The ligation of cell-surface antigen receptors initiates coordinated recruitment of various signalling molecules to the membrane, to form membrane-associated signalling complexes or 'signalosomes' that control lymphocyte proliferation and differentiation. Now, two papers published in Immunity show that, within these signalosomes, CARMA1 (CARD–MAGUK protein 1) acts as a scaffold that coordinates the assembly of the various kinases, adaptors and other effectors that control downstream nuclear factor-κB (NF-κB) activation. Moreover, these studies indicate that phosphorylation of the CARMA1 linker domain by protein kinase C (PKC)-family members provides the missing link between activation of PKC and initiation of the downstream response.

Previous studies have shown that PKCβ (in B cells) and PKCθ (in T cells) are crucial for activation of the inhibitor-of-NF-κB kinase (IKK) complex, which controls NF-κB activation, but the downstream targets of these PKC isoforms that make the link to IKK-complex activation are unknown. However, the recent identification of the proteins CARMA1, BCL-10 and MALT1, which act downstream of PKC-family members in immunoreceptor-dependent NF-κB activation, prompted the authors to assess whether these proteins were targeted by PKC isoforms.

By western-blot analysis, Sommer et al. first confirmed that, after ligation of the B-cell receptor (BCR), PKCβ, CARMA1, BCL-10 and the IKK complex are recruited to immunoreceptor-containing lipid rafts. Using a PKCβ-specific inhibitor, they showed that the lipid-raft recruitment of the signalosome components was PKCβ dependent. Although some PKCβ was shown to constitutively associate with CARMA1, cell activation markedly increased this interaction. Importantly, after BCR ligation, this interaction resulted in PKCβ-mediated phosphorylation of CARMA1, but not of BCL-10. Similarly, Matsumoto et al. showed that CARMA1 but not BCL-10 or MALT1, was inducibly phosphorylated by PKCθ after T-cell activation through T-cell-receptor ligation and CD28 co-stimulation.

Next, both groups carried out studies to identify the sites of PKC-dependent phosphorylation in CARMA1. CARMA1 is composed of five domains: an amino-terminal caspase-recruitment domain (CARD), a coiled-coil, a PDZ domain, and a carboxy-terminal SRC homology 3 domain and guanylate kinase (GUK)-like domain. And between the coiled-coil and PDZ domains is a linker domain. By generating a series of CARMA1 mutants, both groups showed that serine residues in the linker domain are targeted by PKCβ or PKCθ. Reconstitution of cells with CARMA1 mutants that lacked these serine residues did not affect recruitment of CARMA1 to lipid rafts but impaired subsequent recruitment of BCL-10 and IKK, and inhibited NF-κB activation.

On the basis of these observations, the authors proposed that phosphorylation of the CARMA1 linker domain induced a conformational change that exposed the CARD for recruitment of BCL-10. Indeed, Sommer et al. showed that in its unphosphorylated (inactive) state, the linker domain of CARMA1 interacts with the CARD, blocking accessibility of BCL-10 to the CARD. Antigen-receptor-triggered PKC-dependent linker phosphorylation is required to release this inhibition, thereby allowing BCL-10 recruitment and signal propagation to the IKK complex. Consistent with this model, deletion of the linker promoted constitutive IKK-complex activation and PKC-independent NF-κB activation.