Multistage T cell-dendritic cell interactions control optimal CD4 T cell activation through the ADAP-SKAP55-signaling module.

The Ag-specific interactions between T cells and dendritic cells progress through dynamic contact stages in vivo consisting of early long-term stable contacts and later confined, yet motile, short-lived contacts. The signaling pathways that control in vivo interaction dynamics between T cells and dendritic cells during priming remain undefined. Adhesion and degranulation promoting adapter protein (ADAP) is a multifunctional adapter that regulates "inside-out" signaling from the TCR to integrins. Using two-photon microscopy, we demonstrate that, in the absence of ADAP, CD4 T cells make fewer early-stage stable contacts with Ag-laden dendritic cells, and the interactions are characterized by brief repetitive contacts. Furthermore, ADAP-deficient T cells show reduced contacts at the late motile contact phase and display less confinement around dendritic cells. The altered T cell interaction dynamics in the absence of ADAP are associated with defective early proliferation and attenuated TCR signaling in vivo. Regulation of multistage contact behaviors and optimal T cell signaling involves the interaction of ADAP with the adapter src kinase-associated phosphoprotein of 55 kDa (SKAP55). Thus, integrin activation by the ADAP-SKAP55-signaling module controls the stability and duration of T cell-dendritic cell contacts during the progressive phases necessary for optimal T cell activation.

The pleckstrin homology domain in the SKAP55 adapter protein defines the ability of the adapter protein ADAP to regulate integrin function and NF-kappaB activation.

Adhesion and degranulation promoting adapter protein (ADAP) is a multifunctional 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 Src kinase-associated phosphoprotein of 55 kDa (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 after 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 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 pleckstrin homology domain of SKAP55, and this recruitment restricts the ability of ADAP to interact with the NF-κB signalosome and regulate NF-κB activation.

NF-kappaB activation in T cells requires discrete control of IkappaB kinase alpha/beta (IKKalpha/beta) phosphorylation and IKKgamma ubiquitination by the ADAP adapter protein.

NF-kappaB activation following engagement of the antigen-specific T cell receptor involves protein kinase C-theta-dependent assembly of the CARMA1-BCL10-MALT1 (CBM) signalosome, which coordinates downstream activation of IkappaB 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-theta. ADAP is not required for TAK1 activation. Although both the TAK1 and the CARMA1 binding sites in ADAP are essential for IkappaB alpha phosphorylation and degradation and NF-kappaB 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 IKKgamma. Thus, distinct sites within ADAP control two key activation responses that are required for NF-kappaB activation in T cells.

Distinct regulation of integrin-dependent T cell conjugate formation and NF-kappa B activation by the adapter protein ADAP.

Following TCR stimulation, T cells utilize the hematopoietic specific adhesion and degranulation-promoting adapter protein (ADAP) to control both integrin adhesive function and NF-kappaB 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-x(L), 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-kappaB 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-kappaB activation.

Type II phosphatidylinositol 4-kinase beta associates with TCR-CD3 zeta chain in Jurkat cells.

Phosphatidylinositol lipid signaling cascades are integral part of TCR-CD3 signaling. The mechanisms by which phosphatidylinositol kinases are coupled to TCR-CD3 complex remain elusive. Here we report an association of type II PtdIns 4-kinase with TCR-CD3 zeta chain upon cross-linking. Mapping studies have revealed that the C-terminal ITAM is critical for docking of the enzyme on the zeta chain. The association is shown to be tyrosyl phosphorylation dependent as mutation of Y-151 and Y-142 on the C-terminal ITAM disrupts interaction of the two proteins. Identification of the associated type II PtdIns 4-kinase revealed that the beta isoform of the enzyme interacts with the zeta chain in vivo.

A type II phosphatidylinositol 4-kinase associates with T cell receptor zeta chain in Con A stimulated splenic lymphocytes through tyrosyl phosphorylation-dependent mechanisms.

T cells show rapid reorganization of cytoskeleton in response to antigenic stimulation. The molecular mechanisms by which TCR-CD3 regulates actin cytoskeleton are not well defined. Here we show that a type II PtdIns 4-kinase associates with cytoskeletal fraction in splenic lymphocytes in response to Con A. Protein tyrosyl phosphorylation of type II PtdIns 4-kinase appears to be the mechanism for its association with cytoskeleton. Over-lay blots suggest that the enzyme binds to TCR-CD3 zeta chain in the cytoskeletal fraction. Anti-TCR-CD3 zeta antibodies competitively inhibit PtdIns 4-kinase association with TCR-CD3 zeta chain. Immunodepletion of TCR-CD3 zeta decreases PtdIns 4-kinase activity in the cytoskeletal fraction with a concomitant increase in PtdIns 4-kinase activity in anti-TCR-CD3 zeta immunoprecipitates. We propose that the association of type II PtdIns 4-kinase with TCR-CD3 zeta chain may bring the enzyme into close proximity of actin and a possible regulation of actin polymerization through localized production of PtdIns4P and PtdIns(4,5)P2.

Resveratrol inhibits type II phosphatidylinositol 4-kinase: a key component in pathways of phosphoinositide turn over.

Resveratrol has anti-inflammatory, cardio protective and cancer chemopreventive properties. The molecular targets for resveratrol in early signaling cascades are not well understood. Resveratrol inhibits type II PtdIns 4-kinase but not PtdIns 3-kinase activity in vitro. Resveratrol directly binds to the enzyme with a Kd of 7.2 microM. Kinetic studies show that resveratrol competes with PtdIns binding. Inhibition of PtdIns 4-kinase activity by resveratrol/phenylarsine oxide reduces Jurkat cell adhesion to matrigel/fibronectin coated surfaces, suggesting a role for type II PtdIns 4-kinase in lymphocyte infiltration to the sites of inflammation.

ADAP regulates cell cycle progression of T cells via control of cyclin E and Cdk2 expression through two distinct CARMA1-dependent signaling pathways.

Adhesion and degranulation-promoting adapter protein (ADAP) is a multifunctional scaffold that regulates T cell receptor-mediated activation of integrins via association with the SKAP55 adapter and the NF-κB pathway through interactions with both the CARMA1 adapter and serine/threonine kinase transforming growth factor ß-activated kinase 1 (TAK1). ADAP-deficient T cells exhibit impaired proliferation following T cell receptor stimulation, but the contribution of these distinct functions of ADAP to this defect is not known. We demonstrate that loss of ADAP results in a G1-S transition block in cell cycle progression following T cell activation due to impaired accumulation of cyclin-dependent kinase 2 (Cdk2) and cyclin E. The CARMA1-binding site in ADAP is critical for mitogen-activated protein (MAP) kinase kinase 7 (MKK7) phosphorylation and recruitment to the protein kinase C θ (PKCθ) signalosome and subsequent c-Jun kinase (JNK)-mediated Cdk2 induction. Cyclin E expression following T cell receptor stimulation of ADAP-deficient T cells is transient and associated with enhanced cyclin E ubiquitination. Both the CARMA1- and TAK1-binding sites in ADAP are critical for restraining cyclin E ubiquitination and turnover independently of ADAP-dependent JNK activation. T cell receptor-mediated proliferation was most dramatically impaired by the loss of ADAP interactions with CARMA1 or TAK1 rather than SKAP55. Thus, ADAP coordinates distinct CARMA1-dependent control of key cell cycle proteins in T cells.

Regulation of NF-kappaB activation in T cells via association of the adapter proteins ADAP and CARMA1.

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.