The N terminus of SKAP55 enables T cell adhesion to TCR and integrin ligands via distinct mechanisms

Abstract

The T cell receptor (TCR) triggers the assembly of "SLP-76 microclusters," which mediate signals required for T cell activation. In addition to regulating integrin activation, we show that Src kinase-associated phosphoprotein of 55 kD (SKAP55) is required for microcluster persistence and movement, junctional stabilization, and integrin-independent adhesion via the TCR. These functions require the dimerization of SKAP55 and its interaction with the adaptor adhesion and degranulation-promoting adaptor protein (ADAP). A "tandem dimer" containing two ADAP-binding SKAP55 Src homology 3 (SH3) domains stabilized SLP-76 microclusters and promoted T cell adhesion via the TCR, but could not support adhesion to integrin ligands. Finally, the SKAP55 dimerization motif (DM) enabled the coimmunoprecipitation of the Rap1-dependent integrin regulator Rap1-GTP-interacting adaptor molecule (RIAM), the recruitment of talin into TCR-induced adhesive junctions, and "inside-out" signaling to β1 integrins. Our data indicate that SKAP55 dimers stabilize SLP-76 microclusters, couple SLP-76 to the force-generating systems responsible for microcluster movement, and enable adhesion via the TCR by mechanisms independent of RIAM, talin, and β1 integrins.

Figure 1.

SKAP55 is recruited into microclusters by SLP-76 and ADAP. (A) Immunofluorescent staining for endogenous SKAP55 in J14.SY cells fixed 5 min after plating on anti-CD3 coated, BSA-blocked stimulatory coverslips. (B) J14.SY cells expressing either SKAP55.TRT (top) or TRT (bottom) were stimulated on coverslips and imaged for 5 min. MOT projections depict the trajectories of microclusters over time (n = 20). (C) MOT images of J14 cells coexpressing SKAP55.mRFP1 and either mYFP (top) or SLP-76.mYFP (bottom) (n = 3). (D) MOT images of ADAP-deficient JDAP cells coexpressing SKAP55.3×Flag.mCFP, SLP-76.YFP, and either 3×Flag.TRT (top) or TRT.3×Flag.TRT.ADAP-120 (bottom) (n = 4). (E) Kymographs depicting the lateral movements of SLP-76, SKAP55, and ADAP microclusters along a narrow region of interest spanning the center of the cell. Bars: (A–D) 10 µm; (E) 5 µm × 60 s.

Figure 2.

SKAP55 is required for microcluster persistence and movement, and for contact stability. (A) Confirmation of the efficacy of SKAP55 knockdown and add-back in stable SKAP55 knockdown cells (JSKAP.SY) with or without transient reconstitution (n = 3). (B) J14.SY and JSKAP.SY cells expressing mRFP1 or SKAP55.mRFP1 were stimulated on coverslips and imaged for at least 5 min. Representative MOT images and kymographs are shown. Bars: (MOT images) 10 µm; (kymographs) 5 µm × 60 s. See Table 2 for microcluster properties and experiment numbers. (C) Composite microcluster traces for the conditions examined in B. Numbers in parentheses indicate the total number of cells examined. Line intensity corresponds to the fraction of microclusters surviving; arrowheads identify points of half-maximal microcluster dissociation. (D) Fraction of the contact area engaged in fluctuation (see Fig. S1, F and G; n = 3). (E) Fraction of cells scored as displaying unstable contacts (see Fig. S1, I and J; n = 5). (F) Surface expression of CD69 in J14.SY and JSKAP.SY cells, after normalization to a TCR-stimulated J14.SY control (n = 4). (G) Kinetics of Erk1/2 phosphorylation in J14.SY and JSKAP.SY stimulated for the indicated time points (n = 3). Error bars indicate mean ± SEM. From parental J14.SY cells (with or without mRFP1): **, P < 0.01. From JSKAP.SY (with or without mRFP1): ##, P < 0.01.

Figure 3.

