Stefan Feske, M.D.

Relative contributions of stromal interaction molecule 1 and CalDAG-GEFI to calcium-dependent platelet activation and thrombosis
Ahmad, F.; Boulaftali, Y.; Greene, T. K.; Ouellette, T. D.; Poncz, M.; Feske, S.; Bergmeier, W.
2011 OCT ;9(10):2077-2086, Journal of thrombosis & haemostasis : JTH
Background: Stromal interaction molecule 1 (STIM1) was recently identified as a critical component of store-operated calcium entry (SOCE) in platelets. We previously reported the Ca(2+)-sensing guanine nucleotide exchange factor CalDAG-GEFI as a critical molecule in Ca(2+) signaling in platelets. Objective: To evaluate the contribution of STIM1/SOCE to Ca(2+)-dependent platelet activation and thrombosis, we here compared the activation responses of platelets lacking STIM1 and platelets lacking CalDAG-GEFI. Methods: The murine Stim1 gene was conditionally deleted in the megakaryocyte/platelet lineage. CalDAG-GEFI(-/-) and Stim1(fl/fl) PF4-Cre mice, along with littermate control mice, were used for in vitro and in vivo experiments under flow as well as static conditions. Results: Integrin alpha(IIb)beta(3)-mediated aggregation was markedly impaired in CalDAG-GEFI-deficient but not STIM1-deficient platelets, under both static and flow conditions. In contrast, deficiency in either STIM1 or CalDAG-GEFI significantly impaired the ability of platelets to express phosphatidylserine on the cell surface. When subjected to a laser injury thrombosis model, mice lacking STIM1 in platelets were characterized by the formation of unstable platelet-rich thrombi and delayed and reduced fibrin generation in injured arterioles. In CalDAG-GEFI(-/-) mice, fibrin generation was also delayed and reduced, but platelet accumulation was almost abolished. Conclusions: Our studies suggest that: (i) STIM1/SOCE is critical for the procoagulant activity but not the proadhesive function of platelets; and (ii) at the site of vascular injury, STIM1 and CalDAG-GEFI are critical for the first wave of thrombin generation mediated by procoagulant platelets
— id: 139587, year: 2011, vol: 9, page: 2077, stat: Journal Article,

Immunodeficiency due to defects in store-operated calcium entry
Feske, Stefan
2011 Nov;1238(1):74-90, Annals of the New York Academy of Sciences
Mutations in genes encoding the calcium-release activated calcium (CRAC) channel abolish calcium influx in cells of the immune system and cause severe congenital immunodeficiency. Patients with autosomal recessive mutations in the CRAC channel gene ORAI1, its activator stromal interaction molecule 1 (STIM1), and mice with targeted deletion of Orai1, Stim1, and Stim2 genes reveal important roles for CRAC channels in adaptive and innate immune responses to infection and in autoimmunity. Because CRAC channels have important functions outside the immune system, deficiency of either ORAI1 or STIM1 is associated with a unique clinical phenotype. This review will give an overview of CRAC channel function in the immune system, examine the consequences of CRAC channel deficiency for immunity in human patients and mice, and discuss genetic defects in immunoreceptor-associated signaling molecules that compromise calcium influx and cause immunodeficiency
— id: 141995, year: 2011, vol: 1238, page: 74, stat: Journal Article,

Polarized but Differential Localization and Recruitment of STIM1, Orai1 and TRPC Channels in Secretory Cells
Hong, Jeong Hee; Li, Qin; Kim, Min Seuk; Shin, Dong Min; Feske, Stefan; Birnbaumer, Lutz; Cheng, Kwong Tai; Ambudkar, Indu S; Muallem, Shmuel
2011 Feb;12(2):232-245, Traffic
Polarized Ca(2+) signals in secretory epithelial cells are determined by compartmentalized localization of Ca(2+) signaling proteins at the apical pole. Recently the ER Ca(2+) sensor STIM1 (stromal interaction molecule 1) and the Orai channels were shown to play a critical role in store-dependent Ca(2+) influx. STIM1 also gates the transient receptor potential-canonical (TRPC) channels. Here, we asked how cell stimulation affects the localization, recruitment and function of the native proteins in polarized cells. Inhibition of Orai1, STIM1, or deletion of TRPC1 reduces Ca(2+) influx and frequency of Ca(2+) oscillations. Orai1 localization is restricted to the apical pole of the lateral membrane. Surprisingly, cell stimulation does not lead to robust clustering of native Orai1, as is observed with expressed Orai1. Unexpectedly, cell stimulation causes polarized recruitment of native STIM1 to both the apical and lateral regions, thus to regions with and without Orai1. Accordingly, STIM1 and Orai1 show only 40% colocalization. Consequently, STIM1 shows higher colocalization with the basolateral membrane marker E-cadherin than does Orai1, while Orai1 showed higher colocalization with the tight junction protein ZO1. TRPC1 is expressed in both apical and basolateral regions of the plasma membrane. Co-IP of STIM1/Orai1/IP(3) receptors (IP(3) Rs)/TRPCs is enhanced by cell stimulation and disrupted by 2-aminoethoxydiphenyl borate (2APB). The polarized localization and recruitment of these proteins results in preferred Ca(2+) entry that is initiated at the apical pole. These findings reveal that in addition to Orai1, STIM1 likely regulates other Ca(2+) permeable channels, such as the TRPCs. Both channels contribute to the frequency of [Ca(2+) ] oscillations and thus impact critical cellular functions
— id: 119228, year: 2011, vol: 12, page: 232, stat: Journal Article,

InsP(3) receptors and Orai channels in pancreatic acinar cells: co-localization and its consequences
Lur, Gyorgy; Sherwood, Mark W.; Ebisui, Etsuko; Haynes, Lee; Feske, Stefan; Sutton, Robert; Burgoyne, Robert D.; Mikoshiba, Katsuhiko; Petersen, Ole H.; Tepikin, Alexei V.
2011 JUN 1 ;436(23):231-239, Biochemical journal
Orai1 proteins have been recently identified as subunits of SOCE (store-operated Ca2+ entry) channels. In primary isolated PACs (pancreatic acinar cells), Orai 1 showed remarkable co-localization and co-immunoprecipitation with all three subtypes of IP(3)Rs (InsP(3) receptors). The co-localization between Orai 1 and IP(3)Rs was restricted to the apical part of PACs. Neither colocalization nor co-immunoprecipitation was affected by Ca2+ store depletion. Importantly we also characterized Orai 1 in basal and lateral membranes of PACs. The basal and lateral membranes of PACs have been shown previously to accumulate STIM1 (stromal interaction molecule 1) puncta as a result of Ca2+ store depletion. We therefore conclude that these polarized secretory cells contain two pools of Orai 1: an apical pool that interacts with IP(3)Rs and a basolateral pool that interacts with STIM1 following the Ca2+ store depletion. Experiments on IP3R knockout animals demonstrated that the apical Orai 1 localization does not require IP(3)Rs and that IP(3)Rs are not necessary for the activation of SOCE. However, the InsP(3)-releasing secretagogue ACh (acetylcholine) produced a negative modulatory effect on SOCE, suggesting that activated IP(3)Rs could have an inhibitory effect on this Ca2+ entry mechanism
— id: 134510, year: 2011, vol: 436, page: 231, stat: Journal Article,

