Selective 14-3-3γ induction quenches p-β-catenin Ser37/Bax-enhanced cell death in cerebral cortical neurons during ischemia.
Lai X J,Ye S Q,Zheng L,Li L,Liu Q R,Yu S B,Pang Y,Jin S,Li Q,Yu A C H,Chen X Q
Cell death & disease
Ischemia-induced cell death is a major cause of disability or death after stroke. Identifying the key intrinsic protective mechanisms induced by ischemia is critical for the development of effective stroke treatment. Here, we reported that 14-3-3γ was a selective ischemia-inducible survival factor in cerebral cortical neurons reducing cell death by downregulating Bax depend direct 14-3-3γ/p-β-catenin Ser37 interactions in the nucleus. 14-3-3γ, but not other 14-3-3 isoforms, was upregulated in primary cerebral cortical neurons upon oxygen-glucose deprivation (OGD) as measured by quantitative PCR, western blot and fluorescent immunostaining. The selective induction of 14-3-3γ in cortical neurons by OGD was verified by the in vivo ischemic stroke model. Knocking down 14-3-3γ alone or inhibiting 14-3-3/client interactions was sufficient to induce cell death in normal cultured neurons and exacerbate OGD-induced neuronal death. Ectopic overexpression of 14-3-3γ significantly reduced OGD-induced cell death in cultured neurons. Co-immunoprecipitation and fluorescence resonance energy transfer demonstrated that endogenous 14-3-3γ bound directly to more p-β-catenin Ser37 but not p-Bad, p-Ask-1, p-p53 and Bax. During OGD, p-β-catenin Ser37 but not p-β-catenin Ser45 was increased prominently, which correlated with Bax elevation in cortical neurons. OGD promoted the entry of 14-3-3γ into the nuclei, in correlation with the increase of nuclear p-β-catenin Ser37 in neurons. Overexpression of 14-3-3γ significantly reduced Bax expression, whereas knockdown of 14-3-3γ increased Bax in cortical neurons. Abolishing β-catenin phosphorylation at Ser37 (S37A) significantly reduced Bax and cell death in neurons upon OGD. Finally, 14-3-3γ overexpression completely suppressed β-catenin-enhanced Bax and cell death in neurons upon OGD. Based on these data, we propose that the 14-3-3γ/p-β-catenin Ser37/Bax axis determines cell survival or death of neurons during ischemia, providing novel therapeutic targets for ischemic stroke as well as other related neurological diseases.
Canonical Wnt signalling activates TAZ through PP1A during osteogenic differentiation.
Byun M R,Hwang J-H,Kim A R,Kim K M,Hwang E S,Yaffe M B,Hong J-H
Cell death and differentiation
TAZ, a transcriptional modulator, has a key role in cell proliferation, differentiation and stem cell self-renewal. TAZ activity is regulated by several signalling pathways, including Hippo, GPCR and Wnt signalling, but the regulatory mechanisms of TAZ activation are not yet clearly understood. In this report, we show that TAZ is regulated by canonical Wnt signalling during osteogenic differentiation. Wnt3a increases TAZ expression and an inhibitor of GSK3β, a downstream effector of Wnt signalling, induces TAZ. Wnt3a facilitates the dephosphorylation of TAZ, which stabilises TAZ and prevents it from binding 14-3-3 proteins, thus inducing the nuclear localisation of TAZ. Dephosphorylation of TAZ occurs via PP1A, and depletion of PP1A blocks Wnt3a-induced TAZ stabilisation. Wnt3a-induced TAZ activates osteoblastic differentiation and siRNA-induced TAZ depletion decreases Wnt3a-induced osteoblast differentiation. Taken together, these results show that TAZ mediates Wnt3a-stimulated osteogenic differentiation through PP1A, suggesting that the Wnt signal regulates the Hippo pathway.
High-Throughput Image-Based Screening to Identify Chemical Compounds Capable of Activating FOXO.
Barradas Marta,Link Wolfgang,Megias Diego,Fernandez-Marcos Pablo J
Methods in molecular biology (Clifton, N.J.)
FOXO proteins are transcription factors with important roles in the regulation of the expression of genes involved in cell growth, proliferation, differentiation, and longevity. FOXO proteins are active in the nucleus but, upon post-translational modification they form a docking site for 14-3-3 proteins and are translocated to the cytoplasm where they are inactive.We make use of this regulatory mechanism of FOXO proteins to develop an image-based high-throughput screening platform to detect compounds that regulate FOXO3 subcellular localization. This system has proven a powerful tool to isolate inhibitors of proteins upstream of FOXO, such as PI3K inhibitors.
14-3-3ε acts as a proviral factor in highly pathogenic porcine reproductive and respiratory syndrome virus infection.
Cao Shengliang,Cong Fangyuan,Tan Min,Ding Guofei,Liu Jiaqi,Li Li,Zhao Yuzhong,Liu Sidang,Xiao Yihong
The highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) emerged in 2006 in China and caused great economic losses for the swine industry because of the lack of an effective vaccine. 14-3-3 proteins are generating significant interest as potential drug targets by allowing the targeting of specific pathways to elicit therapeutic effects in human diseases. In a previous study, 14-3-3s were identified to interact with non-structural protein 2 (NSP2) of PRRSV. In the present study, the specific subtype 14-3-3ε was confirmed to interact with NSP2 and play a role in the replication of the HP-PRRSV TA-12 strain. Knockdown of 14-3-3ε in Marc-145 cells and porcine alveolar macrophages (PAMs) caused a significant decrease in TA-12 replication, while stable overexpression of 14-3-3ε caused a significant increase in the replication of TA-12 and low pathogenic PRRSV (LP-PRRSV) CH-1R. The 14-3-3 inhibitor difopein also decreased TA-12 and CH-1R replication in Marc-145 cells and PAMs. These findings are consistent with 14-3-3ε acting as a proviral factor and suggest that 14-3-3ε siRNA and difopein are therapeutic candidates against PRRSV infection.
AKT and 14-3-3 regulate Notch4 nuclear localization.
Ramakrishnan Gopalakrishnan,Davaakhuu Gantulga,Chung Wen Cheng,Zhu He,Rana Ajay,Filipovic Aleksandra,Green Andrew R,Atfi Azeddine,Pannuti Antonio,Miele Lucio,Tzivion Guri
Members of the Notch family of transmembrane receptors, Notch1-4 in mammals, are involved in the regulation of cell fate decisions and cell proliferation in various organisms. The Notch4 isoform, which is specific to mammals, was originally identified as a viral oncogene in mice, Int3, able to initiate mammary tumors. In humans, Notch4 expression appears to be associated with breast cancer stem cells and endocrine resistance. Following ligand binding, the Notch4 receptor undergoes cleavage at the membrane and the Notch4-intracellular domain (ICD), translocates to the nucleus and regulates gene transcription. Little is known on the mechanisms regulating Notch4-ICD and its nuclear localization. Here, we describe the identification of four distinct AKT phosphorylation sites in human Notch4-ICD and demonstrate that AKT binds Notch4-ICD and phosphorylates all four sites in vitro and in vivo. The phosphorylation in cells is regulated by growth factors and is sensitive to phosphatidyl inositol-3 kinase (PI3K) inhibitors. This phosphorylation generates binding sites to the 14-3-3 regulatory proteins, which are involved in the regulation of nucleocytoplasmic shuttling of target proteins, restricting phosphorylated Notch4-ICD to the cytoplasm. Our findings provide a novel mechanism for Notch4-ICD regulation, suggesting a negative regulatory role for the PI3K-AKT pathway in Notch4 nuclear signaling.
14-3-3σ regulates keratinocyte proliferation and differentiation by modulating Yap1 cellular localization.
