Association between SAP and FynT: Inducible SH3 domain-mediated interaction controlled by engagement of the SLAM receptor - PubMed (original) (raw)

Association between SAP and FynT: Inducible SH3 domain-mediated interaction controlled by engagement of the SLAM receptor

Riyan Chen et al. Mol Cell Biol. 2006 Aug.

Abstract

SAP is an intracellular adaptor molecule composed almost exclusively of an SH2 domain. It is mutated in patients with X-linked lymphoproliferative disease, a human immunodeficiency. Several immune abnormalities were also identified in SAP-deficient mice. By way of its SH2 domain, SAP interacts with tyrosine-based motifs in the cytoplasmic domain of SLAM family receptors. SAP promotes SLAM family receptor-induced protein tyrosine phosphorylation, due to its capacity to recruit the Src-related kinase FynT. This unusual property relies on the existence of a second binding surface in the SAP SH2 domain, centered on arginine 78 of SAP, that binds directly to the FynT SH3 domain. Herein, we wanted to further understand the mechanisms controlling the interaction between SLAM-SAP and FynT. Our experiments showed that, unlike conventional associations mediated by SH3 domains, the interaction of the FynT SH3 domain with SLAM-SAP was strictly inducible. It was absolutely dependent on engagement of SLAM by extracellular ligands. We obtained evidence that this inducibility was not due to increased binding of SLAM to SAP following SLAM engagement. Furthermore, it could occur independently of any appreciable SLAM-dependent biochemical signal. In fact, our data indicated that the induced association of the FynT SH3 domain with SLAM-SAP was triggered by a change in the conformation of SLAM-associated SAP caused by SLAM engagement. Together, these data elucidate further the events initiating SLAM-SAP signaling in immune cells. Moreover, they identify a strictly inducible interaction mediated by an SH3 domain.

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Figures

FIG. 1.

FIG. 1.

The association of SLAM-SAP with FynT is regulated by SLAM engagement. BI-141 T cells expressing Tac-SLAM in the absence or in the presence of SAP (lanes 3 to 6) were stimulated or not for 5 min at 37°C with anti-Tac and the relevant secondary antibody, as detailed in Materials and Methods. Cells expressing full-length SLAM without or with SAP (lanes 1 and 2) were left unstimulated. After lysis, Tac-SLAM and SLAM were immunoprecipitated with anti-Tac or anti-SLAM, respectively, and probed with antiphosphotyrosine (P.tyr) antibodies (first panel). The associations of Tac-SLAM and SLAM with SAP and FynT were assessed by probing parallel immunoprecipitates with anti-SAP (second panel) or anti-FynT (third panel), respectively. The abundance of Tac-SLAM and SLAM in the immunoprecipitates was verified by reprobing with anti-SLAM (fourth panel). The abundance of SAP and FynT in cells was determined by immunoblotting of total cell lysates with anti-SAP (fifth panel) and anti-FynT (sixth panel), respectively. I.P., immunoprecipitate.

FIG. 2.

FIG. 2.

Binding of SLAM-SAP to the FynT SH3 domain, but not the FynT SH2 domain, is inducible. (A) Inducible binding of SLAM-SAP to the FynT SH3 domain. Lysates derived from the experiment for Fig. 1 were incubated with immobilized GST fusion proteins encompassing or not the FynT SH3 domain. After washes were performed, associated SLAM-SAP complexes were detected by immunoblotting with anti-SLAM (first panel). The presence of equivalent amounts of GST fusion proteins was verified by migrating representative aliquots in SDS-polyacrylamide gel electrophoresis (PAGE) gels and staining with Coomassie blue (second panel). (B) The FynT SH2 domain does not contribute detectably to the association with the SLAM-SAP complex. Lysates from BI-141 T cells expressing full-length SLAM and SAP were incubated with the indicated fusion proteins. Following washes, the association with SLAM-SAP was detected by immunoblotting with anti-SLAM (left panel). The presence of equivalent amounts of GST fusion proteins was verified by migrating representative aliquots in SDS-PAGE gels and staining with Coomassie blue (right panel). (C) Impact of the SAP R78A mutation on binding of SLAM to the FynT SH3 domain. Cells expressing full-length SLAM alone or with wild-type SAP or SAP R78A were lysed. The association of SLAM with SAP was assessed by immunoblotting of anti-SLAM immunoprecipitates with anti-SAP (first panel). The ability of GST FynT SH3 domains to bind SLAM was determined as for Fig. 2A (second panel). The expression levels of SLAM and SAP were verified by probing total cell lysates with anti-SLAM (third panel) and anti-SAP (fourth panel), respectively. I.P., immunoprecipitate.

