Disease-Causing SAP Mutants Are Defective in Ligand Binding and Protein Folding

Liu, Chengjun; Iosef, Cristiana; Jia, Christina Y.H.; Gkourasas, Theofanis; Han, V. K. M.; Li, Shawn Shun‐Cheng

doi:10.1021/bi034798l

Cited by 25 publications

(20 citation statements)

References 27 publications

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“…The GST-SAP(R55L) mutant also exhibits reduced Fyn binding (Figure 2D and (41)), (although the extent of this reduction was more variable), consistent with the inducible formation of a SLAM-SAP-Fyn complex (40). In contrast, GST-SAP(R55L) was able to pull-down PKC-θ from lysates of untreated or PV stimulated cells at similar levels as GST-SAP.…”

Section: Resultssupporting

confidence: 59%

Biochemical and Genetic Evidence for a SAP-PKC-θ Interaction Contributing to IL-4 Regulation

Cannons

Gómez-Rodríguez

et al. 2010

The Journal of Immunology

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SAP, an adaptor molecule that recruits Fyn to the SLAM-family of immunomodulatory receptors, is mutated in X-linked lymphoproliferative disease. CD4+ T cells from SAP-deficient mice have defective TCR-induced IL-4 production and impaired T cell-mediated help for germinal center formation; however, the downstream intermediates contributing to these defects remain unclear. We previously found that SAP-deficient CD4+ T cells exhibit decreased PKC-θ recruitment upon TCR stimulation. We demonstrate here using GST-pulldowns and co-immunoprecipitation studies that SAP constitutively associates with PKC-θ in T cells. SAP-PKC-θ interactions required R78 of SAP, a residue previously implicated in Fyn recruitment, yet SAP’s interactions with PKC-θ occurred independent of phosphotyrosine binding and Fyn. Overexpression of SAP in T cells increased and sustained PKC-θ recruitment to the immune synapse and elevated IL-4 production in response to TCR plus SLAM-mediated stimulation. Moreover, PKC-θ, like SAP, was required for SLAM-mediated increases in IL-4 production and conversely, membrane-targeted PKC-θ mutants rescued IL-4 expression in SAP−/− CD4+ T cells, providing genetic evidence that PKC-θ is a critical component of SLAM/SAP-mediated pathways that influence TCR-driven IL-4 production.

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Section: Resultssupporting

confidence: 59%

Biochemical and Genetic Evidence for a SAP-PKC-θ Interaction Contributing to IL-4 Regulation

Cannons

Gómez-Rodríguez

et al. 2010

The Journal of Immunology

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“…4C). Conversely, in the same assay SAP mutants unable to bind either SH3-domains (SAP-R 78 A) or both tyrosine-phosphorylated proteins and SH3 domains (SAP-R 55 L) (3, 35) failed to inhibit DGKα (Fig. 4C).…”

Section: Resultsmentioning

confidence: 91%

SAP-Mediated Inhibition of Diacylglycerol Kinase α Regulates TCR-Induced Diacylglycerol Signaling

Baldanzi

Pighini

Bettio

et al. 2011

The Journal of Immunology

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Diacylglycerol kinases (DGKs) metabolize diacylglycerol (DAG) to phosphatidic acid (PA). In T lymphocytes, DGKα acts as a negative regulator of TCR signaling by decreasing diacylglycerol levels and inducing anergy. Here, we show that upon co-stimulation of the TCR with CD28 or SLAM, DGKα, but not DGKζ, exit from the nucleus and undergoes rapid negative regulation of its enzymatic activity. Inhibition of DGKα is dependent on the expression of SAP, an adaptor protein mutated in X-linked lymphoproliferative disease (XLP), which is essential for SLAM-mediated signaling and contributes to TCR/CD28-induced signaling and T cell activation. Accordingly, over-expression of SAP is sufficient to inhibit DGKα, while SAP mutants unable to bind either phospho-tyrosine residues or SH3 domain are ineffective. Moreover phospholipase C activity and calcium, but not Src-family tyrosine kinases, are also required for negative regulation of DGKα. Finally, inhibition of DGKα in SAP-deficient cells partially rescues defective TCR/CD28 signaling, including Ras and ERK-1/2 activation, PKCθ membrane recruitment, induction of NF-AT transcriptional activity and IL-2 production. Thus SAP-mediated inhibition of DGKα sustains diacylglycerol signaling, thereby regulating T cell activation and may represent a novel pharmacological strategy for XLP treatment.

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“…To test whether such binding is required for SAP-mediated control of T:B conjugation, we examined the SAP R55L mutant that prevents SAP from binding SLAM (Calpe et al, 2008; Chen et al, 2004; Li et al, 2003; Ma et al, 2007). We also evaluated the SAP R78A mutant, which shows impaired Fyn binding but can still associate with SLAM family members and rescue GC formation in Sh2d1a −/− mice (Cannons et al, 2006; McCausland et al, 2007).…”

Section: Resultsmentioning

confidence: 99%

Optimal Germinal Center Responses Require a Multistage T Cell:B Cell Adhesion Process Involving Integrins, SLAM-Associated Protein, and CD84

et al. 2010

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CD4+ T cells deficient in signaling lymphocyte activation molecule (SLAM)-associated protein (SAP) exhibit a selective impairment in adhesion to antigen presenting B cells but not dendritic cells (DC), resulting in defective germinal center formation. However, the nature of this selective adhesion defect remained unclear. We found that whereas T:DC interactions were primarily integrin-dependent, T:B cell interactions had both an early integrin-dependent phase and a sustained phase that also required SAP. We further found that the SLAM family member, CD84, was required for prolonged T:B cell contact, optimal T follicular helper function, and germinal center formation in vivo. Moreover, both CD84 and another SLAM member, Ly108, mediated T cell adhesion and participated in stable T:B cell interactions in vitro. Our results reveal insight into the dynamic regulation of T:B cell interactions and identify SLAM family members as critical components of sustained T:B cell adhesion required for productive humoral immunity.

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Disease-Causing SAP Mutants Are Defective in Ligand Binding and Protein Folding

Cited by 25 publications

References 27 publications

Biochemical and Genetic Evidence for a SAP-PKC-θ Interaction Contributing to IL-4 Regulation

Biochemical and Genetic Evidence for a SAP-PKC-θ Interaction Contributing to IL-4 Regulation

SAP-Mediated Inhibition of Diacylglycerol Kinase α Regulates TCR-Induced Diacylglycerol Signaling

Optimal Germinal Center Responses Require a Multistage T Cell:B Cell Adhesion Process Involving Integrins, SLAM-Associated Protein, and CD84

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