Dimethyl fumarate (DMF) is an electrophilic drug that is used to treat autoimmune conditions, including multiple sclerosis and psoriasis. The mechanism of action of DMF is unclear, but may involve the covalent modification of proteins or DMF serving as a pro-drug that is converted to monomethyl fumarate (MMF). Here, we found that DMF, but not MMF, blocked the activation of primary human and mouse T cells. Using a quantitative, site-specific chemical proteomic platform, we determined the DMF-sensitivity of > 2400 cysteine residues in human T cells. Cysteines sensitive to DMF, but not MMF, were identified in several proteins with established biochemical or genetic links to T cell function, including protein kinase C θ (PKCθ). Furthermore, DMF blocked the association of PKCθ with the costimulatory receptor CD28 by perturbing a CXXC motif in the C2 domain of this kinase. Mutation of these DMF-sensitive cysteines also impaired PKCθ-CD28 interactions and T cell activation, designating the C2 domain of PKCθ as a key functional, electrophile-sensing module important for T cell biology.

Sumoylation regulates many cellular processes, but its role in signaling via the T cell antigen receptor (TCR) remains unknown. We found that the kinase PKC-θ was sumoylated upon costimulation with antigen or via the TCR plus the coreceptor CD28, with Lys325 and Lys506 being the main sumoylation sites. We identified the SUMO E3 ligase PIASxβ as a ligase for PKC-θ. Analysis of primary mouse and human T cells revealed that sumoylation of PKC-θ was essential for T cell activation. Desumoylation did not affect the catalytic activity of PKC-θ but inhibited the association of CD28 with PKC-θ and filamin A and impaired the assembly of a mature immunological synapse and central co-accumulation of PKC-θ and CD28. Our findings demonstrate that sumoylation controls TCR-proximal signaling and that sumoylation of PKC-θ is essential for the formation of a mature immunological synapse and T cell activation.

TNF receptor-associated factor 6 (TRAF6) is an essential ubiquitin E3 ligase in immune responses, but its function in adaptive immunity is not well understood. Here we show that TRAF6 is recruited to the peripheral ring of the T cell immunological synapse in Jurkat T cells or human primary CD4+ T cells conjugated with SEE-pulsed B cells. This recruitment depends on TRAF6 interacting with linker for activation of T cells (LAT) via its TRAF domain. Although LAT was indispensable for TCR/CD28-induced TRAF6 ubiquitination and its ligase activity, RNA interference-induced TRAF6 knockdown in T cells decreased TCR/CD28-induced LAT ubiquitination, tyrosine-phosphorylation and association with tyrosine kinase ZAP70. Overexpression of TRAF6 or its catalytically inactive form C70A promoted and decreased, respectively, LAT tyrosine phosphorylation upon stimulation. Moreover, LAT was ubiquitinated at Lysine-88 by TRAF6 via K63-linked chain. In addition, TRAF6 was required for and synergized with LAT to promote the TCR/CD28-induced activation of NFAT. These results reveal a novel function and mechanism of TRAF6 action in the TCR-LAT signaling pathway distinct from its role in TCR-induced NF-κB activation, indicate LAT also play an adapter role in TCR/CD28-induced activation of TRAF6.

Protein kinase C-θ (PKCθ) is an important component of proximal T cell receptor (TCR) signaling. We previously identified the amino-terminal C2 domain of PKCθ as a phosphotyrosine (pTyr)–binding domain. Using a mutant form of PKCθ that cannot bind pTyr (PKCθHR2A), we showed that pTyr binding by PKCθ was required for TCR-induced T cell activation, proliferation, and TH2 cell differentiation but not for T cell development. Using tandem mass spectrometry and coimmunoprecipitation, we identified the kinase ζ-associated protein kinase of 70 kDa (Zap70) as a binding partner of the PKCθ pTyr-binding pocket. Tyr126 of Zap70 directly bound to PKCθ, and the interdomain B residues Tyr315 and Tyr319 were indirectly required for binding to PKCθ, reflecting their role in promoting the open conformation of Zap70. PKCθHR2A-expressing CD4+ T cells displayed defects not only in known PKCθ-dependent signaling events, such as nuclear factor κB (NF-κB) activation and TH2 cell differentiation, but also in full activation of Zap70 itself and in the activating phosphorylation of linker of activation of T cells (LAT) and phospholipase C-γ1 (PLCγ1), signaling proteins that are traditionally considered to be activated independently of PKC. These findings demonstrate that PKCθ plays an important role in a positive feedback regulatory loop that modulates TCR-proximal signaling and, moreover, provide a mechanistic explanation for earlier reports that documented an important role for PKCθ in T cell Ca2+ signaling. This PKCθ-Zap70 interaction could potentially serve as a promising and highly selective immunosuppressive drug target in autoimmunity and organ transplantation.

