Author response: STRIPAK directs PP2A activity toward MAP4K4 to promote oncogenic transformation of human cells (2020)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Alterations involving serine-threonine phosphatase PP2A subunits occur in a range of human cancers, and partial loss of PP2A function contributes to cell transformation. Displacement of regulatory B subunits by the SV40 Small T antigen (ST) or mutation/deletion of PP2A subunits alters the abundance and types of PP2A complexes in cells, leading to transformation. Here, we show that ST not only displaces common PP2A B subunits but also promotes A-C subunit interactions with alternative B subunits (B''', striatins) that are components of the Striatin-interacting phosphatase and kinase (STRIPAK) complex. We found that STRN4, a member of STRIPAK, is associated with ST and is required for ST-PP2A-induced cell transformation. ST recruitment of STRIPAK facilitates PP2A-mediated dephosphorylation of MAP4K4 and induces cell transformation through the activation of the Hippo pathway effector YAP1. These observations identify an unanticipated role of MAP4K4 in transformation and show that the STRIPAK complex regulates PP2A specificity and activity. eLife digest Cells maintain a fine balance of signals that promote or counter cell growth and division. Two sets of enzymes – called kinases and phosphatases – contribute to this balance. In general, kinases "switch on" other proteins by tagging them with a phosphate molecule. This process is called phosphorylation. Phosphatases, on the other hand, dephosphorylate these proteins, switching them off. Cancer cells often have mutations that activate kinases to drive cancer growth. The same cells can have mutations that inactivate the phosphatases or reduce their abundance. The roles of phosphatases in cancer are still being studied. One major hurdle in this research is that it is not always clear how they recognize the proteins they dephosphorylate. Protein phosphatase 2A (or PP2A for short) is one of the phosphatases that is often mutated or deleted in human cancers. Even just reduced levels of PP2A can promote cancer. Kim, Berrios, Kim, Schade et al. used an experimental trick to decrease the phosphatase activity of PP2A in human cells growing in a dish. Biochemical analysis of these cells showed that, as expected, many proteins were now in their phosphorylated states. Unexpectedly, however, some proteins were dephosphorylated under these conditions. One of these proteins was called MAP4K4. In the case of MAP4K4, the dephosphorylated state contributes to the growth of the cancer cell. Kim et al. carried out further genetic and biochemical experiments to show that, in these cells, PP2A and MAP4K4 stay physically connected to one another. This connection was enabled by a group of proteins called the STRIPAK complex. The STRIPAK proteins directed the remaining PP2A towards MAP4K4. Low levels or activity of PP2A could, therefore, promote cancer in a different way. Taken together, PP2A is not a single phosphatase that always turns proteins off, but rather is a dual switch that turns off some proteins while turning on others. Future experiments will explore to what extent these findings also apply in tumors. Information about how mutations in PP2A affect human cancers could suggest new targets for cancer drugs. Introduction Protein phosphorylation plays a regulatory role in nearly all biological processes and dysregulation of protein phosphorylation contributes to many diseases. Both kinases and phosphatases have been implicated in the pathogenesis of specific cancers, and several small molecule kinase inhibitors are standard treatments in such cancers. In addition, several phosphatases have been identified as tumor suppressors (Sablina and Hahn, 2007; Lawrence et al., 2014). PP2A, an abundant serine/threonine phosphatase in mammalian cells, is comprised of three subunits: A (structural), B (regulatory), and C (catalytic). The A and C subunits form the core enzyme and interact with different B regulatory subunits to create many distinct PP2A enzymes (Pallas et al., 1990; Chen et al., 2007; Cho et al., 2007; Shi, 2009; Sents et al., 2013). Moreover, there are two A and two C isoforms, and at least four classes of B subunits B, B', B'', and B''' (striatins), each of which exist as several different isoforms. Although the prevailing view is that the B subunits