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1.
Stimulated dendritic cells (DCs) mature and migrate to lymphoid organs to prime naive T cells. DC maturation augments antigen-presentation capacity of DCs by increasing peptide loading, half-life, and cell surface localization of MHC molecules. Activated SWAP-70(-/-) DCs fail to properly localize MHCII molecules in the plasma membrane, are strongly impaired in T-cell activation, and are altered in F-actin rearrangement. MHCII synthesis, invariant chain removal, and MHCII internalization, however, are unaffected. MHCII surface localization is known to require RhoGTPases. Surprisingly, SWAP70, hitherto known to bind F-actin and Rac, also binds RhoA-GTP. In SWAP-70(-/-) DCs, RhoA and RhoB are stimulus-independent and constitutively active. Surface localization of MHCII molecules and T-cell activation can be restored by blocking RhoA and RhoB before but not during DC activation. Thus, contrasting positive regulation of Rac, SWAP-70 negatively regulates RhoA and-indirectly-RhoB, preventing premature RhoA/RhoB activation. Through RhoA/RhoB regulation, SWAP-70 defines a new pathway to control surface localization of MHCII, a critical element in DC-dependent immune responses. 相似文献
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cAMP-dependent protein kinase phosphorylation of the acid-sensing ion channel-1 regulates its binding to the protein interacting with C-kinase-1 总被引:1,自引:0,他引:1
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Leonard AS Yermolaieva O Hruska-Hageman A Askwith CC Price MP Wemmie JA Welsh MJ 《Proceedings of the National Academy of Sciences of the United States of America》2003,100(4):2029-2034
The acid-sensing ion channel-1 (ASIC1) contributes to synaptic plasticity and may influence the response to cerebral ischemia and acidosis. We found that cAMP-dependent protein kinase phosphorylated heterologously expressed ASIC1 and endogenous ASIC1 in brain slices. ASIC1 also showed significant phosphorylation under basal conditions. Previous studies showed that the extreme C-terminal residues of ASIC1 bind the PDZ domain of the protein interacting with C-kinase-1 (PICK1). We found that protein kinase A phosphorylation of Ser-479 in the ASIC1 C terminus interfered with PICK1 binding. In contrast, minimizing phosphorylation or mutating Ser-479 to Ala enhanced PICK1 binding. Phosphorylation-dependent disruption of PICK1 binding reduced the cellular colocalization of ASIC1 and PICK1. Thus, the ASIC1 C terminus contains two sites that influence its binding to PICK1. Regulation of this interaction by phosphorylation provides a mechanism to control the cellular localization of ASIC1. 相似文献
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Endothelial cell (EC) migration is a complex process requiring exquisitely coordinated focal adhesion assembly and disassembly. Protein kinase C (PKC) is known to regulate focal adhesion formation. Because lysophosphatidylcholine (lysoPC), a major lipid constituent of oxidized low-density lipoprotein, can activate PKC and inhibit EC migration, we explored the signaling cascade responsible for this inhibition. LysoPC increased PKCdelta activity, measured by in vitro kinase activity assay, and increased PKCdelta phosphorylation. Decreasing PKCdelta activation, using pharmacological inhibitors or antisense oligonucleotides, diminished the antimigratory effect of lysoPC. LysoPC-induced PKCdelta activation was followed by increased phosphorylation of the transmembrane proteoglycan, syndecan-4, and decreased binding of PKCalpha to syndecan-4, with a concomitant decrease in PKCalpha activity. A reciprocal relationship was noted between the interaction of PKCalpha and alpha-actinin with syndecan-4. These changes were temporally related to the observed changes in cell morphology and the inhibition of migration of ECs incubated with lysoPC. The data suggested that generalized activation of PKCdelta by lysoPC initiated a cascade of events, including phosphorylation of syndecan-4, displacement and decreased activity of PKCalpha, binding of alpha-actinin to syndecan-4, and disruption of the time- and site-specific regulation of focal adhesion complex assembly and disassembly required for normal cell migration. 相似文献
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Masat L Caldwell J Armstrong R Khoshnevisan H Jessberger R Herndier B Wabl M Ferrick D 《Proceedings of the National Academy of Sciences of the United States of America》2000,97(5):2180-2184
