) to show the inputs (middle two panels). Anti-Flag antibody was used to immunoprecipitate (IP) Hpo-Flag or HpoKD-Flag proteins for an in vitro kinase assay, in which GST-Mats or GST proteins were used as substrates (the bottom panel; CB: Coomassie blue staining). Hpo autophosphorylation and Mats phosphorylation by Hpo/HpoKD are shown in the top panel. (B) GST was not a substrate of Hpo kinase (lane 1). Hpo (lane 2) but not HpoKD (lane 3) can phosphorylate human Mats1 in vitro. (C) A rat Hpo ortholog, Mst2 kinase, can phosphorylate GST-Mats (lane 3) but not GST (lane 2) in vitro. Like Hpo, Mst2 mediates autophosphorylation (lanes 1–3). (D) In an in vitro pull-down assay, Mats was shown to associate with Hpo. Bacterially produced GST-Mats (lanes 1 and 3) or GST (lane 2) proteins were tested for their ability to interact with Hpo-Flag (lanes 2 and 3) produced in transfected S2 cells. (E) Human Mats1 was co-immunoprecipitated with the endogenous human Mst1. pCMV-hMats1 and pCMV-GFP were transfected into HEK293T cells as indicated. Inputs of hMats1 (lane 2), GFP (lane 3) and hMst1 (lanes 1–3) proteins are shown in panels on the left. Immunoprecipitations were carried out using anti-hMst1 antibodies to isolate the endogenous hMst1 protein (lanes 4-6, the bottom-right panel), and hMats1 (lane 5) but not GFP (lane 6) was co-immunoiprecipitated (top two panels on the right).
Figure 4
Figure 4
Hpo phosphorylation of Mats increases the affinity between Mats and Wts and the ability of Mats to activate Wts. (A) Equal amounts of bacterially produced GST-Mats were preincubated for cold phosphorylation with Hpo or HpoKD immobilized on agarose beads, and then collected from supernatant and mixed with Myc-Wts for in vitro kinase reactions using [γ-32P]ATP. Myc-Wts was produced in transfected HEK293T cells, which were not treated with OA. GST-Yki was used as a substrate of Wts kinase. To ensure that immobilized Hpo kinase proteins were not carried over to the subsequent Wts-mediated phosphorylation reaction, [γ-32P]ATP was added in the Hpo-mediated kinase reaction. Whereas phospho-GST-Mats was detected in both supernatant and pellet (lanes 7 and 8), Hpo protein was exclusively found only in the pellet (lane 8). Thus, phosphorylation of Yki (lane 2) must be mediated by Wts kinase. Indeed, Yki phosphorylation was not detectable when WtsKD was used (lane 5). (B) Phosphorylation of Mats by Hpo increases the affinity between Wts and Mats proteins. Equal amounts of Myc-Wts produced in HEK293T cells treated without (lanes 1 and 2) or with (lanes 3 and 4) OA were linked to anti-Myc agarose beads, and GST-Mats treated with Hpo (lanes 1 and 3) or HpoKD (lanes 2 and 4) was tested for their ability to associate with Wts.
Figure 5
Figure 5
Mats is a phosphoprotein and Mats is critical for Hpo-mediated Wts activation in living cells. (A) OA treatment as well as coexpression of Mats increased the catalytic activity of Wts kinase as shown in an in vitro kinase assay. Equal amounts of Myc-wts and HA-mats DNA were transfected into HEK293T cells. Myc-Wts was immunoprecipitated from cells that were cotransfected without (lanes 1 and 3) or with (lanes 2 and 4) HA-mats in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of OA treatment as indicated. (B) Both Mats and Wts are phosphoproteins in living cells. Using a TAP method, the HA-Mats protein complex was highly purified and examined by SDS–PAGE analysis. Sypro Ruby staining was used to detect all proteins that include HA-Mats and other proteins in the HA-Mats complex. Pro-Q staining identified phosphoproteins. HEK293T cells were either single or double-transfected with or without OA treatment as indicated. (C) Hpo activates Wts kinase and Mats is critical for Hpo-mediated Wts activation in living cells. HEK293T cells were either double or triple-transfected as indicated. After immunoprecipitating Myc-Wts or Myc-WtsKD protein complex using anti-Myc antibodies, HA-Mats, Myc-Wts/Myc-WtsKD and Hpo-Flag/HpoKD-Flag proteins were examined by Western blot analysis. Equal amounts of Myc-Wts/Myc-WtsKD proteins were used for in vitro kinase reactions, and catalytic activities of Wts and Hpo for autophosphorylation were measured. Moreover, bacterially produced GST-Yki protein was added to determine Wts kinase activity. Hpo does not appear to phosphorylate Yki (lane 4 and data not shown). The cells were not treated with OA.
Figure 6
Figure 6
A model for growth inhibition by Hpo, Wts and Mats tumor suppressors. Hpo kinase, acting downstream of Mer and Ex, directly activates Mats via phosphorylation in addition to targeting Wts. Consequently, Mats becomes a potent activator of Wts kinase to phosphorylate a downstream transcription cofactor Yki for down-regulation. Hpo-mediated phosphorylation is indicated by small blue circle, and Wts-mediated phosphorylation by small red circles.
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References

    1. Bichsel SJ, Tamaskovic R, Stegert MR, Hemmings BA (2004) Mechanism of activation of NDR (nuclear Dbf2-related) protein kinase by the hMOB1 protein. J Biol Chem 279: 35228–35235 - PubMed
    1. Bothos J, Tuttle RL, Ottey M, Luca FC, Halazonetis TD (2005) Human LATS1 is a mitotic exit network kinase. Cancer Res 65: 6568–6575 - PubMed
    1. Brand A, Perrimon N (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118: 401–415 - PubMed
    1. Chan EH, Nousiainen M, Chalamalasetty RB, Schafer A, Nigg EA, Sillje HH (2005) The Ste20-like kinase Mst2 activates the human large tumor suppressor kinase Lats1. Oncogene 24: 2076–2086 - PubMed
    1. Edgar BA (2006) From cell structure to transcription: Hippo forges a new path. Cell 124: 267–273 - PubMed

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