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蛋白激酶 CK2 是一種具有多功能的絲胺酸/蘇胺酸蛋白激酶，大量表現於哺乳類動物的腦中，對於調控細胞週期的發展、基因表現、訊息傳遞以及抗細胞凋亡機制扮演相當重要的角色。許多研究顯示 CK2 也參與調節許多神經系統功能，包括神經保護及神經存活，但是其中調控機制目前尚未釐清。DARPP-32 (dopamine- and cAMP- regulated phosphoprotein with a molecular mass of 32 kDa) 主要表現在紋狀體中型多刺狀神經元中，過去研究已證實 DARPP-32 Ser102 胺基酸是CK2 的磷酸化作用受質。雖然DARPP-32 被發現主要透過抑制蛋白磷酸水解酶 PP1 參與藥物成癮的細胞調控機制，但近年研究指出DARPP-32 也參與抗細胞凋亡作用。PP1 是真核細胞的絲胺酸/蘇胺酸磷酸水解酶，能調節多種細胞功能，如轉錄、細胞訊息傳遞及細胞凋亡。過去文獻已指出 PP1 可以調節 Bcl-x 基因的 pre-mRNA 選擇性剪切，再經由轉譯過程合成抗細胞凋亡 Bcl-xL 異構蛋白，研究也發現抑制 PP1 可以防止細胞週期的停滯及細胞凋亡，強調細胞在壓力的情況下，PP1 扮演了相當關鍵性的角色。因此論文以人類神經母細胞瘤 SH-SY5Y 為實驗模式，探討透過 CK2 調控 DARPP-32 Ser102 的磷酸化是否具有抑制 PP1 的活性並促進細胞存活的作用。實驗結果顯示，抑制 CK2或DARPP-32 蛋白含量會導致細胞存活率下降，轉染 CK2 siRNA 會降低 DARPP-32 Ser102 的磷酸化現象、Bcl-xL 的蛋白質表現；轉染DARPP-32 siRNA 及突變型DARPP-32 S102A DNA 質體也會降低 Bcl-xL 的蛋白質表現，PP1 活性則會因轉染突變型DARPP-32 S102A DNA 質體而增加；此外，給予 PP1 抑制劑的實驗結果發現會促進 Bcl-xL/Bcl-xS mRNA 的比例以及 Bcl-xL 的蛋白質表現量。利用過氧化氫誘導細胞造成氧化壓力狀況下，同時給予 PP1 抑制劑，發現 Bcl-xL 的蛋白質表現量會回復以及促進細胞存活。轉染 CK2-EGFP 或 DARPP-32 S102D DNA 質體可以顯著回復Bcl-xL 的蛋白質表現量及Bcl-xL/Bcl-xS mRNA 的比例，轉染 DARPP-32 S102D DNA 質體亦可降低 PP1 的活性。論文的實驗結果提供 CK2 調節抗細胞凋亡基因表現的新機制，是經由促進 DARPP-32 Ser102 磷酸化作用進而抑制 PP1 活性，此條細胞訊息傳遞路徑將可提供應用於在氧化壓力下提升神經存活的臨床治療。 / Protein kinase casein kinase II (CK2) is a multifunctional serine/threonine protein kinase and is highly abundant expression in the mammalian brain. CK2 plays an important role in the regulation of the cell cycle, gene expression, signal transduction and anti-apoptotic mechanisms. A number of studies have indicated that CK2 is involved in several neuronal functions including the neuroprotection and neuron survival, but its cellular mechanisms are not well-studied. The DARPP-32 (dopamine- and cAMP-regulated phosphoprotein with a molecular mass of 32 kDa) is highly enriched in the striatal medium spiny neurons and the Ser102 residue is identified as the phosphorylation site for CK2. Although DARPP-32 is known as a prominent cellular mediator of drug abuse through the inhibition of protein phosphatase 1 (PP1), the recent studies indicate that DARPP-32 may also be involved in the anti-apoptotic effects. Protein phosphatase PP1 is a major eukaryotic serine/threonine phosphatase that regulates diverse cellular functions such as transcription, cell signaling and apoptosis. PP1 is indicated to regulate the pre-mRNA alternative splicing of Bcl-x gene to encode the anti-apoptotic Bcl-xL isoform. Inhibition of PP1 prevents the induction of cell cycle arrest and apoptosis, underlines the crucial role of PP1 in the cellular response to the stress. In my thesis study, the neuroblastoma SH-SY5Y cell line system was used to investigate whether the promotion of cell survival by PP1 inhibition is through the signaling pathway of DARPP-32 Ser102 phosphorylation by CK2. The current results reveals that the cell viability is decreased under down-regulations of CK2 and DARPP-32. The Ser102 phosphorylation status of DARPP-32, Bcl-xL mRNA and protein level are decreased by CK2 siRNA transfection. Transfection of either DARPP-32 siRNA or mutant DARPP-32 S102A plasmid DNA decreased the Bcl-xL protein level. The PP1 activity was increased by mutant DARPP-32 S102A plasmid DNA transfection. Furthermore, the PP1 inhibitor treatment increased the Bcl-xL/Bcl-xS mRNA ratio and Bcl-xL protein level. Under oxidative stress, inhibition of PP1 activity can reverse the H2O2-induced decrease in Bcl-xL protein level and promote the cell viability. The transfection of CK2-EGFP or DARPP-32 S102D plasmid DNA both can antagonize the effects of H2O2 on Bcl-xL protein level and the Bcl-xL/Bcl-xS mRNA ratio. The DARPP-32 S102D plasmid DNA transfection also attenuated the induction of PP1 activity under oxidative stress. These findings provide another insight for the regulation of anti-apoptotic gene expression by inhibition of PP1 activity through DARPP-32 phosphorylation on Ser102 by CK2. This signaling pathway might be applied in the clinical therapy for neuronal survival under oxidative stress.