ORAI1-mediated calcium influx is required for human cytotoxic lymphocyte degranulation and target cell lysis
Maul-Pavicic, Andrea; Chiang, Samuel C C; Rensing-Ehl, Anne; Jessen, Birthe; Fauriat, Cyril; Wood, Stephanie M; Sjoqvist, Sebastian; Hufnagel, Markus; Schulze, Ilka; Bass, Thilo; Schamel, Wolfgang W; Fuchs, Sebastian; Pircher, Hanspeter; McCarl, Christie-Ann; Mikoshiba, Katsuhiko; Schwarz, Klaus; Feske, Stefan; Bryceson, Yenan T; Ehl, Stephan
2011 Feb 22;108(8):3324-3329, Proceedings of the National Academy of Sciences of the United States of America
Lymphocytes mediate cytotoxicity by polarized release of the contents of cytotoxic granules toward their target cells. Here, we have studied the role of the calcium release-activated calcium channel ORAI1 in human lymphocyte cytotoxicity. Natural killer (NK) cells obtained from an ORAI1-deficient patient displayed defective store-operated Ca(2+) entry (SOCE) and severely defective cytotoxic granule exocytosis leading to impaired target cell lysis. Similar findings were obtained using NK cells from a stromal interaction molecule 1-deficient patient. The defect occurred at a late stage of the signaling process, because activation of leukocyte functional antigen (LFA)-1 and cytotoxic granule polarization were not impaired. Moreover, pharmacological inhibition of SOCE interfered with degranulation and target cell lysis by freshly isolated NK cells and CD8(+) effector T cells from healthy donors. In addition to effects on lymphocyte cytotoxicity, synthesis of the chemokine macrophage inflammatory protein-1beta and the cytokines TNF-alpha and IFN-gamma on target cell recognition was impaired in ORAI1-deficient NK cells, as previously described for T cells. By contrast, NK cell cytokine production induced by combinations of IL-12, IL-15, and IL-18 was not impaired by ORAI1 deficiency. Taken together, these results identify a critical role for ORAI1-mediated Ca(2+) influx in granule exocytosis for lymphocyte cytotoxicity as well as for cytokine production induced by target cell recognition
— id: 132236, year: 2011, vol: 108, page: 3324, stat: Journal Article,

STIM1 Deficiency Results In Impaired Platelet Procoagulant Activity and Protection From Arterial Thrombosis
Ahmad, Firdos; Stefanini, Lucia; Ouellette, Timothy Daniel; Greene, Teshell K.; Feske, Stefan; Bergmeiter, Wolfgang
2010 NOV 19 ;116(21):215-216, Blood
— id: 130859, year: 2010, vol: 116, page: 215, stat: Journal Article,

Whole-exome sequencing-based discovery of STIM1 deficiency in a child with fatal classic Kaposi sarcoma
Byun, Minji; Abhyankar, Avinash; Lelarge, Virginie; Plancoulaine, Sabine; Palanduz, Ayse; Telhan, Leyla; Boisson, Bertrand; Picard, Capucine; Dewell, Scott; Zhao, Connie; Jouanguy, Emmanuelle; Feske, Stefan; Abel, Laurent; Casanova, Jean-Laurent
2010 Oct 25;207(11):2307-2312, Journal of experimental medicine
Classic Kaposi sarcoma (KS) is exceedingly rare in children from the Mediterranean Basin, despite the high prevalence of human herpesvirus-8 (HHV-8) infection in this region. We hypothesized that rare single-gene inborn errors of immunity to HHV-8 may underlie classic KS in childhood. We investigated a child with no other unusually severe infectious or tumoral phenotype who died from disseminated KS at two years of age. Whole-exome sequencing in the patient revealed a homozygous splice-site mutation in STIM1, the gene encoding stromal interaction molecule 1, which regulates store-operated Ca(2+) entry. STIM1 mRNA splicing, protein production, and Ca(2+) influx were completely abolished in EBV-transformed B cell lines from the patient, but were rescued by the expression of wild-type STIM1. Based on the previous discovery of STIM1 deficiency in a single family with a severe T cell immunodeficiency and the much higher risk of KS in individuals with acquired T cell deficiencies, we conclude that STIM1 T cell deficiency precipitated the development of lethal KS in this child upon infection with HHV-8. Our report provides the first evidence that isolated classic KS in childhood may result from single-gene defects and provides proof-of-principle that whole-exome sequencing in single patients can decipher the genetic basis of rare inborn errors
— id: 140042, year: 2010, vol: 207, page: 2307, stat: Journal Article,

Roles of store-operated Ca2+ entry mediated by STIM1 and ORAI1 in immunity
Feske S.
2010 ;107:47-47, Basic & Clinical Pharmacology & Toxicology
Store-operated Ca²⁺ entry (SOCE) through Ca²⁺ -release activated Ca²⁺ (CRAC) channels is arguably the most important mechanism to generate Ca²⁺ signals in lymphocytes and other cells of the immune system. ORAI1 functions as the pore subunit of the CRAC channel and is activated by stromal interaction molecules (STIM) 1 and 2 in response to depletion of ER Ca²⁺ stores. The pathway is essential for functional immune responses involving T cells, mast cells and potentially other cells in the immune system. This presentation will discuss mechanisms of CRAC channel activation, phenotypes of human patients and mice lacking functional ORAI1 and STIM1 and the potential role of SOCE, ORAI1 and STIM1 in inflammatory diseases
— id: 111601, year: 2010, vol: 107, page: 47, stat: Journal Article,

CRAC channelopathies
Feske, Stefan
2010 Jul;460(2):417-435, Pflugers archiv = European journal of physiology
Store-operated Ca2+ entry (SOCE) is an important Ca2+ influx pathway in many non-excitable and some excitable cells. It is regulated by the filling state of intracellular Ca2+ stores, notably the endoplasmic reticulum (ER). Reduction in [Ca2+]ER results in activation of plasma membrane Ca2+ channels that mediate sustained Ca2+ influx which is required for many cell functions as well as refilling of Ca2+ stores. The Ca2+ release activated Ca2+ (CRAC) channel is the best characterized SOC channel with well-defined electrophysiological properties. In recent years, the molecular components of the CRAC channel, long mysterious, have been defined. ORAI1 (or CRACM1) acts as the pore-forming subunit of the CRAC channel in the plasma membrane. Stromal interaction molecule (STIM) 1 is localized in the ER, senses [Ca2+]ER, and activates the CRAC channel upon store depletion by binding to ORAI1. Both proteins are widely expressed in many tissues in both human and mouse consistent with the widespread prevalence of SOCE and CRAC channel currents in many cells types. CRAC channelopathies in human patients with mutations in STIM1 and ORAI1 are characterized by abolished CRAC channel currents, lack of SOCE and-clinically-immunodeficiency, congenital myopathy, and anhydrotic ectodermal dysplasia. This article reviews the role of ORAI and STIM proteins for SOCE and CRAC channel function in a variety of cell types and tissues and compares the phenotypes of ORAI1 and STIM1-deficient human patients and mice with targeted deletion of Orai and Stim genes
— id: 110092, year: 2010, vol: 460, page: 417, stat: Journal Article,

