Striatin-interacting Phosphatase And Kinase Research Articles

Striatin plays a major role in angiotensin II-induced cardiomyocyte and cardiac hypertrophy in mice in vivo.

The three striatins (STRN, STRN3, STRN4) form the core of STRiatin-Interacting Phosphatase and Kinase (STRIPAK) complexes. These place protein phosphatase 2A (PP2A) in proximity to protein kinases thereby restraining kinase activity and regulating key cellular processes. Our aim was to establish if striatins play a significant role in cardiac remodelling associated with cardiac hypertrophy and heart failure. All striatins were expressed in control human hearts, with upregulation of STRN and STRN3 in failing hearts. We used mice with global heterozygote gene deletion to assess the roles of STRN and STRN3 in cardiac remodelling induced by angiotensin II (AngII; 7 days). Using echocardiography, we detected no differences in baseline cardiac function or dimensions in STRN+/- or STRN3+/- male mice (8 weeks) compared with wild-type littermates. Heterozygous gene deletion did not affect cardiac function in mice treated with AngII, but the increase in left ventricle mass induced by AngII was inhibited in STRN+/- (but not STRN3+/-) mice.Histological staining indicated that cardiomyocyte hypertrophy was inhibited. To assess the role of STRN in cardiomyocytes, we converted the STRN knockout line for inducible cardiomyocyte-specific gene deletion. There was no effect of cardiomyocyte STRN knockout on cardiac function or dimensions, but the increase in left ventricle mass induced by AngII was inhibited. This resulted from inhibition of cardiomyocyte hypertrophy and cardiac fibrosis. The data indicate that cardiomyocyte striatin is required for early remodelling of the heart by AngII and identify the striatin-based STRIPAK system as a signalling paradigm in the development of pathological cardiac hypertrophy.

Mst4, a novel cardiac STRIPAK complex–associated kinase, regulates cardiomyocyte growth and survival and is upregulated in human cardiomyopathy

Myocardial failure is associated with adverse remodeling which includes apoptotic loss of cardiomyocytes, hypertrophy as well as alterations in cell-cell contacts. Striatin-interacting phosphatase and kinase (STRIPAK) complexes and their kinase Mst4 have been linked to the development of different diseases. The role and targets of Mst4 in cardiomyocytes have not been investigated, yet.Multi tissue immunoblot experiments show highly enriched Mst4-expression in rodent hearts. Analyses of human biopsy samples from patients suffering from dilated cardiomyopathy revealed that Mst4 is upregulated (5,8-fold p<0.001) compared with non-failing controls. Increased abundance of Mst4 could also be detected in mouse models of cardiomyopathy. We confirmed that Mst4 interacts with STRIPAK components in neonatal rat ventricular cardiomyocytes, indicating that STRIPAK is present in the heart. Immunofluorescence stainings and molecular interaction studies revealed that Mst4 is localized to the intercalated disc and interacts with several intercalated disc proteins. Overexpression of Mst4 in neonatal rat cardiomyocytes results in hypertrophy compared with controls. In adult rat cardiomyocytes, Mst4-overexpression increases cellular and sarcomeric fractional shortening (p<0.05), indicating enhanced contractility. Overexpression of Mst4 also inhibits apoptotic cell death shown by reduction of cleaved Caspase3 (-69%, p<0.0001) and Caspase7 (-80%, p<0.0001) as well as cleaved Parp1 (-27%, p<0.001). To elucidate potential Mst4 targets, we performed phosphoproteomics analyses in neonatal rat cardiomyocytes after Mst4 overexpression and inhibition. The results revealed several target candidates of Mst4 at the intercalated disc and sarcolemma. ConclusionWe identified Mst4 as a novel cardiac kinase that is upregulated in human cardiomyopathy and regulates cardiomyocyte growth and survival.

Cellular Impacts of Striatins and the STRIPAK Complex and Their Roles in the Development and Metastasis in Clinical Cancers (Review).