SWAP-70 is a component of an enzyme complex that recombines Ig switch regions in vitro. We report here the cloning of the human cDNA and its B lymphocyte-specific expression. Although its sequence contains three nuclear localization signals, in small resting B cells, SWAP-70 is mainly found in the cytoplasm. On stimulation, SWAP-70 translocates to the nucleus. In activated, class-switching B cell cultures, it is associated with membrane IgG, but not IgM. The membrane Ig association requires a functional pleckstrin homology domain and is controlled by the C terminus. We suggest that SWAP-70 is involved not only in nuclear events but also in signaling in B cell activation. 相似文献
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Protein tyrosine phosphatase receptor-type O truncated (PTPROt) regulates SYK phosphorylation, proximal B-cell-receptor signaling, and cellular proliferation
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The strength and duration of B-cell-receptor (BCR) signaling depends upon the balance between protein tyrosine kinase (PTK) activation and protein tyrosine phosphatase (PTP) inhibition. BCR-dependent activation of the SYK PTK initiates downstream signaling events and amplifies the original BCR signal. Although BCR-associated SYK phosphorylation is clearly regulated by PTPs, SYK has not been identified as a direct PTP substrate. Herein, we demonstrate that SYK is a major substrate of a tissue-specific and developmentally regulated PTP, PTP receptor-type O truncated (PTPROt). PTPROt is a member of the PTPRO family (also designated GLEPP, PTP-?, PTP-oc, and PTPu2), a group of highly conserved receptor-type PTPs that are thought to function as tumor suppressor genes. The overexpression of PTPROt inhibited BCR-triggered SYK tyrosyl phosphorylation, activation of the associated adaptor proteins SHC and BLNK, and downstream signaling events, including calcium mobilization and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) activation. PTPROt overexpression also inhibited lymphoma cell proliferation and induced apoptosis in the absence of BCR cross-linking, suggesting that the phosphatase modulates tonic BCR signaling. 相似文献
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Decreased susceptibility of a 70-kDa protein to cathepsin L after phosphorylation by protein kinase C. 总被引:2,自引:0,他引:2
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S Laumas M Abdel-Ghany K Leister R Resnick A Kandrach E Racker 《Proceedings of the National Academy of Sciences of the United States of America》1989,86(9):3021-3025
A 70-kDa protein is phosphorylated in cell-free preparations from rat or mouse fibroblasts by an endogenous protein kinase. This protein is immunologically related to a group of 68-kDa to 87-kDa proteins described in the literature as substrates for protein kinase C (PK-C). Although the phosphorylation of the 70-kDa protein by isolated plasma membranes takes place in the presence of EGTA, we conclude that the reaction is catalyzed by PK-C based on its inhibition by staurosporin. As shown previously, pure PK-C phosphorylates a synthetic random polymer of arginine and serine in the absence of Ca2+ and lipids, a reaction markedly stimulated by an endogenous unidentified activator of PK-C. When the 70-kDa protein from normal fibroblasts was exposed to the cytosol of chemically or ras-transformed fibroblasts, it disappeared as measured by phosphorylation by added PK-C. Cytosol of normal fibroblasts was much less effective (ca. 20%). Cathepsin L purified from rat kidney or from the medium of transformed cells had an effect similar to that of the cytosol of transformed cells. When the 70-kDa protein was phosphorylated by PK-C prior to exposure to cathepsin L or to the cytosol of transformed cells, there was a marked protection of the 70-kDa protein. We conclude that the 70-kDa protein is degraded by cathepsin L as ascertained by both immunological and biochemical assays and that it is protected by prior phosphorylation with PK-C. The possible role of this effect in signal transduction is discussed. 相似文献
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Seong-Hye Hwang Seung-Hyun Jung Saseong Lee Susanna Choi Seung-Ah Yoo Ji-Hwan Park Daehee Hwang Seung Cheol Shim Laurent Sabbagh Ki-Jo Kim Sung Hwan Park Chul-Soo Cho Bong-Sung Kim Lin Leng Ruth R. Montgomery Richard Bucala Yeun-Jun Chung Wan-Uk Kim 《Proceedings of the National Academy of Sciences of the United States of America》2015,112(47):E6535-E6543
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Clinical significance of ZAP-70 protein expression in B-cell chronic lymphocytic leukemia 总被引:7,自引:1,他引:6
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Del Principe MI Del Poeta G Buccisano F Maurillo L Venditti A Zucchetto A Marini R Niscola P Consalvo MA Mazzone C Ottaviani L Panetta P Bruno A Bomben R Suppo G Degan M Gattei V de Fabritiis P Cantonetti M Lo Coco F Del Principe D Amadori S 《Blood》2006,108(3):853-861
