A Protease-Independent Function for SPPL3 in NFAT Activation

Makowski, Stefanie L.; Wang, Zhaoquan; Pomerantz, Joel L.

doi:10.1128/mcb.01124-14

Cited by 24 publications

(22 citation statements)

References 83 publications

(85 reference statements)

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“…At 40 h after transfection, cells were stimulated in 1 ml medium alone or medium supplemented with 1 g/ml each of mouse anti-human CD3 (catalog number 555329; BD Pharmingen), mouse anti-human CD28 (catalog number 555725; BD Pharmingen), and anti-mouse IgG1 (catalog number 553440; BD Pharmingen) for 4 to 5 h. For tumor necrosis factor alpha (TNF-␣) stimulation assays, 75 ng/ml TNF-␣ (catalog number T6674; Sigma) was used. Nuclear factor of activated T cell (NFAT) assays used 1,500 ng of the NFAT 4 -IFN-LUC reporter (38) and anti-CD3 in the absence of anti-human CD28 during stimulation. Samples were harvested in 150 l Promega lysis buffer and assayed for luciferase and ␤-Gal activity as previously described (15).…”

Section: Bret-based Interaction Cloning (Bric)mentioning

confidence: 99%

Negative Regulation of CARD11 Signaling and Lymphoma Cell Survival by the E3 Ubiquitin Ligase RNF181

Pedersen

Chan

Jattani

et al. 2016

Molecular and Cellular Biology

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NF-B activation downstream of antigen receptor engagement is a highly regulated event required for lymphocyte activation during the adaptive immune response. The pathway is often dysregulated in lymphoma, leading to constitutive NF-B activity that supports the aberrant proliferation of transformed lymphocytes. To identify novel regulators of antigen receptor signaling to NF-B, we developed bioluminescence resonance energy transfer-based interaction cloning (BRIC), a screening strategy that can detect protein-protein interactions in live mammalian cells in a high-throughput manner. Using this strategy, we identified the RING finger protein RNF181 as an interactor of CARD11, a key signaling scaffold in the antigen receptor pathway. We present evidence that RNF181 functions as an E3 ubiquitin ligase to inhibit antigen receptor signaling to NF-B downstream of CARD11. The levels of the obligate signaling protein Bcl10 are reduced by RNF181 even prior to signaling, and Bcl10 can serve as a substrate for RNF181 E3 ligase activity in vitro. Furthermore, RNF181 limits the proliferation of human diffuse large B cell lymphoma cells that depend upon aberrant CARD11 signaling to NF-B for growth and survival in culture. Our results define a new regulatory checkpoint that can modulate the output of CARD11 signaling to NF-B in both normal and transformed lymphocytes.T he activation of the NF-B transcription factor downstream of antigen receptor engagement is a critical event required for lymphocyte activation during the adaptive immune response (1, 2). In humans and mice, genetic defects in components of the antigen receptor signaling pathway result in immunodeficiency syndromes in which the failure to mount an effective immune response confers enhanced susceptibilities to infection and impaired clearance of pathogens (3, 4).CARD11 is a critical signaling scaffold protein that functions to transmit signals from triggered antigen receptors to the IB kinase (IKK) complex, which inducibly phosphorylates inhibitory IB proteins, leading to their ubiquitinylation and degradation, which allows NF-B heterodimers to stably translocate to the nucleus (5-14). During antigen receptor signaling, CARD11 undergoes a transition from a closed, inactive state to an open, active scaffold that recruits several signaling cofactors into a complex that induces IKK kinase activity. CARD11 is kept inactive prior to receptor engagement by an inhibitory domain (ID) that participates in intramolecular interactions that involve the CARD, LATCH, and coiled-coil (CC) domains (15, 16). Triggering of the T cell receptor (TCR) or B cell receptor (BCR) results in the phosphorylation of the ID on specific serines (17-21), which neutralizes its inhibitory activity and allows the signal-induced recruitment of obligate signaling cofactors to CARD11, including Bcl10, MALT1, TRAF6, IKK␥, and caspase-8 (15). The formation and stability of the CARD11-nucleated multiprotein complex dictate the extent and duration of IKK kinase activity and NF-B activation following antig...

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Section: Bret-based Interaction Cloning (Bric)mentioning

confidence: 99%

Negative Regulation of CARD11 Signaling and Lymphoma Cell Survival by the E3 Ubiquitin Ligase RNF181

Pedersen

Chan

Jattani

et al. 2016

Molecular and Cellular Biology

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“…Both protease-independent and protease-dependent functions for SPPL3 have recently been described (20–22). The phenotypes observed after expression of the protease-dead SPPL3 D271A allele in NK cells reveal that NK cell maturation relies on the proteolytic activity of SPPL3, and suggest that the protease-independent function of SPPL3 in facilitating store-operated calcium entry is not required in this context.…”

Section: Discussionmentioning

confidence: 99%

“…Both protease-dependent and protease-independent functions have very recently been described for SPPL3. In a protease-independent manner, SPPL3 functions in the endoplasmic reticulum (ER) in T cells to promote store-operated calcium entry in response to T cell receptor engagement by enhancing the interaction between STIM1 and Orai1 (20). SPPL3 has also been shown to function as a protease in cell lines and murine embryonic fibroblasts (MEFs) to regulate the extent of constitutive complex glycosylation by targeting several glycosylation enzymes for cleavage and shedding from the ER or Golgi (21, 22).…”