Striatins (STRNs) are generally considered to be cytoplasmic proteins, with lower expression observed in the nucleus and at cell-cell contact regions. Together with protein phosphatase 2A (PP2A), STRNs form the core region of striatin-interacting phosphatase and kinase (STRIPAK) complexes through the coiled-coil region of STRN proteins, which is crucial for substrate recruitment. Over the past two decades, there has been an increasing amount of research into the biological and cellular functions of STRIPAK members. STRNs and the constituent members of the STRIPAK complex have been found to regulate several cellular functions, such as cell cycle control, cell growth, and motility. Dysregulation of these cellular events is associated with cancer development. Importantly, their roles in cancer cells and clinical cancers are becoming recognised, with several STRIPAK components found to have elevated expression in cancerous tissues compared to healthy tissues. These molecules exhibit significant diagnostic and prognostic value across different cancer types and in metastatic progression. The present review comprehensively summarises and discusses the current knowledge of STRNs and core STRIPAK members, in cancer malignancy, from both cellular and clinical perspectives.

Orchestration of autophagosome fusion by STRIPAK complex components in muscle tissue.

Autophagy is a central process responsible for the disposal of normal as well as damaged cellular proteins and organelles. Proper regulation of multiple steps - including initiation and the fusion between autophagosomes and lysosomes - is essential for the completion of cargo disposal. While the function of many proteins that mediate canonical autophagy has been characterized, the identification of new autophagy regulators may shed light on differences between tissues and/or responses to cellular stresses. In this punctum, we discuss our recent findings about how the Striatin-Interacting Phosphatase and Kinase (STRIPAK)-NUAK-Starvin (Stv) complex coordinately regulates autophagy in the muscle tissue of Drosophila melanogaster.

Abstract P1181: Targeted Loss Of Sarcolemmal Membrane Associated Protein Isoform 3 (slmap3) In Cardiac Progenitors Stunts The Embryonic Growth Of Myocardium

Sarcolemmal membrane associated proteins (SLMAPs) belong to the superfamily of tail anchored membrane proteins known to regulate variety of functions including vesicle transport. SLMAP3 is the largest isoform which is ubiquitously expressed and has been linked to Brugada syndrome and sodium channel activity. SLMAP3 is expressed during cardiogenesis, and we examined its role by nullifying its expression in mouse cardiac progenitors using the Nkx2.5-cre-lox system. Hearts from SLMAP3-knockout (KO) mouse embryos at different stages of cardiogenesis, e9.5, e12.5 and e16.5, were smaller (p&lt;0.05) than wildtype (Wt) littermates. Histological analysis of heart wall measurements from the left ventricles revealed thinner walls in SLMAP3-KO from e9.5 (-25.16%, p&lt;0.05), e12.5 (-29.36%, p&lt;0.05) and e16.5 (-40.27%, p&lt;0.05) compared to Wt although normal chamber formations was observed at these developmental stages. To evaluate the potential reasons for the early difference in cardiac growth, we examined myocardial cell numbers and size with markers of proliferation and hypertrophy. Immunofluorescent analysis of e12.5 hearts with proliferative markers (pH3, Ki67) was not significantly (-5%, p&gt;0.05) altered in KO hearts while cardiomyocyte size differences assessed by immunofluorescence with anti-Troponin C, revealed that KO cardiomyocytes were significantly (-19.4%, p&lt;0.05) smaller than Wt. However, the phospho-to total ratios of AKT1(1.45%, p&gt;0.05) and MTOR1(-12%, p&gt;0.05) were not significantly altered in KO hearts when compared to Wt. SLMAP3 has been implicated in Hippo signaling via the STRIPAK (striatin interacting phosphatase and kinase) complex to impact YAP/TAZ and cell growth. Western blot analysis of e12.5 Wt and KO hearts showed no change in phosphorylation of MST1/2(-6.64%, p&gt;0.05) or YAP-Ser 127(-8.84%, p&gt;0.05) ruling out involvement of Hippo signaling. Interestingly, the expression of the other SLMAP isoforms, SLMAP1 and SLMAP2 remained unaffected during cardiac development in SLMAP3 KO hearts. Thus, SLMAP expression is critical for normal development since the loss of SLMAP3 isoform leads to stunted growth of the embryonic heart. Supported by CIHR.

