SAV1 promotes Hippo kinase activation through antagonizing the PP2A phosphatase STRIPAK

Abstract

The Hippo pathway controls tissue growth and homeostasis through a central MST-LATS kinase cascade. The scaffold protein SAV1 promotes the activation of this kinase cascade, but the molecular mechanisms remain unknown. Here, we discover SAV1-mediated inhibition of the PP2A complex STRIPAK^SLMAP as a key mechanism of MST1/2 activation. SLMAP binding to autophosphorylated MST2 linker recruits STRIPAK and promotes PP2A-mediated dephosphorylation of MST2 at the activation loop. Our structural and biochemical studies reveal that SAV1 and MST2 heterodimerize through their SARAH domains. Two SAV1-MST2 heterodimers further dimerize through SAV1 WW domains to form a heterotetramer, in which MST2 undergoes trans-autophosphorylation. SAV1 directly binds to STRIPAK and inhibits its phosphatase activity, protecting MST2 activation-loop phosphorylation. Genetic ablation of SLMAP in human cells leads to spontaneous activation of the Hippo pathway and alleviates the need for SAV1 in Hippo signaling. Thus, SAV1 promotes Hippo activation through counteracting the STRIPAK^SLMAP PP2A phosphatase complex.

Keywords: Escherichia coli; cell biology; human; human 293FT cell; human MCF10A cell.

Figure 1.. Feedback inhibition of MST2 activation by SLMAP binding to autophosphorylated MST2 linker.

(A) Domain organization of human MST2 and seven phospho-TM sites in the linker region. (B) Identification of the TM motifs in the MST2 linker critical for inhibition of MST2 T180 phosphorylation. Anti-FLAG and anti-pMST2 (T180) blots of lysates of 293FT cells transfected with the indicated FLAG-MST2 plasmids. Anti-FLAG blot was used to evaluate protein expression levels. (C) Anti-FLAG, anti-pMST2 (T180), anti-pMST2 (T336), and anti-pMST2 (T378) blots of lysates of 293FT cells transfected with the indicated FLAG-MST2 plasmids. (D) Anti-Myc and anti-SLMAP blots of cell lysates (input) and anti-FLAG immunoprecipitates (IP) of 293FT cells transfected with the indicated plasmids. WT, wild type; ΔFHA, mutant with FHA deleted. (E) Immunoblots of cell lysates (input) and anti-FLAG IP of 293FT cells transfected with the indicated plasmids. Endogenous SLAMP was detected by the anti-SLMAP blot. Asterisk designates a non-specific band. (F) Immunoblots of cell lysates (input) and anti-FLAG IP of 293FT cells transfected with the indicated plasmids. Asterisk designates a non-specific band. (G) ITC thermogram (top) and isotherm (middle) of the binding between purified recombinant human SLMAP FHA and the pT378-MST2^373-382 (pMST2) peptide with the binding affinity (K_d) indicated. DP, differential power. (H) Superposition of the crystal structures of human SLMAP FHA (cyan) and SLMAP FHA in complex with the pMST2 peptide (wheat). pMST2 is colored yellow and shown as sticks. The pMST2 residues are labeled in magenta. All structure figures are generated by PyMOL (Schrödinger, LLC; http://www.pymol.org). (I) Surface drawing of the pT-binding site of SLMAP FHA. The surface is colored according to the electrostatic potential, with blue, red, and white representing positive, negative, and neutral charges, respectively. pMST2 is shown as yellow sticks with residues labeled.

Figure 1—figure supplement 1.. Feedback inhibition of MST2 activation by SLMAP binding to autophosphorylated MST2 linker.

(A and B) Anti-FLAG and anti-pMST2 (T180) blots of lysates of 293FT cells transfected with the indicated FLAG-MST2 plasmids. (C) Anti-FLAG, anti-pMST2 (T336), and anti-pMST2 (T378) blots of lysates of 293FT cells transfected with the indicated FLAG-MST2 plasmids. (D) In vitro kinase assay of MST2 kinase domain using GST-MST2 D146N as the substrate. Immunoblots of the kinase reaction mixtures were shown. Anti-GST blot was used to evaluate protein levels. (E) The in vitro kinase assays of MST2-FL and MST2-∆L using myelin basic protein (MBP) as substrate. The reaction mixtures were separated on SDS-PAGE and analyzed by a phosphoimager (top) and Coomassie blue staining (middle). FL, full-length; ΔL, linker deletion. The kinetic profiles of MBP phosphorylation by activated MST2 FL and ∆L as monitored by ³²P incorporation (bottom). The relative ³²P-MBP signal intensities, normalized to those of MST2 FL and ∆L at 60 min (100%), respectively, are plotted against time. Means ± range for two independent experiments are plotted. (F) Coomassie-stained gel of the indicated MST2 proteins bound to GST-SLMAP FHA (residues 1–140) beads. FL, full-length; KD, kinase domain; ΔL, linker deletion.