Sambandam Sumitha A T,Kasetti Ramesh B,Xue Lei,Dean Douglas C,Lu Qingxian,Li Qiutang
The Journal of investigative dermatology
The homozygous repeated epilation (Er/Er) mouse mutant of the gene encoding 14-3-3σ displays an epidermal phenotype characterized by hyperproliferative keratinocytes and undifferentiated epidermis. Heterozygous Er/+ mice develop spontaneous skin tumors and are highly sensitive to tumor-promoting 7,12-dimethylbenzanthracene/12-O-tetradecanoyl-phorbol-13-acetate induction. The molecular mechanisms underlying 14-3-3σ regulation of epidermal proliferation, differentiation, and tumor formation have not been well elucidated. In this study, we found that Er/Er keratinocytes failed to sequester Yap1 in the cytoplasm, leading to its nuclear localization during epidermal development in vivo and under differentiation-inducing culture conditions in vitro. In addition, enhanced Yap1 nuclear localization was also evident in 7,12-dimethylbenzanthracene/12-O-tetradecanoyl-phorbol-13-acetate-induced tumors from Er/+ skin. Furthermore, short hairpin RNA (shRNA) knockdown of Yap1 expression in Er/Er keratinocytes inhibited their proliferation, suggesting that YAP1 functions as a downstream effector of 14-3-3σ controlling epidermal proliferation. We then demonstrated that keratinocytes express all seven 14-3-3 protein isoforms, some of which form heterodimers with 14-3-3σ, either full-length wild type (WT) or the mutant form found in Er/Er mice. However, Er 14-3-3σ does not interact with Yap1, as demonstrated by coimmunoprecipitation. We conclude that Er 14-3-3σ disrupts the interaction between 14-3-3 and Yap1, and thus fails to block Yap1 nuclear transcriptional function, causing continued progenitor expansion and inhibition of differentiation in the Er/Er epidermis.
Homeostatic control of Hippo signaling activity revealed by an endogenous activating mutation in YAP.
Chen Qian,Zhang Nailing,Xie Rui,Wang Wei,Cai Jing,Choi Kyung-Suk,David Karen Kate,Huang Bo,Yabuta Norikazu,Nojima Hiroshi,Anders Robert A,Pan Duojia
Genes & development
The Hippo signaling pathway converges on YAP to regulate growth, differentiation, and regeneration. Previous studies with overexpressed proteins have shown that YAP is phosphorylated by its upstream kinase, Lats1/2, on multiple sites, including an evolutionarily conserved 14-3-3-binding site whose phosphorylation is believed to inhibit YAP by excluding it from the nucleus. Indeed, nuclear localization of YAP or decreased YAP phosphorylation at this site (S168 in Drosophila, S127 in humans, and S112 in mice) is widely used in current literature as a surrogate of YAP activation even though the physiological importance of this phosphorylation event in regulating endogenous YAP activity has not been defined. Here we address this question by introducing a Yap(S112A) knock-in mutation in the endogenous Yap locus. The Yap(S112A) mice are surprisingly normal despite nuclear localization of the mutant YAP protein in vivo and profound defects in cytoplasmic translocation in vitro. Interestingly, the mutant Yap(S112A) mice show a compensatory decrease in YAP protein levels due to increased phosphorylation at a mammalian-specific phosphodegron site on YAP. These findings reveal a robust homeostatic mechanism that maintains physiological levels of YAP activity and caution against the assumptive use of YAP localization alone as a surrogate of YAP activity.
Genetic Analysis Reveals a Longevity-Associated Protein Modulating Endothelial Function and Angiogenesis.
Villa Francesco,Carrizzo Albino,Spinelli Chiara C,Ferrario Anna,Malovini Alberto,Maciąg Anna,Damato Antonio,Auricchio Alberto,Spinetti Gaia,Sangalli Elena,Dang Zexu,Madonna Michele,Ambrosio Mariateresa,Sitia Leopoldo,Bigini Paolo,Calì Gaetano,Schreiber Stefan,Perls Thomas,Fucile Sergio,Mulas Francesca,Nebel Almut,Bellazzi Riccardo,Madeddu Paolo,Vecchione Carmine,Puca Annibale A
RATIONALE:Long living individuals show delay of aging, which is characterized by the progressive loss of cardiovascular homeostasis, along with reduced endothelial nitric oxide synthase activity, endothelial dysfunction, and impairment of tissue repair after ischemic injury. OBJECTIVE:Exploit genetic analysis of long living individuals to reveal master molecular regulators of physiological aging and new targets for treatment of cardiovascular disease. METHODS AND RESULTS:We show that the polymorphic variant rs2070325 (Ile229Val) in bactericidal/permeability-increasing fold-containing-family-B-member-4 (BPIFB4) associates with exceptional longevity, under a recessive genetic model, in 3 independent populations. Moreover, the expression of BPIFB4 is instrumental to maintenance of cellular and vascular homeostasis through regulation of protein synthesis. BPIFB4 phosphorylation/activation by protein-kinase-R-like endoplasmic reticulum kinase induces its complexing with 14-3-3 and heat shock protein 90, which is facilitated by the longevity-associated variant. In isolated vessels, BPIFB4 is upregulated by mechanical stress, and its knock-down inhibits endothelium-dependent vasorelaxation. In hypertensive rats and old mice, gene transfer of longevity-associated variant-BPIFB4 restores endothelial nitric oxide synthase signaling, rescues endothelial dysfunction, and reduces blood pressure levels. Furthermore, BPIFB4 is implicated in vascular repair. BPIFB4 is abundantly expressed in circulating CD34(+) cells of long living individuals, and its knock-down in endothelial progenitor cells precludes their capacity to migrate toward the chemoattractant SDF-1. In a murine model of peripheral ischemia, systemic gene therapy with longevity-associated variant-BPIFB4 promotes the recruitment of hematopoietic stem cells, reparative vascularization, and reperfusion of the ischemic muscle. CONCLUSIONS:Longevity-associated variant-BPIFB4 may represent a novel therapeutic tool to fight endothelial dysfunction and promote vascular reparative processes.
Protein kinase Cδ regulates nuclear export of FOXO1 through phosphorylation of the chaperone 14-3-3ζ.
Gerst Felicia,Kaiser Gabriele,Panse Madhura,Sartorius Tina,Pujol Anna,Hennige Anita M,Machicao Fausto,Lammers Reiner,Bosch Fatima,Häring Hans-Ulrich,Ullrich Susanne
AIMS/HYPOTHESIS:Forkhead box protein O1 (FOXO1) is a transcription factor essential for beta cell fate. Protein kinase B-dependent phosphorylation of FOXO1 at S256 (P-FOXO1) enables its binding to 14-3-3 dimers and nuclear export. Dephosphorylated FOXO1 enters nuclei and activates pro-apoptotic genes. Since our previous observations suggest that protein kinase C delta (PKCδ) induces nuclear accumulation of FOXO1, the underlying mechanism was examined. METHODS:In human islets, genetically modified mice and INS-1E cells apoptosis was assessed by TUNEL staining. Subcellular translocation of proteins was examined by confocal microscopy and signalling pathways were analysed by western blotting and overlay assay. RESULTS:In PKCδ-overexpressing (PKCδ-tg) mouse islet cells and INS-1E cells FOXO1 accumulated in nuclei, surprisingly, as P-FOXO1. PKCδ-tg decelerated IGF-1-dependent stimulation of nuclear export, indicating that changes in export caused nuclear retention of P-FOXO1. Nuclear accumulation of P-FOXO1 was accompanied by increased phosphorylation of 14-3-3ζ at S58 and reduced dimerisation of 14-3-3ζ. Palmitic acid further augmented phosphorylation of 14-3-3ζ and triggered nuclear accumulation of FOXO1 in both INS-1E and human islet cells. Furthermore, the overexpression of a phosphomimicking mutant of 14-3-3ζ (S58D) enhanced nuclear FOXO1. In accordance with the nuclear accumulation of P-FOXO1, PKCδ overexpression alone did not increase apoptotic cell death. Additionally, insulin secretion and glucose homeostasis in PKCδ-overexpressing mice remained unaffected. CONCLUSIONS/INTERPRETATION:These results suggest that PKCδ-mediated phosphorylation of 14-3-3ζ contributes to the nuclear retention of FOXO1, even when FOXO1 is phosphorylated as under non-stress conditions. P-FOXO1 does not induce pro-apoptotic genes, but may rather exert beneficial effects on beta cells.
VE-cadherin and endothelial adherens junctions: active guardians of vascular integrity.
Giannotta Monica,Trani Marianna,Dejana Elisabetta
VE-cadherin is a component of endothelial cell-to-cell adherens junctions, and it has a key role in the maintenance of vascular integrity. During embryo development, VE-cadherin is required for the organization of a stable vascular system, and in the adult it controls vascular permeability and inhibits unrestrained vascular growth. The mechanisms of action of VE-cadherin are complex and include reshaping and organization of the endothelial cell cytoskeleton and modulation of gene transcription. Here we review some of the most important pathways through which VE-cadherin modulates vascular homeostasis and discuss the emerging concepts in the overall biological role of this protein.