The clinical course of B-cell chronic lymphocytic leukemia (B-CLL) is variable, and novel biologic parameters need to be added to the clinical staging systems to predict an indolent or aggressive outcome. We investigated the 70-kDa zeta-associated protein (ZAP-70), CD38, soluble CD23 (sCD23), and cytogenetics in 289 patients with B-CLL. Both a shorter progression-free survival (PFS) and overall survival (OS) were observed in ZAP-70(+) (P < .001), in CD38(+) (P < .001) and in sCD23(+) patients (P < .001 and P = .013, respectively). ZAP-70(+)CD38(+) or ZAP-70(+) patients with an unmutated IgV(H) status showed both a shorter PFS (P < .001) and OS (P < .001 and P < .001, respectively) as compared with ZAP-70(-)/CD38(-) or ZAP-70(-) patients with mutated IgV(H) genes. Discordant patients showed an intermediate outcome. Note, ZAP-70(+) patients even if CD38(-) or mutated showed a shorter PFS, whereas ZAP-70(-) patients even if CD38(+) or unmutated had a longer PFS. Furthermore, ZAP-70 positivity was associated with a shorter PFS both within normal karyotype (P < .001) and within the poor-risk cytogenetic subset (P = .02). The predictive value of ZAP-70 expression was confirmed in multivariate analysis. Thus, ZAP-70 protein determined by flow cytometry improves the prognostic significance of cytogenetics and appears to be a better predictor of outcomes than IgV(H) gene mutational status. On this line, we recommend and are also interested in conducting a prospective randomized trial of early intervention versus observation for ZAP-70(+) patients. 相似文献
11.
Yuwei Li Walter G. Gonzalez Andrey Andreev Weiyi Tang Shashank Gandhi Alexandre Cunha David Prober Carlos Lois Marianne E. Bronner 《Proceedings of the National Academy of Sciences of the United States of America》2020,117(44):27400
Individual cell migration requires front-to-back polarity manifested by lamellipodial extension. At present, it remains debated whether and how membrane motility mediates this cell morphological change. To gain insights into these processes, we perform live imaging and molecular perturbation of migrating chick neural crest cells in vivo. Our results reveal an endocytic loop formed by circular membrane flow and anterograde movement of lipid vesicles, resulting in cell polarization and locomotion. Rather than clathrin-mediated endocytosis, macropinosomes encapsulate F-actin in the cell body, forming vesicles that translocate via microtubules to deliver actin to the anterior. In addition to previously proposed local conversion of actin monomers to polymers, we demonstrate a surprising role for shuttling of F-actin across cells for lamellipodial expansion. Thus, the membrane and cytoskeleton act in concert in distinct subcellular compartments to drive forward cell migration.Cell migration is central to embryogenesis, organogenesis, and cancer metastasis (1, 2). Individual migrating cells rapidly change their shape via cycles of protrusion and retraction (3, 4), raising the question of how a cell distributes its limited amount of membrane to maintain polarized morphology without affecting membrane integrity. Early studies, based on observing the movement of cross-linked antigen on the cell surface, proposed a “membrane flow” model to explain the role of membrane recycling during cell locomotion (5–8). This two-dimensional (2D) model posits that cells undergo clathrin-mediated endocytosis in the rear of the cell, which generates anterograde flow of lipid vesicles and retrograde flow of membrane (Fig. 1A). Such a retrograde membrane flow could enable membrane proteins to generate traction forces against the extracellular matrix (ECM) to push the cell forward; thus, this model in theory explains how mechanical forces could drive cell migration (9, 10). However, other research presents conflicting data (11), likely due to the use of different experimental systems and cell tagging reagents. Moreover, most studies of individual cell motility use cultured cells or simple organisms, such that little is known about how membrane and vesicle motion coordinate to influence three-dimensional (3D) morphological changes of migrating cells in higher vertebrates.Open in a separate windowFig. 