Figure 1—figure supplement 2.. Crystal structures of SLMAP FHA and SLMAP FHA bound to pMST2.

(A) Cartoon drawing of the crystal structure of human SLMAP FHA. SLMAP FHA is colored cyan. (B) Cartoon drawing of the crystal structure of human SLMAP FHA in complex with the pMST2 peptide. SLMAP FHA is colored wheat. pMST2 is colored yellow and shown as sticks. The pMST2 residues are labeled. (C) Zoomed-in view of the pT-binding site in the SLMAP FHA-pMST2 complex. The backbones and side chains of SLMAP FHA interface residues are shown as sticks and are labeled. (D) Anti-Myc and anti-SLMAP blots of lysates (input) and anti-FLAG IP of 293FT cells transfected with the indicated plasmids.

Figure 2.. STRIPAK^SLMAP inhibits the Hippo pathway in human cells.

(A) 293FT cells were co-transfected with siSLMAP and the indicated FLAG-MST2 plasmids. The total cell lysates were blotted with the indicated antibodies. Anti-GAPDH blot was used as the loading control. (B) Immunoblots with the indicated antibodies of lysates of 293FT and MCF10A cells with SLMAP deleted. KO, knockout; HM, hydrophobic motif. (C) Quantification of the ratios of pMST/MST, pMOB1/MOB1, pLATS/LATS, and pYAP/YAP signals in (B). The total and phosphorylated protein levels were individually normalized to GAPDH levels. Normalized values were used to calculate the ratios. Data are plotted as mean ± SEM of three biological replicates (*p<0.05; **p<0.01; ***p<0.001). (D) Immunofluorescence staining of YAP localization in control and SLMAP KO MCF10A cells. Cells were fixed, permeabilized, and stained with anti-YAP (red) and DAPI (blue). Scale bars, 10 μm. (E) Quantification of immunofluorescence signal intensities in (D). Approximately 50 cells were counted from 7 random fields. N < C (blue), N = C (grey), and N > C (red) categories indicate YAP localization in cytoplasm, both cytoplasm and nucleus, and nucleus, respectively. Data are plotted as mean ± SEM of three biological replicates. (F) Relative expression of YAP target genes CTGF and CYR61 in control and SLMAP KO MCF10A cells. Data are plotted as mean ± SEM of three biological replicates (****p<0.0001).

Figure 2—figure supplement 1.. STRIPAK^SLMAP inhibits the Hippo pathway in human cells.

(A) 293FT cells were transfected with siSTRIP1 with or without FLAG-STRIP1. The total cell lysates were blotted with the indicated antibodies. (B) Immunoblots with the indicated antibodies of lysates of control MCF10A cells, SLMAP KO cells, and SLMAP KO cells stably expressing GFP-SLMAP WT or ΔFHA. (C) Relative expression of YAP target genes CTGF and CYR61 in control MCF10A cells, SLMAP KO cells, and SLMAP KO cells stably expressing GFP-SLMAP WT or ΔFHA. YAP target gene expression was analyzed by quantitative real-time RT-PCR and normalized to GAPDH. Data are plotted as mean ± SEM of three independent experiments (**p<0.01).

Figure 3.. SAV1 is required for Hippo pathway activation in human cells.

(A) Immunoblots of cell lysates of MCF10A and MCF10A-SAV1 KO cells with or without latrunculin B (LatB) treatment with the indicated antibodies. Both control and SAV1 KO cells were treated with LatB (1 μg/ml) or vehicle for 1 hr before they were harvested. (B) Quantification of the ratios of pMST/MST, pMOB1/MOB1, pLATS/LATS, and pYAP/YAP signals in (A). The total and phosphorylated protein levels were individually normalized to GAPDH levels. Normalized values were used to calculate the ratios. Data are plotted as mean ± SEM of three biological replicates (*p<0.05; **p<0.01; ***p<0.001). (C) Relative expression of YAP target genes CTGF and CYR61 in control and SAV1 KO MCF10A cells with or without LatB treatment. Data are plotted as mean ± SEM of three biological replicates (**p<0.01 and ***p<0.001). (D) Quantitative immunoblots with the indicated antibodies of the in vitro kinase reactions containing human MOB1 and the indicated MST2-FL or MST2-FL/SAV1-∆N198 proteins at the indicated times. The relative pT35-MOB1 signal intensities, normalized to those of MST2 FL and MST2-FL/SAV1-∆N198 at 60 min (100%), respectively, are plotted against time. Means ± range for two biological replicates are plotted.

