FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W3049715207> ?p ?o ?g. }

• W3049715207 endingPage "14221" @default.
• W3049715207 startingPage "14214" @default.
• W3049715207 abstract "T-cell activation is a critical part of the adaptive immune system, enabling responses to foreign cells and external stimulus. In this process, T-cell antigen receptor (TCR) activation stimulates translocation of the downstream kinase PKCθ to the membrane, leading to NF-κB activation and thus transcription of relevant genes. However, the details of how PKCθ is recruited to the membrane remain enigmatic. It is known that annexin A5 (ANXA5), a calcium-dependent membrane-binding protein, has been reported to mediate PKCδ activation by interaction with PKCδ, a homologue of PKCθ, which implicates a potential role of ANXA5 involved in PKCθ signaling. Here we demonstrate that ANXA5 does play a critical role in the recruitment of PKCθ to the membrane during T-cell activation. ANXA5 knockout in Jurkat T cells substantially inhibited the membrane translocation of PKCθ upon TCR engagement and blocked the recruitment of CARMA1-BCL10-MALT1 signalosome, which provides a platform for the catalytic activation of IKKs and subsequent activation of canonical NF-κB signaling in activated T cells. As a result, NF-κB activation was impaired in ANXA5-KO T cells. T-cell activation was also suppressed by ANAX5 knockdown in primary T cells. These results demonstrated a novel role of ANXA5 in PKC translocation and PKC signaling during T-cell activation. T-cell activation is a critical part of the adaptive immune system, enabling responses to foreign cells and external stimulus. In this process, T-cell antigen receptor (TCR) activation stimulates translocation of the downstream kinase PKCθ to the membrane, leading to NF-κB activation and thus transcription of relevant genes. However, the details of how PKCθ is recruited to the membrane remain enigmatic. It is known that annexin A5 (ANXA5), a calcium-dependent membrane-binding protein, has been reported to mediate PKCδ activation by interaction with PKCδ, a homologue of PKCθ, which implicates a potential role of ANXA5 involved in PKCθ signaling. Here we demonstrate that ANXA5 does play a critical role in the recruitment of PKCθ to the membrane during T-cell activation. ANXA5 knockout in Jurkat T cells substantially inhibited the membrane translocation of PKCθ upon TCR engagement and blocked the recruitment of CARMA1-BCL10-MALT1 signalosome, which provides a platform for the catalytic activation of IKKs and subsequent activation of canonical NF-κB signaling in activated T cells. As a result, NF-κB activation was impaired in ANXA5-KO T cells. T-cell activation was also suppressed by ANAX5 knockdown in primary T cells. These results demonstrated a novel role of ANXA5 in PKC translocation and PKC signaling during T-cell activation. T-cell activation is a core part of adaptive immune response, leading to cytokine production and cell proliferation (1Smith-Garvin J.E. Koretzky G.A. Jordan M.S. T cell activation.Annu. Rev. Immunol. 2009; 27 (19132916): 591-61910.1146/annurev.immunol.021908.132706Crossref PubMed Scopus (1366) Google Scholar). Engagement of TCR-CD3 complex with co-receptor CD28 recruits large signaling complexes to signal transduction cascades. The serine/threonine-specific protein kinase C (PKC) activity is required for TCR/CD3-induced T-cell activation (2Gaud G. Lesourne R. Love P.E. Regulatory mechanisms in T cell receptor signalling.Nat. Rev. Immunol. 2018; 18 (29789755): 485-49710.1038/s41577-018-0020-8Crossref PubMed Scopus (237) Google Scholar). PKCθ, a novel calcium-independent member of the PKC family, is proved to selectively mediate several essential functions in TCR-linked signaling, leading to cell activation (3Gerondakis S. Fulford T.S. Messina N.L. Grumont R.J. NF-κB control of T cell development.Nat. Immunol. 2014; 15 (24352326): 15-2510.1038/ni.2785Crossref PubMed Scopus (159) Google Scholar). PKCθ-deficient T cells displayed defects in TCR-induced proliferation and differentiation (4Sun Z. Arendt C.W. Ellmeier W. Schaeffer E.M. Sunshine M.J. Gandhi L. Annes J. Petrzilka D. Kupfer A. Schwartzberg P.L. Littman D.R. PKC-θ is required for TCR-induced NF-κB activation in mature but not immature T lymphocytes.Nature. 2000; 404 (10746729): 402-40710.1038/35006090Crossref PubMed Scopus (788) Google Scholar). PKCθ is usually found in the cytosol when inactive. Upon TCR stimulation, PKCθ rapidly translocates to membrane lipid rafts and activates the downstream signaling, which subsequently results in NF-κB activation (5Bi K. Tanaka Y. Coudronniere N. Sugie K. Hong S. van Stipdonk M.J. Altman A. Antigen-induced translocation of PKC-θ to membrane rafts is required for T cell activation.Nat. Immunol. 