Human mesenchymal stem cells inhibit vascular permeability by modulating vascular endothelial cadherin/β-catenin signaling.
Pati Shibani,Khakoo Aarif Y,Zhao Jing,Jimenez Fernando,Gerber Michael H,Harting Matthew,Redell John B,Grill Raymond,Matsuo Yoichi,Guha Sushovan,Cox Charles S,Reitz Marvin S,Holcomb John B,Dash Pramod K
Stem cells and development
The barrier formed by endothelial cells (ECs) plays an important role in tissue homeostasis by restricting passage of circulating molecules and inflammatory cells. Disruption of the endothelial barrier in pathologic conditions often leads to uncontrolled inflammation and tissue damage. An important component of this barrier is adherens junctions, which restrict paracellular permeability. The transmembrane protein vascular endothelial (VE)-cadherin and its cytoplasmic binding partner β-catenin are major components of functional adherens junctions. We show that mesenchymal stem cells (MSCs) significantly reduce endothelial permeability in cocultured human umbilical vascular endothelial cells (HUVECs) and following exposure to vascular endothelial growth factor, a potent barrier permeability-enhancing agent. When grown in cocultures with HUVECs, MSCs increased VE-cadherin levels and enhanced recruitment of both VE-cadherin and β-catenin to the plasma membrane. Enhanced membrane localization of β-catenin was associated with a decrease in β-catenin-driven gene transcription. Disruption of the VE-cadherin/β-catenin interaction by overexpressing a truncated VE-cadherin lacking the β-catenin interacting domain blocked the permeability-stabilizing effect of MSCs. Interestingly, a conditioned medium from HUVEC-MSC cocultures, but not from HUVEC or MSC cells cultured alone, significantly reduced endothelial permeability. In addition, intravenous administration of MSCs to brain-injured rodents reduced injury-induced enhanced blood-brain barrier permeability. Similar to the effect on in vitro cultures, this stabilizing effect on blood-brain barrier function was associated with increased expression of VE-cadherin. Taken together, these results identify a putative mechanism by which MSCs can modulate vascular EC permeability. Further, our results suggest that the mediator(s) of these vascular protective effects is a secreted factor(s) released as a result of direct MSC-EC interaction.
YAP and TAZ regulate adherens junction dynamics and endothelial cell distribution during vascular development.
Neto Filipa,Klaus-Bergmann Alexandra,Ong Yu Ting,Alt Silvanus,Vion Anne-Clémence,Szymborska Anna,Carvalho Joana R,Hollfinger Irene,Bartels-Klein Eireen,Franco Claudio A,Potente Michael,Gerhardt Holger
Formation of blood vessel networks by sprouting angiogenesis is critical for tissue growth, homeostasis and regeneration. How endothelial cells arise in adequate numbers and arrange suitably to shape functional vascular networks is poorly understood. Here we show that YAP/TAZ promote stretch-induced proliferation and rearrangements of endothelial cells whilst preventing bleeding in developing vessels. Mechanistically, YAP/TAZ increase the turnover of VE-Cadherin and the formation of junction associated intermediate lamellipodia, promoting both cell migration and barrier function maintenance. This is achieved in part by lowering BMP signalling. Consequently, the loss of YAP/TAZ in the mouse leads to stunted sprouting with local aggregation as well as scarcity of endothelial cells, branching irregularities and junction defects. Forced nuclear activity of TAZ instead drives hypersprouting and vascular hyperplasia. We propose a new model in which YAP/TAZ integrate mechanical signals with BMP signaling to maintain junctional compliance and integrity whilst balancing endothelial cell rearrangements in angiogenic vessels.
The actin-binding protein EPS8 binds VE-cadherin and modulates YAP localization and signaling.
Giampietro Costanza,Disanza Andrea,Bravi Luca,Barrios-Rodiles Miriam,Corada Monica,Frittoli Emanuela,Savorani Cecilia,Lampugnani Maria Grazia,Boggetti Barbara,Niessen Carien,Wrana Jeff L,Scita Giorgio,Dejana Elisabetta
The Journal of cell biology
Vascular endothelial (VE)-cadherin transfers intracellular signals contributing to vascular hemostasis. Signaling through VE-cadherin requires association and activity of different intracellular partners. Yes-associated protein (YAP)/TAZ transcriptional cofactors are important regulators of cell growth and organ size. We show that EPS8, a signaling adapter regulating actin dynamics, is a novel partner of VE-cadherin and is able to modulate YAP activity. By biochemical and imaging approaches, we demonstrate that EPS8 associates with the VE-cadherin complex of remodeling junctions promoting YAP translocation to the nucleus and transcriptional activation. Conversely, in stabilized junctions, 14-3-3-YAP associates with the VE-cadherin complex, whereas Eps8 is excluded. Junctional association of YAP inhibits nuclear translocation and inactivates its transcriptional activity both in vitro and in vivo in Eps8-null mice. The absence of Eps8 also increases vascular permeability in vivo, but did not induce other major vascular defects. Collectively, we identified novel components of the adherens junction complex, and we introduce a novel molecular mechanism through which the VE-cadherin complex controls YAP transcriptional activity.
A dual phosphorylation switch controls 14-3-3-dependent cell surface expression of TASK-1.
Kilisch Markus,Lytovchenko Olga,Arakel Eric C,Bertinetti Daniela,Schwappach Blanche
Journal of cell science
The transport of the K(+) channels TASK-1 and TASK-3 (also known as KCNK3 and KCNK9, respectively) to the cell surface is controlled by the binding of 14-3-3 proteins to a trafficking control region at the extreme C-terminus of the channels. The current model proposes that phosphorylation-dependent binding of 14-3-3 sterically masks a COPI-binding motif. However, the direct effects of phosphorylation on COPI binding and on the binding parameters of 14-3-3 isoforms are still unknown. We find that phosphorylation of the trafficking control region prevents COPI binding even in the absence of 14-3-3, and we present a quantitative analysis of the binding of all human 14-3-3 isoforms to the trafficking control regions of TASK-1 and TASK-3. Surprisingly, the affinities of 14-3-3 proteins for TASK-1 are two orders of magnitude lower than for TASK-3. Furthermore, we find that phosphorylation of a second serine residue in the C-terminus of TASK-1 inhibits 14-3-3 binding. Thus, phosphorylation of the trafficking control region can stimulate or inhibit transport of TASK-1 to the cell surface depending on the target serine residue. Our findings indicate that control of TASK-1 trafficking by COPI, kinases, phosphatases and 14-3-3 proteins is highly dynamic.
14-3-3 Proteins regulate Akt Thr308 phosphorylation in intestinal epithelial cells.
Gómez-Suárez M,Gutiérrez-Martínez I Z,Hernández-Trejo J A,Hernández-Ruiz M,Suárez-Pérez D,Candelario A,Kamekura R,Medina-Contreras O,Schnoor M,Ortiz-Navarrete V,Villegas-Sepúlveda N,Parkos C,Nusrat A,Nava P
Cell death and differentiation
Akt activation has been associated with proliferation, differentiation, survival and death of epithelial cells. Phosphorylation of Thr308 of Akt by phosphoinositide-dependent kinase 1 (PDK1) is critical for optimal stimulation of its kinase activity. However, the mechanism(s) regulating this process remain elusive. Here, we report that 14-3-3 proteins control Akt Thr308 phosphorylation during intestinal inflammation. Mechanistically, we found that IFNγ and TNFα treatment induce degradation of the PDK1 inhibitor, 14-3-3η, in intestinal epithelial cells. This mechanism requires association of 14-3-3ζ with raptor in a process that triggers autophagy and leads to 14-3-3η degradation. Notably, inhibition of 14-3-3 function by the chemical inhibitor BV02 induces uncontrolled Akt activation, nuclear Akt accumulation and ultimately intestinal epithelial cell death. Our results suggest that 14-3-3 proteins control Akt activation and regulate its biological functions, thereby providing a new mechanistic link between cell survival and apoptosis of intestinal epithelial cells during inflammation.
Mitochondria-Mediated Protein Regulation Mechanism of Polymorphs-Dependent Inhibition of Nanoselenium on Cancer Cells.
