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Immune adaptor protein SKAP1 (SKAP-55) forms homodimers as mediated by the N-terminal region

Objective Immune cell adaptor protein SKAP1 couples the antigen-receptor (TCR/CD3) with the activation of LFA-1 adhesion in T-cells. Previous work by ourselves and others have shown that SKAP1 can directly bind to other adaptors such as ADAP and RapL. However, it has been unclear whether SKAP1 can form homodimers with itself and the regions within SKAP1 that mediated homodimer formation. Results Here, we show that SKAP1 and SKAP2 form homodimers in cells. Homodimer formation of immune adaptor protein SKAP1 (SKAP-55) are mediated by residues A17 to L21 in the SKAP1 N-terminal region. SKAP1 dimer formation was not needed for its binding to RapL. These data indicate that the pathway linking SKAP1 to RapL is not dependent on the homo-dimerization of SKAP1.

In this study, we have assessed whether SKAP1 and SKAP2 can form homodimers and the region involved in the dimerization. We show biochemically that SKAP1 and SKAP2 can form homodimers in the generation of signals in T-cells. Homodimer formation of immune adaptor protein SKAP1 (SKAP-55) are mediated by the N-terminal region.

Cell culture
293T cells were grown in DMEM culture medium with 10% fetal calf serum (FCS), 2 mM l-glutamine, penicillin, and streptomycin.

Constructs and transfection
The constructs of SKAP1 and SKAP2 were inserted into a pGEX5x-3 (GE Healthcare) and into a 3×Flag-tagged as well as the vectors encoding EGFP-tagged pcDNA3.1-Hygro (Invitrogen). Site-directed mutagenesis was conducted using QuickChange protocol and Pfu Ultra II Fusion HS DNA Polymerase (Stratagene). Transfections were conducted using BTX ECM 830 electroporator as described [18].

Immunoblotting
Precipitations were conducted by lysis in Triton X-100 lysis buffer followed by the incubation with antibody for 1-2 h at 4 °C and purification of complexes using protein G-Sepharose beads (10% w/v) as described [16][17][18]. For blotting, material on gels transferred onto nitrocellulose filters (Schleicher and Schuell) and detected using horseradish peroxidase-conjugated rabbit anti-mouse antibody together with enhanced chemiluminescence (ECL, Amersham Biosciences).

GST pull down assay
The expression of recombinant GST-proteins was induced in Escherichia coli BL21 cells at 37 °C for 2 h by the addition of 1 mM IPTG. GST-fused proteins were purified with the Cell Lytic B protocol (Sigma #B7435). Cell lysates were incubated with GST fusion proteins for 3 h followed by analysis in SDS-PAGE and western blotting as described [18].