2001; 2 (11376344): 556-56310.1038/88765Crossref PubMed Scopus (266) Google Scholar, 6Ruland J. Hartjes L. CARD-BCL-10-MALT1 signalling in protective and pathological immunity.Nat. Rev. Immunol. 2019; 19 (30467369): 118-13410.1038/s41577-018-0087-2Crossref PubMed Scopus (91) Google Scholar). PKCθ translocation to lipid rafts plays a pivotal role in T-cell activation, but the molecular basis of PKCθ translocation has not been elucidated. There have been some investigations on the molecular events of triggering the membrane-bound PKCθ in T-cell activation. PKCθ has no obvious raft-targeting motif, so the lipid raft localization of PKC needs the association with another raft-targeted signaling protein. The cysteine-rich C1 domain of PKCθ can bind membrane-containing second messenger DAG upon stimulation (7Carrasco S. Merida I. Diacylglycerol-dependent binding recruits PKCθ and RasGRP1 C1 domains to specific subcellular localizations in living T lymphocytes.Mol. Biol. Cell. 2004; 15 (15064353): 2932-294210.1091/mbc.e03-11-0844Crossref PubMed Scopus (107) Google Scholar). Phosphoinositide-dependent kinase 1 (PDK1) and LCK have been reported to modulate PKC phosphorylation and its translocation (8Lee K.Y. D'Acquisto F. Hayden M.S. Shim J.H. Ghosh S. PDK1 nucleates T cell receptor-induced signaling complex for NF-κB activation.Science. 2005; 308 (15802604): 114-11810.1126/science.1107107Crossref PubMed Scopus (203) Google Scholar, 9Liu Y. Witte S. Liu Y.C. Doyle M. Elly C. Altman A. Regulation of protein kinase Cθ function during T cell activation by Lck-mediated tyrosine phosphorylation.J. Biol. Chem. 2000; 275 (10652356): 3603-360910.1074/jbc.275.5.3603Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Vav and CD28 have also been reported to mediate the recruitment of PKCθ to immunological synapse by interaction with PKCθ (10Villalba M. Coudronniere N. Deckert M. Teixeiro E. Mas P. Altman A. A novel functional interaction between Vav and PKCθ is required for TCR-induced T cell activation.Immunity. 2000; 12 (10714681): 151-16010.1016/S1074-7613(00)80168-5Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 11Isakov N. Altman A. PKC-θ-mediated signal delivery from the TCR/CD28 surface receptors.Front. Immunol. 2012; 3 (22936936): 27310.3389/fimmu.2012.00273Crossref PubMed Scopus (59) Google Scholar). Although much is known about PKCθ activation, the process of PKCθ translocation and the proteins that regulate it need further identification. The annexin superfamily (Anx) is a calcium (Ca2+)- and phospholipid-binding protein family. Annexin A5 (ANXA5) belongs to the annexin family, which is well-known for its high affinity to phosphatidylserine (PS) and widely used in apoptosis detection (12Volker G. Moss S.E. Annexins: from structure to function.Physiol. Rev. 2002; 82 (11917092): 331-37110.1152/physrev.00030.2001Crossref PubMed Scopus (1626) Google Scholar), even in molecular imaging for disease diagnosis in clinical applications (13Sebastian S. Denise P. Ursula R. Annexins in translational research: hidden treasures to be found.Int. J. Mol. Sci. 2018; 19 (29914106): 1781-179810.3390/ijms19061781Crossref Scopus (45) Google Scholar). ANXA5 is involved in various intra- and extracellular processes, including blood coagulation, anti-inflammatory processes, membrane trafficking, and signal transduction (14Gerke V. Creutz C.E. Moss S.E. Annexins: linking Ca2+ signalling to membrane dynamics.Nat. Rev. Mol. Cell Biol. 2005; 6 (15928709): 449-46110.1038/nrm1661Crossref PubMed Scopus (1130) Google Scholar). However, the biological functions of ANXA5 are believed to depend primarily on its interactions with lipids in membranes. Several annexins have been reported to interact with different PKC isozymes, such as AnxA1, -A2, -A5, and -A6 (15Hoque M. Rentero C. Cairns R. Tebar F. Enrich C. Grewal T. Annexins—scaffolds modulating PKC localization and signaling.Cell. Signal. 2014; 26 (24582587): 1213-122510.1016/j.cellsig.2014.02.012Crossref PubMed Scopus (46) Google Scholar). It has been shown that ANXA5 interacts with PKCδ as an essential step in PKCδ translocation and activation (16Kheifets V. Bright R. Inagaki K. Schechtman D. Mochly-Rosen D. Protein kinase C δ (δPKC)-annexin V interaction: a required step in δPKC translocation and function.J. Biol. Chem. 2006; 281 (16785226): 23218-2322610.1074/jbc.M602075200Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Within the PKC family, PKCδ displays the highest homology with PKCθ. PKCθ is most closely related to PKCδ, because the V1 domains of these two enzymes share 49% homology (17Spitaler M. Cantrell D.A. Protein kinase C and beyond.Nat. Immunol. 