The adapter protein ADAP regulates T lymphocyte adhesion and activation. We present evidence for a previously unrecognized function for ADAP in regulating T cell receptor (TCR)-mediated activation of the transcription factor NF-kappaB. Stimulation of ADAP-deficient mouse T cells with antibodies to CD3 and CD28 resulted in impaired nuclear translocation of NF-kappaB, a reduced DNA binding, and delayed degradation and decreased phosphorylation of IkappaB (inhibitor of NF-kappaB). TCR-stimulated assembly of the CARMA1-BCL-10-MALT1 complex was substantially impaired in the absence of ADAP. We further identified a region of ADAP that is required for association with the CARMA1 adapter and NF-kappaB activation but is not required for ADAP-dependent regulation of adhesion. These findings provide new insights into ADAP function and the mechanism by which CARMA1 regulates NF-kappaB activation in T cells.
Following TCR stimulation, T cells utilize the hematopoietic specific adhesion and degranulation-promoting adapter protein (ADAP) to control both integrin adhesive function and NF-κB transcription factor activation. We have investigated the molecular basis by which ADAP controls these events in primary murine ADAP−/− T cells. Naive DO11.10/ADAP−/− T cells show impaired adhesion to OVAp (OVA aa 323–339)-bearing APCs that is restored following reconstitution with wild-type ADAP. Mutational analysis demonstrates that the central proline-rich domain and the C-terminal domain of ADAP are required for rescue of T:APC conjugate formation. The ADAP proline-rich domain is sufficient to bind and stabilize the expression of SKAP55 (Src kinase-associated phosphoprotein of 55 kDa), which is otherwise absent from ADAP−/− T cells. Interestingly, forced expression of SKAP55 in the absence of ADAP is insufficient to drive T:APC conjugate formation, demonstrating that both ADAP and SKAP55 are required for optimal LFA-1 function. Additionally, the ADAP proline-rich domain is required for optimal Ag-induced activation of CD69, CD25, and Bcl-xL, but is not required for assembly of the CARMA1/Bcl10/Malt1 (caspase-recruitment domain (CARD) membrane-associated guanylate kinase (MAGUK) protein 1/B-cell CLL-lymphoma 10/mucosa-associated lymphoid tissue lymphoma translocation protein 1) signaling complex and subsequent TCR-dependent NF-κB activity. Our results indicate that ADAP is used downstream of TCR engagement to delineate two distinct molecular programs in which the ADAP/SKAP55 module is required for control of T:APC conjugate formation and functions independently of ADAP/CARMA1-mediated NF-κB activation.
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 ...
Adhesion and degranulation-promoting adapter protein (ADAP) is a multi-functional hematopoietic adapter protein that regulates TCR-dependent increases in both integrin function and activation of the NF-κB transcription factor. Activation of integrin function requires both ADAP and the ADAP-associated adapter SKAP55. In contrast, ADAP-mediated regulation of NF-κB involves distinct binding sites in ADAP that promote the inducible association of ADAP, but not SKAP55, with the CARMA1 adapter and the TAK1 kinase. This suggests that the presence or absence of associated SKAP55 defines functionally distinct pools of ADAP. To test this hypothesis, we developed a novel SKAP/ADAP chimeric fusion protein and demonstrated that physical association of ADAP with SKAP55 is both sufficient and necessary for the rescue of integrin function in ADAP-deficient T cells. Similar to wild-type ADAP, the SKAP/ADAP chimera associated with the LFA-1 integrin following TCR stimulation. Although the SKAP/ADAP chimera contains the CARMA1 and TAK1 binding sequences from ADAP, expression of the chimera does not restore NF-κB signaling in ADAP−/− T cells. A single point mutation in the pleckstrin homology (PH) domain of SKAP55 (R131M) blocks the ability of the SKAP/ADAP chimera to restore integrin function and to associate with LFA-1. However, the R131M mutant was now able to restore NF-κB signaling in ADAP-deficient T cells. We conclude that integrin regulation by ADAP involves the recruitment of ADAP to LFA-1 integrin complexes by the PH domain of SKAP55 and this recruitment restricts the ability of ADAP to interact with the NF-κB signalosome and regulate NF-κB activation.
T lymphocyte activation requires physical contact with an antigen-presenting cell and the propagation of signals from the antigen-specific T cell receptor (TCR) that result in proliferation and differentiation. 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 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). 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). 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.The successful progression of T cells through the cell cycle following TCR stimulation involves the temporal induction and activation of cyclins and cyclin-dependent kinases (Cdk's) (47). D-type cyclins, Cdk4, and Cdk6 are induced during the G 1 phase of the cell cycle, followed by the induction of cyclin E and the induction and activation of Cdk2 at the late G 1 restriction point.Expression of cyclin E is controlled by transcriptional regulation of cyclin E as well as by ubiquitin-dependent degradation of cyclin E. Both the cullin-3 E3 ubiquitin ligase (6, 37) and the SCF Fbw7 E3 ubiquitin ligase control cyclin E levels in a manner that is dependent on cyclin E phosphorylation and the association of cyclin E with Cdk2 (20,25,40,48). The signaling pathways that control the induction of cell cycle regulatory proteins in T cells remain incompletely characterized. NF-B has been implicated in the activation of cyclin D1 and cyclin A transcription, and IB kinase (IKK) has been proposed to play a role in cell cycle regulation (1,13,18,19,21,30). The c-Jun kinase (JNK) signaling pathway has also been reported to regulate cell cycle progression of multiple cell types. In fibroblasts, the JNK1 and JNK2 isoforms differentially regulate G 1 -S-phase transition and cell cycle progression via c-Jun, a downstream target of JNK (34). Similar differential functions for JNK1 and JNK2 have also b...
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