1.Circular membrane flow across migrating cells. (A) The 2D “membrane flow” model from top view. Endocytosis at the posterior end produces vesicles that move toward the anterior end (arrows inside the cytoplasm); these vesicles integrate into the lamellipodium and then translate into retrograde membrane flow (arrows outside the cell). (B) The 3D “treadmilling” model from lateral view. F-actin (red) is distributed underneath the plasma membrane. In the basal side of the lamellipodium, actin displays a net polymerization toward the cell’s anterior end. “−” and “+” represents depolymerization and polymerization end of actin, respectively. (C) Schematic illustration of explant culture and imaging. An ∼500-μm-thick transverse slice is dissected through the trunk of virally labeled chicken embryos for confocal time-lapse imaging. Successive movies of individual migrating cells are collected at different positions from the apical to the basal sides of the cell. For quantitative analysis, a coordinate system is used in which 0 denotes the center of the cell body. (D) Live imaging reveals a stereotypical fashion of cell migration (Top). A cell protrudes the lamellipodium (red arrow) and then progressively retracts its body (yellow arrow) toward the anterior end. The cell surface is computationally segmented with each optical slice pseudocolored. The surface area of individual segmented slices is measured, and the results are presented in SI Appendix, Fig. S1 C and D. (Middle) Top view. (Bottom) Lateral view. (Scale bar: 5 µm.) (E–H) Quantification of membrane flow based on the photo-conversion experiment (see SI Appendix, Fig. S1 E–G for details). On the basal side, anterior intensity of the red fluorescence is higher following photo-conversion (t = 15 s) (rank sum test in frame 6, P < 0.001, n = 7 cells) (E), suggesting anterior membrane flow (Schematic, F). This scenario is reversed on the apical side (rank sum test in frame 6, P < 0.001, n = 8 cells) (G and H).In addition to the lipid portion of the plasma membrane that maintains fluidity of the cell boundary, the underlying cytoskeleton provides rigidity to the cell surface (12). A “treadmilling” model was used to explain cytoskeletal regulation of lamellipodial function (13, 14). According to this model, actin polymerization at the cell’s leading edge and actin depolymerization at the back of the network (Fig. 1B) cause relative displacement to the cytosol, which subsequently “pulls” the rounded cell body. Yet, it is unclear whether and how actin turnover on the cell’s basal side is coupled with other actin pools and membrane flow throughout the cells.To address these long-standing cell biology questions in vivo, we directly visualize membrane and cytoskeletal behaviors in migrating neural crest cells at the trunk level of chicken embryos. The neural crest is one of the most migratory of embryonic cell types (15), initiating movement via an epithelial to mesenchymal transition from the neural tube (16). These multipotent cells then migrate throughout the periphery as individuals (17, 18), differentiating into diverse cells types including peripheral neurons, glia, and melanocytes of the skin (19). As neural crest-derived cells are prone to give rise to adult cancers, including melanoma, neuroblastoma, and gliomas, their innate migratory mode appears to be recapitulated during cancer metastasis (16). By combining live imaging with quantitative analysis, we extract dynamic molecular and cellular information about cell migration and utilize perturbation approaches to challenge it. 相似文献
12.
目的 探讨早老素(PSI)蛋白突变体在阿尔茨海默病(AD)发生中的作用和PSI蛋白的正常生理功能及分子伴侣蛋白热休克蛋白70同源蛋白羧基端相互作用蛋白(CHIP)的相互作用.方法 采用酵母双杂交系统筛选与PSI蛋白相互作用的蛋白.在构建pGBKT7-PS1-C203诱饵质粒和含全长CHIP的pACT2一CHIP质粒表达载体后,用p-半乳糖苷酶活性测定法检测两者的相互作用;然后转染哺乳动物细胞293T,用Co-IP和Western blot法测试其相瓦作用. 结果 获得了一个能与PSI蛋白相互作用的蛋白,即CHIP,并通过β-半乳糖甘酶活性测试、免疫共沉淀进一步证实了PSI和CHIP相互作用的特异性. 结论 CHIP既可以和分子伴侣蛋白相互作用,本身又具有泛素连接酶活性,可调控蛋白的折叠和降解,PSI与CHIP相互作用的证实,有助于阐明机体泛素-蛋白酶体系统对PSI的调控和进一步阐明AD的病理机制. 相似文献
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Ca2+/calmodulin-dependent protein kinase II phosphorylation regulates the cardiac ryanodine receptor
The cardiac ryanodine receptor (RyR2)/calcium release channel on the sarcoplasmic reticulum is required for muscle excitation-contraction coupling. Using site-directed mutagenesis, we identified the specific Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation site on recombinant RyR2, distinct from the site for protein kinase A (PKA) that mediates the "fight-or-flight" stress response. CaMKII phosphorylation increased RyR2 Ca2+ sensitivity and open probability. CaMKII was activated at increased heart rates, which may contribute to enhanced Ca2+-induced Ca2+ release. Moreover, rate-dependent CaMKII phosphorylation of RyR2 was defective in heart failure. CaMKII-mediated phosphorylation of RyR2 may contribute to the enhanced contractility observed at higher heart rates. The full text of this article is available online at http://circres.ahajournals.org. 相似文献
14.