2004; 5 (15282562): 785-79010.1038/ni1097Crossref PubMed Scopus (244) Google Scholar, 18Ponting C.P. Parker P.J. Extending the C2 domain family: C2s in PKCs δ, ε, η, θ, phospholipases, GAPs, and perforin.Protein Sci. 1996; 5 (8771209): 162-16610.1002/pro.5560050120Crossref PubMed Scopus (155) Google Scholar). In addition, the V1 domain of PKCδ is required for interaction with ANXA5, so we hypothesize that ANXA5 might be involved in PKCθ membrane translocation and PKCθ-mediated function by interaction with PKCθ. In this study, we demonstrate that ANXA5 is involved in T-cell activation by ANXA5-PKCθ interaction. ANXA5 deficiency selectively inhibited PKCθ-mediated NF-κB activation via blocking the recruitment of CARMA1/BCL10/MALT1 complex. Our results present a novel role of ANXA5 on PKCθ signaling in T-cell activation. To investigate the role of ANXA5 in T-cell activation, we used CRISPR/Cas9 technology to generate ANXA5-KO Jurkat T cells (Fig. 1A). There are three main types of stimulus for T-cell activation: anti-CD3/CD28 co-stimulation, 12-O-tetradecanoylphorbol-13-acetate (TPA), and concanavalin A (ConA) (1Smith-Garvin J.E. Koretzky G.A. Jordan M.S. T cell activation.Annu. Rev. Immunol. 2009; 27 (19132916): 591-61910.1146/annurev.immunol.021908.132706Crossref PubMed Scopus (1366) Google Scholar, 19Chen L. Flies D.B. Molecular mechanisms of T cell co-stimulation and co-inhibition.Nat. Rev. Immunol. 2013; 13 (23470321): 227-24210.1038/nri3405Crossref PubMed Scopus (1804) Google Scholar). With the treatment of each stimulus, T-cell activation was examined in both ANXA5-KO and WT Jurkat T cells. CD69 as a T-cell activation marker is always used to evaluate the degree of T-cell activation by FACS analysis. After 24 h of stimulation, the induction of CD69 expression was readily apparent in WT Jurkat T cells, but not in ANXA5-KO Jurkat T cells (Fig. 1, B and C). By CCK8 assay, ANXA5-KO Jurkat T cells were also shown to be defective in cell proliferation upon anti-CD3/CD28 co-stimulation (Fig. 1D). During T-cell activation, TCR-CD3 engagement leads to increased production of interleukin-2 (IL-2) (20Spolski R. Li P. Leonard W.J. Biology and regulation of IL-2: from molecular mechanisms to human therapy.Nat. Rev. Immunol. 2018; 18 (30089912): 648-65910.1038/s41577-018-0046-yCrossref PubMed Scopus (229) Google Scholar). Consistently, we found that the production of IL-2 was effectively up-regulated in Jurkat T cells responding to various stimulus, but little change was detected in ANXA5-KO cells (Fig. 1E). Collectively, our data suggested that ANXA5 plays an important role in T-cell activation. To explore the signal transduction pathway of ANXA5 in T-cell activation, three major signaling pathways ERK, p38 MAPK, and NF-κB, were examined. In response to anti-CD3/CD28 co-stimulation, the activations of ERK and MAPK pathways were intact in ANXA5-KO Jurkat T cells, but NF-κB activation was impaired (Fig. 2A). The defects on the NF-κB pathway were reconfirmed in two other clones of ANXA5-KO Jurkat T cells (Fig. S1). Similarly, with the treatment of ConA or TPA, ANXA5-KO Jurkat T cells also showed impaired NF-κB activation but normal ERK and p38 MAPK pathways (Fig. 2, B and C). When the expression of ANXA5 was recovered by transfection in ANXA5-KO Jurkat T cells, partial rescue of the IKK activation was observed (Fig. 2D). Together, these results suggested that ANXA5 modulated T-cell activation via the NF-κB signaling pathway. A number of studies have indicated that PKC isozymes play a critical role in mature T-cell activation. We examined the kinase activity of PKC isozymes in ANXA5-KO Jurkat T cells. Upon TPA stimulation, PKC activity was weaker in ANXA5-KO Jurkat T cells compared with the parent Jurkat T cells (Fig. 3A), suggesting that ANXA5 deletion partially suppressed PKC activation. By examination of various PKC isoforms, we found that ANXA5 knockout selectively inhibited PKCθ activation, whereas it had no impact on PKCα and PKCμ activations (Fig. 3B). PKCθ is mainly expressed in T cells and involved in TCR-induced proliferation, cytokine production, and differentiation (21Hayashi K. Altman A. Protein kinase C θ (PKCθ): a key player in T cell life and death.Pharmacol. Res. 2007; 55 (17544292): 537-54410.1016/j.phrs.2007.04.009Crossref PubMed Scopus (153) Google Scholar). Differing from other PKCs in T cells, PKCθ is unique in its translocation to the site of the immunological synapse on the plasma membrane (3Gerondakis S. Fulford T.S. Messina N.L. Grumont R.J. NF-κB control of T cell development.Nat. Immunol. 2014; 15 (24352326): 15-2510.1038/ni.2785Crossref PubMed Scopus (159) Google Scholar). Membrane translocation of cytosolic PKC is the hallmark of PKC activation (22Gould C. Newton A. The life and death of protein kinase C.Curr. Drug Targets. 