STRIPAK directs PP2A activity toward MAP4K4 to promote oncogenic transformation of human cells

  1. Jong Wook Kim
  2. Christian Berrios
  3. Miju Kim
  4. Amy E Schade
  5. Guillaume Adelmant
  6. Huwate Yeerna
  7. Emily Damato
  8. Amanda Balboni Iniguez
  9. Laurence Florens
  10. Michael P Washburn
  11. Kim Stegmaier
  12. Nathanael S Gray
  13. Pablo Tamayo
  14. Ole Gjoerup
  15. Jarrod A Marto
  16. James DeCaprio  Is a corresponding author
  17. William C Hahn  Is a corresponding author
  1. Broad Institute of Harvard and MIT, United States
  2. Dana-Farber Cancer Institute, United States
  3. University of California, San Diego, United States
  4. Harvard University, United States
  5. Brigham and Women’s Hospital and Harvard Medical School, United States
  6. Stowers Institute for Medical Research, United States
  7. University of Kansas Medical Center, United States
  8. Brigham and Women's Hospital and Harvard Medical School, United States
8 figures and 8 additional files

Figures

Figure 1 with 1 supplement
Global phosphoproteomic analysis identifies MAP4K4 dephosphorylation in cells transformed by PP2A perturbation.

(A) Schematic illustrating the global phosphoproteomics experiment. (B) The heatmap depicts phosphopeptides that are either positively or negatively correlated with the transformation phenotype (p<0.…

Figure 1—figure supplement 1
Changes in PP2A levels and AI growth with PP2A knockdown and STRIPAK interactions with ST from HPyV.

(A) PP2A Ca after knockdown using shPP2A Ca1 and shPP2A Ca2 and (B) PP2A B56g after knockdown using shPP2A B56g1 and shPP2A B56g2 as measured by RNAseq (Reads Per Kilobase of transcript, per Million …

Figure 2 with 1 supplement
Partial knockdown of MAP4K4 expression promotes oncogenic transformation and tumor formation.

(A) Schematic of experimental design to reveal binding proteins that when depleted can substitute for ST in transformation (B) Representative image of AI growth induced by ST or MAP4K4 partial …

Figure 2—source data 1

Quantification of soft-agar colony and tumor volume with MAP4K4 knockdown.

https://cdn.elifesciences.org/articles/53003/elife-53003-fig2-data1-v3.xlsx
Figure 2—figure supplement 1
Changes in AI growth with MAP4K4 knockdown.

(A) AI colony count of HEK TER cells expressing the corresponding shRNAs or SV40 ST. Student’s t-test was performed for each shRNA compared to shLuc control. (B) AI colony count of HEK TER cells …

Figure 3 with 1 supplement
ST promotes MAP4K4 interactions with STRIPAK and MAP4K4 dephosphorylation.

(A) Schematic of targeted proteomic analysis of MAP4K4 phosphorylation and interacting proteins in the presence of ST or GFP control. (B) SILAC experiment in which MAP4K4-associated proteins were …

Figure 3—source data 1

Quantification of MAP4K4 interacting proteins and phosphopeptides.

https://cdn.elifesciences.org/articles/53003/elife-53003-fig3-data1-v3.xlsx
Figure 3—figure supplement 1
Changes in MAP4K4-STRIPAK interactions with ST expression.

(A) Co-immunoprecipitation analysis in 293T cells of CTAP-tagged SV40 ST wild-type and PP2A binding mutants. Co-immunoprecipitation of STRN3 and Aα is shown. (B) Co-immunoprecipitation analysis of …

Figure 4 with 1 supplement
Partial inhibition of MAP4K4 kinase activity elicits transformation.

(A) Quantification of MAP4K4 in vitro kinase activity after MAP4K4 was tandem-affinity purified from cells expressing ST or GFP control. (B) Quantification of AI growth after increasing …

Figure 4—source data 1

Quantification of AI growth and in vitro MAP4K4 kinase activity.

https://cdn.elifesciences.org/articles/53003/elife-53003-fig4-data1-v3.xlsx
Figure 4—figure supplement 1
Changes in AI growth and proliferation with inhibition of MAP4K4 kinase activities.

(A) In vitro kinase assay of tandem-affinity purified MAP4K4 (Flag/HA) from HEK TER cells expressing either ST or GFP was performed and MBP was used as a substrate. HA and phospho-MBP were detected …

Figure 5 with 1 supplement
STRN4 is required for ST-mediated transformation and tumor induction.