Wang Ge,Guo Yuming,Yang Gai,Yang Lin,Ma Xiaoming,Wang Kui,Zhu Lin,Sun Jiaojiao,Wang Xiaobing,Zhang Hua
The present study was (i) to prepare two types of selenium nanoparticles, namely an amorphous form of selenium quantum dots (A-SeQDs) and a crystalline form of selenium quantum dots (C-SeQDs); and (ii) to investigate the nano-bio interactions of A-SeQDs and C-SeQDs in MCF-7, HepG2, HeLa, NIH/3T3, L929 cells and BRL-3A cells. It was found that A-SeQDs could induce the mitochondria-mediated apoptosis, necrosis and death of cells, while C-SeQDs had much weaker effects. This polymorphs-dependent anti-proliferative activity of nano-selenium was scarcely reported. Further investigation demonstrated that A-SeQDs could differentially regulate 61 proteins and several pathways related to stress response, protein synthesis, cell migration and cell cycle, including "p38 MAPK Signaling", "p53 Signaling", "14-3-3-mediated Signaling", "p70S6K Signaling" and "Protein Ubiquitination Pathway". This was the first report to demonstrate the involvement of protein synthesis and post-translational modification pathways in the anti-proliferative activity associated with NMs. Compared with previously fragmentary studies, this study use a nanomics approach combining bioinformatics and proteomics to systematically investigate the nano-bio interactions of selenium nanoparticles in cancer cells.
Protein kinase B (AKT) regulates SYK activity and shuttling through 14-3-3 and importin 7.
Mohammad Dara K,Nore Beston F,Gustafsson Manuela O,Mohamed Abdalla J,Smith C I Edvard
The international journal of biochemistry & cell biology
The Protein kinase B (AKT) regulates a plethora of intracellular signaling proteins to fine-tune signaling of multiple pathways. Here, we found that following B-cell receptor (BCR)-induced tyrosine phosphorylation of the cytoplasmic tyrosine kinase SYK and the adaptor BLNK, the AKT/PKB enzyme strongly induced BLNK (>100-fold) and SYK (>100-fold) serine/threonine phosphorylation (pS/pT). Increased phosphorylation promoted 14-3-3 binding to BLNK (37-fold) and SYK (2.5-fold) in a pS/pT-concentration dependent manner. We also demonstrated that the AKT inhibitor MK2206 reduced pS/pT of both BLNK (3-fold) and SYK (2.5-fold). Notably, the AKT phosphatase, PHLPP2 maintained the activating phosphorylation of BLNK at Y84 and increased protein stability (8.5-fold). In addition, 14-3-3 was required for the regulation SYK's interaction with BLNK and attenuated SYK binding to Importin 7 (5-fold), thereby perturbing shuttling to the nucleus. Moreover, 14-3-3 proteins also sustained tyrosine phosphorylation of SYK and BLNK. Furthermore, substitution of S295 or S297 for alanine abrogated SYK's binding to Importin 7. SYK with S295A or S297A replacements showed intense pY525/526 phosphorylation, and BLNK pY84 phosphorylation correlated with the SYK pY525/526 phosphorylation level. Conversely, the corresponding mutations to aspartic acid in SYK reduced pY525/526 phosphorylation. Collectively, these and previous results suggest that AKT and 14-3-3 proteins down-regulate the activity of several BCR-associated components, including BTK, BLNK and SYK and also inhibit SYK's interaction with Importin 7.
Phosphorylation of Janus kinase 1 (JAK1) by AMP-activated protein kinase (AMPK) links energy sensing to anti-inflammatory signaling.
Rutherford Claire,Speirs Claire,Williams Jamie J L,Ewart Marie-Ann,Mancini Sarah J,Hawley Simon A,Delles Christian,Viollet Benoit,Costa-Pereira Ana P,Baillie George S,Salt Ian P,Palmer Timothy M
Adenosine 5'-monophosphate-activated protein kinase (AMPK) is a pivotal regulator of metabolism at cellular and organismal levels. AMPK also suppresses inflammation. We found that pharmacological activation of AMPK rapidly inhibited the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway in various cells. In vitro kinase assays revealed that AMPK directly phosphorylated two residues (Ser and Ser) within the Src homology 2 domain of JAK1. Activation of AMPK enhanced the interaction between JAK1 and 14-3-3 proteins in cultured vascular endothelial cells and fibroblasts, an effect that required the presence of Ser and Ser and was abolished in cells lacking AMPK catalytic subunits. Mutation of Ser and Ser abolished AMPK-mediated inhibition of JAK-STAT signaling stimulated by either the sIL-6Rα/IL-6 complex or the expression of a constitutively active V658F-mutant JAK1 in human fibrosarcoma cells. Clinically used AMPK activators metformin and salicylate enhanced the inhibitory phosphorylation of endogenous JAK1 and inhibited STAT3 phosphorylation in primary vascular endothelial cells. Therefore, our findings reveal a mechanism by which JAK1 function and inflammatory signaling may be suppressed in response to metabolic stress and provide a mechanistic rationale for the investigation of AMPK activators in a range of diseases associated with enhanced activation of the JAK-STAT pathway.
Proteomics Screen Identifies Class I Rab11 Family Interacting Proteins as Key Regulators of Cytokinesis.
Laflamme Carl,Galan Jacob A,Ben El Kadhi Khaled,Méant Antoine,Zeledon Carlos,Carréno Sébastien,Roux Philippe P,Emery Gregory
Molecular and cellular biology
The 14-3-3 protein family orchestrates a complex network of molecular interactions that regulates various biological processes. Owing to their role in regulating the cell cycle and protein trafficking, 14-3-3 proteins are prevalent in human diseases such as cancer, diabetes, and neurodegeneration. 14-3-3 proteins are expressed in all eukaryotic cells, suggesting that they mediate their biological functions through evolutionarily conserved protein interactions. To identify these core 14-3-3 client proteins, we used an affinity-based proteomics approach to characterize and compare the human and Drosophila 14-3-3 interactomes. Using this approach, we identified a group of Rab11 effector proteins, termed class I Rab11 family interacting proteins (Rab11-FIPs), or Rip11 in Drosophila We found that 14-3-3 binds to Rip11 in a phospho-dependent manner to ensure its proper subcellular distribution during cell division. Our results indicate that Rip11 plays an essential role in the regulation of cytokinesis and that this function requires its association with 14-3-3 but not with Rab11. Together, our results suggest an evolutionarily conserved role for 14-3-3 in controlling Rip11-dependent protein transport during cytokinesis.
Structural Basis for the 14-3-3 Protein-Dependent Inhibition of Phosducin Function.
Kacirova Miroslava,Novacek Jiri,Man Petr,Obsilova Veronika,Obsil Tomas
Phosducin (Pdc) is a conserved phosphoprotein that, when unphosphorylated, binds with high affinity to the complex of βγ-subunits of G protein transducin (Gβγ). The ability of Pdc to bind to Gβγ is inhibited through its phosphorylation at S54 and S73 within the N-terminal domain (Pdc-ND) followed by association with the scaffolding protein 14-3-3. However, the molecular basis for the 14-3-3-dependent inhibition of Pdc binding to Gβγ is unclear. By using small-angle x-ray scattering, high-resolution NMR spectroscopy, and limited proteolysis coupled with mass spectrometry, we show that phosphorylated Pdc and 14-3-3 form a complex in which the Pdc-ND region 45-80, which forms a part of Pdc's Gβγ binding surface and contains both phosphorylation sites, is restrained within the central channel of the 14-3-3 dimer, with both 14-3-3 binding motifs simultaneously participating in protein association. The N-terminal part of Pdc-ND is likely located outside the central channel of the 14-3-3 dimer, but Pdc residues 20-30, which are also involved in Gβγ binding, are positioned close to the surface of the 14-3-3 dimer. The C-terminal domain of Pdc is located outside the central channel and its structure is unaffected by the complex formation. These results indicate that the 14-3-3 protein-mediated inhibition of Pdc binding to Gβγ is based on steric occlusion of Pdc's Gβγ binding surface.
The AMP-Related Kinase (AMPK) Induces Ca-Independent Dilation of Resistance Arteries by Interfering With Actin Filament Formation.
