The role of CARMA1 phosphorylation in T cell receptor-induced NF-kappaB activation
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The stimulation of T cell receptor (TCR) and CD3 complexes induces a series of signaling transduction events leading to activation of multiple transcription factors including NF-AT, NF-κB, and AP-1. The costimulation of the TCR/CD3 complex and CD28 receptors is known as CD3-CD28 costimulation. The contact site between antigen-presenting cells (APC) and T cells is termed an immunological synapse or microdomain, and CD3-CD28 co-stimulation initiates the aggregation of functional microdomains containing signaling molecules at this immunological synapse. PKC[straight theta] was suggested to be the major isoform of protein kinase C (PKC) moving into the immunological synapse, and mediates NF-κB activation after CD3-CD28 costimulation in T cells and PKCβ in B cells. Recent studies demonstrated that CARMA1, a membrane-associated guanylate-kinase-like (MAGUK) molecule, is required for CD3-CD28 costimulation-induced NF-κB activation and functions downstream of PKC[straight theta]. However, the mechanism by which PKC[straight theta] links to CARMA1 and leads to the recruitment of downstream signaling molecules remains to be determined. In this study, we found that CARMA1 was phosphorylated, and this phosphorylation was enhanced after TCR stimulation in Jurkat T cells. This phosphorylation was likely induced by PKC[straight theta], since co-expression of a constitutively active form of PKC[straight theta] and CARMA1 in HEK293 cells, led to a significant amount of phosphorylated CARMA1. Our mutagenesis studies indicate that Ser552, Ser637, and Ser645 in the Linker region of CARMA1 are the critical sites for PKC-dependent phosphorylation. In addition to these putative PKC phosphorylation sites, we found that Ser565, which is not within a PKC-consensus site, seems to be an important phosphorylation site by an unknown kinase, since a S565A mutant could not activate NF-κB upon PMA-CD28 stimulation. Further more, using an in vitro kinase assay, we found that PCK[straight theta] phosphorylated the Linker region of CARMA1, and this phosphorylation mainly occurred on Ser 552. The purification of lipid rafts from CARMA1-deficient cells stably reconstituted with wild type or mutant forms of CARMA1 revealed that this phosphorylation is important for the recruitment of NEMO into the immunological synapse, thus leading to the NF-κB activation. Mutation studies of CARMA3, a family member of CARMA1 expressed in nonhematopoietic cells, indicated that CARMA3 also has a conserved critical Ser520 residue, which is aligned with either Ser552 or Se555 of CARMA1. This result suggests that the phosphorylation may be a conserved mechanism for the activation of CARMA family proteins. Since recent studies indicate that CARMA3 functions in G protein-coupled receptor (GPCR) signaling pathways, and is required for the NF-κB activation induced by GPCRs, our results suggest that CARMA3 may also undergo a GPCR-dependent phosphorylation and this phosphorylation may involve Ser520. Together, these studies provide the genetic evidence that the phosphorylation of CARMA family members plays an important role for mediating NF-κB activation.