provide substrate specificity, how B subunits accomplish this regulation remains unclear (Shi, 2009; Hertz et al., 2016). Genome characterization studies of human cancers have identified recurrent mutations and deletions involving PP2A subunits. Indeed, the PP2A Aα (PPP2R1A) subunit ranks among the most recurrently mutated gene across many cancer types (Lawrence et al., 2014). Notably, mutations in Aα occur at high frequency in premalignant endometrial lesions (Anglesio et al., 2017). PP2A is also a target of the Small T antigens (ST) of SV40 and other polyomaviruses including the human oncogenic Merkel cell polyomavirus (Pallas et al., 1990; Chen et al., 2004; Cheng et al., 2017), and this interaction contributes to cell transformation (Hahn et al., 2002). Structural studies have shown that ST disrupts the formation of a functional PP2A holoenzyme by displacing or hindering B subunit access to the PP2A core-enzyme (Chen et al., 2007; Cho et al., 2007). However, ST has a lower binding affinity in vitro for the PP2A core enzyme than B' subunits, which suggests that ST interaction with the core enzyme may either occur prior to the B subunit binding or ST directly inhibits PP2A activity independently of subunit assembly (Chen et al., 2007). Several investigators have used mass spectrometry to identify proteins that interact with PP2A (Goudreault et al., 2009; Herzog et al., 2012). These studies identified a large complex called the Striatin-interacting phosphatase and kinase (STRIPAK) complex (Goudreault et al., 2009). The STRIPAK complex contains striatin family (STRN) proteins, several kinases, scaffolding proteins, and PP2A subunits. Indeed, striatins were initially described as non-canonical PP2A regulatory subunits (B''' subunits) (Moreno et al., 2000). STRIPAK complexes have also been shown to associate with members of the GCKIII kinase subfamily (MST3, STK24, and STK25) (Kean et al., 2011). In addition, mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), a Ste20-like kinase, although not an obligate member of the STRIPAK complex, associates with STRIPAK (Frost et al., 2012; Herzog et al., 2012; Hyodo et al., 2012). We also identified members of the STRIPAK complex, including STRN3, STRN4, STRIP1, and MAP4K4 in complex with SV40 ST (Rozenblatt-Rosen et al., 2012). Although STRIPAK comprises multiple signaling enzymes, it is unclear how disruptions to the biochemical complex integrate with or disrupt phosphorylation cascades; or whether these signaling alterations synergize with ST to mediate cellular transformation. MAP4K4 is a serine/threonine kinase that was initially found to activate the c-Jun N-terminal kinase (JNK) signaling pathway (Yao et al., 1999), downstream of TNF-α. MAP4K4 has also been implicated in a large number of biological processes including insulin resistance, focal adhesion disassembly, as well as cellular invasion and migration (Collins et al., 2006; Tang et al., 2006; Yue et al., 2014; Danai et al., 2015; Vitorino et al., 2015). Recent studies have shown that MAP4K4 phosphorylates LATS1/2, activating the Hippo tumor suppressor pathway, leading to YAP1 inactivation (Mohseni et al., 2014; Meng et al., 2015; Zheng et al., 2015). Here, we investigated the role of the STRIPAK complex and MAP4K4 in human cell transformation driven by SV40 ST and found that kinase inactivation or partial suppression of MAP4K4 replace the expression of ST in the transformation of human cells. Results Identification of MAP4K4 as a candidate phosphoprotein targeted in cells transformed by PP2A perturbation Human embryonic kidney (HEK) epithelial cells expressing SV40 Large T antigen (LT), the telomerase catalytic subunit (hTERT), and oncogenic HRAS (referred to as HEK TER hereafter) have served as a useful model system to identify pathways and protein complexes that can functionally substitute for SV40 ST in promoting transformation, including partial depletion of PP2A (Chen et al., 2004; Sablina et al., 2010). These cells, upon expression of SV40 ST or partial knockdown of PP2A Aα or Cα subunits, become tumorigenic (Hahn et al., 2002; Chen et al., 2004). Prior studies have shown that expression of ST, or partial inhibition of certain PP2A subunits, causes increased phosphorylation of PP2A substrates (Sablina and Hahn, 2008; Sablina et al., 2010). To assess the serine/threonine phosphorylation events that are associated with transformation