<p>  </p> <p>Metastatic breast cancer (MBC) is an extremely recalcitrant disease capable of overcoming targeted therapies and evading immune surveillance via the engagement of complicated signaling networks. Resistance to targeted therapies and therapeutic failure of immune checkpoint blockade (ICB) are two major challenges in treating MBC. To survive in the dynamic tumor microenvironment (TME) during metastatic progression, shared signaling nodes are required for MBC cells to regulate the signaling networks efficiently, which are potential multifunctional therapeutic targets. SH2 containing protein tyrosine phosphatase-2 (SHP2) is a druggable oncogenic phosphatase that is a key shared node in both tumor cells and immune cells. How tumor-cell autonomous SHP2 manages its signaling inputs and outputs to facilitate the growth of tumor cells, drug resistance, immunosuppression, and the limited response of ICB in MBC is not fully understood. Herein, we used inducible genetic depletion and two distinct types of pharmacological inhibitors to investigate anti-tumor effects with immune reprogramming during SHP2 targeting. </p> <p>We first focus on the signaling inputs and outputs of SHP2. We find that phosphorylation of SHP2 at Y542 predicts the survival rates of breast cancer patients and their immune profiles. Phosphorylation of SHP2 at Y542 is elevated with differential activation mechanisms under a growth-factor-induced and extracellular matrix (ECM)-rich culture environment. Phosphorylation of SHP2 at Y542 is also elevated in HER2 positive MBC cells upon acquired resistance to the HER2 kinase inhibitor, neratinib. The resistant cells can be targeted by SHP2 inhibitors. SHP2 inhibitors block ERK1/2 and AKT signaling and readily prevented MBC cell growth induced by multiple growth factors. Inhibition of SHP2 also blocks these signaling events generated from the ECM signaling. In fact, the inhibitory effects of SHP2 blockade are actually enhanced in the ECM-rich culture environment. We utilize the <em>in vitro</em> T-cell killing assays and demonstrate that pretreatment of tumor cells with FGF2 and PDGF reduces the cytotoxicity of CD8+ T cells in a SHP2-dependent manner. Both growth factors and ECM-rich culture environment transcriptionally induce PD-L1 via SHP2. SHP2 inhibition balances MAPK signaling and STAT1 signaling, which prevents growth factor-mediated suppression of INF-γ-induced expression of MHC class I. </p> <p>Next, we evaluate the efficacy of SHP2 inhibitors. Blockade of SHP2 in the adjuvant setting decreased pulmonary metastasis <em>in vivo</em> and extended the survival of systemic tumor-bearing mice. Tumor-cell autonomous depletion of SHP2 reduces pulmonary metastasis and relieves exhaustion markers on CD8+ and CD4+ cells. Meanwhile, both systemic SHP2 inhibition and tumor-cell autonomous SHP2 depletion reduce tumor-infiltrated CD4+ T cells and M2-polarized tumor associated macrophages. </p> <p>Finally, we investigate potential combination therapies with SHP2 inhibitors. The combination of SHP2 inhibitors and FGFR-targeted kinase inhibitors synergistically blocks the growth of MBC cells. Pharmacological inhibition SHP2 sensitizes MBC cells growing in the lung to α-PD-L1 antibody treatment via relieving T cell exhaustion induced by ICB. </p> <p>Overall, our findings support the conclusion that MBC cells are capable of simultaneously engaging several survival pathways and immune-suppressive mechanisms via SHP2 in response to multiple growth factors and ECM signaling. Inhibition of SHP2, potentially in combination with other targeted agents and ICB, holds promise for the therapeutic management of MBC.</p>