Immunodeficiency due to mutations in ORAI1 and STIM1
Feske, Stefan; Picard, Capucine; Fischer, Alain
2010 May;135(2):169-182, Clinical immunology
Lymphocyte activation requires Ca(2+) influx through specialized Ca(2+) channels in the plasma membrane. In T cells the predominant Ca(2+) channel is the Ca(2+) release activated Ca(2+) (CRAC) channel encoded by the gene ORAI1. ORAI1 is activated by stromal interaction molecule (STIM) 1 that is localized in the ER where it senses the concentration of stored Ca(2+). Following antigen binding to immunoreceptors such as the TCR, ER Ca(2+) stores are depleted, STIM1 is activated and ORAI1-CRAC channels open resulting in what is referred to as store-operated Ca(2+) entry (SOCE). Mutations in ORAI1 and STIM1 genes in human patients that lead to expression of non-functional ORAI1 or complete lack of ORAI1 or STIM1 protein are associated with a unique clinical phenotype that is characterized by immunodeficiency, muscular hypotonia and anhydrotic ectodermal dysplasia, as well as, in the case of STIM1 deficiency, autoimmunity and lymphoproliferative disease. The immunodeficiency in these patients is due to a severe defect in T cell activation but not in lymphocyte development. This review describes the immunological and non-immunological phenotypes of patients with defects in SOCE and CRAC channel function and discusses them in the context of similar immunodeficiency diseases and animal models of ORAI1 and STIM1 function
— id: 109210, year: 2010, vol: 135, page: 169, stat: Journal Article,

B Cell Receptor-Mediated Calcium Signaling Is Impaired in B Lymphocytes of Type Ia Patients with Common Variable Immunodeficiency
Foerster, C; Voelxen, N; Rakhmanov, M; Keller, B; Gutenberger, S; Goldacker, S; Thiel, J; Feske, S; Peter, HH; Warnatz, K
2010 JUN 15 ;184(12):7305-7313, Journal of immunology
Several lines of evidence have demonstrated B cell intrinsic activation defects in patients with common variable immunodeficiency (CVID). The rapid increase of intracellular free calcium concentrations after engagement of the BCR represents one crucial element in this activation process. The analysis of 53 patients with CVID for BCR-induced calcium flux identified a subgroup of patients with significantly reduced Ca2+ signals in primary B cells. This subgroup strongly corresponded to the class Ia of the Freiburg classification. Comparison at the level of defined B cell subpopulations revealed reduced Ca2+ signals in all mature B cell populations of patients with CVID class Ia when compared with healthy individuals and other groups of patients with CVID but not in circulating transitional B cells. BCR-induced Ca2+ responses were the lowest in CD21(low) B cells in patients as well as healthy donors, indicating an additional cell-specific mechanism inhibiting the Ca2+ flux. Although proximal BCR signaling events are unperturbed in patients' B cells, including normal phospholipase C gamma 2 phosphorylation and Ca2+ release from intracellular stores, Ca2+ influx from the extracellular space is significantly impaired. CD22, a negative regulator of calcium signals in B cells, is highly expressed on CD21(low) B cells from patients with CVID Ia and might be involved in the attenuated Ca2+ response of this B cell subpopulation. These data from patients with CVID suggest that a defect leading to impaired BCR-induced calcium signaling is associated with the expansion of CD21(low) B cells, hypogammaglobulinemia, autoimmune dysregulation, and lymphadenopathy. The Journal of Immunology, 2010, 184: 7305-7313
— id: 110116, year: 2010, vol: 184, page: 7305, stat: Journal Article,

Protein Kinase C-induced Phosphorylation of Orai1 Regulates the Intracellular Ca2+ Level via the Store-operated Ca2+ Channel
Kawasaki, T; Ueyama, T; Lange, I; Feske, S; Saito, N
2010 AUG 13 ;285(33):25720-25730, Journal of biological chemistry
Ca2+ signals through store-operated Ca2+ (SOC) channels, activated by the depletion of Ca2+ from the endoplasmic reticulum, regulate various physiological events. Orai1 is the pore-forming subunit of the Ca2+ release-activated Ca2+ (CRAC) channel, the best characterized SOC channel. Orai1 is activated by stromal interaction molecule (STIM) 1, a Ca2+ sensor located in the endoplasmic reticulum. Orai1 and STIM1 are crucial for SOC channel activation, but the molecular mechanisms regulating Orai1 function are not fully understood. In this study, we demonstrate that protein kinase C (PKC) suppresses store-operated Ca2+ entry (SOCE) by phosphorylation of Orai1. PKC inhibitors and knockdown of PKC beta both resulted in increased Ca2+ influx. Orai1 is strongly phosphorylated by PKC in vitro and in vivo at N-terminal Ser-27 and Ser-30 residues. Consistent with these results, substitution of endogenous Orai1 with an Orai1 S27A/S30A mutant resulted in increased SOCE and CRAC channel currents. We propose that PKC suppresses SOCE and CRAC channel function by phosphorylation of Orai1 at N-terminal serine residues Ser-27 and Ser-30
— id: 111926, year: 2010, vol: 285, page: 25720, stat: Journal Article,

T-cell-specific deletion of STIM1 and STIM2 protects mice from EAE by impairing the effector functions of Th1 and Th17 cells
Ma, Jian; McCarl, Christie-Ann; Khalil, Sara; Luthy, Kevin; Feske, Stefan
2010 Nov;40(11):3028-3042, European journal of immunology
T-cell function is dependent on store-operated Ca(2+) influx that is activated by the stromal interaction molecules (STIM) 1 and 2. We show that mice with T-cell-specific deletion of STIM1 or STIM2 are protected from EAE, a mouse model of multiple sclerosis (MS). While STIM1- and STIM2-deficient T cells could be successfully primed by autoantigen, they failed to produce the proinflammatory cytokines IL-17 and IFN-gamma. STIM1-deficient T cells showed reduced expression of IL-23R, required for Th17 cell homeostasis, and had impaired chemokine-dependent T-cell migration caused by a lack of chemokine-induced Ca(2+) influx. Autoantigen-specific STIM1- or STIM2-deficient T cells failed to expand and accumulate in the CNS and lymph nodes following adoptive transfer to passively induce EAE, suggesting that autoantigen-specific restimulation or homeostasis of STIM1- and STIM2-deficient T cells are impaired. Combined deletion of both STIM1 and STIM2, previously shown to impair Treg development and function, completely protected mice from EAE. This indicates that, in the absence of Ca(2+) influx, autoreactive T cells are severely dysfunctional rendering Treg dispensable for the prevention of CNS inflammation. Our findings demonstrate that both STIM1 and STIM2 are critical for T-cell function and autoimmunity in vivo
— id: 114192, year: 2010, vol: 40, page: 3028, stat: Journal Article,