A conserved STRIPAK complex is required for autophagy in muscle tissue.

Autophagy is important for cellular homeostasis and to prevent the abnormal accumulation of proteins. While many proteins that comprise the canonical autophagy pathway have been characterized, the identification of new regulators may help understand tissue and/or stress-specific responses. Using an in-silico approach, we identified Striatin interacting protein (Strip), MOB kinase activator 4, and fibroblast growth factor receptor 1 oncogene partner 2 as conserved mediators of muscle tissue maintenance. We performed affinity purification-mass spectrometry (AP-MS) experiments with Drosophila melanogaster Strip as a bait protein and copurified additional Striatin-interacting phosphatase and kinase (STRIPAK) complex members from larval muscle tissue. NUAK family kinase 1 (NUAK) and Starvin (Stv) also emerged as Strip-binding proteins and these physical interactions were verified in vivo using proximity ligation assays. To understand the functional significance of the STRIPAK-NUAK-Stv complex, we employed a sensitized genetic assay combined with RNA interference (RNAi) to demonstrate that both NUAK and stv function in the same biological process with genes that encode for STRIPAK complex proteins. RNAi-directed knockdown of Strip in muscle tissue led to the accumulation of ubiquitinated cargo, p62, and Autophagy-related 8a, consistent with a block in autophagy. Indeed, autophagic flux was decreased in Strip RNAi muscles, while lysosome biogenesis and activity were unaffected. Our results support a model whereby the STRIPAK-NUAK-Stv complex coordinately regulates autophagy in muscle tissue.

STRIP2 motivates non-small cell lung cancer progression by modulating the TMBIM6 stability through IGF2BP3 dependent

BackgroundStriatin interacting protein 2 (STRIP2) is a core component of the striatin-interacting phosphatase and kinase (STRIPAK) complexes, which is involved in tumor initiation and progression via the regulation of cell contractile and metastasis. However, the underlying molecular mechanisms of STRIP2 in non-small cell lung cancer (NSCLC) progression remain largely unknown.MethodsThe expressions of STRIP2 and IGF2BP3 in human NSCLC specimens and NSCLC cell lines were detected using quantitative RT-PCR, western blotting, and immunohistochemistry (IHC) analyses. The roles and molecular mechanisms of STRIP2 in promoting NSCLC progression were investigated in vitro and in vivo.ResultsHere, we found that STRIP2 expression was significantly elevated in NSCLC tissues and high STRIP2 expression was associated with a poor prognosis. Knockdown of STRIP2 suppressed tumor growth and metastasis in vitro and in vivo, while STRIP2 overexpression obtained the opposite effect. Mechanistically, P300/CBP-mediated H3K27 acetylation activation in the promoter of STRIP2 induced STRIP2 transcription, which interacted with insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) and upregulated IGF2BP3 transcription. In addition, STRIP2-IGF2BP3 axis stimulated m6A modification of TMBIM6 mRNA and enhanced TMBIM6 stability. Consequently, TMBIM6 involved NSCLC cell proliferation, migration and invasion dependent on STRIP2 and IGF2BP3. In NSCLC patients, high co-expression of STRIP2, IGF2BP3 and TMBIM6 was associated with poor outcomes.ConclusionsOur findings indicate that STRIP2 interacts with IGF2BP3 to regulate TMBIM6 mRNA stability in an m6A-dependent manner and may represent a potential prognostic biomarker and therapeutic target for NSCLC.

PDCD10 promotes the aggressive behaviors of pituitary adenomas by up-regulating CXCR2 and activating downstream AKT/ERK signaling.