Schubert Kai Michael,Qiu Jiehua,Blodow Stephanie,Wiedenmann Margarethe,Lubomirov Lubomir T,Pfitzer Gabriele,Pohl Ulrich,Schneider Holger
RATIONALE:Decreasing Ca sensitivity of vascular smooth muscle (VSM) allows for vasodilation without lowering of cytosolic Ca. This may be particularly important in states requiring maintained dilation, such as hypoxia. AMP-related kinase (AMPK) is an important cellular energy sensor in VSM. Regulation of Ca sensitivity usually is attributed to myosin light chain phosphatase activity, but findings in non-VSM identified changes in the actin cytoskeleton. The potential role of AMPK in this setting is widely unknown. OBJECTIVE:To assess the influence of AMPK on the actin cytoskeleton in VSM of resistance arteries with regard to potential Ca desensitization of VSM contractile apparatus. METHODS AND RESULTS:AMPK induced a slowly developing dilation at unchanged cytosolic Ca levels in potassium chloride-constricted intact arteries isolated from mouse mesenteric tissue. This dilation was not associated with changes in phosphorylation of myosin light chain or of myosin light chain phosphatase regulatory subunit. Using ultracentrifugation and confocal microscopy, we found that AMPK induced depolymerization of F-actin (filamentous actin). Imaging of arteries from LifeAct mice showed F-actin rarefaction in the midcellular portion of VSM. Immunoblotting revealed that this was associated with activation of the actin severing factor cofilin. Coimmunoprecipitation experiments indicated that AMPK leads to the liberation of cofilin from 14-3-3 protein. CONCLUSIONS:AMPK induces actin depolymerization, which reduces vascular tone and the response to vasoconstrictors. Our findings demonstrate a new role of AMPK in the control of actin cytoskeletal dynamics, potentially allowing for long-term dilation of microvessels without substantial changes in cytosolic Ca.
Amyotrophic lateral sclerosis-related mutant superoxide dismutase 1 aggregates inhibit 14-3-3-mediated cell survival by sequestration into the JUNQ compartment.
Park Ju-Hwang,Jang Hae Rim,Lee In Young,Oh Hye Kyung,Choi Eui-Ju,Rhim Hyangshuk,Kang Seongman
Human molecular genetics
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by motor neuron loss in the spinal cord and brain. Mutations in the superoxide dismutase 1 (SOD1) gene have been linked to familial ALS. To elucidate the role of SOD1 mutations in ALS, we investigated 14-3-3, a crucial regulator of cell death that was identified in patients with familial ALS. In a transgenic mouse model (SOD1-G93A) of ALS, 14-3-3 co-localized with mutant SOD1 aggregates and was more insoluble in the spinal cords of mutant SOD1 transgenic mice than in those of wild-type mice. Immunofluorescence and co-immunoprecipitation experiments showed that the 14-3-3ɛ and θ isoforms interact with mutant SOD1 aggregates in the juxtanuclear quality control compartment of N2a neuroblastoma cells. Fluorescence loss in photobleaching experiments revealed that movement of the isoforms of 14-3-3 was markedly reduced in SOD1 aggregates. Bax translocation into and cytochrome c release from the mitochondria were promoted by the sequestration of 14-3-3 into mutant SOD1 aggregates, increasing cell death. Mutant SOD1 aggregates were dissolved by the Hsp104 chaperone, which increased the interaction of 14-3-3 with Bax, reducing cell death. Our study demonstrates that mutant SOD1 inhibits 14-3-3-mediated cell survival. This information may contribute to the identification of a novel therapeutic target for ALS.
TRIP13 promotes tumor growth and is associated with poor prognosis in colorectal cancer.
Sheng Nengquan,Yan Li,Wu Kai,You Weiqiang,Gong Jianfeng,Hu Landian,Tan Gewen,Chen Hongqi,Wang Zhigang
Cell death & disease
Colorectal cancer (CRC) is one of the most common neoplasms worldwide. However, the mechanisms underlying its development are still poorly understood. Thyroid hormone Receptor Interactor 13 (TRIP13) is a key mitosis regulator, and recent evidence has shown that it is an oncogene. Here, we report that TRIP13, which is overexpressed in CRC, is correlated with the CEA (carcino-embryonic antigen), CA19-9 (carbohydrate antigen 19-9) and pTNM (pathologic primary tumor, lymph nodes, distant metastasis) classification. Multivariate analyses showed that TRIP13 might serve as an independent prognostic marker of CRC. We also found that TRIP13 promoted CRC cell proliferation, invasion and migration in vitro and subcutaneous tumor formation in vivo. Furthermore, the potential mechanism underlying these effects involves the interaction of TRIP13 with a 14-3-3 protein, YWHAZ, which mediates G2-M transition and epithelial-mesenchymal transition (EMT). Together, these findings suggest that TRIP13 may be a potential biomarker and therapeutic target for CRC.
14-3-3 proteins in platelet biology and glycoprotein Ib-IX signaling.
Chen Yunfeng,Ruggeri Zaverio M,Du Xiaoping
Members of the 14-3-3 family of proteins function as adapters/modulators that recognize phosphoserine/phosphothreonine-based binding motifs in many intracellular proteins and play fundamental roles in signal transduction pathways of eukaryotic cells. In platelets, 14-3-3 plays a wide range of regulatory roles in phosphorylation-dependent signaling pathways, including G-protein signaling, cAMP signaling, agonist-induced phosphatidylserine exposure, and regulation of mitochondrial function. In particular, 14-3-3 interacts with several phosphoserine-dependent binding sites in the major platelet adhesion receptor, the glycoprotein Ib-IX complex (GPIb-IX), regulating its interaction with von Willebrand factor (VWF) and mediating VWF/GPIb-IX-dependent mechanosignal transduction, leading to platelet activation. The interaction of 14-3-3 with GPIb-IX also plays a critical role in enabling the platelet response to low concentrations of thrombin through cooperative signaling mediated by protease-activated receptors and GPIb-IX. The various functions of 14-3-3 in platelets suggest that it is a possible target for the treatment of thrombosis and inflammation.
Dynamic Fluctuations in Subcellular Localization of the Hippo Pathway Effector Yorkie In Vivo.
Manning Samuel A,Dent Lucas G,Kondo Shu,Zhao Ziqing W,Plachta Nicolas,Harvey Kieran F
Current biology : CB
The Hippo pathway is an evolutionarily conserved signaling network that integrates diverse cues to control organ size and cell fate. The central downstream pathway protein in Drosophila is the transcriptional co-activator Yorkie (YAP and TAZ in humans), which regulates gene expression with the Scalloped/TEA domain family member (TEAD) transcription factors [1-8]. A central regulatory step in the Hippo pathway is phosphorylation of Yorkie by the NDR family kinase Warts, which promotes Yorkie cytoplasmic localization by stimulating association with 14-3-3 proteins [9-12]. Numerous reports have purported a static model of Hippo signaling whereby, upon Hippo activation, Yorkie/YAP/TAZ become cytoplasmic and therefore inactive, and upon Hippo repression, Yorkie/YAP/TAZ transit to the nucleus and are active. However, we have little appreciation for the dynamics of Yorkie/YAP/TAZ subcellular localization because most studies have been performed in fixed cells and tissues. To address this, we used live multiphoton microscopy to investigate the dynamics of an endogenously tagged Yorkie-Venus protein in growing epithelial organs. We found that the majority of Yorkie rapidly traffics between the cytoplasm and nucleus, rather than being statically localized in either compartment. In addition, discrete cell populations within the same organ display different rates of Yorkie nucleo-cytoplasmic shuttling. By assessing Yorkie dynamics in warts mutant tissue, we found that the Hippo pathway regulates Yorkie subcellular distribution by regulating its rate of nuclear import. Furthermore, Yorkie's localization fluctuates dramatically throughout the cell cycle, being predominantly cytoplasmic during interphase and, unexpectedly, chromatin enriched during mitosis. Yorkie's association with mitotic chromatin is Scalloped dependent, suggesting a potential role in mitotic bookmarking.
mTORC1 Promotes Metabolic Reprogramming by the Suppression of GSK3-Dependent Foxk1 Phosphorylation.
