SAV1 promotes Hippo kinase activation through antagonizing the PP2A phosphatase STRIPAK
- University of Texas Southwestern Medical Center, United States
Figures
Figure 1 with 2 supplements
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. …
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 …
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 …
Figure 2 with 1 supplement
STRIPAKSLMAP 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 …
Figure 2—figure supplement 1
STRIPAKSLMAP 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 …
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 …
Figure 4 with 1 supplement
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 …
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 …
Figure 5 with 2 supplements
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 …
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 …
Figure 6 with 1 supplement
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 …
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 …
Figure 7 with 1 supplement
SAV1 stimulates MST1/2 activation through antagonizing STRIPAK.
(A) SAV1-MST2 forms a complex with STRIPAKSLMAP. 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 …
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.
Tables
Table 1
Summary of the ITC results.
https://doi.org/10.7554/eLife.30278.005| Protein | pMST2 peptide | Kd (µM) | ΔH (kcal/mol) | ΔS (cal/mol·K) |
|---|---|---|---|---|
| MOB133-216 | pT378-MST2373-382 | 9.95 | −4.0 | 9.2 |
| pT378-MST2371-401 | 0.296 | −13.5 | −16.1 | |
| SLMAP FHA | pT325-MST2320-329 | 0.392 | −10.3 | −5.8 |
| pT336-MST2331-340 | 1.14 | −8.2 | −0.7 | |
| pT378-MST2373-382 | 0.160 | −9.0 | 0.5 | |
| pT378-MST2371-401 | 0.393 | −9.5 | −3.2 | |
| SLMAP FHA/R32A | pT378-MST2373-382 | 18.8 | −6.7 | −1.3 |
Table 2
Data collection and refinement statistics for apo-SLMAP FHA and the SLMAP FHA–pMST2 complex.
https://doi.org/10.7554/eLife.30278.006| Data collection | ||
|---|---|---|
| Crystal | Apo | Complex |
| Space group | P212121 | P212121 |
| Wavelength (Å) | 0.97918 | 0.97918 |
| Unit cell | ||
| a, b, c (Å) | 42.90, 51.22, 56.42 | 38.76, 70.53, 91.02 |
| Resolution range (Å) | 50–1.08 (1.10–1.08)* | 38.24–1.55 (1.59–1.55) |
| Unique reflections | 53,830 (2,647) | 36,933 (2,719) |
| Multiplicity | 8.6 (4.5) | 12.5 (10.3) |
| Data completeness (%) | 99.7 (98.5) | 99.8 (97.3) |
| Rmerge (%)† | 5.8 (53.9) | 10.2 (107.2) |
| Rpim (%)‡ | 2.0 (23.6) | 2.9 (31.8) |
| I/σ(I) | 43.9 (2.3) | 39.4 (1.4) |
| CC1/2§ | 0.847 | 0.784 |
| Wilson B-value (Å2) | 9.92 | 22.70 |
| Refinement statistics | ||
| Resolution range (Å) | 20.49–1.08 (1.11–1.08) | 38.24–1.55 (1.59–1.55) |
| No. of reflections Rwork/Rfree | 53,761/2,000 (3,571/139) | 35,078/1,846 (2,583/136) |
| Data completeness (%) | 99.6 (97.0) | 99.7 (97.3) |
| Atoms (non-H protein/solvent/peptide) | 1,325/235/0 | 2,210/203/102 |
| Rwork (%) | 16.4 (26.6) | 16.1 (27.5) |
| Rfree (%) | 17.9 (27.7) | 18.8 (30.3) |
| R.m.s.d. bond length (Å) | 0.007 | 0.009 |
| R.m.s.d. bond angle (°) | 0.935 | 1.005 |
| Mean B-value (Å2) (protein/solvent/peptide) | 12.83/25.96/- | 35.93/39.37/48.18 |
| Ramachandran plot (%) (favored/additional/disallowed)# | 97.1/2.9/0.0 | 97.4/2.6/0.0 |
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*Data for the highest-resolution shell are shown in parentheses.
†Rmerge = 100 ΣhΣi|Ih,i— ⟨Ih⟩|/ΣhΣi⟨Ih,i⟩, where the outer sum (h) is over the unique reflections and the inner sum (i) is over the set of independent observations of each unique reflection.
-
‡Rpim = 100 ΣhΣi [1/(nh - 1)]1/2|Ih,i— ⟨Ih⟩|/ΣhΣi⟨Ih,i⟩, where nh is the number of observations of reflections h.
§CC1/2 values shown are for the highest resolution shell.
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#As defined by the validation suite MolProbity.
Table 3
Data collection and refinement statistics for the MST2–SAV1 complex.
https://doi.org/10.7554/eLife.30278.012| Data collection | |
|---|---|
| Crystal | Native |
| Space group | R32 |
| Wavelength (Å) | 0.97918 |
| Cell dimensions | |
| a, b, c (Å) | 223.68, 223.68, 79.65 |
| α, β, γ (˚) | 90.00, 90.00, 120.00 |
| Resolution range (Å) | 42.27–2.95 (3.00–2.95)* |
| Unique reflections | 15,976 (763) |
| Multiplicity | 19.0 (12.0) |
| Data completeness (%) | 99.9 (98.5) |
| Rmerge (%)† | 12.4 (171.5) |
| Rpim (%)‡ | 2.9 (48.3) |
| I/σ(I) | 28.3 (1.0) |
| CC1/2§ | 0.841 |
| Wilson B-value (Å2) | 48.5 |
| Refinement Statistics | |
| Resolution range (Å) | 42.27–2.95 (3.18–2.95) |
| No. of reflections Rwork/Rfree | 13,199/650 (1,027/52) |
| Data completeness (%) | 82.3 (34.0) |
| Atoms (non-H protein/solvent/metal) | 3,340/31/1 |
| Rwork (%) | 23.5 (32.2) |
| Rfree (%) | 25.2 (27.9) |
| R.m.s.d. bond length (Å) | 0.007 |
| R.m.s.d. bond angle (°) | 0.600 |
| Mean B-value (Å2) (protein/solvent/ions) | 55.4/43.4/33.7 |
| Ramachandran plot (%) (favored/additional/disallowed)# | 94.2/4.5/1.3 |
-
*Data for the highest-resolution shell are shown in parentheses.
†Rmerge = 100 ΣhΣi|Ih,i— ⟨Ih⟩|/ΣhΣi⟨Ih,i⟩, where the outer sum (h) is over the unique reflections and the inner sum (i) is over the set of independent observations of each unique reflection.
-
‡Rpim = 100 ΣhΣi [1/(nh - 1)]1/2|Ih,i— ⟨Ih⟩|/ΣhΣi⟨Ih,i⟩, where nh is the number of observations of reflections h.
§CC1/2 values shown are for the highest resolution shell.
-
#As defined by the validation suite MolProbity.
Additional files
-
Transparent reporting form
- https://doi.org/10.7554/eLife.30278.020