As the second most common primary intracranial neoplasms, about 40% of pituitary adenomas (PAs) exhibit aggressive behaviors and resulting in poor patient prognosis. The molecular mechanisms underlying the aggressive behaviors of PAs are not yet fully understood. Biochemical studies have reported that programmed cell death 10 (PDCD10) is a component of the striatin-interacting phosphatase and kinase (STRIPAK) complex and plays a dual role in cancers in a tissue- or disease-specific manner. In the present study, we report for the first time that the role of PDCD10 in PAs. Cell proliferation, migration and invasion were either enhanced by overexpressing or inhibited by silencing PDCD10 in PA cells. Moreover, PDCD10 significantly promoted epithelial–mesenchymal transition (EMT) of pituitary adenoma cells. Mechanistically, we showed that the expression of CXCR2, together with phosphorylation levels of AKT and ERK1/2 were regulated by PDCD10. Activation of CXCR2 inversed inactivation of AKT/ERK signal pathways and the tumor-suppressive effects induced by PDCD10 silencing. Finally, the pro-oncogenic effect of PDCD10 was confirmed by in vivo tumor grafting. Taken together, we demonstrate for the first time that PDCD10 can induce aggressive behaviors of PAs by promoting cellular proliferation, migration, invasion and EMT through CXCR2-AKT/ERK signaling axis.

Combinatorial targeting of Hippo-STRIPAK and PARP elicits synthetic lethality in gastrointestinal cancers.

The striatin-interacting phosphatase and kinase (STRIPAK) complexes integrate extracellular stimuli that result in intracellular activities. Previously, we discovered that STRIPAK is a key machinery responsible for loss of the Hippo tumor suppressor signal in cancer. Here, we identified the Hippo-STRIPAK complex as an essential player in the control of DNA double-stranded break (DSB) repair and genomic stability. Specifically, we found that the mammalian STE20-like protein kinases 1 and 2 (MST1/2), independent of classical Hippo signaling, directly phosphorylated zinc finger MYND type–containing 8 (ZMYND8) and hence resulted in the suppression of DNA repair in the nucleus. In response to genotoxic stress, the cyclic GMP-AMP synthase/stimulator of IFN genes (cGAS/STING) pathway was determined to relay nuclear DNA damage signals to the dynamic assembly of Hippo-STRIPAK via TANK-binding kinase 1–induced (TBK1-induced) structural stabilization of the suppressor of IKBKE 1– sarcolemma membrane–associated protein (SIKE1-SLMAP) arm. As such, we found that STRIPAK-mediated MST1/2 inactivation increased the DSB repair capacity of cancer cells and endowed these cells with resistance to radio- and chemotherapy and poly(ADP-ribose)polymerase (PARP) inhibition. Importantly, targeting the STRIPAK assembly with each of 3 distinct peptide inhibitors efficiently recovered the kinase activity of MST1/2 to suppress DNA repair and resensitize cancer cells to PARP inhibitors in both animal- and patient-derived tumor models. Overall, our findings not only uncover what we believe to be a previously unrecognized role for STRIPAK in modulating DSB repair but also provide translational implications of cotargeting STRIPAK and PARP for a new type of synthetic lethality anticancer therapy.

SIKE’s Phosphorylation Sites Alter Quaternary Structure

Protein‐protein interactions (PPIs) are a crucial part of intracellular communication and function. The “rules” for specificity and/or selection of a protein‐protein interaction are not completely understood, but protein modifications, such as phosphorylation, have been implicated in this process. Suppressor of IKKepsilon (SIKE) is associated with multiple, distinct proteins including TANK‐binding kinase 1 (TBK1), STRIPAK (striatin‐interacting phosphatase and kinase), and cytoskeletal proteins tubulin and actinin. Although SIKE’s function is not fully defined in these complexes, protein modification has been observed in SIKE (phosphorylated at six serine residues: 133, 185, 187, 188, 190, and 198) that may direct PPI formation: Co‐immunoprecipitation revealed that the tubulin interaction was enhanced with the phosphomimetic SIKE (S133/185/187/188/190/198E). We hypothesize that the quaternary state of SIKE, regulated by phosphorylation, dictates SIKE PPI formation. Using the SIKE phosphomimetic mutant, size exclusion chromatography and chemical crosslinking studies showed a monomeric species, whereas native SIKE separated as a dimer. These studies support SIKE undergoing a phosphorylation‐induced change in quaternary state. Prior computational assessment of dimer interface stability with single phosphomimetic substitutions identified S187E, S190E, and S198E as significantly different from WT on a per residue basis. Phosphomimetic mutants for these sites were created and sequence confirmed. In SEC, the phosphomimetic point mutations shift the elution pattern from primarily dimer to containing more monomeric species. To assess SIKE interactions with tubulin, SIKE constructs were labeled using a maleimide‐thiol conjugation to BODIPY TMR. BODIPY‐labeled SIKE, phosphomimetic point mutations, and S6E SIKE interactions with tubulin were monitored using fluorescence polarization. These PPI assays assess the effect of phosphorylation in the selection of SIKE interaction partners. Together, this work advances our understanding of the role that phosphorylation‐induced changes in quaternary state play in regulating protein interactions.

