Biochemical and genetic evidence for a SAP-PKC-theta interaction contributing to IL-4 regulation.
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.
Cannons
Jennifer L
JL
National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.
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
D000818:Animals; D015703:Antigens, CD / Q000235:genetics* / Q000378:metabolism*; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D005786:Gene Expression Regulation / Q000276:immunology; D006801:Humans; D015847:Interleukin-4 / Q000096:biosynthesis* / Q000172:deficiency / Q000235:genetics; D007527:Isoenzymes / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism*; D019169:Jurkat Cells; D008213:Lymphocyte Activation / Q000235:genetics / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008822:Mice, Transgenic; D011493:Protein Kinase C / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism*; D000074121:Protein Kinase C-theta; D021381:Protein Transport / Q000235:genetics / Q000276:immunology; D011956:Receptors, Cell Surface / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology; D000071177:Signaling Lymphocytic Activation Molecule Family Member 1; D015854:Up-Regulation / Q000235:genetics / Q000276:immunology
D015703:Antigens, CD; D007527:Isoenzymes; D011956:Receptors, Cell Surface; D000071177:Signaling Lymphocytic Activation Molecule Family Member 1; D015847:Interleukin-4; C489593:Prkcq protein, mouse; D011493:Protein Kinase C; D000074121:Protein Kinase C-theta