Regulation and Function of SKAP-55 Non-canonical Motif Binding to the SH3c Domain of Adhesion and Degranulation-promoting Adaptor Protein

Abstract

Modular domains in proteins such as protein-tyrosine kinases, phosphatases and adaptors play central roles in the generation of signals needed for mammalian cell function (1). Of these, Src homology domain 2 (SH2) 3 recognizes phosphotyrosine-based motifs, whereas Src homology domain 3 (SH3) domains recognize proline-based PXXP motifs (2). Since the description of Abelson SH3 domain recognition of the 3BP1 protein (3), numerous SH3 domain-mediated interactions have been documented. Examples include SH3-mediated interactions between the adaptor Grb-2 (growth factor receptor-bound protein-2) and Son-of-Sevenless, Src kinase SH3 domain binding to the p85 subunit of phosphatidylinositol 3-kinase, and Crk SH3 domain binding to Crk SH3 domain-binding guanine nucleotide-releasing factor, among others. SH3 domain binding is involved in subcellular localization, cytoskeletal organization, and signal transduction (4).Structurally, SH3 domains are comprised of two anti-parallel ␤ sheets packed at right angles to one other (2, 4, 5). The core øPXøP motif (where ø represents a hydrophobic residue) interacts with SH3 through two defined consensus sequences: Class I (R/KXXPXXP) and Class II (PXXPXR) (6). Domains can bind ligands in either an N-to C-terminal or C-to N-terminal orientation due to the pseudosymmetrical nature of the polyproline class II helix that is stabilized primarily by hydrophobic and additional electrostatic interactions. Directionality is conferred by the interaction of the arginine or lysine residues with the charged outer face on the SH3 domain, while the tandem prolines bind to two hydrophobic pockets. Binding depends on the two SH3 variable loops, the RT and n-Src loops, that flank a ligand-binding region.In addition to the binding to øPXøP-based motifs, an increasing number of studies have documented SH3 domain binding to non-canonical motifs (7). These include a PX(V/I)(D/N)RXXKP motif that is responsible for STAM2 SH3 domain binding to the deubiquitination enzyme ubiquitin isopeptidase Y/Usp8 (8, 9) and Grb-2 related protein (GADS), SH3 domain binding to SH2-domain-containing leukocyte protein of 76 kDa (SLP-76) (10 -13). In the latter case, the interaction has a 10 -20 times higher affinity than interaction between SH3 domains and their øPXøP motifs. Other examples of non-canonical interactions include amphiphysin SH3 domain binding to dynamin (14), Eps8 SH3 domain binding to the PXXDY motif (15), and HPK1 SH3 domain binding to a RXXK motif (12). We recently identified a novel RKXXYXXY motif that is found in the T-cell adaptor Src kinase-associated protein of 55 kDa (SKAP-55) and that is recognized by the FYN-SH3 and ADAP-SH3c domains (16).In the immune system, SH3 domains are found in Src-related kinases and immune specific adaptor proteins (17, 18). Adaptors lack enzymatic activity and transcription binding domains, and instead are comprised of multiple binding domains and sites that facilitate protein-protein aggrega- Grb2, growth factor receptor-bound protein-2; Gads, Grb-2-related protein; SLP...

The ADAP/SKAP55 Signaling Module Regulates T-Cell Receptor-Mediated Integrin Activation through Plasma Membrane Targeting of Rap1

“…We are aware that our findings are in discordance with a recent report showing that the RKXXY 294 XXY 297 motif of SKAP55 is involved in regulating TCR-mediated adhesion and TCR-mediated activation of an NFAT/AP1-luciferase reporter construct (12). However, in multiple transfection/overexpression experiments we did not observe an obvious effect of SKAP55 or any of its mutants upon TCR-mediated activation of various luciferase-based reporter genes (B. Schraven, unpublished data).…”

Functional Defects of SKAP-55-Deficient T Cells Identify a Regulatory Role for the Adaptor in LFA-1 Adhesion

“…Mutation of YDDV sites in ADAP impairs conjugation, SMAC formation, and cytokine production (39). Further, loss of the SKAP-55 SH3 domain, loss of the ADAP SH3c domain, or over expression of a peptide which binds to the proline-rich region of ADAP ablates adhesion (7,18,39). (Fig.…”

“…This effect occurred under conditions of normal expression of the homologue SKAP-55-related (SKAP-55R) or SKAP-55Hom, indicating a nonredundant function for SKAP-55 in T cells (13). An interaction between ADAP and SKAP-55 is needed for adhesion, since the loss of the SKAP-55 SH3 domain or the ADAP SH3c domain binding sites on SKAP-55 ablates adhesion and conjugate formation (7,18,40). ADAP-SKAP-55 may facilitate the translocation of Rap1 to membranes (18).…”

“…It is enriched in T cells and possesses a unique N-terminal region followed by a pleckstrin homology domain and a C-terminal SH3 domain (23,24). SKAP-55 binds with high stoichiometry to ADAP (4,25), an interaction that is mediated by binding of the SKAP-55 SH3 domain to a proline-rich region in ADAP (23,25) and by weaker binding of the ADAP SH3c domain to a tyrosine-based RKXXYXXY motif in SKAP-55 (7,14). SKAP-55 may also interact with the D1 domain of the phosphatase CD45 (43).…”

SKAP-55, SKAP-55-related and ADAP adaptors modulate integrin-mediated immune-cell adhesion

“…The degree to which the ADAP hSH3 domain binds the RKxxYxxY motif and/or phospholipids is the subject of debate [31,34,35]. Although SKAP-55 SH3 domain interaction with ADAP predominates, mutation of the RKxxYxxY motif in SKAP-55 can also disrupt adaptor function [36].In addition to its binding to SLP-76, SKAP-55 and SKAP-55R, ADAP is connected to the cytoskeleton as a result of the binding of ADAP to the EVH1 domain in Ena and VASP …”