He Long,Gomes Ana P,Wang Xin,Yoon Sang Oh,Lee Gina,Nagiec Michal J,Cho Sungyun,Chavez Andre,Islam Tasnia,Yu Yonghao,Asara John M,Kim Bo Yeon,Blenis John
The mammalian Target of Rapamycin Complex 1 (mTORC1)-signaling system plays a critical role in the maintenance of cellular homeostasis by sensing and integrating multiple extracellular and intracellular cues. Therefore, uncovering the effectors of mTORC1 signaling is pivotal to understanding its pathophysiological effects. Here we report that the transcription factor forkhead/winged helix family k1 (Foxk1) is a mediator of mTORC1-regulated gene expression. Surprisingly, Foxk1 phosphorylation is increased upon mTORC1 suppression, which elicits a 14-3-3 interaction, a reduction of DNA binding, and nuclear exclusion. Mechanistically, this occurs by mTORC1-dependent suppression of nuclear signaling by the Foxk1 kinase, Gsk3. This pathway then regulates the expression of multiple genes associated with glycolysis and downstream anabolic pathways directly modulated by Foxk1 and/or by Foxk1-regulated expression of Hif-1α. Thus, Foxk1 mediates mTORC1-driven metabolic rewiring, and it is likely to be critical for metabolic diseases where improper mTORC1 signaling plays an important role.
Quantitative Analysis Reveals that Actin and Src-Family Kinases Regulate Nuclear YAP1 and Its Export.
Ege Nil,Dowbaj Anna M,Jiang Ming,Howell Michael,Hooper Steven,Foster Charles,Jenkins Robert P,Sahai Erik
The transcriptional regulator YAP1 is critical for the pathological activation of fibroblasts. In normal fibroblasts, YAP1 is located in the cytoplasm, while in activated cancer-associated fibroblasts, it is nuclear and promotes the expression of genes required for pro-tumorigenic functions. Here, we investigate the dynamics of YAP1 shuttling in normal and activated fibroblasts, using EYFP-YAP1, quantitative photobleaching methods, and mathematical modeling. Imaging of migrating fibroblasts reveals the tight temporal coupling of cell shape change and altered YAP1 localization. Both 14-3-3 and TEAD binding modulate YAP1 shuttling, but neither affects nuclear import. Instead, we find that YAP1 nuclear accumulation in activated fibroblasts results from Src and actomyosin-dependent suppression of phosphorylated YAP1 export. Finally, we show that nuclear-constrained YAP1, upon XPO1 depletion, remains sensitive to blockade of actomyosin function. Together, these data place nuclear export at the center of YAP1 regulation and indicate that the cytoskeleton can regulate YAP1 within the nucleus.
Hippo Signaling Pathway Dysregulation in Human Huntington's Disease Brain and Neuronal Stem Cells.
Mueller Kaly A,Glajch Kelly E,Huizenga Megan N,Wilson Remi A,Granucci Eric J,Dios Amanda M,Tousley Adelaide R,Iuliano Maria,Weisman Elizabeth,LaQuaglia Michael J,DiFiglia Marian,Kegel-Gleason Kimberly,Vakili Khashayar,Sadri-Vakili Ghazaleh
The Hippo signaling pathway is involved in organ size regulation and tumor suppression. Although inhibition of Hippo leads to tumorigenesis, activation of Hippo may play a role in neurodegeneration. Specifically, activation of the upstream regulator, mammalian sterile 20 (STE20)-like kinase 1 (MST1), reduces activity of the transcriptional co-activator Yes-Associated Protein (YAP), thereby mediating oxidative stress-induced neuronal death. Here, we investigated the possible role of this pathway in Huntington's disease (HD) pathogenesis. Our results demonstrate a significant increase in phosphorylated MST1, the active form, in post-mortem HD cortex and in the brains of CAG knock-in Hdh mice. YAP nuclear localization was also decreased in HD post-mortem cortex and in neuronal stem cells derived from HD patients. Moreover, there was a significant increase in phosphorylated YAP, the inactive form, in HD post-mortem cortex and in Hdh brain. In addition, YAP was found to interact with huntingtin (Htt) and the chaperone 14-3-3, however this interaction was not altered in the presence of mutant Htt. Lastly, YAP/TEAD interactions and expression of Hippo pathway genes were altered in HD. Together, these results demonstrate that activation of MST1 together with a decrease in nuclear YAP could significantly contribute to transcriptional dysregulation in HD.
The dynamic and stress-adaptive signaling hub of 14-3-3: emerging mechanisms of regulation and context-dependent protein-protein interactions.
Pennington K L,Chan T Y,Torres M P,Andersen J L
14-3-3 proteins are a family of structurally similar phospho-binding proteins that regulate essentially every major cellular function. Decades of research on 14-3-3s have revealed a remarkable network of interacting proteins that demonstrate how 14-3-3s integrate and control multiple signaling pathways. In particular, these interactions place 14-3-3 at the center of the signaling hub that governs critical processes in cancer, including apoptosis, cell cycle progression, autophagy, glucose metabolism, and cell motility. Historically, the majority of 14-3-3 interactions have been identified and studied under nutrient-replete cell culture conditions, which has revealed important nutrient driven interactions. However, this underestimates the reach of 14-3-3s. Indeed, the loss of nutrients, growth factors, or changes in other environmental conditions (e.g., genotoxic stress) will not only lead to the loss of homeostatic 14-3-3 interactions, but also trigger new interactions, many of which are likely stress adaptive. This dynamic nature of the 14-3-3 interactome is beginning to come into focus as advancements in mass spectrometry are helping to probe deeper and identify context-dependent 14-3-3 interactions-providing a window into adaptive phosphorylation-driven cellular mechanisms that orchestrate the tumor cell's response to a variety of environmental conditions including hypoxia and chemotherapy. In this review, we discuss emerging 14-3-3 regulatory mechanisms with a focus on post-translational regulation of 14-3-3 and dynamic protein-protein interactions that illustrate 14-3-3's role as a stress-adaptive signaling hub in cancer.
Caveolin-1 Modulates Mechanotransduction Responses to Substrate Stiffness through Actin-Dependent Control of YAP.
Moreno-Vicente Roberto,Pavón Dácil María,Martín-Padura Inés,Català-Montoro Mauro,Díez-Sánchez Alberto,Quílez-Álvarez Antonio,López Juan Antonio,Sánchez-Álvarez Miguel,Vázquez Jesús,Strippoli Raffaele,Del Pozo Miguel A
The transcriptional regulator YAP orchestrates many cellular functions, including tissue homeostasis, organ growth control, and tumorigenesis. Mechanical stimuli are a key input to YAP activity, but the mechanisms controlling this regulation remain largely uncharacterized. We show that CAV1 positively modulates the YAP mechanoresponse to substrate stiffness through actin-cytoskeleton-dependent and Hippo-kinase-independent mechanisms. RHO activity is necessary, but not sufficient, for CAV1-dependent mechanoregulation of YAP activity. Systematic quantitative interactomic studies and image-based small interfering RNA (siRNA) screens provide evidence that this actin-dependent regulation is determined by YAP interaction with the 14-3-3 protein YWHAH. Constitutive YAP activation rescued phenotypes associated with CAV1 loss, including defective extracellular matrix (ECM) remodeling. CAV1-mediated control of YAP activity was validated in vivo in a model of pancreatitis-driven acinar-to-ductal metaplasia. We propose that this CAV1-YAP mechanotransduction system controls a significant share of cell programs linked to these two pivotal regulators, with potentially broad physiological and pathological implications.
Lysosomotropic drugs activate TFEB via lysosomal membrane fluidization and consequent inhibition of mTORC1 activity.
