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. 2017 Dec 19;21(12):3612-3623.
doi: 10.1016/j.celrep.2017.11.076.

Homeostatic Control of Hpo/MST Kinase Activity Through Autophosphorylation-Dependent Recruitment of the STRIPAK PP2A Phosphatase Complex

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Free PMC article

Homeostatic Control of Hpo/MST Kinase Activity Through Autophosphorylation-Dependent Recruitment of the STRIPAK PP2A Phosphatase Complex

Yonggang Zheng et al. Cell Rep. .
Free PMC article

Abstract

The Hippo pathway controls organ size and tissue homeostasis through a kinase cascade leading from the Ste20-like kinase Hpo (MST1/2 in mammals) to the transcriptional coactivator Yki (YAP/TAZ in mammals). Whereas previous studies have uncovered positive and negative regulators of Hpo/MST, how they are integrated to maintain signaling homeostasis remains poorly understood. Here, we identify a self-restricting mechanism whereby autophosphorylation of an unstructured linker in Hpo/MST creates docking sites for the STRIPAK PP2A phosphatase complex to inactivate Hpo/MST. Mutation of the phospho-dependent docking sites in Hpo/MST or deletion of Slmap, the STRIPAK subunit recognizing these docking sites, results in constitutive activation of Hpo/MST in both Drosophila and mammalian cells. In contrast, autophosphorylation of the Hpo/MST linker at distinct sites is known to recruit Mats/MOB1 to facilitate Hippo signaling. Thus, multisite autophosphorylation of Hpo/MST linker provides an evolutionarily conserved built-in molecular platform to maintain signaling homeostasis by coupling antagonistic signaling activities.

Keywords: Hippo signaling; Hpo; MST1/2; PP2A; SLMAP; STRIPAK; autophosphorylation.

