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Cryo-EM structure of the Hippo signaling integrator human STRIPAK

Nature Structural & Molecular Biology volume 28, pages 290–299 (2021)Cite this article

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Abstract

The striatin-interacting phosphatase and kinase (STRIPAK) complex is a large, multisubunit protein phosphatase 2A (PP2A) assembly that integrates diverse cellular signals in the Hippo pathway to regulate cell proliferation and survival. The architecture and assembly mechanism of this critical complex are poorly understood. Using cryo-EM, we determine the structure of the human STRIPAK core comprising PP2AA, PP2AC, STRN3, STRIP1, and MOB4 at 3.2-Å resolution. Unlike the canonical trimeric PP2A holoenzyme, STRIPAK contains four copies of STRN3 and one copy of each the PP2AA–C heterodimer, STRIP1, and MOB4. The STRN3 coiled-coil domains form an elongated homotetrameric scaffold that links the complex together. An inositol hexakisphosphate (IP6) is identified as a structural cofactor of STRIP1. Mutations of key residues at subunit interfaces disrupt the integrity of STRIPAK, causing aberrant Hippo pathway activation. Thus, STRIPAK is established as a noncanonical PP2A complex with four copies of regulatory STRN3 for enhanced signal integration.

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Fig. 1: Cryo-EM structure of human STRIPAK core.
Fig. 2: Interactions between PP2AA and the STRN3 coiled-coil tetramer.
Fig. 3: IP6 is a structural cofactor of STRIP1.
Fig. 4: STRIP1 scaffolds the STRIPAK core.
Fig. 5: MOB4 tethers the WD40 domain of STRN3 to STRIP1.

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Data Availability

Cryo-EM density maps and atomic coordinates for human STRIPAK have been deposited in the Electron Microscopy Data Bank and wwPDB, respectively, under accession codes EMD-22650 and PDB 7K36. Source data are provided with this paper.

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Acknowledgements

We thank H. Yu for helpful discussion. We thank S. Liu for assistance with mutant analysis. Single-particle cryo-EM data were collected at the University of Texas Southwestern Medical Center (UTSW) Cryo-Electron Microscopy Facility, which is funded by a Cancer Prevention and Research Institute of Texas (CPRIT) Core Facility Support Award (Grant no. RP170644). We thank D. Nicastro for facility access and data acquisition. This study is supported in part by grants from the National Institutes of Health (CA220283 to X.Z.; GM132275 to X.L.), and grants from the Welch Foundation (I-1702 to X.Z.; I-1944 to X.-c.B.; I-1932 to X.L.). X.Z. and X.-c.B. are Virginia Murchison Linthicum Scholars in Medical Research at UTSW.

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  1. These authors contributed equally: Byung-Cheon Jeong, Sung Jun Bae.

Authors and Affiliations

  1. Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Byung-Cheon Jeong, Sung Jun Bae, Lisheng Ni, Xuewu Zhang & Xuelian Luo

  2. Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Xuewu Zhang, Xiao-chen Bai & Xuelian Luo

  3. Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Xiao-chen Bai

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Contributions

B.-C.J. performed protein purification, EM grid screening and preparation, structure docking and refinement, and functional studies in vitro. S.J.B. made the KO cell lines and performed functional studies in human cells. B.-C.J. and S.J.B. contributed to the initial draft of the manuscript. L.N. did the MS analysis. X.Z. assisted with structure determination and refinement. X.-c.B. prepared cryo-EM grids, collected cryo-EM data, and determined the cryo-EM structure. X.-c.B. and X.L. co-supervised the research and analyzed data. X.L. wrote the manuscript with help from all authors.

Corresponding authors

Correspondence to Xiao-chen Bai or Xuelian Luo.

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The authors declare no competing interests.

Additional information

Peer review information Nature Structural & Molecular Biology thanks Lanfen Chen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Florian Ullrich and Inês Chen were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Purification of human STRIPAK complexes and single-particle cryo-EM analysis of the STRIPAK core.

a, Coomassie blue staining of fractions from size exclusion chromatography (SEC) purification of the STRIPAK core (left) and STRIPAK (right) complexes. Highlighted fraction (red) was used for cryo-EM analysis. b, Cryo-EM data processing for initial model generation. c, Cryo-EM density map and atomic model of the STRIPAK core at a resolution of ~3.9-Å. d, Final reconstruction with C1 symmetry colored based on local resolution.

Extended Data Fig. 2 Cryo-EM analyses of the STRIPAK complex.

a, Representative micrograph and 2D classes. b, Final reconstructions with C1 or C2 symmetry colored based on local resolution. c, Gold-standard Fourier shell correlation (FSC) curves of the final 3D reconstruction (C1 in orange and C2 in blue). d, Euler angle distribution of the particles used in the final C1 3D reconstruction.

Extended Data Fig. 3

Flow chart of cryo-EM image processing for the STRIPAK complex.

Extended Data Fig. 4 Cryo-EM map of the STRIPAK complex.

a, Overall cryo-EM map of human STRIPAK at a resolution of 3.5-Å in front and back views. b, Representative cryo-EM densities of the STRIPAK complex. The densities from the cryo-EM maps and the corresponding protein fragments are shown in surface and sticks, respectively. Residues numbers of each sample fragment are labeled. Representative residues from STRN3 WD40 or at the PP2AC–STRIP1 interface are labeled.

