NF-κB Activation in T Cells Requires Discrete Control of IκB Kinase α/β (IKKα/β) Phosphorylation and IKKγ Ubiquitination by the ADAP Adapter Protein

Abstract

NF-B activation following engagement of the antigen-specific T cell receptor involves protein kinase C--dependent assembly of the CARMA1-BCL10-MALT1 (CBM) signalosome, which coordinates downstream activation of IB kinase (IKK).We previously identified a novel role for the adhesion-and degranulation-promoting adapter protein (ADAP) in regulating the assembly of the CBM complex via an interaction of ADAP with CARMA1. In this study, we identify a novel site in ADAP that is critical for association with the TAK1 kinase. ADAP is critical for recruitment of TAK1 and the CBM complex, but not IKK, to protein kinase C-. ADAP is not required for TAK1 activation. Although both the TAK1 and the CARMA1 binding sites in ADAP are essential for IB␣ phosphorylation and degradation and NF-B nuclear translocation, only the TAK1 binding site in ADAP is necessary for IKK phosphorylation. In contrast, only the CARMA1 binding site in ADAP is required for ubiquitination of IKK␥. Thus, distinct sites within ADAP control two key activation responses that are required for NF-B activation in T cells.In the immune system, the NF-B transcription factor pathway plays a central role in T cell activation and survival (1, 2). The canonical NF-B pathway involves activation of the IBkinase (IKK) 3 complex, which consists of the catalytic IKK␣ and IKK␤ subunits and the regulatory IKK␥ (NF-B essential modulator (NEMO)) subunit. Activated IKK mediates phosphorylation of IB␣, resulting in IB␣ degradation and translocation of NF-B to the nucleus. In T cells, stimulation of the T cell receptor and the CD28 co-stimulatory receptor results in activation of the PKC isoform and association of the IKK complex with PKC (3). PKC phosphorylates the membrane-associated guanylate kinase (MAGUK) family member adapter CARMA1 (4, 5), which then associates with the adapters BCL-10 and mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) (6, 7). Activation of the IKK complex is proposed to require both Lys-63-linked ubiquitination of IKK␥ (8, 9) and IKK␣/␤ phosphorylation (10). Recent studies suggest that IKK␥ ubiquitination is dependent on efficient CBM complex formation, whereas phosphorylation of IKK␣/␤ is mediated by TGF␤-activated kinase (TAK1) (11). Both of these regulatory events appear to be required for efficient NF-B activation as loss of expression of CARMA1, BCL-10, MALT1, or TAK1 results in impaired NF-B signaling in T cells (7,(12)(13)(14)(15). The mechanism by which TAK1 is recruited to the PKC signalosome is unclear. Although CARMA1 and TAK1 have been reported to associate with each other (11,16,17), TAK1-mediated IKK␣/␤ phosphorylation is intact in CARMA1-deficient Jurkat T cells (11). This suggests that IKK␥ ubiquitination and IKK␣/␤ phosphorylation are independently controlled.Adhesion-and degranulation-promoting adapter protein (ADAP) is a hematopoietic-specific adapter protein that regulates "inside-out" signaling from the T cell receptor to integrins (18,19). We previously identified a novel function for ADAP in controlling ...

ADAP Regulates Cell Cycle Progression of T Cells via Control of Cyclin E and Cdk2 Expression through Two Distinct CARMA1-Dependent Signaling Pathways

“…Adenovirus expression plasmids and recombinant adenovirus were generated as previously described (5,24). The ADAP⌬CAR (deletion of aa 426 to 541 in mouse ADAP), ADAP⌬TAK (deletion of aa 691 to 708 in mouse ADAP), and ADAP⌬SKAP (deletion of aa 338 to 358 in mouse ADAP) mutants were generated as previously described (5,24,38). Freshly isolated resting hCAR-positive (hCAR ϩ ) control and ADAP Ϫ/Ϫ lymph node T cells were transduced with control virus or ADAP virus and incubated at 37°C for 3 days in complete T cell medium containing 10 ng/ml mouse interleukin-7 (R&D Systems) as previously described (4,5,24,38).…”

“…These inducible interactions facilitate the formation of the CARMA1-Bcl10-Malt1 (CBM) complex and the assembly of the protein kinase C (PKC) signalosome that are required for optimal T cell receptor-mediated activation of NF-B (42). Three discrete sites in ADAP mediate the association of ADAP with SKAP55, CARMA1, and TAK1 (24,38). T cells lacking ADAP exhibit impaired TCR-mediated proliferation (16,27,28), but the contribution of these individual protein interactions with ADAP to this proliferative defect remains undefined.…”

“…These two functions of ADAP are controlled by biochemically and functionally distinct pools of ADAP that are defined by SKAP55, another adapter that constitutively associates with a subset of the total ADAP expressed in a T cell (4,5). The pool of ADAP associated with SKAP55 regulates integrin function, while the pool of ADAP not associated with SKAP55 regulates NF-B via TCR-inducible association with the CARMA1 adapter and the serine/threonine kinase transforming growth factor ␤-activated kinase 1 (TAK1) (4,5,24,38). These inducible interactions facilitate the formation of the CARMA1-Bcl10-Malt1 (CBM) complex and the assembly of the protein kinase C (PKC) signalosome that are required for optimal T cell receptor-mediated activation of NF-B (42).…”

“…Adapter proteins coordinate the assembly of signalosomes that are essential for optimal T cell activation (36). In T cells, adhesion and degranulation-promoting adapter protein (ADAP) positively regulates T cell receptor signaling by facilitating the activation of integrin receptors that enhances T cell contact with antigen-presenting cells and by promoting the activation of NF-B (4,5,16,24,28,38,46). These two functions of ADAP are controlled by biochemically and functionally distinct pools of ADAP that are defined by SKAP55, another adapter that constitutively associates with a subset of the total ADAP expressed in a T cell (4,5).…”

T Cell–Independent Modulation of Experimental Autoimmune Encephalomyelitis in ADAP-Deficient Mice

“…In this study, ADAP is critical for the assembly of the Carma1-Bcl10-Malt1-complex (11). Furthermore, ADAP associates with the adaptor molecule Nck (12).…”