SKAP1 binds to SKAP1 and SKAP2
To assess whether SKAP1 can interact with itself and SKAP2, each was expressed in 293T cells followed by precipitation with anti-Flag ( Fig. 1). Cell lysates or precipitates were then blotted with anti-Flag or GFP. Flag-tagged SKAP1 and GFP-tagged SKAP1 were co-expressed followed by precipitation with anti-Flag. Anti-Flag precipitated Flag-tagged SKAP-1 as well as GFP-tagged SKAP1 from cell lysates (lane 5). This indicates that SKAP1 could form homodimers with itself. Similarly, Flag-tagged SKAP2 and GFP-tagged SKAP2 were co-expressed followed by precipitation with anti-Flag. Anti-Flag precipitated Flag-tagged SKAP2 as well as GFP-tagged SKAP2 To assess whether SKAP1 homodimer formation was dependent on the N-terminal domain, a version of Flagtagged SKAP1 with mutations in residues A17/F20/L21 were co-expressed with GFP-tagged wild-type SKAP1 followed by anti-Flag co-precipitation. Mutation of residues A17/F20/L21 abrogated the homodimeric binding of SKAP1 with itself (lane 6). As a control, the blotting of cell lysates showed the expression of the various Flag and GFP tagged proteins (right panel). These data demonstrate the residues in the region of A17 to L21 of the N-terminal region of SKAP1 mediates dimer formation.
We previously showed that SKAP1 binds to RapL and is needed for RapL binding the GTPase Rap1 and the activation of LFA-1 adhesion. N-terminal SKAP1 domain binds to the C-terminal SARAH domain of Rap1. We also show that SKAP1 is needed for RapL binding to membranes in a manner dependent on the PH domain of SKAP1 and the PI3K pathway [16,17]. Others have reported other components such as Rap1-dependent integrin regulator Rap1-GTP-interacting adaptor molecule (RIAM) in the multimeric complex [18]. We therefore next asked whether SKAP-1 monomer or dimer formation was needed for SKAP1 binding to RapL (Fig. 2). Tagged wildtype or A17/F20/L21 mutant SKAP1 was co-expressed with RapL in 293T cells and assessed for co-precipitation. While anti-Flag precipitated GFP-tagged SKAP1, the dimer failed to co-precipitate RapL (lane 2). Similarly, anti-GFP coprecipitated Flag-SKAP1 but a faint RapL band (lane 3). For unknown reasons, the Flag-tagged SKAP1 bound to GFP-SKAP1 consistently migrated at a lower Mr suggestive of a post-translational change in the protein. Intriguingly, anti-V5 precipitated V5-tagged RapL with only the lower Mr Flag-tagged SKAP1 (lane 4). This suggests that the lower Mr version of SKAP1 preferentially associates with RapL. Intriguingly, the same patterns of co-precipitation were observed Flag-A17/ F20/L21 SKAP1 (lanes 7 and 8). Attempts were made to transfect primary T-cells for expression but were limited by the low levels of expression that precluded an analysis of binding in these cells. These data indicate that SKAP1 dimer formation is not needed for its binding to RapL.
Lastly, we next showed the presence of the SKAP1 dimer by blotting with anti-SKAP1 rather than antibodies to tags on the proteins (Fig. 3). Combinations of Flag and GFP-tagged SKAP1 were expressed in 293T cells (left panel) and subjected to precipitation using anti-Flag and the followed by blotting with anti-SKAP1 (right panel). Anti-Flag precipitation of Flag SKAP or FlagDM-Skap1 co-precipitated co-expressed GFP-SKAP1 as detected by anti-SKAP-1 (lanes 4 and 5; longer exposure below). We attempted to co-express these vectors in primary mouse T-cells from spleen but were unable to obtain sufficiently high levels of expression of both proteins to carry out similar analysis with these cells. These data confirmed that SKAP1 dimer formation as detected with anti-SKAP1.

Discussion
Overall, our study shows that SKAP1 forms homodimers dependent on residues A17/F20/L21 in the N-terminus of SKAP1. We previously showed that this region has alternating leucine residues and shares homology with the coiled-coil domain of SKAP-2 [9]. Both SKAP1 and SKAP2 bind ADAP (FYB) through their SH3 domains and served as substrates for the FYN kinase in T cells [9,10]. SKAP1 also colocalizes with another the ADAP binding protein, SLP-76. Mutation of the YDDV sites (termed M12) that disrupt SLP-76 SH2 domain binding interferes with ADAP binding and decreases conjugation and LFA-1 clustering [13]. SKAP1 Src homology 3 (SH3) domains also stabilizes SLP-76 micro-clusters [19].
By contrast, the N-terminal region of SKAP1 binds to RapL such that a RapL mutation (L224A) abrogates SKAP1 binding and arrests T-cells even in the absence of antigen [17]. We now extend these findings by showing that dimerization is not required for the direct binding of SKAP1 to RapL. In fact, the A17/F20/L21 mutant often bound more to RapL than did wild-type SKAP-1. In this context, it is possible that dimerization limits SKAP1 binding to RapL. This contrasts with the reported requirement for the SKAP dimerization for binding to RIAM [19]. It is possible that dimerization may act on functions that are distinct from SKAP1-RapL activation of LFA-1. In the same manner, while the expression of the  [20], it appears to substitute for SKAP1 in stabilizing the formation of microclusters in Jukat T-cells [19]. The full range of functions mediated by SKAP1 dimerization remain to be demonstrated in future studies.

Limitations
Work restricted to non-lymphoid cells.