(A) Schematic of experimental design to reveal binding proteins, that when depleted, inhibit ST-mediated transformation. (B) Representative images of AI colonies observed after knockdown of STRN4 in …

Figure 5—source data 1

Quantification of soft-agar colony and tumor volume with STRN4 knockdown.

https://cdn.elifesciences.org/articles/53003/elife-53003-fig5-data1-v3.xlsx
Figure 5—figure supplement 1
Changes in AI growth with STRN4 knockdown.

(A) AI colony count of HEK TER ST expressing four different STRN4 shRNAs or shLuc control. Immunoblot below depicts corresponding STRN4 expression after the knockdown. (B) Correlation between the …

Figure 6 with 1 supplement
STRN4 is required for STRIPAK to interact with MAP4K4.

(A) Schematic of proteomic analysis of STRN4 interacting proteins in the presence of ST or a GFP control. (B) Fold change in abundance of STRN4 interacting proteins in the presence of ST compared to …

Figure 6—source data 1

Qunatification of STRN4 interacting proteins and in vitro MAP4K4 kinase activity and AI growth.

https://cdn.elifesciences.org/articles/53003/elife-53003-fig6-data1-v3.xlsx
Figure 6—figure supplement 1
Changes in MAP4K4 kinase activity and STRIPAK interactions with STRN4 knockdown.

(A) Detection of ST from co-immunoprecipitation of STRN4 GFP- and ST-expressing cells through proteomic analysis. The values were normalized to STRN4 counts. ST was not detected (N.D.) in the …

Depletion of MAP4K4 and STRN4 are linked to YAP1 regulation.

(A) Heatmap of Enrichment Scores (ES) from RNA-seq analysis showing that partial suppression of MAP4K4 expression in HEK TER cells upregulates a transcriptional signature closely resembling four …

Figure 7—source data 1

Quantification of CTGF and CYR61 gene expression (TPM).

https://cdn.elifesciences.org/articles/53003/elife-53003-fig7-data1-v3.xlsx
Figure 8 with 1 supplement
YAP1 is necessary for transformation upon MAP4K4 knockdown and rescues transformation in STRN4 knockdown cells.

(A) Quantification of AI growth obtained following partial MAP4K4 suppression alone or when combined with YAP1 suppression (shYAP1) in HEK TER cells. Transformation induced by partial MAP4K4

Figure 8—source data 1

Quantification of AI growth with changes in YAP1 and MAP4K4.

https://cdn.elifesciences.org/articles/53003/elife-53003-fig8-data1-v3.xlsx
Figure 8—figure supplement 1
Changes in YAP1 and MAP4K4 protein levels and a proposed model.

(A) Immunoblot depicting changes in MAP4K4 and YAP1 protein levels upon either depleting YAP1 alone, or in combination with MAP4K4. (B) Immunoblot depicting the overexpression of YAP1 WT or YAP1 S5A …

Additional files

Supplementary file 1

Key Resources Table.

https://cdn.elifesciences.org/articles/53003/elife-53003-supp1-v3.docx
Supplementary file 2

Normalized iTRAQ phosphoproteomic profiles of changes in phosphopetides upon suppression of PP2A Cα, Aα, B56γ or SV40ST expression.

https://cdn.elifesciences.org/articles/53003/elife-53003-supp2-v3.xlsx
Supplementary file 3

Results of the SILAC experiment representing MAP4K4 interacting proteins.

https://cdn.elifesciences.org/articles/53003/elife-53003-supp3-v3.xlsx
Supplementary file 4

Results of the SILAC experiment representing targeted MAP4K4 phospho-profiling.

https://cdn.elifesciences.org/articles/53003/elife-53003-supp4-v3.xlsx
Supplementary file 5

Results of MudPIT experiment showing STRN4 interacting proteins.

https://cdn.elifesciences.org/articles/53003/elife-53003-supp5-v3.xlsx
Supplementary file 6

RNAseq (TPM) profiles of MAP4K4 knockdown (shMAP4K4-82).

https://cdn.elifesciences.org/articles/53003/elife-53003-supp6-v3.xlsx
Supplementary file 7

Genesets used in the study.

https://cdn.elifesciences.org/articles/53003/elife-53003-supp7-v3.xlsx
Transparent reporting form
https://cdn.elifesciences.org/articles/53003/elife-53003-transrepform-v3.pdf

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