The STRIPAK component SipC is involved in morphology and cell-fate determination in the nematode-trapping fungus Duddingtonia flagrans.

The striatin-interacting phosphatase and kinase (STRIPAK) complex is a highly conserved eukaryotic signaling hub involved in the regulation of many cellular processes. In filamentous fungi, STRIPAK controls multicellular development, hyphal fusion, septation, and pathogenicity. In this study, we analyzed the role of the STRIPAK complex in the nematode-trapping fungus Duddingtonia flagrans which forms three-dimensional, adhesive trapping networks to capture Caenorhabditis elegans. Trap networks consist of several hyphal loops which are morphologically and functionally different from vegetative hyphae. We show that lack of the STRIPAK component SipC (STRIP1/2/HAM-2/PRO22) results in incomplete loop formation and column-like trap structures with elongated compartments. The misshapen or incomplete traps lost their trap identity and continued growth as vegetative hyphae. The same effect was observed in the presence of the actin cytoskeleton drug cytochalasin A. These results could suggest a link between actin and STRIPAK complex functions.

MAP4K4 expression in cardiomyocytes: multiple isoforms, multiple phosphorylations and interactions with striatins.

The Ser/Thr kinase MAP4K4, like other GCKIV kinases, has N-terminal kinase and C-terminal citron homology (CNH) domains. MAP4K4 can activate c-Jun N-terminal kinases (JNKs), and studies in the heart suggest it links oxidative stress to JNKs and heart failure. In other systems, MAP4K4 is regulated in striatin-interacting phosphatase and kinase (STRIPAK) complexes, in which one of three striatins tethers PP2A adjacent to a kinase to keep it dephosphorylated and inactive. Our aim was to understand how MAP4K4 is regulated in cardiomyocytes. The rat MAP4K4 gene was not properly defined. We identified the first coding exon of the rat gene using 5′-RACE, we cloned the full-length sequence and confirmed alternative-splicing of MAP4K4 in rat cardiomyocytes. We identified an additional α-helix C-terminal to the kinase domain important for kinase activity. In further studies, FLAG-MAP4K4 was expressed in HEK293 cells or cardiomyocytes. The Ser/Thr protein phosphatase inhibitor calyculin A (CalA) induced MAP4K4 hyperphosphorylation, with phosphorylation of the activation loop and extensive phosphorylation of the linker between the kinase and CNH domains. This required kinase activity. MAP4K4 associated with myosin in untreated cardiomyocytes, and this was lost with CalA-treatment. FLAG-MAP4K4 associated with all three striatins in cardiomyocytes, indicative of regulation within STRIPAK complexes and consistent with activation by CalA. Computational analysis suggested the interaction was direct and mediated via coiled-coil domains. Surprisingly, FLAG-MAP4K4 inhibited JNK activation by H2O2 in cardiomyocytes and increased myofibrillar organisation. Our data identify MAP4K4 as a STRIPAK-regulated kinase in cardiomyocytes, and suggest it regulates the cytoskeleton rather than activates JNKs.