induced by ST or by partial knockdown of PP2A, we performed global Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) phosphoproteomic profiling of HEK TER cells expressing ST (HEK TER ST) or in which expression of the PP2A Aα, Cα, or B56γ subunits were fully or partially suppressed using previously characterized shRNAs (Figure 1A, Figure 1—figure supplement 1A–B) (Sablina et al., 2010). We also confirmed that these genetic perturbations promoted the transformation phenotype as gauged by anchorage-independent (AI) growth assays as previously described (Figure 1—figure supplement 1C) (Sablina et al., 2010). Through mass spectrometry analysis of the phosphopeptides altered across these conditions, we identified 6025 phosphopeptides corresponding to 2428 individual proteins reproducibly detected in two replicate experiments. Processing and normalization of the raw data were performed using corresponding control experiments (GFP control for ST, shLuciferase (shLuc) control for shRNAs against PP2A, see methods for details). We then performed comparative marker selection analysis (Gould et al., 2006) to identify candidate phosphoproteins that were most significantly correlated with the transformation phenotype (Figure 1A). In consonance with previous studies (Ratcliffe et al., 2000; Kuo et al., 2008), we observed an increase in phosphorylation of direct or indirect targets of PP2A, including AKT1S and β-catenin (CTNNB1) in cells which were transformed by either expressing ST or partial knockdown of PP2A Cα subunit in HEK TER cells (Figure 1B) (Sablina et al., 2010). Conversely, we also observed decreased phosphorylation on multiple proteins in cells transformed by ST or by PP2A perturbation (B56γ1, Cα2). Notably, the phosphorylation signature for transformation included four distinct sites on MAP4K4 (T804, S888, S889, S1272, p<0.05, Figure 1B). Figure 1 with 1 supplement see all Download asset Open asset 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<0.05, FDR < 1). Each column represents individual samples that were normalized to shLuc for shPP2A or in the case of ST to GFP control. The sample designations after the normalization and comparative marker selection analysis are shown below the heatmap, with each sample shown in replicates. A selected subset of phosphorylated sites which distinguishes transforming and non-transforming perturbations are shown. Our previous systematic analysis of SV40 ST identified MAP4K4, in addition to PP2A and other STRIPAK components in the same complex (STRN4, STRN3, CTTNBP2NL, FAM40A, MAP2K3, STK24, PPP2R1A) (Rozenblatt-Rosen et al., 2012). To confirm these interactions, we generated lentiviral C-terminal Flag-HA Tandem Affinity Purification (CTAP) constructs for SV40 ST as well as ST from three closely related Human Polyoma Viruses (HPyV) including JCPyV-CY, JCPyV-Mad1, and BKPyV, along with GFP as a negative control. We introduced these viral proteins into HCT116 cells and performed HA-tag immunoprecipitations (IP) from lysates of cells expressing ST from the respective viruses. We confirmed co-complex formation between SV40 ST and MAP4K4, as well as with STRIPAK components PP2A C, STRN3, and STRIP1 (Figure 1—figure supplement 1D). We observed that ST of JCPyV and BKPyV, the two most closely related HPyVs to SV40, also interacted with STRN3, STRIP1, and PP2A C but not MAP4K4, indicating that the interaction of SV40 ST and MAP4K4 was unique to SV40 ST. The association of ST with B''' subunits (striatins) was unexpected, because ST was previously reported to primarily bind PP2A Aα and displace most B subunits (Pallas et al., 1990; Chen et al., 2007; Cho et al., 2007; Sablina et al., 2010). These observations raised the possibility that ST modulates MAP4K4 phosphorylation via PP2A activity associated with the STRIPAK complex. Partial knockdown of MAP4K4 promotes cell transformation To determine if MAP4K4 and other SV40 ST interacting proteins participated in cell transformation, we created and stably expressed two distinct shRNAs targeting each of several SV40 ST interacting proteins, including STRN3, STRN4, STRIP1, MARCKS, MAP4K4, and STK24 in HEK TER cells. We then assessed the ability of each of these shRNAs to promote AI growth, a readout for the transformed phenotype (Figure 2A, Figure 2—figure supplement 1A). As expected, expression of SV40 ST or partial knockdown of PP2A Cα subunit in HEK TER cells induced