Store-Operated Ca2+ Entry through ORAI1 Is Critical for T Cell-Mediated Autoimmunity and Allograft Rejection
McCarl, Christie-Ann; Khalil, Sara; Ma, Jian; Oh-Hora, Masatsugu; Yamashita, Megumi; Roether, Jens; Kawasaki, Takumi; Jairaman, Amit; Sasaki, Yoshiteru; Prakriya, Murali; Feske, Stefan
2010 Nov 15;185(10):5845-5858, Journal of immunology
ORAI1 is the pore-forming subunit of the Ca(2+) release-activated Ca(2+) (CRAC) channel, which is responsible for store-operated Ca(2+) entry in lymphocytes. A role for ORAI1 in T cell function in vivo has been inferred from in vitro studies of T cells from human immunodeficient patients with mutations in ORAI1 and Orai1(-/-) mice, but a detailed analysis of T cell-mediated immune responses in vivo in mice lacking functional ORAI1 has been missing. We therefore generated Orai1 knock-in mice (Orai1(KI/KI)) expressing a nonfunctional ORAI1-R93W protein. Homozygosity for the equivalent ORAI1-R91W mutation abolishes CRAC channel function in human T cells resulting in severe immunodeficiency. Homozygous Orai1(KI/KI) mice die neonatally, but Orai1(KI/KI) fetal liver chimeric mice are viable and show normal lymphocyte development. T and B cells from Orai1(KI/KI) mice display severely impaired store-operated Ca(2+) entry and CRAC channel function resulting in a strongly reduced expression of several key cytokines including IL-2, IL-4, IL-17, IFN-gamma, and TNF-alpha in CD4(+) and CD8(+) T cells. Cell-mediated immune responses in vivo that depend on Th1, Th2, and Th17 cell function were severely attenuated in ORAI1-deficient mice. Orai1(KI/KI) mice lacked detectable contact hypersensitivity responses and tolerated skin allografts significantly longer than wild-type mice. In addition, T cells from Orai1(KI/KI) mice failed to induce colitis in an adoptive transfer model of inflammatory bowel disease. These findings reaffirm the critical role of ORAI1 for T cell function and provide important insights into the in vivo functions of CRAC channels for T cell-mediated immunity
— id: 114180, year: 2010, vol: 185, page: 5845, stat: Journal Article,

R93W mutation in Orai1 causes impaired calcium influx in platelets
Bergmeier, Wolfgang; Oh-Hora, Masatsugu; McCarl, Christie-Ann; Roden, R Claire; Bray, Paul F; Feske, Stefan
2009 Jan 15;113(3):675-678, Blood
The intracellular Ca(2+) concentration of many nonexcitable cells is regulated by calcium store release and store-operated calcium entry (SOCE). In platelets, STIM1 was recently identified as the main calcium sensor expressed in the endoplasmic reticulum. To evaluate the role of the SOC channel moiety, Orai1, in platelet SOCE, we generated mice expressing a mutated, inactive form of Orai1 in blood cells only (Orai1(R93W)). Platelets expressing Orai1(R93W) were characterized by markedly reduced SOCE and impaired agonist-induced increases in [Ca(2+)](i). Orai1(R93W) platelets showed reduced integrin activation and impaired degranulation when stimulated with low agonist concentrations under static conditions. This defect, however, did not significantly affect the ability of Orai1(R93W) platelets to aggregate or to adhere to collagen under arterial flow conditions ex vivo. In contrast, these adherent Orai1(R93W) platelets were defective in surface phosphatidylserine exposure, suggesting that Orai1 is crucial for the platelets' procoagulant response rather than for other Ca(2+)-dependent cellular responses
— id: 96028, year: 2009, vol: 113, page: 675, stat: Journal Article,

ORAI1 and STIM1 deficiency in human and mice: roles of store-operated Ca2+ entry in the immune system and beyond
Feske, Stefan
2009 Sep;231(1):189-209, Immunological reviews
Store-operated Ca2+ entry (SOCE) is a mechanism used by many cells types including lymphocytes and other immune cells to increase intracellular Ca2+ concentrations to initiate signal transduction. Activation of immunoreceptors such as the T-cell receptor, B-cell receptor, or Fc receptors results in the release of Ca2+ ions from endoplasmic reticulum (ER) Ca2+ stores and subsequent activation of plasma membrane Ca2+ channels such as the well-characterized Ca2+ release-activated Ca2+ (CRAC) channel. Two genes have been identified that are essential for SOCE: ORAI1 as the pore-forming subunit of the CRAC channel in the plasma membrane and stromal interaction molecule-1 (STIM1) sensing the ER Ca2+ concentration and activating ORAI1-CRAC channels. Intense efforts in the past several years have focused on understanding the molecular mechanism of SOCE and the role it plays for cell functions in vitro and in vivo. A number of transgenic mouse models have been generated to investigate the role of ORAI1 and STIM1 in immunity. In addition, mutations in ORAI1 and STIM1 identified in immunodeficient patients provide valuable insight into the role of both genes and SOCE. This review focuses on the role of ORAI1 and STIM1 in vivo, discussing the phenotypes of ORAI1- and STIM1-deficient human patients and mice
— id: 102408, year: 2009, vol: 231, page: 189, stat: Journal Article,

"A minimal regulatory domain in the C terminus of STIM1 binds to and activates ORAI1 CRAC channels (vol 385, pg 49, 2009)"
Kawasaki, T; Lange, I; Feske, S
2009 SEP 25 ;387(3):623-623, Biochemical & biophysical research communications
— id: 101945, year: 2009, vol: 387, page: 623, stat: Journal Article,

A minimal regulatory domain in the C terminus of STIM1 binds to and activates ORAI1 CRAC channels
Kawasaki, Takumi; Lange, Ingo; Feske, Stefan
2009 Jul 17;385(1):49-54, Biochemical & biophysical research communications
Store-operated Ca(2+) entry (SOCE) is a universal mechanism to increase intracellular Ca(2+) concentrations in non-excitable cells. It is initiated by the depletion of ER Ca(2+) stores, activation of stromal interaction molecule (STIM) 1 and gating of the Ca(2+) release activated Ca(2+) (CRAC) channel ORAI1 in the plasma membrane. We identified a minimal activation domain in the cytoplasmic region of STIM1 (CCb9) which activated Ca(2+) influx and CRAC currents (I(CRAC)) in the absence of store depletion similar to but more potently than the entire C terminus of STIM1. A STIM1 fragment (CCb7) that is longer by 31 amino acids than CCb9 at its C terminal end showed reduced ability to constitutively activate I(CRAC) consistent with our observation that CCb9 but not CCb7 efficiently colocalized with and bound to ORAI1. Intracellular application of a 31 amino acid peptide contained in CCb7 but not CCb9 inhibited constitutive and store-dependent CRAC channel activation. In summary, these findings suggest that CCb9 represents a minimal ORAI1 activation domain within STIM1 that is masked by an adjacent 31 amino acid peptide preventing efficient CRAC channel activation in cells with replete Ca(2+) stores
— id: 99322, year: 2009, vol: 385, page: 49, stat: Journal Article,