No / Hyperphosphorylated tau plays an important role in the formation of neurofibrillary tangles in brains of patients with Alzheimer's disease (AD) and related tauopathies and is a crucial factor in the pathogenesis of these disorders. Though diverse kinases have been implicated in tau phosphorylation, protein phosphatase 2A (PP2A) seems to be the major tau phosphatase. Using murine primary neurons from wild-type and human tau transgenic mice, we show that the antidiabetic drug metformin induces PP2A activity and reduces tau phosphorylation at PP2A-dependent epitopes in vitro and in vivo. This tau dephosphorylating potency can be blocked entirely by the PP2A inhibitors okadaic acid and fostriecin, confirming that PP2A is an important mediator of the observed effects. Surprisingly, metformin effects on PP2A activity and tau phosphorylation seem to be independent of AMPK activation, because in our experiments (i) metformin induces PP2A activity before and at lower levels than AMPK activity and (ii) the AMPK activator AICAR does not influence the phosphorylation of tau at the sites analyzed. Affinity chromatography and immunoprecipitation experiments together with PP2A activity assays indicate that metformin interferes with the association of the catalytic subunit of PP2A (PP2Ac) to the so-called MID1-alpha4 protein complex, which regulates the degradation of PP2Ac and thereby influences PP2A activity. In summary, our data suggest a potential beneficial role of biguanides such as metformin in the prophylaxis and/or therapy of AD.

[EN] ABSTRACT Abscisic acid (ABA) signaling plays a critical role in regulating root growth and root system architecture. ABA-mediated growth promotion and root tropic response under water stress are key responses for plant survival under limiting water conditions. In this work, we have explored the role of Arabidopsis (Arabidopsis thaliana) PYR/PYL/RCAR receptors (PYRABACTIN RESISTANCE1 (PYR1)/PYR1 LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS) for root ABA signaling. As a result, we discovered that PYL8 plays a nonredundant role for the regulation of root ABA sensitivity. Unexpectedly, given the multigenic nature and partial functional redundancy observed in the PYR/PYL family, the single pyl8 mutant showed reduced sensitivity to ABA-mediated root growth inhibition. This effect was due to the lack of PYL8-mediated inhibition of several clade A phosphatases type 2C (PP2Cs), since PYL8 interacted in vivo with at least five PP2Cs, namely HYPERSENSITIVE TO ABA1 (HAB1), HAB2, ABAINSENSITIVE1 (ABI1), ABI2, and PP2CA/ABA-HYPERSENSITIVE GERMINATION3 as revealed by tandem affinity purification and mass spectrometry proteomic approaches. Membrane-delimited abscisic acid (ABA) signal transduction plays a critical role in early ABA signaling, but the molecular mechanisms linking core signaling components to the plasma membrane are unclear. We show that transient calciumdependent interactions of PYR/PYL/RCAR ABA receptors with membranes are mediated through a 10-member family of C2-domain ABA-related (CAR) proteins in Arabidopsis thaliana. Specifically, we found that PYL4 interacted in an ABA-independent manner with CAR1 in both the plasma membrane and nucleus of plant cells. CAR1 belongs to a plant-specific gene family encoding CAR1 to CAR10 proteins, and bimolecular fluorescence complementation and coimmunoprecipitation assays showed that PYL4-CAR1 as well as other PYR/PYL-CAR pairs interacted in plant cells. The crystal structure of CAR4 was solved, which revealed that, in addition to a classical calcium-dependent lipid binding C2 domain, a specific CAR signature is likely responsible for the interaction with PYR/PYL/RCAR receptors and their recruitment to phospholipid vesicles. This interaction is relevant for PYR/PYL/RCAR function and ABA signaling, since different car triple mutants affected in CAR1, CAR4, CAR5, and CAR9 genes showed reduced sensitivity to ABA in seedling establishment and root growth assays. In summary, we identified