FIG. 3.

FIG. 3.

Induction of SLAM tyrosine phosphorylation by pervanadate is inefficient at triggering binding of SLAM-SAP to the FynT SH3 domain. BI-141 cells expressing Tac-SLAM and SAP were stimulated or not with the protein tyrosine phosphatase pervanadate (100 μM) for 10 min at 37°C or with anti-Tac MAb 7G7 as outlined for Fig. 1. Lysates were then processed as detailed for Fig. 1 and 2A. Tac-SLAM tyrosine phosphorylation was determined by immunoblotting of anti-Tac immunoprecipitates with antiphosphotyrosine (P.tyr) (first panel), whereas the association of Tac-SLAM with SAP was ascertained by probing of parallel immunoprecipitates with anti-SAP (second panel). The presence of equivalent amounts of Tac-SLAM in the immunoprecipitates was verified by reprobing with a rabbit anti-Tac serum (third panel). The ability of the Tac-SLAM-SAP complex to bind FynT SH3 domains was assessed by incubating lysates with immobilized GST-FynT SH3 domain fusion proteins and probing bound proteins with anti-SLAM (fourth panel). The abundance of SAP was verified by probing total cell lysates with anti-SAP (fifth panel). I.P., immunoprecipitate.

FIG. 4.

FIG. 4.

Effects of low temperature on SLAM-SAP signaling and the association of SLAM-SAP with the FynT SH3 domain. (A) BI-141 cells expressing Tac-SLAM and SAP were stimulated for the indicated periods of time with anti-Tac and the relevant secondary antibody at either 4°C (lanes 1 to 4) or 37°C (lanes 5 to 8). Lysates were subsequently obtained and processed as detailed in the legends of Fig. 1 and 2A. Induction of Tac-SLAM tyrosine phosphorylation was assessed by probing of total cell lysates with antiphosphotyrosine (P.tyr) (first panel). The association of Tac-SLAM with SAP was ascertained by immunoblotting of anti-Tac immunoprecipitates with anti-SAP (second panel), and the abundance of Tac-SLAM in these immunoprecipitates was verified by reprobing with a rabbit anti-Tac serum (third panel). Binding of the Tac-SLAM-SAP complex to the FynT SH3 domain was determined by probing proteins bound to immobilized GST-FynT SH3 domains with anti-SLAM (fourth panel). I.P., immunoprecipitate. (B) Quantitation of the results of Fig. 4A was done, using a Phosphorimager. Data are presented as the percentages of the maximum response observed.

FIG. 5.

FIG. 5.

Impact of mutations of the intracytoplasmic tyrosines of SLAM on binding of SLAM-SAP to the FynT SH3 domain. (A) Primary structure of mouse SLAM. SLAM contains an extracellular segment with two Ig-like domains, one variable (V)-like and one constant 2 (C2)-like, a transmembrane region, and a cytoplasmic domain with three tyrosine-based motifs. The motif centered on Y288 (Y1) is implicated in SAP binding, while the motifs centered on Y315 (Y2) and Y335 (Y3) are involved in recruitment of downstream effectors like SHIP-1. (B) Biochemical studies. BI-141 cells expressing the indicated Tac-SLAM polypeptides in the presence of SAP were stimulated at 37°C for 5 min with anti-Tac and the relevant secondary antibody. The cells were then lysed, and the lysates were processed as detailed in the legends of Fig. 1 and 2A. Tac-SLAM tyrosine phosphorylation was assessed by probing of anti-Tac immunoprecipitates with antiphosphotyrosine (P.tyr) (first panel). The association of Tac-SLAM with SAP was tested by immunoblotting of anti-Tac immunoprecipitates with anti-SAP (second panel), while the amount of Tac-SLAM in these immunoprecipitates was verified by reprobing with a rabbit anti-Tac serum (third panel). Binding of Tac-SLAM-SAP complexes to the FynT SH3 domain was determined by probing proteins bound to immobilized GST-FynT SH3 domains with anti-Tac (fourth panel). The amount of SAP was assessed by immunoblotting of total cell lysates with anti-SAP (fifth panel). I.P., immunoprecipitate.