ORAI1 deficiency and lack of store-operated Ca(2+) entry cause immunodeficiency, myopathy, and ectodermal dysplasia
McCarl, Christie-Ann; Picard, Capucine; Khalil, Sara; Kawasaki, Takumi; Rother, Jens; Papolos, Alexander; Kutok, Jeffery; Hivroz, Claire; Ledeist, Francoise; Plogmann, Katrin; Ehl, Stephan; Notheis, Gundula; Albert, Michael H; Belohradsky, Bernd H; Kirschner, Janbernd; Rao, Anjana; Fischer, Alain; Feske, Stefan
2009 Dec;124(6):1311-1318.e7, Journal of allergy & clinical immunology
BACKGROUND: Defects in the development or activation of T cells result in immunodeficiency associated with severe infections early in life. T-cell activation requires Ca(2+) influx through Ca(2+)-release activated Ca(2+) (CRAC) channels encoded by the gene ORAI1. OBJECTIVE: Investigation of the genetic causes and the clinical phenotype of immunodeficiency in patients with impaired Ca(2+) influx and CRAC channel function. METHODS: DNA sequence analysis for mutations in the genes ORAI1, ORAI2, ORAI3, and stromal interaction molecule (STIM) 1 and 2, as well as mRNA and protein expression analysis of ORAI1 in immunodeficient patients. Immunohistochemical analysis of ORAI1 tissue distribution in healthy human donors. RESULTS: We identified mutations in ORAI1 in patients from 2 unrelated families. One patient is homozygous for a frameshift nonsense mutation in ORAI1 (ORAI1-A88SfsX25), and a second patient is compound heterozygous for 2 missense mutations in ORAI1 (ORAI1-A103E/L194P). All 3 mutations abolish ORAI1 expression and impair Ca(2+) influx and CRAC channel function. The clinical syndrome associated with ORAI1 deficiency is characterized by immunodeficiency with a defect in the function but not in the development of lymphocytes, congenital myopathy, and anhydrotic ectodermal dysplasia with a defect in dental enamel calcification. In contrast with the limited clinical phenotype, we found ORAI1 protein expression in a wide variety of cell types and organs. CONCLUSION: Ca(2+) influx through ORAI1 is crucial for lymphocyte function in vivo. Despite almost ubiquitous ORAI1 expression, the channel has a nonredundant role in only a few cell types judging from the limited clinical phenotype in ORAI1-deficient patients
— id: 105929, year: 2009, vol: 124, page: 1311, stat: Journal Article,

Brief Report: STIM1 Mutation Associated with a Syndrome of Immunodeficiency and Autoimmunity
Picard, C; McCarl, CA; Papolos, A; Khalil, S; Luthy, K; Hivroz, C; LeDeist, F; Rieux-Laucat, F; Rechavi, G; Rao, A; Fischer, A; Feske, S
2009 MAY 7 ;360(19):1971-1980, New England journal of medicine
A mutation in ORAI1, the gene encoding the pore-forming subunit of the Ca(sup 2+)-release-activated Ca(sup 2+) (CRAC) channel, abrogates the store-operated entry of Ca(sup 2+) into cells and impairs lymphocyte activation. Stromal interaction molecule 1 (STIM1) in the endoplasmic reticulum activates ORAI1-CRAC channels. We report on three siblings from one kindred with a clinical syndrome of immunodeficiency, hepatosplenomegaly, autoimmune hemolytic anemia, thrombocytopenia, muscular hypotonia, and defective enamel dentition. Two of these patients have a homozygous nonsense mutation in STIM1 that abrogates expression of STIM1 and Ca(sup 2+) influx
— id: 98841, year: 2009, vol: 360, page: 1971, stat: Journal Article,

R93W Mutation in Orai1 Causes Impaired Calcium Influx in Platelets
Bergmeier, W; Oh-Hora, M; McCarl, CA; Roden, RC; Bray, PF; Feske, S
2008 NOV 16 ;112(11):647-647, Blood
— id: 93288, year: 2008, vol: 112, page: 647, stat: Journal Article,

Hair loss and defective T- and B-cell function in mice lacking ORAI1
Gwack, Yousang; Srikanth, Sonal; Oh-Hora, Masatsugu; Hogan, Patrick G; Lamperti, Edward D; Yamashita, Megumi; Gelinas, Curtis; Neems, Daniel S; Sasaki, Yoshiteru; Feske, Stefan; Prakriya, Murali; Rajewsky, Klaus; Rao, Anjana
2008 Sep;28(17):5209-5222, Molecular & cellular biology
ORAI1 is a pore subunit of the store-operated Ca(2+) release-activated Ca(2+) (CRAC) channel. To examine the physiological consequences of ORAI1 deficiency, we generated mice with targeted disruption of the Orai1 gene. The results of immunohistochemical analysis showed that ORAI1 is expressed in lymphocytes, skin, and muscle of wild-type mice and is not expressed in Orai1(-/-) mice. Orai1(-/-) mice with the inbred C57BL/6 background showed perinatal lethality, which was overcome by crossing them to outbred ICR mice. Orai1(-/-) mice were small in size, with eyelid irritation and sporadic hair loss resembling the cyclical alopecia observed in mice with keratinocyte-specific deletion of the Cnb1 gene. T and B cells developed normally in Orai1(-/-) mice, but B cells showed a substantial decrease in Ca(2+) influx and cell proliferation in response to B-cell receptor stimulation. Naive and differentiated Orai1(-/-) T cells showed substantial reductions in store-operated Ca(2+) entry, CRAC currents, and cytokine production. These features are consistent with the severe combined immunodeficiency and mild extraimmunological symptoms observed in a patient with a missense mutation in human ORAI1 and distinguish the ORAI1-null mice described here from a previously reported Orai1 gene-trap mutant mouse which may be a hypomorph rather than a true null
— id: 81086, year: 2008, vol: 28, page: 5209, stat: Journal Article,

Dual functions for the endoplasmic reticulum calcium sensors STIM1 and STIM2 in T cell activation and tolerance
Oh-Hora, Masatsugu; Yamashita, Megumi; Hogan, Patrick G; Sharma, Sonia; Lamperti, Ed; Chung, Woo; Prakriya, Murali; Feske, Stefan; Rao, Anjana
2008 Apr;9(4):432-443, Nature immunology
Store-operated Ca2+ entry through calcium release-activated calcium channels is the chief mechanism for increasing intracellular Ca2+ in immune cells. Here we show that mouse T cells and fibroblasts lacking the calcium sensor STIM1 had severely impaired store-operated Ca2+ influx, whereas deficiency in the calcium sensor STIM2 had a smaller effect. However, T cells lacking either STIM1 or STIM2 had much less cytokine production and nuclear translocation of the transcription factor NFAT. T cell-specific ablation of both STIM1 and STIM2 resulted in a notable lymphoproliferative phenotype and a selective decrease in regulatory T cell numbers. We conclude that both STIM1 and STIM2 promote store-operated Ca2+ entry into T cells and fibroblasts and that STIM proteins are required for the development and function of regulatory T cells
— id: 81087, year: 2008, vol: 9, page: 432, stat: Journal Article,

Calcium signalling in lymphocyte activation and disease
Feske, Stefan
2007 Sep;7(9):690-702, Nature reviews. Immunology
Calcium signals in cells of the immune system participate in the regulation of cell differentiation, gene transcription and effector functions. An increase in intracellular levels of calcium ions (Ca(2+)) results from the engagement of immunoreceptors, such as the T-cell receptor, B-cell receptor and Fc receptors, as well as chemokine and co-stimulatory receptors. The major pathway that induces an increase in intracellular Ca(2+) levels in lymphocytes is through store-operated calcium entry (SOCE) and calcium-release-activated calcium (CRAC) channels. This Review focuses on the role of Ca(2+) signals in lymphocyte functions, the signalling pathways leading to Ca(2+) influx, the function of the recently discovered regulators of Ca(2+) influx (STIM and ORAI), and the relationship between Ca(2+) signals and diseases of the immune system
— id: 73417, year: 2007, vol: 7, page: 690, stat: Journal Article,