robust AI growth (Figure 2—figure supplement 1A). Among the STRIPAK components, we found that one of the two shRNAs targeting MAP4K4 (shMAP4K4-82) elicited a potent transformation phenotype (Figure 2B–C, Figure 2—figure supplement 1A). To ensure that the observed phenotype was specific to targeting MAP4K4 and not due to an off-target effect of this shRNA, we repeated the AI growth assay using eight different MAP4K4-targeting shRNAs including the two shRNAs used in the initial experiment (Figure 2—figure supplement 1B). In addition, we found that the three shRNAs which promoted HEK TER cells to grow in an AI manner (shRNA-82, 92, 93) only partially suppressed MAP4K4 levels (Figure 2—figure supplement 1B). Specifically, we focused on shMAP4K4-82, which promoted the most robust AI growth and knocked down MAP4K4 mRNA levels by 50% (Figure 2—figure supplement 1B–C). In contrast, none of the shRNAs that induced more than 50% knockdown of MAP4K4 expression resulted in AI growth (Figure 2—figure supplement 1B). This relationship between partial knockdown and cell transformation is similar to what has been reported for the knockdown of PP2A Aα and Cα subunits (Chen et al., 2005; Sablina et al., 2010). To further confirm these data in vivo, we performed xenograft experiments to assess tumor formation by subcutaneous injection of immunodeficient mice. Consistent with the in vitro studies, shMAP4K4-82 induced potent tumor formation when compared to the shLuc control (Figure 2D–E). These observations suggest that partial knockdown, but not full depletion, of MAP4K4, promotes both transformation and tumor formation. Figure 2 with 1 supplement see all Download asset Open asset 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 knockdown (Grid shows 5 mm). (C) Quantification of AI growth following expression of MAP4K4 shRNA-82 (shMAP4K4), SV40 ST or corresponding controls (GFP or shLuc). Graph depicting tumor volume as a function of time (D) or an endpoint at day 28 (E) for subcutaneous xenografts expressing shLuc control or shMAP4K4-82 in HEK TER cells (Student's t-test, **p<0.001, ***p<0.0001, ****p<0.00001). 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 Download elife-53003-fig2-data1-v3.xlsx SV40 ST promotes the interaction of MAP4K4 with STRIPAK To understand the mechanism by which the ST/MAP4K4 axis contributes to cell transformation, we first assessed changes in interactions between MAP4K4 and its binding partners upon ST expression. Specifically, we stably expressed NTAP-MAP4K4 in HEK TER cells expressing either ST or GFP as a negative control. We used Stable Isotope Labeling with Amino Acids (SILAC) to encode proteins in each condition (Figure 3A). We found that MAP4K4 interacted with STRIPAK components, including STRIP1, STRN3, STRN4, and the PP2A Aα subunit. The interactions between MAP4K4 and the STRIPAK components were increased by 3–4-fold in cells expressing ST relative to the GFP control (Figure 3B). We tested a series of ST mutants (R21A, W147A, F148A, P132A) that are unable to bind to PP2A Aα (Cho et al., 2007) in 293 T cells, and found that these mutant ST proteins were unable to interact with STRN3, a core component of the STRIPAK complex (Figure 3—figure supplement 1A), demonstrating that this interaction is dependent on ST binding to PP2A Aα subunit. These observations indicated that MAP4K4 interaction with STRIPAK is enhanced in cells expressing SV40 ST. Figure 3 with 1 supplement see all Download asset Open asset 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 assessed in cells expressing ST or a GFP control by SILAC experiments performed as biological replicates (IP1, IP2). The proteins that showed a fold change above one have increased interactions with MAP4K4 in ST expressing cells relative to GFP. All values of the retrieved peptides were normalized to the total number of MAP4K4 peptides, prior to calculating the ratios between ST- versus GFP-expressing cells to account for variations in the amount of MAP4K4 after affinity purification. (C) Immunoblot showing results of a Co-Immunoprecipitation (Co-IP) analysis of components of STRIPAK with MAP4K4 in ST- or GFP-expressing cells. ST induced the association of MAP4K4 with STRIPAK components. (D) Quantification of fold changes in the abundance of MAP4K4 phosphorylation across indicated sites (y-axis) in cells expressing ST relative to the GFP control in two independent