Assessment by Sequence Conservation and Computational Analyses of SIKE's Phosphorylation Sites to Quaternary Structure Stability

Suppressor of IKKepsilon (SIKE), a protein first identified in the antiviral innate immune response associated with TANK-binding kinase 1 (TBK1), is associated with multiple, distinct protein complexes including TBK1, STRIPAK (striatin interacting phosphatase and kinase) and cytoskeletal proteins, tubulin and actinin. Although SIKE's function is not fully defined in these complexes, SIKE does function as a high-affinity substrate of TBK1 with phosphorylation occurring at up to six SIKE serine residues, S133, S185, S187, S188, S190, and S198. The accompanying figure shows a cartoon diagram of SIKE dimer model with the phosphorylation sites shown in spheres (red 187, 190; orange 133; 187 green; 188 and 190 cyan). Using a phosphomimetic mutant (S133/185/187/188/190/198E) of SIKE, size exclusion chromatography and chemical crosslinking studies showed primarily a monomeric species, whereas unmodified SIKE separated as a dimeric species. These observations suggested that SIKE could undergo a phosphorylation-induced change in quaternary structure, but how many of the six phosphorylation sites were necessary? To explore this idea, conservation of the phosphorylation sites was explored. 133 SIKE sequences (Animalia, Fungi) from OrthoDB and Protein Blast were compiled and aligned using Clustal Omega. Positions 187 and 190 were conserved as serine in >90% of sequences whereas position 133 was ~60% serine and 25% aspartate or glutamate with a 9:10 bias towards aspartate. The remaining positions had 75% or less retaining serine with position 188 registering ~32% lysine. Using a model of the SIKE dimer where the phosphorylation sites line part of the dimer interface, phosphorylated S187/190 in each subunit would be positioned to repulse one another. To gain further insight into the role of individual phosphorylation sites on the theoretical dimer interface, phosphomimetic mutants at each phosphorylation site were computationally created (PyMOL), energy minimized (GalaxyRefineComplex) to yield 10 models, and the interface stability of each model for each mutant assessed on a per residue basis (HawkDock MM/GBSA). These mutant data were then compared to a similar analysis of the wild type SIKE model to identify residues with significant changes in total free energy of binding (dimer interface). S133E showed no significant changes from WT whereas S185/187/188/190/198E individually revealed a similar pattern of residues with altered total free energy of binding adjacent to the phosphorylation sites that differed in the number (19-30) of residues affected in the region. The sequence conservation suggested that positions 187 and 190 could serve as key phosphorylation sites to trigger a dimer to monomer transition. Although the computational analyses has not resolved the effect of multiple phosphorylation events, the primary alteration to dimer stability is localized to the regions adjacent to the phosphorylation sites.

Characterization of Strip1 Expression in Mouse Cochlear Hair Cells.

Striatin-interacting protein 1 (Strip1) is a core component of the striatin interacting phosphatase and kinase (STRIPAK) complex, which is involved in embryogenesis and development, circadian rhythms, type 2 diabetes, and cancer progression. However, the expression and role of Strip1 in the mammalian cochlea remains unclear. Here we studied the expression and function of Strip1 in the mouse cochlea by using Strip1 knockout mice. We first found that the mRNA and protein expression of Strip1 increases as mice age starting from postnatal day (P) 3 and reaches its highest expression level at P30 and that the expression of Strip1 can be detected by immunofluorescent staining starting from P14 only in cochlear HCs, and not in supporting cells (SCs). Next, we crossed Strip1 heterozygous knockout (Strip +/−) mice to obtain Strip1 homozygous knockout (Strip1−/−) mice for studying the role of Strip1 in cochlear HCs. However, no Strip1−/− mice were obtained and the ratio of Strip +/− to Strip1+/+ mice per litter was about 2:1, which suggested that homozygous Strip1 knockout is embryonic lethal. We measured hearing function and counted the HC number in P30 and P60 Strip +/− mice and found that they had normal hearing ability and HC numbers compared to Strip1+/+ mice. Our study suggested that Strip1 probably play important roles in HC development and maturation, which needs further study in the future.

Pack a STRIPAK with hubs inside a hub

The highly conserved striatin-interacting phosphatase and kinase (STRIPAK) multimeric complex regulates the Hippo signaling pathway through phosphatase activity. A recent structure of the core STRIPAK hub reveals how striatins tetramerize to serve as a scaffolding platform for the assembly of an intricate architecture, which is distinct from that of all other protein phosphatase 2A (PP2A) complexes.