Signalling to transcription: Store-operated Ca(2+) entry and NFAT activation in lymphocytes
Gwack, Yousang; Feske, Stefan; Srikanth, Sonal; Hogan, Patrick G; Rao, Anjana
2007 Aug;42(2):145-156, Cell calcium
In cells of the immune system that are stimulated by antigen or antigen-antibody complexes, Ca(2+) entry from the extracellular medium is driven by depletion of endoplasmic reticulum Ca(2+) stores and occurs through specialized store-operated Ca(2+) channels known as Ca(2+)-release-activated Ca(2+) (CRAC) channels. The process of store-operated Ca(2+) influx is essential for short-term as well as long-term responses by immune-system cells. Short-term responses include mast cell degranulation and killing of target cells by effector cytolytic T cells, whereas long-term responses typically involve changes in gene transcription and include T and B cell proliferation and differentiation. Transcription downstream of Ca(2+) influx is in large part funneled through the transcription factor nuclear factor of activated T cells (NFAT), a heavily phosphorylated protein that is cytoplasmic in resting cells, but that enters the nucleus when dephosphorylated by the calmodulin-dependent serine/threonine phosphatase calcineurin. The importance of the Ca(2+)/calcineurin/NFAT signalling pathway for lymphocyte activation is underscored by the finding that the underlying defect in a family with a hereditary severe combined immune deficiency (SCID) syndrome is a defect in CRAC channel function, store-operated Ca(2+) entry, NFAT activation and transcription of cytokines, chemokines and many other NFAT target genes whose transcription is essential for productive immune defence. We recently used a two-pronged genetic approach to identify Orai1 as the pore subunit of the CRAC channel. On the one hand, we initiated a positional cloning approach in which we utilised genome-wide single nucleotide polymorphism (SNP) mapping to identify the genomic region linked to the mutant gene in the SCID family described above. In parallel, we used a genome-wide RNAi screen in Drosophila to identify critical regulators of NFAT nuclear translocation and store-operated Ca(2+) entry. These approaches, together with subsequent mutational and electrophysiological analyses, converged to identify human Orai1 as a pore subunit of the CRAC channel and as the gene product mutated in the SCID patients.
— id: 72942, year: 2007, vol: 42, page: 145, stat: Journal Article,

Biochemical and functional characterization of Orai proteins
Gwack, Yousang; Srikanth, Sonal; Feske, Stefan; Cruz-Guilloty, Fernando; Oh-hora, Masatsugu; Neems, Daniel S; Hogan, Patrick G; Rao, Anjana
2007 Jun 1;282(22):16232-16243, Journal of biological chemistry
Stimulation of immune cells triggers Ca2+ entry through store-operated Ca2+ release-activated Ca2+ channels, promoting nuclear translocation of the transcription factor NFAT. Through genome-wide RNA interference screens in Drosophila, we and others identified olf186-F (Drosophila Orai, dOrai) and dStim as critical components of store-operated Ca2+ entry and showed that dOrai and its human homologue Orai1 are pore subunits of the Ca2+ release-activated Ca2+ channel. Here we report that Orai1 is predominantly responsible for store-operated Ca2+ influx in human embryonic kidney 293 cells and human T cells and fibroblasts, although its paralogue Orai3 can partly compensate in the absence of functional Orai1. All three mammalian Orai are widely expressed at the mRNA level, and all three are incorporated into the plasma membrane. In human embryonic kidney 293 cells, Orai1 is glycosylated at an asparagine residue in the predicted second extracellular loop, but mutation of the residue does not compromise function. STIM1 and Orai1 colocalize after store depletion, but Orai1 does not associate detectably with STIM1 in glycerol gradient centrifugation or coimmunoprecipitation experiments. Glutamine substitutions in two conserved glutamate residues, located within predicted transmembrane helices of Drosophila Orai and human Orai1, greatly diminish store-operated Ca2+ influx, and primary T cells ectopically expressing mutant E106Q and E190Q Orai1 proteins show reduced proliferation and cytokine secretion. Together, these data establish Orai1 as a predominant mediator of store-operated calcium entry, proliferation, and cytokine production in T cells.
— id: 72943, year: 2007, vol: 282, page: 16232, stat: Journal Article,

A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function
Feske, Stefan; Gwack, Yousang; Prakriya, Murali; Srikanth, Sonal; Puppel, Sven-Holger; Tanasa, Bogdan; Hogan, Patrick G; Lewis, Richard S; Daly, Mark; Rao, Anjana
2006 May 11;441(7090):179-185, Nature
Antigen stimulation of immune cells triggers Ca2+ entry through Ca2+ release-activated Ca2+ (CRAC) channels, promoting the immune response to pathogens by activating the transcription factor NFAT. We have previously shown that cells from patients with one form of hereditary severe combined immune deficiency (SCID) syndrome are defective in store-operated Ca2+ entry and CRAC channel function. Here we identify the genetic defect in these patients, using a combination of two unbiased genome-wide approaches: a modified linkage analysis with single-nucleotide polymorphism arrays, and a Drosophila RNA interference screen designed to identify regulators of store-operated Ca2+ entry and NFAT nuclear import. Both approaches converged on a novel protein that we call Orai1, which contains four putative transmembrane segments. The SCID patients are homozygous for a single missense mutation in ORAI1, and expression of wild-type Orai1 in SCID T cells restores store-operated Ca2+ influx and the CRAC current (I(CRAC)). We propose that Orai1 is an essential component or regulator of the CRAC channel complex.
— id: 72945, year: 2006, vol: 441, page: 179, stat: Journal Article,

A genome-wide Drosophila RNAi screen identifies DYRK-family kinases as regulators of NFAT
Gwack, Yousang; Sharma, Sonia; Nardone, Julie; Tanasa, Bogdan; Iuga, Alina; Srikanth, Sonal; Okamura, Heidi; Bolton, Diana; Feske, Stefan; Hogan, Patrick G; Rao, Anjana
2006 Jun 1;441(7093):646-650, Nature
Precise regulation of the NFAT (nuclear factor of activated T cells) family of transcription factors (NFAT1-4) is essential for vertebrate development and function. In resting cells, NFAT proteins are heavily phosphorylated and reside in the cytoplasm; in cells exposed to stimuli that raise intracellular free Ca2+ levels, they are dephosphorylated by the calmodulin-dependent phosphatase calcineurin and translocate to the nucleus. NFAT dephosphorylation by calcineurin is countered by distinct NFAT kinases, among them casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3). Here we have used a genome-wide RNA interference (RNAi) screen in Drosophila to identify additional regulators of the signalling pathway leading from Ca2+-calcineurin to NFAT. This screen was successful because the pathways regulating NFAT subcellular localization (Ca2+ influx, Ca2+-calmodulin-calcineurin signalling and NFAT kinases) are conserved across species, even though Ca2+-regulated NFAT proteins are not themselves represented in invertebrates. Using the screen, we have identified DYRKs (dual-specificity tyrosine-phosphorylation regulated kinases) as novel regulators of NFAT. DYRK1A and DYRK2 counter calcineurin-mediated dephosphorylation of NFAT1 by directly phosphorylating the conserved serine-proline repeat 3 (SP-3) motif of the NFAT regulatory domain, thus priming further phosphorylation of the SP-2 and serine-rich region 1 (SRR-1) motifs by GSK3 and CK1, respectively. Thus, genetic screening in Drosophila can be successfully applied to cross evolutionary boundaries and identify new regulators of a transcription factor that is expressed only in vertebrates.
— id: 72946, year: 2006, vol: 441, page: 646, stat: Journal Article,