2008; 9 (18691009): 614-62510.2174/138945008785132411Crossref PubMed Scopus (109) Google Scholar). Based on the above finding, we speculated a possibility that ANXA5 acts as a PKCθ membrane target. To test this hypothesis, we performed a co-immunoprecipitation assay in Jurkat T cells. There was ANXA5-PKCθ interaction in normal T cells, and their interaction was significantly enhanced in activated T cells (Fig. 3C), suggesting that ANXA5 is involved in T-cell activation by association with PKCθ. The Ca2+ increase is an early signaling following the engagement of TCR (2Gaud G. Lesourne R. Love P.E. Regulatory mechanisms in T cell receptor signalling.Nat. Rev. Immunol. 2018; 18 (29789755): 485-49710.1038/s41577-018-0020-8Crossref PubMed Scopus (237) Google Scholar). ANXA5 can rapidly translocate from the cytosol to the plasma membrane upon Ca2+ elevation (the elevation of calcium ion concentration) (15Hoque M. Rentero C. Cairns R. Tebar F. Enrich C. Grewal T. Annexins—scaffolds modulating PKC localization and signaling.Cell. Signal. 2014; 26 (24582587): 1213-122510.1016/j.cellsig.2014.02.012Crossref PubMed Scopus (46) Google Scholar). There was no obvious difference in the increase of Ca2+ initiated by anti-CD3/CD28 co-stimulation between ANXA5-KO and WT Jurkat T cells (Fig. 3D). Next, we tested whether ANXA5 is required for PKCθ translocation. By immunofluorescence observation, ANXA5 and PKCθ were distributed in the cytoplasm in resting Jurkat T cells and rapidly translocated and co-localized in the plasma membrane upon anti-CD3/CD28 co-stimulation (Fig. 3E). However, in ANXA5-KO Jurkat T cells, PKCθ was predominately located in the cytosol, and there was almost no localization on the membrane in response to TCR stimulus, supporting ANXA5 as a binding target for PKCθ translocation in the process of T-cell activation (Fig. 3E). Further confirmation was performed using cellular fractionation for Western blotting analysis. Consistent with the immunofluorescence observation, PKCθ was not detected in the membrane fraction, indicating that PKCθ translocation was lost in ANXA5-KO Jurkat T cells. In contrast, the presence of ANXA5 in Jurkat T cells led to membrane-bound PKCθ following anti-CD3/CD28 co-stimulation (Fig. 3F). Together, our data suggested that ANXA5 was a PKCθ-binding protein required for PKCθ translocation on the membrane. Recent reports have shown that PKCθ cooperates with CARMA1-Bcl10-MALT1 complex to activate the NF-κB pathway. To validate the importance of ANXA5-PKCθ association for NF-κB activation, we generated PKCθ knockout cells on the basis of Jurkat T cells for investigation (Fig. S2A). As expected, NF-κB activation was inhibited, but ERK and p38 activations were unaffected in PKCθ-KO Jurkat T cells (Fig. 4A and Fig. S4), just like the same phenotype in ANXA5-KO Jurkat T cells. Next, we examined the effect of ANXA5 on PKCθ-mediated function. The PKCθ-mediated CARMA1 phosphorylation is crucial for the assembly of CARMA1-Bcl10-MALT1 (CBM) signaling complex in T cells (23David L. Li Y. Ma J. Garner E. Zhang X. Wu H. Assembly mechanism of the CARMA1-BCL10-MALT1-TRAF6 signalosome.Proc. Natl. Acad. Sci. U. S. A. 2018; 115 (29382759): 1499-150410.1073/pnas.1721967115Crossref PubMed Scopus (60) Google Scholar). Consistent with this report, the phosphorylation of CARMA1 was actually inhibited in PKCθ-KO Jurkat T cells (Fig. 4B), which was reconfirmed by another clone of PKCθ-KO cells (Fig. S2B). Similar to PKCθ-KO Jurkat T cells, ANXA5-KO Jurkat T cells also showed the inhibition on the phosphorylation of CARMA1 in response to anti-CD3/CD28 co-stimulation (Fig. 4C) or TPA (Fig. 4D) or ConA treatment (Fig. 4E). Phosphorylated CARMA1 acts as a seed for CBM complex assembly in TCR-mediated cell activation. Consistently, we found that there were normal translocations of CARMA1, Bcl10, and MALT1 from the soluble to the cell membrane fraction in WT Jurkat T cells, but little in ANXA5-KO Jurkat T cells (Fig. 4F). Furthermore, we validated the involvement of ANXA5 in the CBM complex formation by a co-immunoprecipitation assay. The result showed that the formation of the CBM complex was weakened in the absence of ANXA5 but strengthened in the presence of ANXA5, especially in activated T cells (Fig. 4G). Finally, we verified the role of ANXA5 in primary T-cell activation. T lymphocytes isolated from lymph nodes were electrotransfected with ANXA5 siRNAs to knock down endogenous ANXA5 level. With about 16–20% transfection efficiency, the reduced endogenous ANXA5 was detected by Western blotting (Fig. S3). Then these ANXA5 siRNA–treated T cells were activated with anti-CD3/CD28 co-stimulation and analyzed for CD69 expression by FACS. In both CD4+ and CD8+ T cells, the increased CD69 expression induced by TCR stimulation was clearly inhibited by ANXA5 knockdown and, importantly, was rescued by the recovery expression of ANXA5 (Fig. 4H). These results demonstrate that ANXA5 is an important regulator linked to T-cell activation. T-cell activation is accompanied by the clustering of lipid rafts to the site of T-cell engagement and the recruitment of different intracellular signaling proteins into these rafts. Lipid raft recruitment is required for PKCθ to activate NF-κB (5Bi K. Tanaka Y. Coudronniere N. Sugie K. Hong S. van Stipdonk M.J. Altman A. Antigen-induced translocation of PKC-θ to membrane rafts is required for T cell activation.Nat. Immunol. 