Zhitomirsky Benny,Yunaev Anna,Kreiserman Roman,Kaplan Ariel,Stark Michal,Assaraf Yehuda G
Cell death & disease
Transcription factor EB (TFEB) is a master transcriptional regulator playing a key role in lysosomal biogenesis, autophagy and lysosomal exocytosis. TFEB activity is inhibited following its phosphorylation by mammalian target of rapamycin complex 1 (mTORC1) on the surface of the lysosome. Phosphorylated TFEB is bound by 14-3-3 proteins, resulting in its cytoplasmic retention in an inactive state. It was suggested that the calcium-dependent phosphatase calcineurin is responsible for dephosphorylation and subsequent activation of TFEB under conditions of lysosomal stress. We have recently demonstrated that TFEB is activated following exposure of cancer cells to lysosomotropic anticancer drugs, resulting in lysosome-mediated cancer drug resistance via increased lysosomal biogenesis, lysosomal drug sequestration, and drug extrusion through lysosomal exocytosis. Herein, we studied the molecular mechanism underlying lysosomotropic-drug-induced activation of TFEB. We demonstrate that accumulation of lysosomotropic drugs results in membrane fluidization of lysosome-like liposomes, which is strictly dependent on the acidity of the liposomal lumen. Lysosomal accumulation of lysosomotropic drugs and the consequent fluidization of the lysosomal membrane, facilitated the dissociation of mTOR from the lysosomal membrane and inhibited the kinase activity of mTORC1, which is necessary and sufficient for the rapid translocation of TFEB to the nucleus. We further show that while lysosomotropic drug sequestration induces Ca release into the cytoplasm, facilitating calcineurin activation, chelation of cytosolic Ca, or direct inhibition of calcineurin activity, do not interfere with drug-induced nuclear translocation of TFEB. We thus suggest that lysosomotropic drug-induced activation of TFEB is mediated by mTORC1 inhibition due to lysosomal membrane fluidization and not by calcineurin activation. We further postulate that apart from calcineurin, other constitutively active phosphatase(s) partake in TFEB dephosphorylation and consequent activation. Moreover, a rapid export of TFEB from the nucleus to the cytosol occurs upon relief of mTORC1 inhibition, suggesting that dephosphorylated TFEB constantly travels between the nucleus and the cytosol, acting as a rapidly responding sensor of mTORC1 activity.
Nigrostriatal pathology with reduced astrocytes in LRRK2 S910/S935 phosphorylation deficient knockin mice.
Zhao Ye,Keshiya Shikara,Atashrazm Farzaneh,Gao Jianqun,Ittner Lars M,Alessi Dario R,Halliday Glenda M,Fu Yuhong,Dzamko Nicolas
Neurobiology of disease
Leucine-rich repeat kinase 2 (LRRK2) is genetically implicated in both familial and sporadic Parkinson's disease (PD). Moreover, LRRK2 has emerged as a compelling therapeutic target for the treatment of PD. Consequently, there is much interest in understanding LRRK2 and its role in PD pathogenesis. LRRK2 is constitutively phosphorylated on two serines, S910 and S935, that are required for interaction of LRRK2 with members of the 14-3-3 family of scaffolding proteins. Pathogenic LRRK2 missense mutations impair the phosphorylation of LRRK2 at these sites, but whether this contributes to PD pathology is unclear. To better understand how loss of LRRK2 phosphorylation relates to PD pathology, we have studied double knockin mice in which Lrrk2's serine 910 and 935 have both been mutated to alanine and can therefore no longer be phosphorylated. Nigrostriatal PD pathology was assessed in adult mice, aged mice, and mice inoculated with α-synuclein fibrils. Under all paradigms there was evidence of early PD pathology in the striatum of the knockin mice, namely alterations in dopamine regulating proteins and accumulation of α-synuclein. Striatal pathology was accompanied by a significant decrease in the number of astrocytes in the knockin mice. Despite striatal pathology, there was no degeneration of dopamine neurons in the substantia nigra and no evidence of a PD motor phenotype in the knockin mice. Our results suggest that modulation of LRRK2 serine 910 and 935 phosphorylation sites may have implications for dopamine turnover and astrocyte function, but loss of phosphorylation at these residues is not sufficient to induce PD neurodegeneration.
14-3-3 Proteins regulate mutant LRRK2 kinase activity and neurite shortening.
Lavalley Nicholas J,Slone Sunny R,Ding Huiping,West Andrew B,Yacoubian Talene A
Human molecular genetics
Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common known cause of inherited Parkinson's disease (PD), and LRRK2 is a risk factor for idiopathic PD. How LRRK2 function is regulated is not well understood. Recently, the highly conserved 14-3-3 proteins, which play a key role in many cellular functions including cell death, have been shown to interact with LRRK2. In this study, we investigated whether 14-3-3s can regulate mutant LRRK2-induced neurite shortening and kinase activity. In the presence of 14-3-3θ overexpression, neurite length of primary neurons from BAC transgenic G2019S-LRRK2 mice returned back to wild-type levels. Similarly, 14-3-3θ overexpression reversed neurite shortening in neuronal cultures from BAC transgenic R1441G-LRRK2 mice. Conversely, inhibition of 14-3-3s by the pan-14-3-3 inhibitor difopein or dominant-negative 14-3-3θ further reduced neurite length in G2019S-LRRK2 cultures. Since G2019S-LRRK2 toxicity is likely mediated through increased kinase activity, we examined 14-3-3θ's effects on LRRK2 kinase activity. 14-3-3θ overexpression reduced the kinase activity of G2019S-LRRK2, while difopein promoted the kinase activity of G2019S-LRRK2. The ability of 14-3-3θ to reduce LRRK2 kinase activity required direct binding of 14-3-3θ with LRRK2. The potentiation of neurite shortening by difopein in G2019S-LRRK2 neurons was reversed by LRRK2 kinase inhibitors. Taken together, we conclude that 14-3-3θ can regulate LRRK2 and reduce the toxicity of mutant LRRK2 through a reduction of kinase activity.
Mechanism of action of a WWTR1(TAZ)-CAMTA1 fusion oncoprotein.
Tanas M R,Ma S,Jadaan F O,Ng C K Y,Weigelt B,Reis-Filho J S,Rubin B P
The WWTR1 (protein is known as TAZ)-CAMTA1 (WC) fusion gene defines epithelioid hemangioendothelioma, a malignant vascular cancer. TAZ (transcriptional coactivator with PDZ binding motif) is a transcriptional coactivator and end effector of the Hippo tumor suppressor pathway. It is inhibited by phosphorylation by the Hippo kinases LATS1 and LATS2. Such phosphorylation causes cytoplasmic localization, 14-3-3 protein binding and the phorphorylation of a terminal phosphodegron promotes ubiquitin-dependent degradation (the phosphorylation of the different motifs has several effects). CAMTA1 is a putative tumor suppressive transcription factor. Here we demonstrate that TAZ-CAMTA1 (TC) fusion results in its nuclear localization and constitutive activation. Consequently, cells expressing TC display a TAZ-like transcriptional program that causes resistance to anoikis and oncogenic transformation. Our findings elucidate the mechanistic basis of TC oncogenic properties, highlight that TC is an important model to understand how the Hippo pathway can be inhibited in cancer, and provide approaches for targeting this chimeric protein.
Binding of 14-3-3 reader proteins to phosphorylated DNMT1 facilitates aberrant DNA methylation and gene expression.
Estève Pierre-Olivier,Zhang Guoqiang,Ponnaluri V K Chaithanya,Deepti Kanneganti,Chin Hang Gyeong,Dai Nan,Sagum Cari,Black Karynne,Corrêa Ivan R,Bedford Mark T,Cheng Xiaodong,Pradhan Sriharsa
Nucleic acids research
Mammalian DNA (cytosine-5) methyltransferase 1 (DNMT1) is essential for maintenance methylation. Phosphorylation of Ser143 (pSer143) stabilizes DNMT1 during DNA replication. Here, we show 14-3-3 is a reader protein of DNMT1pSer143. In mammalian cells 14-3-3 colocalizes and binds DNMT1pSer143 post-DNA replication. The level of DNMT1pSer143 increased with overexpression of 14-3-3 and decreased by its depletion. Binding of 14-3-3 proteins with DNMT1pSer143 resulted in inhibition of DNA methylation activity in vitro. In addition, overexpression of 14-3-3 in NIH3T3 cells led to decrease in DNMT1 specific activity resulting in hypomethylation of the genome that was rescued by transfection of DNMT1. Genes representing cell migration, mobility, proliferation and focal adhesion pathway were hypomethylated and overexpressed. Furthermore, overexpression of 14-3-3 also resulted in enhanced cell invasion. Analysis of TCGA breast cancer patient data showed significant correlation for DNA hypomethylation and reduced patient survival with increased 14-3-3 expressions. Therefore, we suggest that 14-3-3 is a crucial reader of DNMT1pSer143 that regulates DNA methylation and altered gene expression that contributes to cell invasion.
Signaling-mediated cooperativity between glycoprotein Ib-IX and protease-activated receptors in thrombin-induced platelet activation.
