Transcriptional target-based expression cloning of immunoregulatory molecules

Lamason, Rebecca; Lew, Stefanie M.; Pomerantz, Joel L.

doi:10.1007/s12026-009-8148-z

Cited by 2 publications

(2 citation statements)

References 13 publications

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“…Because of the requirement of the CARD domain for intramolecular ID association, we hypothesized that some CARD domain residues would mediate interactions that maintain CARD11 in the inactive state prior to ID neutralization by antigen receptor signaling. To test this hypothesis, we adapted a screening methodology previously used to identify molecules that signal to NF-B (11) or that enhance or suppress the signaling activity of CARD11 (25,33). We generated a library of CARD11 variants in the context of a mammalian expression vector in which residues between M1 and L138 of murine CARD11 were randomly mutated by error-prone PCR.…”

Section: Identification Of Novel Gain-of-function Mutations In Card11mentioning

confidence: 99%

A Quantitative Signaling Screen Identifies CARD11 Mutations in the CARD and LATCH Domains That Induce Bcl10 Ubiquitination and Human Lymphoma Cell Survival

Chan

Schaffer

Pomerantz

2013

Molecular and Cellular Biology

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103

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Antigen receptor signaling to NF-B, essential for normal lymphocyte activation, is dysregulated in several types of lymphoma. During normal signaling, the multidomain adapter CARD11 transitions from a closed, inactive state to an open, active scaffold that assembles a multiprotein complex, leading to NF-B activation. The regulation of CARD11 scaffold function is bypassed by lymphoma-associated oncogenic CARD11 mutations that induce spontaneous signaling. We report an unbiased high-throughput quantitative signaling screen that identifies new CARD11 hyperactive variants and defines a LATCH domain that functions with the CARD to promote CARD11 autoinhibition. Gain-of-function mutations in the LATCH or CARD disrupt inhibitory domain binding, promote Bcl10 association, and induce Bcl10 ubiquitination, NF-B activation, and human lymphoma cell survival. Our results identify CARD11 mutations with oncogenic potential, provide a mechanistic explanation for their signaling potency, and offer a straightforward method for the discovery of variants that promote the tumorigenesis of NF-B-dependent lymphomas.

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Section: Identification Of Novel Gain-of-function Mutations In Card11mentioning

confidence: 99%

A Quantitative Signaling Screen Identifies CARD11 Mutations in the CARD and LATCH Domains That Induce Bcl10 Ubiquitination and Human Lymphoma Cell Survival

Chan

Schaffer

Pomerantz

2013

Molecular and Cellular Biology

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103

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“…To identify unrecognized modulators of the signaling pathway to NFAT, we used a transcriptional target-based expression cloning approach (37,38). Pools of clones from a mouse thymus cDNA expression library were cotransfected at a complexity of 100 cDNAs per pool with an NFAT-responsive luciferase reporter, NFAT 4 -IFN-LUC, into HEK293T cells and were assayed for the ability to activate the reporter 3-fold or more relative to the empty expression vector.…”

Section: Sppl3 Is a Specific Nfat Activatormentioning

confidence: 99%

A Protease-Independent Function for SPPL3 in NFAT Activation

Makowski

Wang

Pomerantz

2015

Molecular and Cellular Biology

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The signal peptide peptidase (SPP)-related intramembrane aspartyl proteases are a homologous group of polytopic membrane proteins, some of which function in innate or adaptive immunity by cleaving proteins involved in antigen presentation or intracellular signaling. Signal peptide peptidase-like 3 (SPPL3) is a poorly characterized endoplasmic reticulum (ER)-localized member of this family, with no validated cellular substrates. We report here the isolation of SPPL3 in a screen for activators of NFAT, a transcription factor that controls lymphocyte development and function. We find that SPPL3 is required downstream of T cell receptor engagement for maximal Ca 2؉ influx and NFAT activation. Surprisingly, the proteolytic activity of SPPL3 is not required for its role in this pathway. SPPL3 enhances the signal-induced association of stromal interaction molecule 1 (STIM1) and Orai1 and is even required for the full activity of constitutively active STIM1 variants that bind Orai1 independently of ER Ca 2؉ release. SPPL3 associates with STIM1 through at least two independent domains, the transmembrane region and the CRAC activation domain (CAD), and can promote the association of the STIM1 CAD with Orai1. Our results assign a function in lymphocyte signaling to SPPL3 and highlight the emerging importance of nonproteolytic functions for members of the intramembrane aspartyl protease family. The NFAT family of transcription factors regulates a variety of cellular functions by initiating new programs of gene expression in response to changes in intracellular Ca 2ϩ levels. NFAT plays a critical role in the immune and nervous systems, in heart and bone development, and in other tissues (1, 2). In the adaptive immune system, NFAT regulates genes that control thymocyte development, T cell activation, T helper differentiation, and selftolerance (3) and thus serves as a major determinant of how the immune system responds to pathogens and distinguishes between self and nonself.T cell receptor (TCR) signaling activates NFAT activity through the Ca 2ϩ -dependent phosphatase calcineurin, which dephosphorylates NFAT in the cytoplasm and allows NFAT to translocate to the nucleus to regulate target genes. TCR signaling elevates cytoplasmic Ca 2ϩ concentrations by inducing store-operated Ca 2ϩ entry (SOCE), a process in which inositol-1,4,5-triphosphate (IP 3 )-mediated release of Ca 2ϩ from the endoplasmic reticulum (ER) leads to the activation of Ca 2ϩ channels in the plasma membrane, resulting in Ca 2ϩ influx (4). During SOCE, the drop in the ER Ca 2ϩ concentration causes conformational changes in the EF hand and SAM domains of stromal interaction molecule 1 (STIM1), which reside in the ER lumen (5-9). These changes enhance STIM1 oligomerization and propagate across the transmembrane region into conformational changes that involve several cytoplasmic domains, resulting in the extension of coiled-coil domains, the exposure of the STIM1 Ca 2ϩ release-activated Ca 2ϩ (CRAC) activation domain (CAD; also called SOAR and Ccb9), which bind...

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Transcriptional target-based expression cloning of immunoregulatory molecules

Cited by 2 publications

References 13 publications

A Quantitative Signaling Screen Identifies CARD11 Mutations in the CARD and LATCH Domains That Induce Bcl10 Ubiquitination and Human Lymphoma Cell Survival

A Quantitative Signaling Screen Identifies CARD11 Mutations in the CARD and LATCH Domains That Induce Bcl10 Ubiquitination and Human Lymphoma Cell Survival

A Protease-Independent Function for SPPL3 in NFAT Activation

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