The SUMO modification system plays an important role in T cell activation, yet how sumoylation regulates TCR-proximal signaling remains largely unknown. We show here that Phospholipase C-γ1 (PLC-γ1) is conjugated by SUMO1 at K54 and K987 upon TCR stimulation and that K54 sumoylation is pivotal for PLC-γ1-mediated T cell activation. We further demonstrate that TCR-induced K54 sumoylation of PLC-γ1 significantly promotes the formation of PLC-γ1 microclusters and the association of PLC-γ1 with the adaptor proteins SLP76 and Gads, but only slightly affects the phosphorylation of PLC-γ1 on Y783, which determines the enzyme catalytic activity. Moreover, upon TCR stimulation, the SUMO E3 ligases PIASxβ and PIAS3 both interact with PLC-γ1 and cooperate to sumoylate PLC-γ1, facilitating the assembly of PLC-γ1 microclusters. Together, our findings reveal a critical role of PLC-γ1 K54 sumoylation in PLC-γ1 microcluster assembly that controls PLC-γ1-mediated T cell activation, suggesting that sumoylation may have an important role in the microcluster assembly of TCR-proximal signaling proteins.

PKCθ plays a key role in T cell activation, but the mechanisms that control its activation are not fully understood. We previously reported that the PKCθ N-terminal C2 region is a novel regulatory phosphotyrosine (pTyr)-binding domain. However, the physiological pTyr-containing C2-binding ligand has remained unknown. Using a GST-PKCθ-C2 recombinant fusion protein in pull-down assays, we detected a ~75-kDa pTyr-containing protein that associated with wild-type PKCθ-C2, but not with a pTyr non-binding PKCθ-C2 mutant after TCR/CD28 costimulation. Mass spectrometry analysis identified this protein as ZAP70 kinase. The stimulation-induced association of ZAP70 with full-length PKCθ or PKCθ-C2 in T cells was confirmed by co-immunoprecipitation assays, and was mediated by residues pY315 and pY319 in interdomain B of ZAP70. This transient association upon T cell activation required Lck kinase, followed by rapid complex dissociation that led to ZAP70 phosphorylation on Y493, resulting in full activation of ZAP70 and downstream signals. Mutation of two PKCθ-C2 residues essential for pTyr binding abolished PKCθ’s ability to rescue TCR- and CD28-induced IL-2 production, proliferation and Th2 differentiation in PKCθ-deficient T cells, without affecting T cell development or Th1 differentiation. These data suggest anchoring of PKCθ-C2 domain to phosphorylated interdomain B of ZAP70 promotes the activation of both kinases, thereby provided a novel pharmaceutical target for immunosuppression.

TNF receptor-associated factor 6 (TRAF6) is an essential ubiquitin E3 ligase in immune responses, but its function in adaptive immunity is not well understood. Here we show that TRAF6 is recruited to the peripheral ring of the T cell immunological synapse in Jurkat T cells or human primary CD4+ T cells conjugated with SEE-pulsed B cells. This recruitment depends on TRAF6 interacting with linker for activation of T cells (LAT) via its TRAF domain. Although LAT was indispensable for TCR/CD28-induced TRAF6 ubiquitination and its ligase activity, RNA interference-induced TRAF6 knockdown in T cells decreased TCR/CD28-induced LAT ubiquitination, tyrosine-phosphorylation. Overexpression of TRAF6 or its catalytically inactive form C70A promoted and decreased, respectively, LAT tyrosine-191 phosphorylation upon stimulation. Moreover, LAT was ubiquitinated at Lysine-88 by TRAF6 via K63-linked chain. In addition, TRAF6 was required for and synergized with LAT to promote the TCR/CD28-induced activation of NFAT. These results reveal a novel function and mechanism of TRAF6 action in the TCR-LAT signaling pathway distinct from its role in TCR-induced NF-κB activation, and indicate LAT also play an adapter role in TCR/CD28-induced activation of TRAF6.

As an essential ubiquitin E3 ligase in immune responses, the function and mechanism of TNF receptor-associated factor 6 (TRAF6) in adaptive immunity has not been illustrated as well as in innate immunity. Here we show that upon activation of T cells, TRAF6 is recruited to the peripheral ring of the T cells immunological synapse (IS), and is invovled in CD3/CD28-induced NFAT activation. The underlying mechanism is under study.