Estevez Brian,Kim Kyungho,Delaney M Keegan,Stojanovic-Terpo Aleksandra,Shen Bo,Ruan Changgeng,Cho Jaehyung,Ruggeri Zaverio M,Du Xiaoping
Thrombin-induced cellular response in platelets not only requires protease-activated receptors (PARs), but also involves another thrombin receptor, the glycoprotein Ib-IX complex (GPIb-IX). It remains controversial how thrombin binding to GPIb-IX stimulates platelet responses. It was proposed that GPIb-IX serves as a dock that facilitates thrombin cleavage of protease-activated receptors, but there are also reports suggesting that thrombin binding to GPIb-IX induces platelet activation independent of PARs. Here we show that GPIb is neither a passive thrombin dock nor a PAR-independent signaling receptor. We demonstrate a novel signaling-mediated cooperativity between PARs and GPIb-IX. Low-dose thrombin-induced PAR-dependent cell responses require the cooperativity of GPIb-IX signaling, and conversely, thrombin-induced GPIb-IX signaling requires cooperativity of PARs. This mutually dependent cooperativity requires a GPIb-IX-specific 14-3-3-Rac1-LIMK1 signaling pathway, and activation of this pathway also requires PAR signaling. The cooperativity between GPIb-IX signaling and PAR signaling thus drives platelet activation at low concentrations of thrombin, which are important for in vivo thrombosis.
14-3-3 Proteins regulate K 5.1 surface expression on T lymphocytes.
Fernández-Orth Juncal,Ehling Petra,Ruck Tobias,Pankratz Susann,Hofmann Majella-Sophie,Landgraf Peter,Dieterich Daniela C,Smalla Karl-Heinz,Kähne Thilo,Seebohm Guiscard,Budde Thomas,Wiendl Heinz,Bittner Stefan,Meuth Sven G
Traffic (Copenhagen, Denmark)
K 5.1 channels (also called TASK-2 or Kcnk5) have already been shown to be relevant in the pathophysiology of autoimmune disease because they are known to be upregulated on peripheral and central T lymphocytes of multiple sclerosis (MS) patients. Moreover, overexpression of K 5.1 channels in vitro provokes enhanced T-cell effector functions. However, the molecular mechanisms regulating intracellular K 5.1 channel trafficking are unknown so far. Thus, the aim of the study is to elucidate the trafficking of K 5.1 channels on T lymphocytes. Using mass spectrometry analysis, we have identified 14-3-3 proteins as novel binding partners of K 5.1 channels. We show that a non-classical 14-3-3 consensus motif (R-X-X-pT/S-x) at the channel's C-terminus allows the binding between K 5.1 and 14-3-3. The mutant K 5.1/S266A diminishes the protein-protein interaction and reduces the amplitude of membrane currents. Application of a non-peptidic 14-3-3 inhibitor (BV02) significantly reduces the number of wild-type channels in the plasma membrane, whereas the drug has no effect on the trafficking of the mutated channel. Furthermore, blocker application reduces T-cell effector functions. Taken together, we demonstrate that 14-3-3 interacts with K 5.1 and plays an important role in channel trafficking.
A Tbc1d1 -knockin mutation partially impairs AICAR- but not exercise-induced muscle glucose uptake in mice.
Chen Qiaoli,Xie Bingxian,Zhu Sangsang,Rong Ping,Sheng Yang,Ducommun Serge,Chen Liang,Quan Chao,Li Min,Sakamoto Kei,MacKintosh Carol,Chen Shuai,Wang Hong Yu
AIMS/HYPOTHESIS:TBC1D1 (tre-2/USP6, BUB2, cdc16 domain family member 1) is a Rab GTPase-activating protein (RabGAP) that has been implicated in regulating GLUT4 trafficking. TBC1D1 can be phosphorylated by the AMP-activated protein kinase (AMPK) on Ser, which consequently interacts with 14-3-3 proteins. Given the key role for AMPK in regulating insulin-independent muscle glucose uptake, we hypothesised that TBC1D1-Ser phosphorylation and/or 14-3-3 binding may mediate AMPK-governed glucose homeostasis. METHODS:Whole-body glucose homeostasis and muscle glucose uptake were assayed in mice bearing a Tbc1d1 -knockin mutation or harbouring skeletal muscle-specific Ampkα1/α2 (also known as Prkaa1/2) double-knockout mutations in response to an AMPK-activating agent, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR). Exercise-induced muscle glucose uptake and exercise capacity were also determined in the Tbc1d1 -knockin mice. RESULTS:Skeletal muscle-specific deletion of Ampkα1/a2 in mice prevented AICAR-induced hypoglycaemia and muscle glucose uptake. The Tbc1d1 -knockin mutation also attenuated the glucose-lowering effect of AICAR in mice. Glucose uptake and cell surface GLUT4 content were significantly lower in muscle isolated from the Tbc1d1 -knockin mice upon stimulation with a submaximal dose of AICAR. However, this Tbc1d1 -knockin mutation neither impaired exercise-induced muscle glucose uptake nor affected exercise capacity in mice. CONCLUSIONS/INTERPRETATION:TBC1D1-Ser phosphorylation and/or 14-3-3 binding partially mediates AMPK-governed glucose homeostasis and muscle glucose uptake in a context-dependent manner.
Simulated photoperiod influences testicular activity in quail via modulating local GnRHR-GnIHR, GH-R, Cnx-43 and 14-3-3.
Banerjee Somanshu,Chaturvedi Chandra Mohini
Journal of photochemistry and photobiology. B, Biology
The hypothalamo-hypophyseal-gonadal axis mediated differential photosexual responses in quail kept under different simulated photoperiodic conditions have been studied in details. Local testicular GnRH-GnIH and their receptor system has been hypothesized to be modulated in quail showing different photo-sexual responses and thus influence the testicular activity and steroidogenesis through local (paracrine and autocrine) action. To validate this hypothesis, we studied the expression of gonadotropin releasing hormone receptor (GnRH-R), gonadotropin inhibiting hormone receptor (GnIH-R) mRNA, growth hormone receptor (GH-R), proliferating cell nuclear antigen (PCNA), 14-3-3, Connexin-43 (Cnx-43), steroidogenic factor-1 (SF-1), Steroidogenic Acute Regulatory protein (StAR), steroidogenic enzyme (3β HSD) in testis as well as androgen receptor (AR) in testis and epididymis of photosensitive (PS), scotorefractory (SR), photorefractory (PR) and scotosensitive (SS) quail. Experimental findings clearly indicate the increased expression of GnIH-R mRNA and suppression of GnRH-R, GH-R, PCNA, 14-3-3, Connexin-43, SF-1, StAR, 3β HSD in testis as well as AR in testis and epididymis of PR and SS quail, while PS and SR quail exhibited the opposite results i.e., significantly decreased expression of GnIH-R mRNA and increased expression of GnRH-R, GH-R, PCNA, 14-3-3, Cnx-43, SF-1, StAR, 3β HSD in testis as well as AR in testis and epididymis. The significantly increased intra-testicular testosterone has been observed in the PS and SR quail while, PR and SS quail showed opposite results. Hence, we conclude that PS and SR quail showed significantly increased testicular activity and steroidogenesis while opposite pattern was observed in PR and SS quail.
Modulators of 14-3-3 Protein-Protein Interactions.
Stevers Loes M,Sijbesma Eline,Botta Maurizio,MacKintosh Carol,Obsil Tomas,Landrieu Isabelle,Cau Ylenia,Wilson Andrew J,Karawajczyk Anna,Eickhoff Jan,Davis Jeremy,Hann Michael,O'Mahony Gavin,Doveston Richard G,Brunsveld Luc,Ottmann Christian
Journal of medicinal chemistry
Direct interactions between proteins are essential for the regulation of their functions in biological pathways. Targeting the complex network of protein-protein interactions (PPIs) has now been widely recognized as an attractive means to therapeutically intervene in disease states. Even though this is a challenging endeavor and PPIs have long been regarded as "undruggable" targets, the last two decades have seen an increasing number of successful examples of PPI modulators, resulting in growing interest in this field. PPI modulation requires novel approaches and the integrated efforts of multiple disciplines to be a fruitful strategy. This perspective focuses on the hub-protein 14-3-3, which has several hundred identified protein interaction partners, and is therefore involved in a wide range of cellular processes and diseases. Here, we aim to provide an integrated overview of the approaches explored for the modulation of 14-3-3 PPIs and review the examples resulting from these efforts in both inhibiting and stabilizing specific 14-3-3 protein complexes by small molecules, peptide mimetics, and natural products.