PYR/PYL/RCAR-interacting partners that mediate a transient Ca2+-dependent interaction with phospholipid vesicles, which affects PYR/PYL/RCAR subcellular localization and positively regulates ABA signaling. / [ES] RESUMEN La señalización por la hormona vegetal ácido abscísico (ABA) desempeña un papel crítico en la regulación del crecimiento de la raíz y en la arquitectura del sistema radical. La promoción de crecimiento de la raíz en condiciones de estrés hídrico mediada por ABA es clave para la supervivencia de las plantas bajo condiciones limitantes de agua. En este trabajo, hemos explorado el papel de los receptores PYR/PYL/RCAR (PYRABACTIN RESISTANCE1 (PYR1)/PYR1 LIKE (PYL)/ REGULATORY COMPONENTS OF ABA RECEPTORS) de Arabidopsis (Arabidopsis thaliana) en la ruta de señalización de ABA en raíz. Así, hemos descubierto que el receptor de ABA PYL8 juega un papel no redundante en la regulación de la percepción de ABA en raíz. Inesperadamente, dada la naturaleza multigénica y la redundancia funcional parcial observada en la familia PYR/PYL/RCAR, el mutante pyl8 fue el único mutante sencillo de pérdida de función de los receptores PYR/PYL/RCAR que mostraba una sensibilidad reducida a la inhibición del crecimiento mediada por ABA en raíz. Este efecto se debe a la falta de inhibición mediada por PYL8 de varias fosfatasas del grupo A tipo 2C (PP2Cs), ya que PYL8 es capaz de interactuar in vivo con al menos cinco PP2Cs, denominadas HYPERSENSITIVE TO ABA1 (HAB1), HAB2, ABAINSENSITIVE1 (ABI1), ABI2, and PP2CA/ABA-HYPERSENSITIVE GERMINATION3 según lo han revelado la purificación por afinidad en tándem (TAP por sus siglas en inglés) y estudios proteómicos de espectrometría de masas. La transducción de la señal del ABA localizada en la membrana plasmática celular juega un papel crucial en los pasos iniciales de la señalización de la fitohormona, pero los mecanismos moleculares que unen los componentes básicos de la señalización y la membrana plasmática no están claros. Estudiando las interacciones de los receptores del ABA PYR/PYL/RCAR con la membrana plasmática hemos encontrado que éstos pueden interaccionar transitoriamente con ella de forma dependiente de calcio gracias a una familia de proteínas con dominios C2 relacionadas con la ruta de señalización de ABA (denominadas C2-domain ABA-related (CAR) proteins). Específicamente, se encontró que PYL4 interacciona de manera independiente de ABA con CAR1 tanto en la membrana plasmática como en el núcleo de las células vegetales. La proteína CAR1 pertenece a una familia multigénica constituida por 10 miembros en Arabidopsis thaliana, desde CAR1 hasta CAR10, y que solo se encuentra en plantas. Los ensayos de complementación bi-molecular de fluorescencia y de co-immunoprecipitación confirmaron la interacción en células vegetales tanto de PYL4-CAR1 como de otras parejas de PYR/PYL-CAR. La cristalización de la proteína CAR4 reveló que, además de un dominio C2 clásico de unión a lípidos dependiente de calcio, las proteínas de la familia CAR presentan un dominio específico que probablemente es responsable de la interacción con los receptores PYR/PYL/RCAR y de su posterior reclutamiento a las vesículas de fosfolípidos. Esta interacción es relevante para la función de los receptores PYR/PYL/RCAR en la señalización del ABA, ya que diferentes mutantes triples car de pérdida de función, que tienen afectados los genes CAR1, CAR4, CAR5, y CAR9, demostraron una reducción de la sensibilidad al ABA en ensayos de establecimiento de plántula y crecimiento de la raíz. En resumen, hemos identificado nueva familia de proteínas que son capaces mediar las interacciones transitorias dependientes de Ca2+ con vesículas de fosfolípidos, lo que a su vez afecta localización de PYR/PYL/RCAR y regula positivamente la señalización de ABA. / [CA] RESUM La senyalització per l'hormona vegetal àcid abcíssic (ABA) exerceix un paper crític en