Kim PM Aizawa H Kim PS Huang AS Wickramasinghe SR Kashani AH Barrow RK Huganir RL Ghosh A Snyder SH 《Proceedings of the National Academy of Sciences of the United States of America》2005,102(6):2105-2110
Serine racemase (SR), localized to astrocytic glia that ensheathe synapses, converts L-serine to D-serine, an endogenous ligand of the NMDA receptor. We report the activation of SR by glutamate neurotransmission involving alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors via glutamate receptor interacting protein (GRIP) and the physiologic regulation of cerebellar granule cell migration by SR. GRIP physiologically binds SR, augmenting SR activity and D-serine release. GRIP infection of neonatal mouse cerebellum in vivo enhances granule cell migration. Selective degradation of D-serine by D-amino acid oxidase and pharmacologic inhibition of SR impede migration, whereas D-serine activates the process. Thus, in neuronal migration, glutamate stimulates Bergmann glia to form and release D-serine, which, together with glutamate, activates NMDA receptors on granule neurons, chemokinetically enhancing migration. 相似文献
15.
A protein phosphorylation/dephosphorylation network regulates a plant potassium channel 总被引:1,自引:0,他引:1
Lee SC Lan WZ Kim BG Li L Cheong YH Pandey GK Lu G Buchanan BB Luan S 《Proceedings of the National Academy of Sciences of the United States of America》2007,104(40):15959-15964
Potassium (K(+)) is an essential nutrient for plant growth and development. Plants often adapt to low K(+) conditions by increasing their K(+) uptake capability. Recent studies have led to the identification of a calcium signaling pathway that enables plants to act in this capacity. Calcium is linked to two calcineurin B-like calcium sensors (CBLs) and a target kinase (CBL-interacting protein kinase 23 or CIPK23) that, in turn, appears to phosphorylate and activate the potassium channel, Arabidopsis K(+) transporter 1 (AKT1), responsible for K(+) uptake in roots. Here, we report evidence that this regulatory mechanism is more elaborate than earlier envisaged. The recently described pathway is part of an extensive network whereby several CBLs interact with multiple CIPKs in the activation of the potassium channel, AKT1. The physical interactions among the CBL, CIPK, and AKT1 components provide a mechanism for specifying the members of the CBL and CIPK families functional in AKT1 regulation. The interaction between the CIPKs and AKT1 was found to involve the kinase domain of the CIPK component and the ankyrin repeat domain of the channel. Furthermore, we identified a 2C-type protein phosphatase that physically interacts and inactivates the AKT1 channel. These findings provide evidence that the calcium-sensitive CBL and CIPK families together with 2C-type protein phosphatases form a protein phoshporylation/dephosphorylation network that regulates the AKT1 channel for K(+) transport in plants. 相似文献
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The leukocyte sialyloglycoprotein CD43 exhibits features of a signal transducing molecule and is thought to be important for T-cell activation and adhesion. However, cellular biochemical events in which CD43 participates remain poorly understood. Here we provide evidence that CD43 regulates tyrosine phosphorylation of a specific substrate in T cells. A 93-kD tyrosine phosphoprotein was identified specifically in the CD43+ T-cell line CEM, but not in their CD43-deficient counterparts derived by gene targeting. The 93-kD phosphoprotein was detected in the CD43-deficient CEM cells after transfection with CD43 cDNA, and it could be specifically phosphorylated in lysates from the CD43-deficient cells by incubation with a CD43 immunoprecipitate obtained from the CD43+ cells. Expression of CD43 in HeLa cell transfectants was associated with the appearance of novel phosphoproteins including one with a molecular weight of approximately 93 kD, confirming that tyrosine phosphorylation of cellular substrates results specifically from CD43 expression. We conclude that CD43 regulates tyrosine phosphorylation of a 93-kD T-cell substrate. 相似文献