Extended Data Fig. 5 Mutational studies of STRN3 at the STRN3-PP2AA interface.

a, Effects of mutations in STRN3 on STRIPAK formation. Mock vector or FLAG-STRN3 mutants were transfected into control or STRN1/3/4-KO 293A cells as indicated. Lysates and FLAG IPs were subjected to immunoblotting. b, Effects of mutations in STRN3 on the ratios of pMOB1/MOB1, pLATS/LATS, and pYAP/YAP in control or STRN1/3/4-KO 293A cells transfected with mock vector or indicated plasmids. c, Immunofluorescence staining of YAP localization in 293A control or STRN1/3/4-KO cells transfected with mock vector or indicated plasmids. Scale bar, 10 µm. d, Relative expression of YAP target genes CYR61 in 293A control or STRN1/3/4-KO cells transfected with mock vector or indicated plasmids. Data in b,d are plotted as mean ± SEM of three independent experiments. Results were evaluated by Two-tailed unpaired t tests (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; ns, non-significant). Source data for graphs are available online.

Source data

Extended Data Fig. 6 Functional validation of PP2AC-interacting residues of STRIP1.

a, Effects of mutations in STRIP1 on the ratios of pMOB1/MOB1, pLATS/LATS, and pYAP/YAP in control or STRIP1-KO 293A cells transfected with mock vector or indicated plasmids. b, Immunofluorescence staining of YAP localization in 293A control or STRIP1-KO cells transfected with mock vector or indicated plasmids. Scale bar, 10 µm. Data in a are plotted as mean ± SEM of three independent experiments. Results were evaluated by Two-tailed unpaired t tests (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; ns, non-significant). Source data for graphs are available online.

Source data

Extended Data Fig. 7 Functional validation of the C-terminal tail (CTT) of STRIP1.

a, Effects of mutation in STRIP1 on STRIPAK formation. Control or STRIP1-KO 293A cells were transfected with mock vector, FLAG-STRIP1 WT or ∆CTT mutant plasmid as indicated. Lysates and FLAG IPs were subjected to immunoblotting. b, Immunoblot of lysates of control or STRIP1-KO 293A cells transfected with mock vector, STRIP1 WT or ∆CTT mutant plasmid as indicated. HM indicates hydrophobic motif in LATS1/2. c, Quantification of the ratios of phospho- and total proteins in b. d, Immunofluorescence staining of YAP localization in 293A control or STRIP1-KO cells transfected with mock vector, STRIP1 WT or ∆CTT mutant plasmids. Scale bar, 10 µm. Data in c are plotted as mean ± SEM of three independent experiments. Results were evaluated by Two-tailed unpaired t tests (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; ns, non-significant). Source data for graphs are available online.

Source data

Extended Data Fig. 8 MOB4 interacts with STRIP1 and the WD40 domain of STRN3.

a, Cartoon representation of MOB4. The N-terminal extension (NTE) and core of MOB4 are colored in slate and yellow, respectively. b, Sequence alignment of human MOB NTEs. The N-terminal α helices (Nαs) and residue numbers of MOB1 and MOB4 are shown above and below the sequences, respectively. c, Superposition of MOB4 with the crystal structure of unphosphorylated MOB1 (colored in grey; PDB 5B5V). The NTE and core of MOB1 are colored in teal and grey, respectively. d, Superposition of MOB4–STRN3 WD40 (this study) and pMOB1–LATS1 (colored in grey and salmon, respectively; PDB 5BRK). The NTE of pMOB1 is colored in red. e, Interface between MOB4 and STRIP1 NTD (colored in yellow and wheat, respectively). Potential hydrogen bonds are indicated with red dashed lines. f, Interface between MOB4 core and STRN3 WD40 (colored in yellow and blue, respectively). g, Interface between MOB4 Nα1 and STRN3 WD40.

Extended Data Fig. 9 Interactions between MOB4 and STRN3 WD40.

a,b, Overall structure of the STRN3 WD40 domain with cartoon (a) and surface representation (b) in the same view. Residues at the MOB4–STRIP1 interface are colored salmon. The seven blades of the WD40 domain are numbered from 1 to 7, and the strands of each blade are numbered A-D from the innermost strand to the outermost strand. The N- and C-termini are indicated. c, Immunoblot of lysates of control or MOB4-KO 293A cells transfected with mock vector, MOB4 WT or ∆N40 mutant plasmids. d, Quantification of the ratios of phospho- and total proteins in c. Data in d are plotted as mean ± SEM of three independent experiments. Results were evaluated by Two-tailed unpaired t tests (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; ns, non-significant). Source data for graphs are available online.

Source data

Extended Data Fig. 10 Interactions between STRN3 WD40 and MOB4.

a, Immunoblot of lysates of control or STRN1/3/4-KO 293A cells transfected with mock vector or indicated plasmids. b, Quantification of the ratios of phospho- and total proteins in a. c, Quantification of YAP localization in control or STRN1/3/4-KO 293A cells transfected with mock vector or indicated plasmids, derived from immunofluorescence staining analysis of YAP. Approximately 100 cells were counted for quantification. d, Relative expression of YAP target genes CTGF and CYR61 in control or STRN1/3/4-KO 293A cells transfected with mock vector or indicated plasmids. Data in b,d are plotted as mean ± SEM of three independent experiments. Results were evaluated by Two-tailed unpaired t tests (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; ns, non-significant). Source data for graphs are available online.

Source data

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Jeong, BC., Bae, S.J., Ni, L. et al. Cryo-EM structure of the Hippo signaling integrator human STRIPAK. Nat Struct Mol Biol 28, 290–299 (2021). https://doi.org/10.1038/s41594-021-00564-y

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