experiments (IP1, IP2). The phosphosites with fold changes below one show a decrease of phosphorylation of MAP4K4 in ST expressing cells relative to GFP. All values of the retrieved peptides were normalized to the total number of MAP4K4 peptides, prior to calculating the ratios between ST- versus GFP-expressing cells to account for variations in the amount of MAP4K4 after affinity purification. (E) After immunoprecipitation of STRN4 from ST- versus GFP-expressing cells, in vitro PP2A activity was measured with synthetic MAP4K4 peptides (S771;S775, S876, or S1251) identified in the targeted phosphoproteomic experiments (x-axis). Relative phosphatase activity in ST- relative to GFP-expressing HEK TER cells is shown for each phosphopeptide (y-axis). Okadaic acid (OA) was used to inhibit PP2A activity in parallel conditions (Student's t-test, **p<0.001, ***p<0.0001, ****p<0.00001). Figure 3—source data 1 Quantification of MAP4K4 interacting proteins and phosphopeptides. https://cdn.elifesciences.org/articles/53003/elife-53003-fig3-data1-v3.xlsx Download elife-53003-fig3-data1-v3.xlsx To corroborate these observations, we performed IP of endogenous STRN3, STRN4, STRIP1, and MAP4K4 and compared the interactions of components of the STRIPAK complex with MAP4K4 in HEK TER cells expressing either ST or GFP. Consistent with the proteomic results, we observed that the interaction of MAP4K4 with the STRIPAK complex was significantly enhanced in the presence of ST (Figure 3C). We also performed these experiments in normal human fibroblasts (IMR90) expressing ST or GFP (negative control) and confirmed the enhanced binding of MAP4K4 to STRN4 and STRIP1 (Figure 3—figure supplement 1B). These observations indicate that interactions between MAP4K4 and STRIPAK components, including STRIP1, STRN3, and STRN4 are enhanced in the presence of SV40 ST. We next analyzed the enriched phosphopeptides from affinity-purified MAP4K4 (Figure 3A) to better interrogate the full phosphorylation landscape on the kinase. In two independent experiments, we quantified 17 MAP4K4 phosphorylation sites (Figure 3D). The majority of these sites exhibited reduced phosphorylation in cells expressing ST. These findings further demonstrate that ST mediates dephosphorylation of several distinct MAP4K4 sites. To evaluate if MAP4K4 dephosphorylation is mediated by the STRIPAK complex, we isolated STRN4 from cells expressing ST or GFP (Figure 3—figure supplement 1C) and measured PP2A-specific dephosphorylation activity using synthetic phosphopeptides encompassing MAP4K4 sites S771;S775, S876, or S1251. We selected these sites because they exhibited the largest change in phosphorylation upon ST expression (Figure 3D). As a we parallel samples with acid a potent and specific PP2A As expected, we observed that phosphatase activity under all conditions (Figure In contrast, of MAP4K4 phosphopeptides with STRN4 complexes from cells to dephosphorylation of the and phosphopeptides by than compared to GFP while we found a but increase of dephosphorylation of the (Figure These observations suggest that ST promotes PP2A-mediated dephosphorylation of MAP4K4 in the STRN4 complex. of MAP4K4 kinase activity is associated with transformation MAP4K4 phosphorylation at several sites was in the presence of ST, we assessed whether this decrease in MAP4K4 phosphorylation MAP4K4 activity by an in vitro kinase assay using MAP4K4 from cells that expressed ST or GFP control. We found that the activity of MAP4K4 was reduced in cells compared to cells that expressed GFP control (Figure Figure supplement 1A). Figure 4 with 1 supplement see all Download asset Open asset Partial inhibition of MAP4K4 kinase activity transformation. (A) Quantification of MAP4K4 in vitro kinase activity after MAP4K4 was from cells expressing ST or GFP control. (B) Quantification of AI growth after of the MAP4K4 (C) Quantification of AI growth after expression of MAP4K4 MAP4K4 ST, or GFP in HEK TER cells (Student's t-test, **p<0.001, ***p<0.0001, not Figure data 1 Quantification of AI growth and in vitro MAP4K4 kinase activity. Download To assess the of MAP4K4 kinase activity to the transformation we tested the of or genetic inhibition of MAP4K4 on AI growth. Specifically, we HEK TER cells with a previously described small molecule of MAP4K4 et al., a range of and assessed MAP4K4 activity (Figure supplement 1B) and AI cell growth (Figure In consonance