FIG. 6.

FIG. 6.

Effect of FynT deficiency on the association of SLAM-SAP with recombinant FynT SH3 domains. (A) Association of SLAM-SAP with FynT SH3 domains. Thymocytes were isolated from the indicated mouse strains and lysed. Lysates were then incubated with GST alone or GST encompassing the SH3 domain of FynT. After washing was performed, binding of SLAM-SAP complexes was detected by immunoblotting with anti-SLAM (first panel). The abundance of SLAM, FynT, and SAP in cells was verified by immunoblotting of total cell lysates with anti-SLAM (second panel), anti-FynT (third panel), or anti-SAP (fourth panel), respectively. (B) Association of SLAM with SAP. The extent of association of SLAM with SAP in thymocytes from the indicated mice was determined by probing anti-SLAM immunoprecipitates (lanes 2 and 4) with anti-SAP. As a negative control, lysates were immunoprecipitated with anti-CD4 MAb GK1.5 (lanes 1 and 3).

FIG. 7.

FIG. 7.

Potential model explaining the inducible association of the SLAM-SAP complex with FynT. (A) In the absence of self engagement of its extracellular domain, SLAM is associated in a phospho-independent fashion with SAP (via the first cytoplasmic tyrosine of SLAM). However, there is little or no association with FynT. (B) Engagement of the extracellular region of SLAM triggers a change in the conformation of SLAM, transmitted to SAP by an as yet unknown mechanism. This conformational alteration is independent of SLAM-SAP signaling. It results in an increased affinity and/or accessibility of the arginine 78-based motif of SAP for the FynT SH3 domain, thereby enabling stable binding of the SLAM-SAP complex to FynT. (C) Binding of the SLAM-SAP complex to the FynT SH3 domain causes a change in the conformation of FynT that leads to FynT enzymatic activation and subsequent tyrosine phosphorylation of SLAM (on the second and third cytoplasmic tyrosines). Tyrosine phosphorylation of SLAM triggers the recruitment of downstream effectors such as the 5′ inositol phosphatase SHIP-1.

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References

    1. Abraham, N., M. C. Miceli, J. R. Parnes, and A. Veillette. 1991. Enhancement of T-cell responsiveness by the lymphocyte-specific tyrosine protein kinase p56lck. Nature 350:62-66. - PubMed
    1. Berry, D. M., P. Nash, S. K. Liu, T. Pawson, and C. J. McGlade. 2002. A high-affinity Arg-X-X-Lys SH3 binding motif confers specificity for the interaction between Gads and SLP-76 in T cell signaling. Curr. Biol. 12:1336-1341. - PubMed
    1. Cannons, J. L., L. J. Yu, B. Hill, L. A. Mijares, D. Dombroski, K. E. Nichols, A. Antonellis, G. A. Koretzky, K. Gardner, and P. L. Schwartzberg. 2004. SAP regulates T(H)2 differentiation and PKC-theta-mediated activation of NF-kappaB1. Immunity 21:693-706. - PubMed
    1. Castro, A. G., T. M. Hauser, B. G. Cocks, J. Abrams, S. Zurawski, T. Churakova, F. Zonin, D. Robinson, S. G. Tangye, G. Aversa, K. E. Nichols, J. E. de Vries, L. L. Lanier, and A. O'Garra. 1999. Molecular and functional characterization of mouse signaling lymphocytic activation molecule (SLAM): differential expression and responsiveness in Th1 and Th2 cells. J. Immunol. 163:5860-5870. - PubMed
    1. Chan, B., A. Lanyi, H. K. Song, J. Griesbach, M. Simarro-Grande, F. Poy, D. Howie, J. Sumegi, C. Terhorst, and M. J. Eck. 2003. SAP couples Fyn to SLAM immune receptors. Nat. Cell Biol. 5:155-160. - PubMed

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