la regulació del creixement de l'arrel i també en l'arquitectura del sistema radical. La promoció del creixement de l'arrel en condicions d'estrés hídric, regulada per ABA és clau per la supervivència de les plantes sota condicions limitants d'aigua. Amb aquest treball, hem investigat el paper dels receptors PYR/PYL/RCAR (PYRABACTIN RESISTANCE1 (PYR1)/PYR1 LIKE (PYL)/ REGULATORY COMPONENTS OF ABA RECEPTORS) d'Arabidopsis (Arabidopsis thaliana) en el camí de senyalització d'ABA en arrel. Així, hem descobert que el receptor d'ABA PYL8 exerceix un paper no redundant en la regulació de la percepció d'ABA en arrel. Inesperadament, donada la naturalesa multigènica i la redundància funcional parcial que s'observa en la família PYR/PYL/RCAR, el mutant pyl8 va ser l'únic mutant senzill de pèrdua de funció dels receptors PYR/PYL/RCAR que mostrava una sensibilitat reduïda a la inhibició del creixement mitjançada per l'ABA en l'arrel. Doncs aquest efecte es deu a la falta d'inhibició regulada per PYL8 de diverses fosfatases del grup A tipus 2C (PP2Cs), ja que PYL8 té la capacitat d'interactuar in vivo almenys amb cinc PP2Cs, anomenades HYPERSENSITIVE TO ABA1 (HAB1), HAB2, ABAINSENSITIVE1 (ABI1), ABI2, and PP2CA/ABAHYPERSENSITIVE GERMINATION3 segons ho han revelat per una banda la purificació per afinitat en tàndem (TAP són les seues sigles en anglés) i per altra banda, estudis proteòmics d'espectrometria de masses. Pel que fa a la transducció del senyal del l'ABA, la qual es localitza en la membrana plasmàtica cel¿lular, juga un paper molt important en els primers instants de la senyalització de la fitohormona, no obstant això els mecanismes moleculars que uneixen els components bàsics d'aquesta senyalització amb la membrana plasmàtica, no es troben del tot clars. Per tant, s'han estudiat les interaccions que tenen els receptors del ABA PYR/PYL/RCAR amb la membrana plasmàtica, i hem trobat que aquests tenen la capacitat d'interaccionar transitòriament amb la membrana de forma dependent al calci, gràcies a una família de proteïnes amb domini C2, les quals es troben relacionades amb la ruta de senyalització d'ABA(anomenades C2domain ABArelated (CAR) proteins).Específicament, es va trobar que PYL4 interacciona d'una manera independent al ABA amb CAR1, tant en la membrana plasmàtica, com en el nucli de les cèl¿lules vegetals. La proteïna CAR1 pertany a la família multigènica constituïda per 10 components en Arabidopsis thaliana, des de CAR1 fins CAR10, que tan sols es troba en plantes. Els assajos de complementació bimolecular de fluorescència i de co-immunoprecipitació, van confirmar la interacció en cèl¿lules vegetals, tant de PYL4CAR1 com d'altres parelles de PYR/PYL-CAR. La cristal¿lització de la proteïna CAR4 va revelar que, a més d'un domini C2 clàssic de unió a lípids dependent del calci, les proteïnes de la família CAR presenten un domini PYR/PYL/RCAR, i del seu posterior reclutament a les vesícules fosfolipídiques. Doncs, aquesta interacció és rellevant en la funció dels receptors PYR/PYL/RCAR, ja que participa en la senyalització del l'ABA. Aquesta interacció es clau per a la funció dels receptors, ja que diferents mutants triples car de pèrdua de funció, els quals posseïxen afectats els gens CAR1, CAR4, CAR5 i CAR9, van mostrar una reducció de la sensibilitat a l'ABA en assajos d'establiment de plàntula i creixement de l'arrel. En conclusió, hem identificat una nova família de proteïnes amb la capacitat d'organitzar les interaccions transitòries dependents del calci amb vesícules de fosfolípids, fet que al seu torn afecta la localització de PYR/PYL/RCAR i regula positivament la senyalització d'ABA. / Rodríguez Solovey, LN. (2015). IDENTIFICATION OF TARGETS AND AUXILIARY PROTEINS OF PYR/PYL/RCAR ABA RECEPTORS: PROTEIN PHOSPHATASES TYPE 2C (PP2Cs) AND C2-DOMAIN ABA-RELATED PROTEINS (CARs) [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/58862