Section: Introductionmentioning

confidence: 99%

NK Cell Maturation and Cytotoxicity Are Controlled by the Intramembrane Aspartyl Protease SPPL3

Hamblet

Makowski

Tritapoe

et al. 2016

The Journal of Immunology

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NK cell maturation is critical for normal effector function and the innate immune response to tumors and pathogens. However, the molecular pathways that control NK cell maturation remain largely undefined. Here, we investigate the role of SPPL3, an intramembrane aspartyl protease, in murine NK cell biology. We find that deletion of SPPL3 in the hematopoietic system reduces numbers of peripheral NK cells, clearance of MHC Class I-deficient tumors in vivo, and cytotoxicity against tumor cells in vitro. This phenotype is concomitant with reduced numbers of CD27+CD11b+ and CD27−CD11b+ NK cells, indicating a requirement for SPPL3 in efficient NK cell maturation. NK cell-specific deletion of SPPL3 results in the same deficiencies, revealing a cell-autonomous role for SPPL3 in these processes. CRISPR/Cas9 genomic editing in murine zygotes was used to generate knock-in mice with a catalytically compromised SPPL3 D271A allele. Mice engineered to express only SPPL3 D271A in NK cells phenocopy mice deleted for SPPL3, indicating a requirement for SPPL3 protease activity in NK cell biology. Our results identify SPPL3 as a cell-autonomous molecular determinant of NK cell maturation and expand the role of intramembrane aspartyl proteases in innate immunity.

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“…Following up altered cellular N-glycosylation patterns associated with elevated or reduced SPPL3 expression in vitro and in vivo, Golgi-resident glycosyltransferases including the N-glycan branching enzyme N-acetylglucosaminyltransferase V (GnT-V) were recently uncovered as the first physiological type II membrane protein substrates of SPPL3 (9). Meanwhile, SPPL3 has additionally been shown to modulate cytosolic Ca 2ϩ release and NFAT (nuclear factor of activated T cells) signaling following T-cell receptor engagement apparently in a nonproteolytic fashion (10).…”

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confidence: 99%

Secretome Analysis Identifies Novel Signal Peptide Peptidase-Like 3 (SPPL3) Substrates and Reveals a Role of SPPL3 in Multiple Golgi Glycosylation Pathways*

Kuhn

Voß

Haug-Kröper

et al. 2015

Molecular & Cellular Proteomics

111

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Signal peptide peptidase-like 3 (SPPL3) is a Golgi-resident intramembrane-cleaving protease that is highly conserved among multicellular eukaryotes pointing to pivotal physiological functions in the Golgi network which are only beginning to emerge. Recently, SPPL3 was shown to control protein N-glycosylation, when the key branching enzyme N-acetylglucosaminyltransferase V (GnT-V) and other medial/trans Golgi glycosyltransferases were identified as first physiological SPPL3 substrates. SPPL3-mediated endoproteolysis releases the catalytic ectodomains of these enzymes from their type II membrane anchors. Protein glycosylation is a multistep process involving numerous type II membrane-bound enzymes, but it remains unclear whether only few of them are SPPL3 substrates or whether SPPL3 cleaves many of them and thereby controls protein glycosylation at multiple levels. Therefore, to systematically identify SPPL3 substrates we used Sppl3-deficient and SPPL3-overexpression cell culture models and analyzed them for changes in secreted membrane protein ectodomains using the proteomics "secretome protein enrichment with click sugars (SPECS)" method. SPECS analysis identified numerous additional new SPPL3 candidate glycoprotein substrates, several of which were biochemically validated as SPPL3 substrates. All novel SPPL3 substrates adopt a type II topology. The majority localizes to the Golgi network and is implicated in Golgi functions. Importantly, most of the novel SPPL3 substrates catalyze the modification of Nlinked glycans. Others contribute to O-glycan and in particular glycosaminoglycan biosynthesis, suggesting that SPPL3 function is not restricted to N-glycosylation, but also functions in other forms of protein glycosylation. Hence, SPPL3 emerges as a crucial player of Golgi function and the newly identified SPPL3 substrates will be instrumental to investigate the molecular mechanisms underlying the physiological function of SPPL3 in the Golgi network and in vivo. Data are available via ProteomeXchange with identifier PXD001672. Molecular

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A Protease-Independent Function for SPPL3 in NFAT Activation

Cited by 24 publications

References 83 publications

Negative Regulation of CARD11 Signaling and Lymphoma Cell Survival by the E3 Ubiquitin Ligase RNF181

Negative Regulation of CARD11 Signaling and Lymphoma Cell Survival by the E3 Ubiquitin Ligase RNF181

NK Cell Maturation and Cytotoxicity Are Controlled by the Intramembrane Aspartyl Protease SPPL3

Secretome Analysis Identifies Novel Signal Peptide Peptidase-Like 3 (SPPL3) Substrates and Reveals a Role of SPPL3 in Multiple Golgi Glycosylation Pathways*

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