Figure 4.. Crystal structure and binding interface of human MST2-SAV1.

(A) Schematic drawing of domains and motifs of human SAV1. (B) Cartoon drawing of the crystal structure of the MST2-SAV1 complex. MST2 is colored green, and SAV1 is colored blue. Side chains of K56 and E70, and AMP-PNP are shown as sticks. Mg²⁺ is shown as a magenta sphere. The disordered T-loop is drawn as a green dashed line. (C) Cartoon drawing of the crystal structure of the MST2 SARAH-RASSF5 SARAH heterodimer (PDB ID: 4LGD). MST2 and RASSF5 are colored in light green and orange, respectively. The interface residues are shown as sticks. (D) Cartoon drawing of the crystal structure of the MST2 SARAH-SAV1 SARAH heterodimer (this study). MST2 and SAV1 are colored in green and blue, respectively. The interface residues are shown as sticks. SAV1 residues whose mutations cause defective MST2 binding are labeled in magenta. (E) Sequence alignment of SARAH domains of human SAV1 and RASSF5. The highly conserved interface residues are shaded in yellow. Seven interface residues whose mutations cause defective MST2 binding are marked by asterisks. The secondary structural elements of SAV1 SARAH are shown above the sequences and colored blue. The secondary structural elements of RASSF5 SARAH are shown below the sequences and colored orange. (F) Quantification of the relative binding intensity between MST2 SARAH domain (residue 431–491) and SAV1 SARAH domain (residue 321–383) and its mutants, derived from the pull-down experiments. Relative MST2 binding of the indicated SAV1 mutant is normalized against SAV1 wild type (WT; 100%). SAV1 mutants that lost or retained MST2 binding are colored red and gray, respectively. (G) Sedimentation velocity analytical ultracentrifugation analysis of MST2-FL (black), MST2-FL/SAV1-ΔN198 (red), and MST2-FL/SAV1-ΔN290 (blue). ‘s_20,w’, sedimentation coefficient corrected to standard conditions; S, Svedberg units (10⁻¹³ seconds). (H) Schematic model of the MST2-SAV1 heterotetramer.

Figure 4—figure supplement 1.. SARAH and WW domains of SAV1 mediate the formation of the MST2-SAV1 heterotetramer.

(A) Binding between GST-MST2-SARAH and in vitro translated SAV1 SARAH proteins. SAV1 mutants that are defective in MST2-binding are labeled red. (B) UV traces of molecular weight standards (dashed line), MST2-FL (black line), MST2-FL/SAV1-ΔN320 (purple line), MST2-FL/SAV1-ΔN290 (blue line), and MST2-FL/SAV1-ΔN198 (red line) fractionated on a Superdex 200 gel filtration column. (C) Cartoon drawing of the solution structure of the mouse SAV1 WW2 homodimer (PDB ID: 2DWV) (Ohnishi et al., 2007). Monomer A is colored cyan and monomer B is colored blue. (D) UV traces of human SAV1 WW1 (blue line), and WW2 (red line) fractionated on a Superdex 75 gel filtration column. The molecular weight standard is indicated. (E) Anti-HA and anti-FLAG blots of cell lysates (input) and anti-FLAG IP of 293FT cells co-transfected with HA-SAV1 ∆SARAH and the indicated FLAG-SAV1 plasmids.

Figure 5.. All discernable domains of SAV1 are required for MST2 activation.