Orai1 is an essential pore subunit of the CRAC channel
Prakriya, Murali; Feske, Stefan; Gwack, Yousang; Srikanth, Sonal; Rao, Anjana; Hogan, Patrick G
2006 Sep 14;443(7108):230-233, Nature
Stimulation of immune cells causes depletion of Ca2+ from endoplasmic reticulum (ER) stores, thereby triggering sustained Ca2+ entry through store-operated Ca2+ release-activated Ca2+ (CRAC) channels, an essential signal for lymphocyte activation and proliferation. Recent evidence indicates that activation of CRAC current is initiated by STIM proteins, which sense ER Ca2+ levels through an EF-hand located in the ER lumen and relocalize upon store depletion into puncta closely associated with the plasma membrane. We and others recently identified Drosophila Orai and human Orai1 (also called TMEM142A) as critical components of store-operated Ca2+ entry downstream of STIM. Combined overexpression of Orai and Stim in Drosophila cells, or Orai1 and STIM1 in mammalian cells, leads to a marked increase in CRAC current. However, these experiments did not establish whether Orai is an essential intracellular link between STIM and the CRAC channel, an accessory protein in the plasma membrane, or an actual pore subunit. Here we show that Orai1 is a plasma membrane protein, and that CRAC channel function is sensitive to mutation of two conserved acidic residues in the transmembrane segments. E106D and E190Q substitutions in transmembrane helices 1 and 3, respectively, diminish Ca2+ influx, increase current carried by monovalent cations, and render the channel permeable to Cs+. These changes in ion selectivity provide strong evidence that Orai1 is a pore subunit of the CRAC channel.
— id: 72944, year: 2006, vol: 443, page: 230, stat: Journal Article,

Defective nuclear translocation of nuclear factor of activated T cells and extracellular signal-regulated kinase underlies deficient IL-2 gene expression in Wiskott-Aldrich syndrome
Cianferoni, Antonella; Massaad, Michel; Feske, Stefan; de la Fuente, Miguel A; Gallego, Lola; Ramesh, Narayanaswamy; Geha, Raif S
2005 Dec;116(6):1364-1371, Journal of allergy & clinical immunology
BACKGROUND: Proliferation and IL-2 production in response to T-cell receptor ligation are impaired in patients with Wiskott-Aldrich syndrome (WAS). The transcription factors nuclear factor-kappaB (NF-kappaB), nuclear factor of activated T cells (NF-AT), and activating protein-1 (AP-1) play a critical role in IL-2 gene expression. OBJECTIVE: To investigate the mechanisms of impaired IL-2 production after T-cell receptor ligation in T cells deficient in WAS protein (WASP). METHODS: T cells from WASP-/- mice were stimulated with anti-CD3 and anti-CD28. Nuclear NF-kappaB, NF-AT, and AP-1 DNA-binding activity was examined by electroshift mobility assay. NF-ATp dephosphorylation and nuclear localization were examined by Western blot and indirect immunofluorescence. Phosphorylation of the mitogen-activated protein kinases Erk and Jnk, and of their nuclear substrates Elk-1 and c-Jun, was examined by Western blot. Expression of mRNA for IL-2 and the NF-kappaB-dependent gene A20 and of the AP-1 components c-fos and c-Jun was examined by quantitative RT-PCR. RESULTS: Nuclear translocation and activity of NF-kappaB were normal in T cells from WASP-/- mice. In contrast, NF-ATp dephosphorylation and nuclear localization, nuclear AP-1 binding activity, and expression of c-fos, but not c-Jun, were all impaired. Phosphorylation of Jnk, c-Jun, and Erk were normal. However, nuclear translocation of phosphorylated Erk and phosphorylation of its nuclear substrate Elk1, which activates the c-fos promoter, were impaired. CONCLUSION: These results suggest that WASP is essential for NF-ATp activation, and for nuclear translocation of p-Erk, Elk1 phosphorylation, and c-fos gene expression in T cells. These defects underlie defective IL-2 expression and T-cell proliferation in WAS.
— id: 72948, year: 2005, vol: 116, page: 1364, stat: Journal Article,

A severe defect in CRAC Ca2+ channel activation and altered K+ channel gating in T cells from immunodeficient patients
Feske, Stefan; Prakriya, Murali; Rao, Anjana; Lewis, Richard S
2005 Sep 5;202(5):651-662, Journal of experimental medicine
Engagement of the TCR triggers sustained Ca(2+) entry through Ca(2+) release-activated Ca(2+) (CRAC) channels, which helps drive gene expression underlying the T cell response to pathogens. The identity and activation mechanism of CRAC channels at a molecular level are unknown. We have analyzed ion channel expression and function in T cells from SCID patients which display 1-2% of the normal level of Ca(2+) influx and severely impaired T cell activation. The lack of Ca(2+) influx is not due to deficient regulation of Ca(2+) stores or expression of several genes implicated in controlling Ca(2+) entry in lymphocytes (kcna3/Kv1.3, kcnn4/IKCa1, trpc1, trpc3, trpv6, stim1). Instead, electrophysiologic measurements show that the influx defect is due to a nearly complete absence of functional CRAC channels. The lack of CRAC channel activity is correlated with diminished voltage sensitivity and slowed activation kinetics of the voltage-dependent Kv1.3 channel. These results demonstrate that CRAC channels provide the major, if not sole, pathway for Ca(2+) entry activated by the TCR in human T cells. They also offer evidence for a functional link between CRAC and Kv1.3 channels, and establish a model system for molecular genetic studies of the CRAC channel.
— id: 72950, year: 2005, vol: 202, page: 651, stat: Journal Article,

