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. 2020 Jul 7;32(1):107860.
doi: 10.1016/j.celrep.2020.107860.

MAP4K Interactome Reveals STRN4 as a Key STRIPAK Complex Component in Hippo Pathway Regulation

Gayoung Seo  1 , Han Han  1 , Rebecca Elizabeth Vargas  1 , Bing Yang  1 , Xu Li  2 , Wenqi Wang  3
Affiliations

MAP4K Interactome Reveals STRN4 as a Key STRIPAK Complex Component in Hippo Pathway Regulation

Gayoung Seo et al. Cell Rep. .

Abstract

Mitogen-activated protein kinase kinase kinase kinases (MAP4Ks) constitute a mammalian STE20-like serine/threonine kinase subfamily. Recent studies provide substantial evidence for MAP4K family kinases in the Hippo pathway regulation, suggesting a broad role of MAP4Ks in human physiology and diseases. However, a comprehensive analysis of the regulators and effectors for this key kinase family has not been fully achieved. Using a proteomic approach, we define the protein-protein interaction network for human MAP4K family kinases and reveal diverse cellular signaling events involving this important kinase family. Through it, we identify a STRIPAK complex component, STRN4, as a generic binding partner for MAP4Ks and a key regulator of the Hippo pathway in endometrial cancer development. Taken together, the results of our study not only generate a rich resource for further characterizing human MAP4K family kinases in numerous biological processes but also dissect the STRIPAK-mediated regulation of MAP4Ks in the Hippo pathway.

Keywords: Hippo pathway; MAP4K; STRIPAK; STRN4; YAP; endometrial cancer; proteomics.

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Conflict of interest statement

Declaration of Interests The authors declare no competing financial interests.