2001; 2 (11376344): 556-56310.1038/88765Crossref PubMed Scopus (266) Google Scholar). The details of PKCθ translocation are not completely defined in T-cell activation. DAG weakly recruited PKCθ to the membrane, and the PS binding of PKCθ phosphorylation was reported to enhance its binding to DAG, resulting in PKCθ activation (21Hayashi K. Altman A. Protein kinase C θ (PKCθ): a key player in T cell life and death.Pharmacol. Res. 2007; 55 (17544292): 537-54410.1016/j.phrs.2007.04.009Crossref PubMed Scopus (153) Google Scholar). ANXA5 has high PS-binding ability and translocates to membranes, dependent on the increased intracellular Ca2+ levels (14Gerke V. Creutz C.E. Moss S.E. Annexins: linking Ca2+ signalling to membrane dynamics.Nat. Rev. Mol. Cell Biol. 2005; 6 (15928709): 449-46110.1038/nrm1661Crossref PubMed Scopus (1130) Google Scholar, 24Moss S.E. Morgan R.O. The annexins.Genome Biol. 2004; 5 (15059252): 21910.1186/gb-2004-5-4-219Crossref PubMed Scopus (405) Google Scholar). In this study, we reveal an important role of ANXA5 in T-cell activation and provide evidence that ANXA5 may act as an early sensor in PKCθ translocation and activation. As we all know, TCR-stimulated PLCγ1 activity stimulates Ca2+-permeable ion channel receptors (IP3R) on the endoplasmic reticulum membrane, leading to the release of endoplasmic reticulum Ca2+ stores into the cytoplasm. We found that ANXA5 translocated to the membrane along with Ca2+ elevation during T-cell activation, which was essential for the lipid raft recruitment of PKCθ. ANXA5 was previously reported to play a prominent scaffolding role for PKCδ in various signal transduction pathways relevant in cancer (16Kheifets V. Bright R. Inagaki K. Schechtman D. Mochly-Rosen D. Protein kinase C δ (δPKC)-annexin V interaction: a required step in δPKC translocation and function.J. Biol. Chem. 2006; 281 (16785226): 23218-2322610.1074/jbc.M602075200Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Here we demonstrate that ANXA5 interacts with PKCθ and contributes to the membrane translocation of PKCθ in Jurkat T cells (Fig. 3). Our studies indicate that the ANXA5-PKCθ interaction precedes PKCθ translocation and is an essential step in the function of PKCθ. ANXA5 knockout inhibited TCR-induced PKCθ activity. Like the downstream signal transduction of PKCθ, the assembly of CARMA1-Bcl10-MALT1 complex and NF-κB activation were both inhibited by ANXA5 knockout in T-cell activation (Fig. 4). Consistently, the formation of CARMA1-Bcl10-MALT1 complex in the membrane-bound state was undetected in ANXA5-KO Jurkat T cells. Furthermore, there were the same phenotypes between ANXA5-KO and PKCθ-KO T cells, such as the defective NF-κB activation and intact ERK and p38 MAPK pathways, suggesting a functional link between ANXA5 and PKCθ on NF-κB signaling in T cell activation. Recent studies have highlighted the essential role of PKCθ in activating the NF-κB signaling pathway in T cells. PKCθ, but not other PKCs, mediates the activation of the NF-κB complex induced by TCR/CD28 co-stimulation via selective activation of IκB kinase β (IKKβ) (25Xin L. O'Mahony A. Yajun M. Romas G. Warner C. Protein kinase C-θ participates in NF-κB activation induced by CD3-CD28 costimulation through selective activation of IκB kinase β.Mol. Cell. Biol. 2000; 20 (10733597): 2933-294010.1128/mcb.20.8.2933-2940.2000Crossref PubMed Scopus (230) Google Scholar). Thus, NF-κB activation represents the most critical target of PKCθ in the TCR signal leading to production of IL-2, a major T-cell growth factor. In ANXA5-KO T cells, IL-2 production was significantly inhibited, supporting the involvement of ANXA5 in TCR-induced PKCθ activity. Notably, the phosphorylation of PKCθ, not PKCα and PKCμ, was inhibited in the absence of ANXA5, suggesting the selective regulation of ANXA5 on PKCθ function. Studies on PKCθ-deficient mice showed apparently relieved symptoms of multiple sclerosis, inflammatory bowel disease, arthritis, and asthma (26Healy A.M. Izmailova E. Fitzgerald M. Walker R. Hattersley M. Silva M. Siebert E. Terkelsen J. Picarella D. Pickard M.D. LeClair B. Chandra S. Jaffee B. PKC-θ-deficient mice are protected from Th1-dependent antigen-induced arthritis.J. Immunol. 