(A) Immunoblots of lysates of 293FT cells co-transfected with the indicated Myc-MST2 and HA-SAV1 or HA-RASSF1A plasmids. (B) Immunoblots of lysates of 293FT cells co-transfected with Myc-SAV1 and the indicated FLAG-MST2 plasmids. (C) Immunoblots of lysates of 293FT cells co-transfected with Myc-MST2 and the indicated FLAG-SAV1 plasmids. (D) Immunoblots of cell lysates of 293FT cells co-transfected with FLAG-MST2 and the indicated Myc-SAV1 plasmids. (E) Total MST2 and pT180 blots of the auto-kinase reactions by the indicated MST2-SAV1 complexes at the indicated time points (left panel). The kinetic profiles of T180 autophosphorylation of MST2-FL/SAV1-∆N198 (red line) and MST2-FL/SAV1-∆N268 (black line) are shown on the right. The relative anti-pT180 intensities were normalized against MST2-FL without PP2A phosphatase treatment (100%). Data are representative of at least two independent experiments. (F) Immunoblots of lysates of 293FT cells co-transfected with FLAG-MST2 and the indicated N-terminal truncation of Myc-SAV1 constructs. (G) 293FT cells were co-transfected with FLAG-MST2 and Myc-SAV1 plasmids. The total cell lysates were blotted with the indicated antibodies. (H) 293FT cells were co-transfected with the indicated plasmids encoding FLAG-SLMAP, HA-MST2 and Myc-SAV1. Anti-FLAG IP and the total cell lysates (input) were blotted with the indicated antibodies.

Figure 5—figure supplement 1.. The N-terminal region of SAV1 is required for MST2 activation.

Immunoblots of cell lysates of 293FT cells co-transfected with FLAG-MST2 and the indicated N-terminal truncation of Myc-SAV1 constructs. FBM, FERM-binding motif.

Figure 5—figure supplement 2.. SAV1 and MST2 are present in the cytosol.

FLAG-MST2 and Myc-SAV1 were co-transfected into 293FT cells. Cells were collected after 24 hr and subjected to cytosol-membrane fractionation using the membrane protein extraction kit (Thermo Scientific). The cytosol and membrane fractions were blotted with the indicated antibodies. Tubulin, NF2 and Pan Cadherin were used as markers for cytosolic, peripheral membrane, and integral membrane proteins, respectively.

Figure 6.. The N-terminal region of SAV1 directly inhibits PP2A.

(A and B) Association of PP2A A-C with the N terminal region of SAV1. 293FT cells were mock transfected or transfected with the indicated FLAG-SAV1 plasmids. The total cell lysates (input) and anti-FLAG IP were blotted with the indicated antibodies. Asterisk designates IgG. (C) UV traces of the PP2A A and SAV1^1-90 complex (blue line), PP2A A alone (magenta line), SAV1^1-90 alone (green line), and molecular weight standard (dashed line, corresponding molecular weight indicated in kDa) fractionated on a Superdex 200 gel filtration column, respectively. The underlined fractions were separated on SDS-PAGE and stained with Coomassie. (D) The N-terminal region of SAV1 directly inhibits dephosphorylation of MST2 pT180 by PP2A. Quantitative MST2 pT180 and total MST2 immunoblotting of phosphatase reactions containing human MST2 and SAV1^1-90 at the indicated concentrations. The relative anti-pT180 intensities were normalized against MST2 without PP2A phosphatase treatment (100%). Data are representative of at least two independent experiments.

Figure 6—figure supplement 1.. The N-terminal 90 residues of SAV1 associates with PP2A_A subunit in human cells.

Control and SLMAP KO 293FT cells were transfected with the indicated FLAG-SAV1 plasmids. Anti-FLAG IP and cell lysates (input) were blotted with the indicated antibodies. Asterisk designates non-specific bands.

Figure 7.. SAV1 stimulates MST1/2 activation through antagonizing STRIPAK.

(A) SAV1-MST2 forms a complex with STRIPAK^SLMAP. 293FT cells were co-transfected with FLAG-SAV1 and the indicated Myc-MST2 plasmids. The total cell lysates (input) and anti-FLAG IP were blotted with the indicated antibodies. (B) Immunoblots with the indicated antibodies of lysates of control MCF10A cells and MCF10A cells with SLMAP, SAV1, or both deleted. Asterisk denotes a non-specific band. (C) Relative expression of YAP target genes CTGF and CYR61 in control, SLMAP KO, SAV1 KO, and SLMAP/SAV1 DKO MCF10A cells. The gene expression was measured by quantitative real-time RT-PCR and normalized to GAPDH. Data are plotted as mean ± SEM of three biological replicates (*p<0.05; **p<0.01; and ***p<0.001). (D) Model for SAV1-dependent MST2 activation during Hippo signaling.

Figure 7—figure supplement 1.. CRISPR/Cas9-induced indel mutations in MCF10A and 293FT cells.

(A and B) Intact genomic sequences of SLMAP and SAV1 are shown in the top panels. The indel mutations in SLMAP KO or SAV1 KO cells are shown in the bottom panels.