Cryo-EM structure of the Hippo signaling integrator human STRIPAK.

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.

MEKK2 and MEKK3 orchestrate multiple signals to regulate Hippo pathway

The Hippo pathway is an evolutionarily conserved signaling pathway that controls organ size in animals via the regulation of cell proliferation and apoptosis. It consists of a kinase cascade, in which MST1/2 and MAP4Ks phosphorylate and activate LATS1/2, which in turn phosphorylate and inhibit YAP/TAZ activity. A variety of signals can modulate LATS1/2 kinase activity to regulate Hippo pathway. However, the full mechanistic details of kinase-mediated regulation of Hippo pathway signaling remain elusive. Here, we report that TNF activates LATS1/2 and inhibits YAP/TAZ activity through MEKK2/3. Furthermore, MEKK2/3 act in parallel to MST1/2 and MAP4Ks to regulate LATS1/2 and YAP/TAZ in response to various signals, such as serum and actin dynamics. Mechanistically, we show that MEKK2/3 interact with LATS1/2 and YAP/TAZ and phosphorylate them. In addition, Striatin-interacting phosphatase and kinase (STRIPAK) complex associates with MEKK3 via CCM2 and CCM3 to inactivate MEKK3 kinase activity. Upstream signals of Hippo pathway trigger the dissociation of MEKK3 from STRIPAK complex to release MEKK3 activity. Our work has uncovered a previous unrecognized regulation of Hippo pathway via MEKK2/3 and provides new insights into molecular mechanisms for the interplay between Hippo-YAP and NF-κB signaling and the pathogenesis of cerebral cavernous malformations.

MBRS-39. MAP4K4 CONTROLS PRO-INVASIVE SIGNALING IN MEDULLOBLASTOMA CELLS

The molecular mechanisms contributing to distant dissemination and local recurrence of medulloblastoma, the most common malignant brain tumor in childhood, are poorly understood and no targeted anti-invasion therapies exist till date. We explored regulators and effectors of MAP4K4, a pro-invasive kinase overexpressed in MB and associated with metastatic progression in different solid malignancies. MAP4K4 is upregulated both at mRNA and protein levels in primary pediatric brain tumors compared to normal cerebellum. MAP4K4 is required for growth factor- and irradiation-induced migration and invasion of medulloblastoma cells. It furthermore promotes turnover and activation of the receptor tyrosine kinase c-Met and of the ß1 integrin adhesion receptor 1. To characterize these clinically relevant consequences and to identify druggable targets of MAP4K4 function, we profiled the interactome of MAP4K4 in starved and growth factor stimulated medulloblastoma cells. To systematically address MAP4K4 impact on receptor expression and turnover, we determined the MAP4K4-dependent surface proteome in medulloblastoma cells. We found that MAP4K4 is part of the striatin-interacting phosphatase and kinase (STRIPAK) complex and that STRIPAK component striatin 4 is controlling cell motility and invasiveness in medulloblastoma cells. Invasiveness of medulloblastoma cells is abrogated by a truncation mutant of MAP4K4 lacking the striatin 4 interaction domain. We furthermore found that MAP4K4 mediates growth factor-induced surface expression of solute carriers and immunomodulatory proteins involved in chemoresistance and immune evasion. Thus, our study identified MAP4K4 as a missing link between pro-tumorigenic growth factor signaling and tumor cell functions relevant for disease progression. It may help identifying druggable vulnerabilities in medulloblastoma cells to restrict tumor growth and dissemination. 1. Tripolitsioti, D. et al., Oncotarget9, 23220–23236 (2018).