2006; 177 (16849501): 1886-189310.4049/jimmunol.177.3.1886Crossref PubMed Scopus (98) Google Scholar, 27Tan S.L. Zhao J. Bi C. Chen X.C. Hepburn D.L. Wang J. Sedgwick J.D. Chintalacharuvu S.R. Na S. Resistance to experimental autoimmune encephalomyelitis and impaired IL-17 production in protein kinase Cθ-deficient mice.J. Immunol. 2006; 176 (16493044): 2872-287910.4049/jimmunol.176.5.2872Crossref PubMed Scopus (124) Google Scholar, 28Salek-Ardakani S. So T. Halteman B.S. Altman A. Croft M. Protein kinase Cθ controls Th1 cells in experimental autoimmune encephalomyelitis.J. Immunol. 2005; 175 (16301673): 7635-764110.4049/jimmunol.175.11.7635Crossref PubMed Scopus (95) Google Scholar, 29Salek-Ardakani S. So T. Halteman B.S. Altman A. Croft M. Differential regulation of Th2 and Th1 lung inflammatory responses by protein kinase Cθ.J. Immunol. 2004; 173 (15528385): 6440-644710.4049/jimmunol.173.10.6440Crossref PubMed Scopus (112) Google Scholar, 30Curnock A. Bolton C. Chiu P. Doyle E. Fraysse D. Hesse M. Jones J. Weber P. Jimenez J.M. Selective protein kinase Cθ (PKCθ) inhibitors for the treatment of autoimmune diseases.Biochem. Soc. Trans. 2014; 42 (25399564): 1524-152810.1042/BST20140167Crossref PubMed Scopus (11) Google Scholar). Future research on ANXA5 in modulating PKCθ activation might serve as a new target for the selective regulation of PKC signaling in health and disease. Several annexins have been reported in immunological process, such as ANXA1, ANXA2, and ANXA6 (31Bruschi M. Petretto A. Vaglio A. Santucci L. Candiano G. Ghiggeri G.M. Annexin A1 and autoimmunity: from basic science to clinical applications.Int. J. Mol. Sci. 2018; 19 (29751523): 134810.3390/ijms19051348Crossref Scopus (35) Google Scholar, 32Marlin R. Pappalardo A. Kaminski H. Willcox C.R. Pitard V. Netzer S. Khairallah C. Lomenech A.M. Harly C. Bonneville M. Moreau J.F. Scotet E. Willcox B.E. Faustin B. Déchanet-Merville J. Sensing of cell stress by human γδ TCR-dependent recognition of annexin A2.Proc. Natl. Acad. Sci. U. S. A. 2017; 114 (28270598): 3163-316810.1073/pnas.1621052114Crossref PubMed Scopus (75) Google Scholar, 33Cornely R. Pollock A.H. Rentero C. Norris S.E. Alvarez-Guaita A. Grewal T. Mitchell T. Enrich C. Moss S.E. Parton R.G. Rossy J. Gaus K. Annexin A6 regulates interleukin-2-mediated T-cell proliferation.Immunol. Cell Biol. 2016; 94 (26853809): 543-55310.1038/icb.2016.15Crossref PubMed Scopus (21) Google Scholar). However, little attention has been paid to the immunological role of ANXA5. Our study fills a gap in this knowledge about an important role of ANXA5 in T-cell activation. ANXA5 as a PKCθ association partner will provide new clues to the complicated molecular mechanism of PKCθ function during T-cell activation. The human acute T-cell leukemia cell line Jurkat (ATCC TIB-152™) was cultured in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Gibco), 1% (v/v) penicillin–streptomycin (Gibco). Primary T cells were prepared from lymph node of mice. Cells were grown at 37 °C in 5% CO2. All experiments were approved by the State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University. Gene knockout was conducted with a CRISPR/Cas9 system as described previously (34Qian X. Li X. Shi Z. Xia Y. Cai Q. Xu D. Tan L. Du L. Zheng Y. Zhao D. Zhang C. Lorenzi P.L. You Y. Jiang B.H. Jiang T. et al.PTEN suppresses glycolysis by dephosphorylating and inhibiting autophosphorylated PGK1.Mol. Cell. 2019; 76 (31492635): 516-527.e710.1016/j.molcel.2019.08.006Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Single guide RNAs targeting the ANXA5 (5′-AGGGTACTACCAGCGGATGT-3′) and PKCθ (5′-GCCGCCATGTTTACCGACAC-3′) genes were designed using an online CRISPR design platform (https://zlab.bio/guide-design-resources), and each was cloned into pLentiCRISPRv2. Briefly, HEK-293T cells were co-transfected with constructed pLentiCRISPRv2 plasmid and lentiviral envelope plasmids (PL3, PL4, and PL5). The viruses were harvested by ultracentrifugation 3 days after transfection. Viruses were then added into the cultures of Jurkat T cells, followed by selection with puromycin (2 μg/ml). Finally, single-cell clones were separated by serial dilutions in a 96-well plate and then transferred to 6-well plates. The knockout cell clones were identified by Western blotting. For the recovery expression in ANXA5-KO Jurkat T cells, human ANXA5 cDNA was cloned into the plenti6/v5-D-Topo expression vector (Invitrogen). Then ANXA5 expression plasmid and lentiviral envelope plasmids (PL3, PL4, and PL5) were co-transfected into HEK-293T cells. The viruses were harvested on day 3. Then ANXA5-KO cells were treated with lentiviral transduction. 