Figures

Figure 1
Figure 1. The linker region of Hpo contains two types of autophosphorylation sites
(A) GST pull-down assay of binding between Mats and Hpo variants with mutated linker autophosphorylation sites. Left: a schematic of Hpo protein showing the five autophosphorylation sites and sequence of the linker region. Mats-binding site is labeled by red, non-Mats-binding sites by blue, and the hydrophobic motif recognized by Mats (HFM) by orange. Right: cell lysates were prepared from S2R+ cells transfected with the indicated Hpo variants and then incubated with GST-Mats in GST pull-down assay. Hpo was pulled down by GST-Mats (lane 2), but not GST alone (lane 1). Also note the comparable binding between GST-Mats and wildtype Hpo (lane 2) and Hpo4A/431T (lane 8), and reduced binding between GST-Mats and the remaining Hpo mutants (lanes 3–7). (B) A schematic illustration of the FLP-out expression system. A FRT y+ FRT cassette containing a transcription STOP signal is inserted between Tubulin α1 promoter and hpo cDNA to prevent hpo expression. A tissue-specific source of FLP is used to excise the FRT y+ FRT cassette through recombination between the two FRT sequences, leading to hpo expression. (C–F) Images of adult wings expressing the indicated Hpo mutants in the engrailed (en)-expression posterior compartment by the FLP-out expression system. Note that the size of the posterior compartment (highlighted by green color) was greatly decreased by Hpo4A/431T (F). Scale bars are 0.2mm. (G) Quantification of the size of the posterior compartment relative to the anterior compartment for experiments described in (C–F) (mean ± SEM, n = 15). Wildtype Hpo slightly and Hpo4A/431T greatly decreased the size of the en-expression domain, while Hpo431A expression had no effect. (H–K) Third instar wing imaginal discs corresponding to experiments described in (C–F) and stained with anti-Diap1 antibodies. Note the reduction of Diap1 protein levels by Hpo4A/431T, but not wildtype or Hpo431A in the GFP-positive en-expression domain (arrows). Scale bars represent 50μm.
Figure 2
Figure 2. The non-Mats-binding sites in the Hpo linker bind to Slmap in a phospho-dependent manner
(A) The indicated FLAG-Hpo variants were expressed in S2R+ cells, partially purified by anti-FLAG IP and blotted for T195 phosphorylation. The p-Hpo level over total Hpo level was quantified relative to the wildtype Hpo and indicated below the blot. Note the increased p-Hpo level for Hpo4A/431T compared to wildtype Hpo. (B) Reciprocal co-IP between Myc-Hpo and HA-Slmap expressed in S2R+ cells. (C) S2R+ cells expressing the indicted constructs were subjected to co-IP. Note the absence of interaction between Slmap and HpoK71R. (D) The FHA domain of Slmap is required and sufficient for Hpo binding. Left: co-IP assay showing interaction between Hpo and wildtype Slmap or SlmapΔTM, but not SlmapΔFHA. Right: GST pull-down assay showing binding between GST-FHA and FLAG-Hpo expressed in S2R+ cells. No binding was detected between GST alone and FLAG-Hpo. (E) Identification of 356T as a strong Slmap binding site in the Hpo linker. S2R+ cells expressing the indicated constructs were analyzed by anti-FLAG immunoprecipitation. Note the absence of binding between Hpo5A and Slmap (lane 3), very weak binding between Hpo356A to Slmap (lane 4), and mostly restored binding between Hpo4A/356T and Slmap (lane 5). (F) Characterization of additional Slmap binding sites in the Hpo linker. S2R+ cells expressing the indicated constructs were analyzed by anti-FLAG immunoprecipitation. Both short (S) and Long (L) exposures of the same gel were shown to reveal weak binding. Besides the strong binder Hpo4A/356T (lane 6), Hpo4A/421T (lane 3), 4A/387T (lane 4) and 4A/374T (lane 5) also showed slightly higher binding than Hpo5A (lane 1) and Hpo4A/431T (lane 2).
Figure 3
Figure 3. Slmap mediates the association between Hpo and the STRIPAK PP2A phosphatase complex in Drosophila
(A–F) Wing imaginal discs expressing UAS-slmap RNAi by the en-Gal4 UAS-GFP driver were examined for the expression of Diap1 (A–B), diap1-LacZ (C–D) and ex-LacZ (E–F). Note the downregulation of these reporters in the en-expression domain (GFP-positive). Scale bars represent 50μm. (G) The indicated subunits of the STRIPAK complex were depleted by RNAi in S2R+ cells and the cell lysates were examined for T195 phosphorylation of endogenous Hpo. Note increased phospho-T195 level upon RNAi of Cka, Slmap, Fgop2, Strip, or Mob4, but not Cttnbp2. (H) Eye-antennal imaginal discs were dissected from third instar larvae of wildtype, rassf36/rassf44.2 trans-heterozygotes or slmap4 homozygotes and lysed in 2×SDS loading buffer and examined for phospho-Hpo (T195) level. Note increased p-Hpo level in slmap but not rassf mutant tissues. rassf36 and rassf44.2 are null alleles (Polesello et al., 2006). (I) S2R+ cells co-expressing HA-Slmap and the indicated FLAG-tagged proteins were analyzed by anti-FLAG immunoprecipitation. Note interactions between Slmap and Fgop2. (J) Myc-Hpo was co-expressed with the indicated FLAG-tagged subunits of the STRIPAK complex in S2R+ cells, with (lane 9) or without HA-Slmap (lanes 1–8). The interactions between Hpo and these subunits was assayed by anti-FLAG IP. Note strong interactions between Hpo and Slmap, and weak interactions between Hpo and Fgop2, Cka or Mts. Also note the markedly enhanced interaction between Hpo and Fgop2 in the presence of HA-Slmap (compare lane 9 to lane 3). (K) Tissue lysates from wildtype or slamp4 homozygous larvae, both carrying the same tub-FLAG-cka transgene, were subjected to anti-FLAG immunoprecipitation. Note the presence of co-purified endogenous Hpo from wildtype not slmap mutant animals.
Figure 4
Figure 4. Phospho-dependent recruitment of SLMAP by MST2 in mammalian cells
(A) HEK293 cells transfected with the indicated constructs were analyzed by anti-FLAG immunoprecipitation. Note the interaction between SLMAP and wildtype MST2, but not MST2-7A or MST2D164A. (B) Control and SLMAP mutant HAP1 cells were probed for phosphorylation of MST1/2 (T183/180), YAP (S127 and S397) and MOB1 (T35). Note increased phosphorylation of these proteins in the SLMAP mutant cells, which was completely suppressed by XMU-MP-1. (C–D) Subcellular localization of YAP in HAP1 cells. Note that YAP was evenly distributed between cytosol and nucleus in the wildtype HAP1 cells, but mostly concentrated in the cytosol in the SLMAP mutant HAP1 cells. Scale bars are 20μm. (E) Identification of 325T, 336T and 378T as SLMAP binding sites in the MST2 linker. HEK293 cells expressing V5-SLMAP and the indicated linker site mutants of MST2 were subjected to immunoprecipitation and analyzed by the indicated antibodies. The phospho-T180 over total MST2 level was also quantified relative to the wildtype MST2 and indicated below the p-MST2 blot. Note the absence of SLMAP-binding for MST2-7A (lane 3), the significant restoration of SLAMP-binding for MST2-6A/325T (lane 4) and MST2-6A/378T (lane 10), and the weak restoration of SLMAP-binding for MST2-6A/336T (lane 5). Also note the reverse correlation between p-MST2 level and the strength of SLMAP-binding. (F) Further characterization of 325T, 336T and 378T as SLMAP binding sites. Similar to (E) except that different MST2 mutants were analyzed. Note the absence of SLMAP-binding for MST2-3A and MST2-7A, and the comparable SLMAP-binding for wildtype and MST2-3T. Also note that phospho-T180 level of MST2-3A or MST2-3T is higher than that of wildtype MST2 but lower than MST2-7A. (G–L) Subcellular localization of MST2 and SLMAP. HeLa cells expressing the indicated FLAG-MST2 variants (red) with or without V5-SLMAP (green) were visualized by FLAG and V5 immunostaining. Note the ubiquitous cytosolic distribution of MST, MST2-7A or MST2D164A in the absence of co-expressed SLMAP (G, I and K). Also note the co-localization of MST2, but not MST2-7A or MST2D164A, to punctate structures in the presence of co-expressed SLMAP (compare H with J and L). Scale bars are 25μm.

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