Figures

Figure 1.
Figure 1.. Proteomic Analysis of MAP4K Protein Interaction Network
(A) Schematic illustration of the protein domains for the MAP4K family kinases. (B) Schematic illustration of the major steps used in TAP-MS analysis of human MAP4K-associated protein complexes. MAP4Ks were constructed into a C-terminal SFB-tag-fused lentiviral vector. HEK293T cells stably expressing each bait protein were generated by lentiviral infection and puromycin selection. The purified protein complexes were identified by MS analysis, and final interacting proteins were generated using the MUSE statistical model. (C) Localization of MAP4Ks in the HEK293A cells stably expressing each MAP4K family member. Immunofluorescent staining was performed by using FLAG antibody. Scale bar, 20 μm. (D) Summary of the MAP4K TAP-MS analysis. Experiment information and total numbers of peptides and proteins identified in the MS analysis are shown. A MUSE score of ≥0.8 was used as the cutoff to identify HCIPs. (E) The total spectral counts (TSCs) and corresponding numbers of HCIPs for MAP4Ks are shown. (F) Data reproducibility between two biological TAP-MS experiments is evaluated using the number of peptide spectrum matches (PSMs). (G and H) GO annotations of the identified MAP4Ks-HCIPs. Cellular localization (G) and cellular functions (H) of the MAP4Ks-HCIPs are shown. See also Tables S1, S2, S3, and S4.
Figure 2.
Figure 2.. Hierarchical Clustering Analysis of the MAP4K-HCIPs
A heatmap was generated from hierarchical clustering of 221 HCIPs for MAP4K family kinases. Five prominent HCIP clusters were manually selected and enlarged below. The color of squares in the heatmap indicates the identified HCIP peptide number for each MAP4K protein. The shared STRIPAK complex components are shown in red. See also Table S3.
Figure 3.
Figure 3.. Integrated Interaction Map of the Human MAP4K Protein Interaction Network
As for each MAP4K family member, HCIPs are grouped based on their cellular functions, as indicated by GO analysis and literature search. The different circle colors indicate different cellular functions. The shared HCIP groups are connected by dot-lines. The identified HCIPs are compared to the known MAP4K-binding proteins reported in the BioGrid database and are indicated in different colors. See also Tables S3 and S4.
Figure 4.
Figure 4.. Validation of MAP4K Protein Interaction Network
(A) A summary of reciprocal TAP-MS analyses for the selected MAP4K-HCIPs. (B) Validation of the interaction between MAP4K2 and GABARAP family proteins. The bacterially purified GST-GABARAP family proteins were subjected to the pull-down assay. CBS, Coomassie blue staining. (C) Validation of the interaction between MAP4K3 and SH3GL1. HEK293T cells were transfected with the indicated constructs and subjected to the pull-down assay. (D and E) Validation of the interaction between MAP4K5 and CTTN. HEK293T cells were transfected with the indicated constructs and subjected to the pull-down assay (D). The co-localization between MAP4K5 and CTTN in lamellipodia was indicated by arrows (E). Scale bar, 20 μm. (F and G) Validation of the interaction between MAP4K family kinases and the STRIPAK complex components. HEK293T cells were transfected with the indicated constructs and subjected to the pull-down assay (F). The pull-down experiment results were summarized (G).
Figure 5.
Figure 5.. STRN4 Negatively Regulates the Hippo Pathway
(A) Binding specificity for STRN4 and SLMAP are respectively examined within the Hippo pathway components. HEK293T cells were transfected with the indicated constructs and subjected to the pull-down assay. (B–F) Depletion of STRN4 inactivates YAP. Loss of STRN4 (B) increased the phosphorylation of LATS1 and YAP (C), which can be rescued by re-expressing STRN4 in the knockout cells. Loss of STRN4 suppressed YAP downstream gene transcription (mean ± SD, n = 3 biological replicates) (D) and induced YAP cytoplasmic translocation (E). STRN4 knockout cells (~200 cells in total) were randomly selected and quantified for YAP localization (F). Western blotting was performed with the indicated antibodies. **p < 0.01 (Student’s t test). Scale bar, 20 μm. (G–I) Reconstituting STRN4 rescues YAP activity in the STRN4 knockout cells. SFB-tagged STRN4 was re-expressed in the STRN4 knockout cells, where YAP downstream gene transcription (mean ± SD, n = 3 biological replicates) (G) and YAP cellular localization were examined (H and I). FLAG-positive cells (~200 cells in total) were randomly selected and quantified for YAP localization (I). **p < 0.01, ***p < 0.001 (Student’s t test). ΔWD, STRN4 WD-repeat-deletion mutant. Scale bar, 20 μm. (J–M) The WD-repeat region is required for the association between STRN4 and MAP4Ks. A series of STRN4 truncation and deletion mutants used in this study were illustrated (J). HEK293T cells were transfected with the indicated constructs and subjected to the pull-down assay for MAP4K1 (K), MAP4K2 (L), and MAP4K4 (M). (N–P) Overexpression of STRN4 but not its ΔWD mutant induces YAP oncogenic activity in MCF10A cells. The indicated MCF10A stable cells were subjected to examination of YAP downstream gene transcription (mean ± SD, n = 3 biological replicates) (N) and acini formation. The representative acini were shown (O) and the relative acini size was quantified (~50 acini were randomly selected in different views) (mean ± SD, n = 3 biological replicates) (P). ***p < 0.001 (Student’s t test). ns, no significance. Scale bar, 200 μm. See also Figure S1 and Table S5.
Figure 6.
Figure 6.. STRN4 Is a Potential Oncoprotein in Human Endometrial Cancer
(A) STRN4 is highly expressed in human endometrial cancer. The alteration frequency of STRN4 is analyzed through the cBioportal database (https://www.cbioportal.org). (B) STRN4 expression is examined in a panel of endometrial cancer cell lines by western blotting. (C) YAP cellular localization is examined in a panel of endometrial cancer cell lines by immunofluorescent staining. Scale bar, 20 μm. (D) Immunohistochemical staining of STRN4 and YAP were performed in endometrial cancer tissue microarrays, where the indicated regions in the box were shown 2.5 times enlarged. Brown staining indicates positive immunoreactivity. Correlation analyses between STRN4 and YAP in human normal endometrial tissue and endometrial adenocarcinoma samples are shown as tables. Statistical significance was determined by chi-square test. R, correlation coefficient; N, nuclear localization; C, cytoplasmic localization. Scale bar, 100 μm.
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