Abstract 15577: Mst 4, a Novel Stripak Associated Kinase is Upregulated in Human Cardiomyopathy and Regulates Cardiomyocyte Hypertrophy, Contractility and Apoptosis

Heart failure is one of the major causes of death worldwide. Its pathophysiology is complex and involves alterations in calcium cycling and hypertrophic signaling, as well as subsequent loss of cardiomyocytes due to apoptosis, cardiac fibrosis and progressive contractile dysfunction. The Striatin-interacting phosphatase and kinase (STRIPAK) complex is a mammalian multiprotein complex that consists of phosphatases, kinases and various adaptor proteins. Yet, the STRIPAK complex has so far not been studied in cardiomyocytes. Now we show in cardiomyocytes that the STRIPAK associated kinase MST4 directly interacts with a recently described novel cardiac STRIPAK protein, Myoscape/STRIP2. Multi tissue immunoblot experiments showed that Mst4 abundance is also highly enriched in heart and skeletal muscle. Immunoblot analyses in human biopsy samples form patients with dilated (DCM n=10) or ischemic cardiomyopathy (ICM n=10) revealed that MST4 is strongly upregulated in DCM (12-fold p&lt;0.001) and ICM (9.6 fold p&lt;0.05) vs. controls. Conversely, upon biomechanical stretch we observed a significant downregulation of MST4 in vitro by 30% (p&lt;0.05) as well as in vivo (-45%, n=6; P&lt;0.001) in a murine model for pressure overload (experimental aortic constriction, (TAC) vs. Sham) under Moreover, we also observed downregulation of MST4 in a porcine myocardial infarction model (-48% vs. porcine controls; n=5 p&lt;0.05). Adenoviral overexpression of MST4 in NRVCM results in significant cellular hypertrophy (485μm2 vs 421μm2; p&lt;0.001) compared to LacZ. Overexpression of MST4 also increases cellular and sarcomeric fractional shortening in adult rat cardiac myocytes vs. LacZ (p&lt;0.05). Finally, MST4 overexpression also protects from cardiac apoptosis as shown by 90% reduced PARP cleavage as well as significantly reduced expression of Caspase 3 and 7 (n=3; p&lt;0.05). Taken together we show that MST 4 is a cardiac enriched Kinase that regulates cardiomyocyte hypertrophy, contractility and apoptosis and may thus be a target for future therapeutic intervention

The STRIPAK signaling complex regulates dephosphorylation of GUL1, an RNA-binding protein that shuttles on endosomes.

The striatin-interacting phosphatase and kinase (STRIPAK) multi-subunit signaling complex is highly conserved within eukaryotes. In fungi, STRIPAK controls multicellular development, morphogenesis, pathogenicity, and cell-cell recognition, while in humans, certain diseases are related to this signaling complex. To date, phosphorylation and dephosphorylation targets of STRIPAK are still widely unknown in microbial as well as animal systems. Here, we provide an extended global proteome and phosphoproteome study using the wild type as well as STRIPAK single and double deletion mutants (Δpro11, Δpro11Δpro22, Δpp2Ac1Δpro22) from the filamentous fungus Sordaria macrospora. Notably, in the deletion mutants, we identified the differential phosphorylation of 129 proteins, of which 70 phosphorylation sites were previously unknown. Included in the list of STRIPAK targets are eight proteins with RNA recognition motifs (RRMs) including GUL1. Knockout mutants and complemented transformants clearly show that GUL1 affects hyphal growth and sexual development. To assess the role of GUL1 phosphorylation on fungal development, we constructed phospho-mimetic and -deficient mutants of GUL1 residues. While S180 was dephosphorylated in a STRIPAK-dependent manner, S216, and S1343 served as non-regulated phosphorylation sites. While the S1343 mutants were indistinguishable from wild type, phospho-deficiency of S180 and S216 resulted in a drastic reduction in hyphal growth, and phospho-deficiency of S216 also affects sexual fertility. These results thus suggest that differential phosphorylation of GUL1 regulates developmental processes such as fruiting body maturation and hyphal morphogenesis. Moreover, genetic interaction studies provide strong evidence that GUL1 is not an integral subunit of STRIPAK. Finally, fluorescence microscopy revealed that GUL1 co-localizes with endosomal marker proteins and shuttles on endosomes. Here, we provide a new mechanistic model that explains how STRIPAK-dependent and -independent phosphorylation of GUL1 regulates sexual development and asexual growth.