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Biochemical and genetic evidence for a SAP-PKC-theta interaction contributing to IL-4 regulation.

ABSTRACT: Signaling lymphocytic activation molecule-associated protein (SAP), an adaptor molecule that recruits Fyn to the signaling lymphocytic activation molecule (SLAM) family of immunomodulatory receptors, is mutated in X-linked lymphoproliferative disease. CD4(+) T cells from SAP-deficient mice have defective TCR-induced and follicular Th cell 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 protein kinase C (PKC)-theta recruitment upon TCR stimulation. We demonstrate in this paper using GST pulldowns and coimmunoprecipitation studies that SAP constitutively associates with PKC- in T cells. SAP-PKC-theta interactions required R78 of SAP, a residue previously implicated in Fyn recruitment, yet SAP's interactions with PKC-theta occurred independent of phosphotyrosine binding and Fyn. Overexpression of SAP in T cells increased and sustained PKC-theta recruitment to the immune synapse and elevated IL-4 production in response to TCR plus SLAM-mediated stimulation. Moreover, PKC-theta, like SAP, was required for SLAM-mediated increases in IL-4 production, and, conversely, membrane-targeted PKC-theta mutants rescued IL-4 expression in SAP(-/-) CD4(+) T cells, providing genetic evidence that PKC-theta is a critical component of SLAM/SAP-mediated pathways that influence TCR-driven IL-4 production.

SUBMITTER: Cannons JL

PROVIDER: S-EPMC3422635 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Biochemical and genetic evidence for a SAP-PKC-theta interaction contributing to IL-4 regulation.

Cannons Jennifer L JL Wu Julie Z JZ Gomez-Rodriguez Julio J Zhang Jinyi J Dong Baoxia B Liu Yin Y Shaw Stephen S Siminovitch Katherine A KA Schwartzberg Pamela L PL

Journal of immunology (Baltimore, Md. : 1950) 20100728 5

Signaling lymphocytic activation molecule-associated protein (SAP), an adaptor molecule that recruits Fyn to the signaling lymphocytic activation molecule (SLAM) family of immunomodulatory receptors, is mutated in X-linked lymphoproliferative disease. CD4(+) T cells from SAP-deficient mice have defective TCR-induced and follicular Th cell IL-4 production and impaired T cell-mediated help for germinal center formation; however, the downstream intermediates contributing to these defects remain unc ...[more]

PMID: 20668219

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Project description:It has been shown that sphingolipid activator proteins (SAPs) 1 and 2 are encoded on the same gene along with two other putative activator proteins [Fürst, Machleidt & Sandhoff (1988) Biol. Chem. Hoppe-Seyler 369, 317-328 and O'Brien, Kretz, Dewji, Wenger, Esch & Fluharty (1988) Science 241, 1098-1101]. We have undertaken further biochemical investigations on a patient and fetal sibling, who were previously shown to have a unique sphingolipid storage disorder associated with an SAP-2 deficiency [Harzer, Paton, Poulos, Kustermann-Kuhn, Roggendorf, Grisar & Popp (1989) Eur. J. Pediatr. 149, 31-39]. The severity of their disorder suggested that other products of the SAP precursor or prosaposin gene may also be deficient. The turnover of cerebroside sulphate and globotriaosylceramide were investigated and were both impaired in fibroblasts from the patient and fetus. However, the activities of cerebroside sulphate sulphatase and globotriaosylceramide alpha-galactosidase in vitro were normal in cells from the fetus and patient respectively. In addition, there was an increase in cerebroside sulphate concentration in the kidney of the affected fetus. These results indicate that, in addition to the SAP-2 deficiency, there was a defect in SAP-1 function in this disorder. Additional increases in the concentration of monohexosyl- and dihexosyl-ceramide in the fetal kidney probably reflect the deficiency of SAP-2 in the case of monohexosylceramides, and the combined activator deficiency in the case of dihexosylceramides. Lactosylceramide-loading studies confirmed that there was a defect in the turnover of this lipid in fibroblasts from the affected patient and fetus but not from a patient with an isolated SAP-1 deficiency, or from patients with Krabbe disease, GM1 gangliosidosis or galactosialidosis. It has been suggested [Potier, Lamontagne, Michaud & Tranchemontagne (1990) Biochem. Biophys. Res. Commun. 173, 449-456] that the prosaposin gene also codes for lysosomal neuroaminidase. However, we found normal neuraminidase activity in fibroblasts from our patient, using assay conditions which are diagnostic for sialidosis patients. The role of prosaposin gene products in sphingolipid metabolism is discussed in view of our biochemical findings in this genetic disorder.

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Biochemical and genetic evidence for a SAP-PKC-theta interaction contributing to IL-4 regulation.

Publications

Biochemical and genetic evidence for a SAP-PKC-theta interaction contributing to IL-4 regulation.

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