The Pleckstrin Homology Domain in the SKAP55 Adapter Protein Defines the Ability of the Adapter Protein ADAP To Regulate Integrin Function and NF-κB Activation

Abstract

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.

Analysis of Phosphorylation-dependent Protein Interactions of Adhesion and Degranulation Promoting Adaptor Protein (ADAP) Reveals Novel Interaction Partners Required for Chemokine-directed T cell Migration

“…1A) (10,11), whereas another PRS (FPPPP) is responsible for the interaction with the actin regulator Ena/ VASP-like protein (EVL) (12). Membrane binding of the ADAP-SKAP55 complex is conferred by the PH domain of SKAP55 and to a lower extend by the C-terminal hSH3 domain (hSH3 C ) of ADAP (13)(14)(15)(16). Moreover, ADAP is strongly tyrosine-phosphorylated upon TCR stimulation and thereby serves as a hub for SH2 domain-containing proteins such as SLP76, FYN and NCK (Fig.…”

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

“…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).…”

D120 and K152 within the PH Domain of T Cell Adapter SKAP55 Regulate Plasma Membrane Targeting of SKAP55 and LFA-1 Affinity Modulation in Human T Lymphocytes

“…Analysis of ADAP Ϫ/Ϫ T cells revealed defects in LFA-1-mediated adhesion, avidity modulation, and, consequently, an impaired interaction of T cells with APCs in vitro and in vivo (9)(10)(11)(12)(13). ADAP possesses a central proline-rich region, two helical SH3 domains, and one Ena/VASP homology 1 (EVH1) binding domain (8).…”

“…Consequently, ADAP Ϫ/Ϫ T cells are also deficient for SKAP55 (11,16). Similar to ADAP, the loss of SKAP55 in T cells leads to defective TCR-mediated LFA-1 function and attenuated T cell/APC interactions and is termed here the ADAP/SKAP55 module (11,12,16,17).…”

“…Two studies reported that a deletion of the PH domain or mutation of arginine 131 (R131) within the PH domain of SKAP55 impairs adhesion and conjugate formation of T cells with APCs (12,21). In contrast, two other reports showed that neither the deletion of the PH domain within full-length SKAP55 nor the overexpression of the isolated PH domain of SKAP55 alters TCRmediated adhesion (16,18).…”