24 h after transduction, 10 μg/ml blasticidin was added into the medium for selection. Finally, the expression of ANAX5 was detected by Western blotting. Cells were cytospun, fixed with 3.75% formaldehyde/PBS, and permeabilized with 0.1% (v/v) Triton X-100. After blocking with 10% goat serum, sections were incubated with anti-human PKCθ and ANXA5 primary antibodies (1:50; Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. After washing, slides were incubated with appropriate fluorochrome-conjugated secondary antibody (1:1000; Invitrogen) for 1 h at room temperature in the dark. Slides were counterstained with 4′,6-diamidino-2-phenylindole (Invitrogen). All images were visualized and captured by a fluorescence microscope (Zeiss AX10, Carl Zeiss AG, Jena, Germany). Cells were seeded at a density of 1000 cells/plate and were maintained in culture for 0, 48, 72, and 108 h. Cell proliferation was detected by a CCK8 cell proliferation kit (Beyotime, Shanghai, China). Cell activation was measured by a flow cytometer (FACSCalibur, BD Biosciences, Mississauga, Canada) equipped with Cell Quest software (BD Biosciences). Antibodies against human CD69 or mouse CD69 were purchased from BD Pharmingen (San Diego, CA). Cells were fractionated using a membrane protein extraction kit (Beyotime). Briefly, cells were lysed using lysis buffer A provided by the kit and then homogenized in ice. The lysates were centrifuged at 700 × g for 10 min at 4 °C, and the supernatant was collected and spun at 14,000 × g for 30 min at 4 °C. The pellets were suspended using extraction buffer B and incubated for 20 min. After centrifugation at 14,000 × g for 5 min at 4 °C, the supernatant was used as the membranous fraction. The samples were then analyzed by Western blotting. Total RNA was extracted using TRIzol (Invitrogen Life Technologies). Reverse transcription was accomplished with a PrimeScript RT reagent kit (Takara). Quantitative PCR was performed with SYBR Green PCR Master Mix according to the manufacturer's instructions (Vazyme) on a StepOne/StepOne PlusTM real-time PCR system (Applied Biosystems). Sequence-specific primers for human IL-2 (forward primer, 5′-TACAAGAATCCCAAACTCACCAG-3′; reverse primer, 5′-GGCACAAAAAGAATCATAAAAGA-3′) and human actin (forward primer, 5′-TGGTGATGGAGGAGGTTTAGTAAGT-3′; reverse primer, 5′-AACCAATAAAACCTACTCCTCCCTTAA-3′) were used. 6–12-week-old mice were purchased from the Model Animal Research Center of Nanjing University. All the animal experiments were approved by the Nanjing University Animal Care and Use Committee. The mice were sacrificed and sterilized with 75% ethanol. Lymph nodes were isolated and prepared for single-cell suspension. Primary murine T cells were maintained in RPMI 1640 medium (Gibco) supplemented with 100 mg/ml streptomycin, 100 units/ml penicillin, and 10% fetal calf serum. For primary murine T-cell transfection, the mouse T Cell NucleofectorTM kit (Lonza) was used for electrotransfection with the Amaxa transfection device NucleofectorTM II according to the manufacturer's instructions. In brief, 1 × 107 cells were resuspended in 100 μl of room temperature Nucleofector® solution (Lonza) and electroporated with 40 pmol of negative control siRNAs, 40 pmol of ANXA5 siRNA, or 40 pmol of ANXA5 siRNAs together with 2 μg of plasmid pCMV-ANXA5, respectively. CD69 expression was examined by flow cytometer 24 h after electrotransfection. The transfection of 2 μg of pmax-GFP (Lonza) was used as a positive control for indicating the transfection efficiency measured by flow cytometer. Negative control siRNA (5′-UUCUCCGAACGUGUCACGUTT-3′) and mouse ANXA5 siRNA (5′-AUGCUCCGAAUAGACUUCACGTT-3′) were purchased from GenePharma. The plasmid of pCMV-ANXA5 was constructed and stored by our laboratory. All experiments were conducted at least three times. The experimental data were processed by GraphPad Prism 7.00 and are presented as mean ± S.D. A p value of <0.05 shows that there is a statistically significant difference, marked with an asterisk. p values of <0.01 and <0.001 are marked with two and three asterisks, respectively. All data are included in the article. We thank Ben Li (School of Life Sciences, Nanjing University, Nanjing, China) for helpful discussion on the manuscript. Download .pdf (2.64 MB) Help with pdf files T-cell receptor protein kinase C phosphatidylserine 12-O-tetradecanoylphorbol-13-acetate concanavalin A interleukin extracellular signal–regulated kinase mitogen-activated protein kinase CARMA1-Bcl10-MALT1 knockout." @default.
• W3049715207 created "2020-08-21" @default.
• W3049715207 creator A5017947172 @default.
• W3049715207 creator A5029491821 @default.
• W3049715207 creator A5036921991 @default.