with what we observed with partially knocked down MAP4K4 of this MAP4K4 to an increase in the number of AI it 2 when MAP4K4 kinase activity was more than as measured by in vitro kinase assays Figure Figure supplement 1A). We found that induced on cell the range of tested (Figure supplement Consistent with the results from the genetic experiments (Figure we observed that partial inhibition of MAP4K4 activity to increased AI growth. We also tested whether MAP4K4 by expressing a MAP4K4 promoted transformation. The MAP4K4 has previously been to as a mutant et al., 2013). We created HEK TER cells stably expressing or the of MAP4K4 and confirmed the loss of kinase activity for the MAP4K4 mutant (Figure supplement we performed AI growth we observed that the of MAP4K4 but not MAP4K4 induced cell transformation (Figure these observations demonstrate that partial depletion or inhibition of MAP4K4 activity ST in transformation and that of MAP4K4 kinase activity is associated with cell transformation. STRN4 is required for transformation of MAP4K4 levels and activity was to drive transformation in the of therefore, we also investigated whether members of the STRIPAK complex were required for oncogenic transformation (Figure Specifically, we assessed the of components of STRIPAK in TER ST cell and found that knockdown of STRN4 to a in transformation (Figure We tested 4 shRNAs and observed in AI in a manner that significantly correlated with the of STRN4 knockdown (Figure supplement To confirm that these findings were not due to an off-target effect of we created a STRN4 to the and expressed this in HEK TER ST cells (Figure supplement We found that expression of this STRN4 the of STRN4 on AI growth (Figure We also deleted STRN4 using gene and further confirmed that STRN4 expression was required for cell transformation (Figure supplement 1D). We assessed the of down STRN4 in and found that STRN4 knockdown significantly reduced tumor formation of HEK TER ST cells (Figure these observations demonstrate that STRN4 is required for transformation and tumor formation. Figure 5 with 1 supplement see all Download asset Open asset STRN4 is required for transformation and tumor (A) Schematic of experimental design to reveal binding proteins, that when inhibit transformation. (B) Representative of AI observed after knockdown of STRN4 in HEK TER ST cells with (C) Quantification of the number of AI following the of or shLuc control. (D) Quantification of the number of AI after expression of in the presence or of an STRN4 volume as a function of time (E) or at the endpoint at day for subcutaneous xenografts expressing shLuc control or STRN4 in HEK TER ST cells (Student's t-test, **p<0.001, ****p<0.00001). Figure data 1 Quantification of soft-agar colony and tumor volume with STRN4 knockdown. Download STRN4 is required for the STRIPAK complex to associate with MAP4K4 To assess whether ST modulates interactions involving STRN4, we isolated endogenous STRN4 from cells expressing either ST or a GFP control and performed a proteomic analysis of associated proteins (Figure We found that STRN4 interactions with MAP4K4 were increased while interactions with the PP2A C subunits not in cells expressing SV40 ST relative to GFP control (Figure Figure supplement 1A). Figure with 1 supplement see all Download asset Open asset 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) change in abundance of STRN4 interacting proteins in the presence of ST compared to the GFP control. All values were normalized to STRN4 levels to account for variations in the total amount of STRN4 isolated from cells expression ST or GFP control. was to in the amount of STRN4 interacting proteins between cells expressing ST relative to GFP. of a number of proteins in the STRIPAK complex, including PP2A C, MAP4K4, CTTNBP2NL, and with STRN4, were increased in cells relative to the GFP control. (C) Immunoblot showing a analysis of STRIPAK core components and MAP4K4 after knockdown of STRN4 using STRN4 is required for the STRIPAK component STRIP1 to interact with MAP4K4 and PP2A (D) Quantification of MAP4K4 in vitro kinase activity after STRN4 knockdown (E) Quantification of AI growth after expression of STRN4 with or of MAP4K4 in HEK TER ST cells. of MAP4K4 expression the transformation from STRN4 All experiments were performed in and the were performed relative to the controls (Student's t-test, Figure data 1 of STRN4