A molecular dissection of lymphocyte unresponsiveness induced by sustained calcium signalling
Heissmeyer, Vigo; Macian, Fernando; Varma, Rajat; Im, Sin-Hyeog; Garcia-Cozar, Francisco; Horton, Heidi F; Byrne, Michael C; Feske, Stefan; Venuprasad, K; Gu, Hua; Liu, Yun-Cai; Dustin, Michael L; Rao, Anjana
2005 ;267:165-174, Novartis foundation symposium
In lymphocytes, integration of Ca2+ and other signalling pathways results in productive activation, while unopposed Ca2+ signalling leads to decreased responsiveness to subsequent stimulation (anergy). The Ca(2+)-regulated transcription factor NFAT has an integral role in both aspects of lymphocyte function. NFAT cooperates with the transcription factor AP-1 (Fos/Jun) to up-regulate genes involved in productive activation of lymphocytes. However, in the absence of AP-1, NFAT imposes an opposing genetic programme that leads to lymphocyte anergy. Anergy is implemented at least partly through proteolytic degradation of the key signalling proteins PKCtheta and PLCgamma1. Sustained Ca(2+)-calcineurin signalling increases mRNA and protein levels of the E3 ubiquitin ligases Itch, CblB and Grail and induces expression of Tsg1O1, the ubiquitin-binding component of the ESCRT1 endosomal sorting complex. Subsequent stimulation or homotypic cell adhesion promotes membrane translocation of Itch and the related protein Nedd4, resulting in PKCtheta and PLCgamma1 degradation. T cells from Itch- and CblB-deficient mice are resistant to anergy induction. Anergic T cells show impaired calcium mobilization after TCR triggering and are unable to maintain a mature immunological synapse. Thus Ca(2+)-calcineurin-NFAT signalling links gene transcription to a multi-step programme that leads to impaired signal transduction in anergic T cells
— id: 68165, year: 2005, vol: 267, page: 165, stat: Journal Article,

Perforin triggers a plasma membrane-repair response that facilitates CTL induction of apoptosis
Keefe, Dennis; Shi, Lianfa; Feske, Stefan; Massol, Ramiro; Navarro, Francisco; Kirchhausen, Tomas; Lieberman, Judy
2005 Sep;23(3):249-262, Immunity
Perforin delivers granzymes to induce target-cell apoptosis. At high concentrations, perforin multimerizes in the plasma membrane to form pores. However, whether granzymes enter target cells via membrane pores is uncertain. Here we find that perforin at physiologically relevant concentrations and during cell-mediated lysis creates pores in the target-cell membrane, transiently allowing Ca(2+) and small dyes into the cell. The Ca(2+) flux triggers a wounded membrane-repair response in which internal vesicles, including lysosomes and endosomes, donate their membranes to reseal the damaged membrane. Perforin also triggers the rapid endocytosis of granzymes into large EEA-1-staining vesicles. The restoration of target-cell membrane integrity by triggering the repair response is necessary for target cells subjected to cytotoxic T lymphocyte attack to avoid necrosis and undergo the slower process of programmed cell death. Thus, the target cell actively participates in determining its own fate during cell-mediated death.
— id: 72949, year: 2005, vol: 23, page: 249, stat: Journal Article,

A 10-aa-long sequence in SLP-76 upstream of the Gads binding site is essential for T cell development and function
Kumar, Lalit; Feske, Stefan; Rao, Anjana; Geha, Raif S
2005 Dec 27;102(52):19063-19068, Proceedings of the National Academy of Sciences of the United States of America
The adapter SLP-76 is essential for T cell development and function. SLP-76 binds to the src homology 3 domain of Lck in vitro. This interaction depends on amino acids 185-194 of SLP-76. To examine the role of the Lck-binding region of SLP-76 in T cell development and function, SLP-76(-/-) mice were reconstituted with an SLP-76 mutant that lacks amino acids 185-194. Double and single positive thymocytes from reconstituted mice were severely reduced in numbers and exhibited impaired positive selection and increased apoptosis. Peripheral T cells were also reduced in numbers, exhibited impaired phospholipase C-gamma1 and Erk phosphorylation, and failed to flux calcium, secrete IL-2, and proliferate in response to T cell antigen receptor ligation. Delayed cutaneous hypersensitivity responses and Ab responses to T cell-dependent antigen were severely impaired. These results indicate that the Lck binding region of SLP-76 is essential for T cell antigen receptor signaling and normal T cell development and function.
— id: 72947, year: 2005, vol: 102, page: 19063, stat: Journal Article,

Calcineurin imposes T cell unresponsiveness through targeted proteolysis of signaling proteins
Heissmeyer, Vigo; Macian, Fernando; Im, Sin-Hyeog; Varma, Rajat; Feske, Stefan; Venuprasad, K; Gu, Hua; Liu, Yun-Cai; Dustin, Michael L; Rao, Anjana
2004 Mar;5(3):255-265, Nature immunology
Sustained calcium signaling induces a state of anergy or antigen unresponsiveness in T cells, mediated through calcineurin and the transcription factor NFAT. We show here that Ca(2+)-induced anergy is a multistep program that is implemented at least partly through proteolytic degradation of specific signaling proteins. Calcineurin increased mRNA and protein of the E3 ubiquitin ligases Itch, Cbl-b and GRAIL and induced expression of Tsg101, the ubiquitin-binding component of the ESCRT-1 endosomal sorting complex. Subsequent stimulation or homotypic cell adhesion promoted membrane translocation of Itch and the related protein Nedd4, resulting in degradation of two key signaling proteins, PKC-theta and PLC-gamma1. T cells from Itch- and Cbl-b-deficient mice were resistant to anergy induction. Anergic T cells showed impaired calcium mobilization after TCR triggering and were unable to maintain a mature immunological synapse, instead showing late disorganization of the outer ring containing lymphocyte function-associated antigen 1. Our results define a complex molecular program that links gene transcription induced by calcium and calcineurin to a paradoxical impairment of signal transduction in anergic T cells
— id: 44918, year: 2004, vol: 5, page: 255, stat: Journal Article,

The cell surface receptor SLAM controls T cell and macrophage functions
Wang, Ninghai; Satoskar, Abhay; Faubion, William; Howie, Duncan; Okamoto, Susumu; Feske, Stefan; Gullo, Charles; Clarke, Kareem; Sosa, Miriam Rodriguez; Sharpe, Arlene H; Terhorst, Cox
2004 May 3;199(9):1255-1264, Journal of experimental medicine
Signaling lymphocyte activation molecule (SLAM), a glycoprotein expressed on activated lymphocytes and antigen-presenting cells, has been shown to be a coregulator of antigen-driven T cell responses and is one of the two receptors for measles virus. Here we show that T cell receptor-induced interleukin (IL)-4 secretion by SLAM(-/-) CD4(+) cells is down-regulated, whereas interferon gamma production by CD4(+) T cells is only slightly up-regulated. Although SLAM controls production of IL-12, tumor necrosis factor, and nitric oxide in response to lipopolysaccharide (LPS) by macrophages, SLAM does not regulate phagocytosis and responses to peptidoglycan or CpG. Thus, SLAM acts as a coreceptor that regulates signals transduced by the major LPS receptor Toll-like receptor 4 on the surface of mouse macrophages. A defective macrophage function resulted in an inability of SLAM(-/-) C57Bl/6 mice to remove the parasite Leishmania major. We conclude that the coreceptor SLAM plays a central role at the interface of acquired and innate immune responses.
— id: 72951, year: 2004, vol: 199, page: 1255, stat: Journal Article,

Ca2+/calcineurin signalling in cells of the immune system
Feske, Stefan; Okamura, Heidi; Hogan, Patrick G; Rao, Anjana
2003 Nov 28;311(4):1117-1132, Biochemical & biophysical research communications
Calcineurin is a serine-threonine - phosphatase that is expressed in a wide variety of tissues and has particularly critical functions in neurons, cardiac and skeletal muscle cells, and lymphocytes. This review focuses on recent studies elucidating the role of Ca(2+)/calcineurin signalling of the immune system.
— id: 72952, year: 2003, vol: 311, page: 1117, stat: Journal Article,