• W3049715207 creator A5049841636 @default.
• W3049715207 creator A5058965019 @default.
• W3049715207 creator A5059817966 @default.
• W3049715207 creator A5062813777 @default.
• W3049715207 creator A5064761273 @default.
• W3049715207 creator A5067290584 @default.
• W3049715207 creator A5072479022 @default.
• W3049715207 creator A5088335237 @default.
• W3049715207 date "2020-10-01" @default.
• W3049715207 modified "2023-10-12" @default.
• W3049715207 title "Annexin A5 is essential for PKCθ translocation during T-cell activation" @default.
• W3049715207 cites W1524506477 @default.
• W3049715207 cites W1585445496 @default.
• W3049715207 cites W1894231348 @default.
• W3049715207 cites W1932669647 @default.
• W3049715207 cites W1988811776 @default.
• W3049715207 cites W1999500196 @default.
• W3049715207 cites W2015080165 @default.
• W3049715207 cites W2021911222 @default.
• W3049715207 cites W2025848946 @default.
• W3049715207 cites W2027731275 @default.
• W3049715207 cites W2046445536 @default.
• W3049715207 cites W2046887284 @default.
• W3049715207 cites W2058811157 @default.
• W3049715207 cites W2073394241 @default.
• W3049715207 cites W2080692650 @default.
• W3049715207 cites W2090757010 @default.
• W3049715207 cites W2093853778 @default.
• W3049715207 cites W2100412095 @default.
• W3049715207 cites W2120812662 @default.
• W3049715207 cites W2123383768 @default.
• W3049715207 cites W2130473166 @default.
• W3049715207 cites W2138375420 @default.
• W3049715207 cites W2138651971 @default.
• W3049715207 cites W2322953082 @default.
• W3049715207 cites W2331807942 @default.
• W3049715207 cites W2593763775 @default.
• W3049715207 cites W2802580172 @default.
• W3049715207 cites W2803300773 @default.
• W3049715207 cites W2804321178 @default.
• W3049715207 cites W2887245816 @default.
• W3049715207 cites W2901928825 @default.
• W3049715207 cites W2972278366 @default.
• W3049715207 cites W4244376495 @default.
• W3049715207 cites W4254666598 @default.
• W3049715207 doi "https://doi.org/10.1074/jbc.ra120.015143" @default.
• W3049715207 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/7549025" @default.
• W3049715207 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/32796034" @default.
• W3049715207 hasPublicationYear "2020" @default.
• W3049715207 type Work @default.
• W3049715207 sameAs 3049715207 @default.
• W3049715207 citedByCount "6" @default.
• W3049715207 countsByYear W30497152072021 @default.
• W3049715207 countsByYear W30497152072022 @default.
• W3049715207 countsByYear W30497152072023 @default.
• W3049715207 crossrefType "journal-article" @default.
• W3049715207 hasAuthorship W3049715207A5017947172 @default.
• W3049715207 hasAuthorship W3049715207A5029491821 @default.
• W3049715207 hasAuthorship W3049715207A5036921991 @default.
• W3049715207 hasAuthorship W3049715207A5049841636 @default.
• W3049715207 hasAuthorship W3049715207A5058965019 @default.
• W3049715207 hasAuthorship W3049715207A5059817966 @default.
• W3049715207 hasAuthorship W3049715207A5062813777 @default.
• W3049715207 hasAuthorship W3049715207A5064761273 @default.
• W3049715207 hasAuthorship W3049715207A5067290584 @default.
• W3049715207 hasAuthorship W3049715207A5072479022 @default.
• W3049715207 hasAuthorship W3049715207A5088335237 @default.
• W3049715207 hasBestOaLocation W30497152071 @default.
• W3049715207 hasConcept C104317684 @default.
• W3049715207 hasConcept C138626823 @default.
• W3049715207 hasConcept C153911025 @default.
• W3049715207 hasConcept C185592680 @default.
• W3049715207 hasConcept C195794163 @default.
• W3049715207 hasConcept C55493867 @default.
• W3049715207 hasConcept C62478195 @default.
• W3049715207 hasConcept C86803240 @default.
• W3049715207 hasConcept C95444343 @default.
• W3049715207 hasConceptScore W3049715207C104317684 @default.
• W3049715207 hasConceptScore W3049715207C138626823 @default.
• W3049715207 hasConceptScore W3049715207C153911025 @default.
• W3049715207 hasConceptScore W3049715207C185592680 @default.
• W3049715207 hasConceptScore W3049715207C195794163 @default.
• W3049715207 hasConceptScore W3049715207C55493867 @default.
• W3049715207 hasConceptScore W3049715207C62478195 @default.
• W3049715207 hasConceptScore W3049715207C86803240 @default.
• W3049715207 hasConceptScore W3049715207C95444343 @default.
• W3049715207 hasFunder F4320321001 @default.
• W3049715207 hasFunder F4320335774 @default.
• W3049715207 hasIssue "41" @default.
• W3049715207 hasLocation W30497152071 @default.
• W3049715207 hasLocation W30497152072 @default.
• W3049715207 hasLocation W30497152073 @default.
• W3049715207 hasOpenAccess W3049715207 @default.

• next