The SKAP55 SH3 domain is necessary but not sufficient for stabilization of SLP-76 microclusters. (A) Domain structures of the wild-type (WT), SH3 mutant (W333R, “WR”), and SH3-only SKAP55 chimeras (SH3). (B) mRFP1 expression levels in J14.SY cells transiently transfected with the indicated SKAP55.mRFP1 chimeras (n = 3). (C) Cells from B were stimulated, imaged, and presented as in Fig. 2 B. See Table 1 for microcluster properties and experiment numbers. Bars: (MOT images) 10 µm; (kymographs) 5 µm × 60 s. (D) Composite microcluster traces for the conditions examined in C. Numbers in parentheses indicate the total number of cells examined. Line intensity corresponds to the fraction of microclusters surviving; arrowheads identify points of half-maximal microcluster dissociation. (E) SKAP55.3×Flag.mCFP chimeras were expressed in J14.SY cells, immunoprecipitated with anti-Flag, and Western blotted for mCFP or ADAP (n = 4). (F) Fraction of cells scored as displaying unstable contacts (n = 4). Error bars indicate mean ± SEM. From parental J14.SY cells expressing mRFP1: **, P < 0.01. From J14.SY cells expressing SKAP55.WT.mRFP1: ††, P < 0.01.

Figure 4.

The SKAP55 PH domain and linker tyrosines are dispensable for recruitment into and stabilization of SLP-76 microclusters, and for T cell adhesion. (A) Domain structures of the ΔPH, 3YF, R131M, and PH-alone SKAP55 chimeras. (B) J14.SY cells were transiently transfected with the indicated SKAP55.mRFP1 chimeras; expression levels were determined by Western blotting (n = 3). (C) Cells from B were stimulated, imaged, and presented as in Fig. 2 B. See Table 1 for microcluster properties and experiment numbers. (D) Composite microcluster traces for the conditions examined in C; numbers in parentheses indicate the total number of cells examined. Line intensity corresponds to the fraction of microclusters surviving; arrowheads identify points of half-maximal microcluster dissociation. (E) Fraction of cells scored as displaying unstable contacts (n = 4). (F) JSKAP.SY cells transiently transfected with the indicated SKAP55.mRFP1 chimeras were stimulated, imaged, and presented as in Fig. 2 B (n = 3). (G) JSKAP.SY cells transfected with Akt.PH.mCFP and SKAP55.mRFP1 chimeras were imaged as in Fig. 2 B. Still images acquired at and 4 µm above the coverslip are shown (n = 3). (H) Fractional retention of unstimulated or TCR-stimulated cells on fibronectin-coated coverslips was calculated by dividing the post-wash signal by the prewash signal and normalizing to the corresponding PMA control (n = 3). (I) Fractional retention of T cells on uncoated or anti-CD3 coated coverslips was calculated by dividing the post-wash signal by the prewash signal (n = 3). Bars: (C and F, MOT images; and G) 10 µm; (C and F, kymographs) 5 µm × 60 s. Error bars indicate mean ± SEM. From parental J14.SY cells (with or without mRFP1): *, P < 0.05. From JSKAP.SY (with or without mRFP1): #, P < 0.05.

Figure 5.

SKAP-Hom heterodimerizes with and is functionally redundant with SKAP55. (A) Domain structures of the SKAP55 and SKAP-Hom chimeras. (B) Cells were lysed and Western blotted to determine relative expression of the SKAP55 (WT) and SKAP-Hom (Hom) chimeras (n = 3). (C) J14.SY and JSKAP.SY cells transiently expressing mRFP1 or SKAP-Hom.mRFP1 were stimulated, imaged, and presented as in Fig. 2 B. See Tables 1 and 2 for microcluster properties and experiment numbers. Bars: (MOT and conventional images) 10 µm; (kymographs) 5 µm × 60 s. (D) Composite microcluster traces for the conditions examined in C; numbers in parentheses indicate the total number of cells examined. Line intensity corresponds to the fraction of microclusters surviving; arrowheads identify points of half-maximal microcluster dissociation. (E) Fraction of cells scored as displaying unstable contacts (n = 3). (F and G) Fractional retention on fibronectin-coated or anti-CD3-coated coverslips, as described in Fig. 4 (n = 3). Error bars indicate mean ± SEM. From parental J14.SY cells (with or without mRFP1): *, P < 0.05; **, P < 0.01. From JSKAP.SY (with or without mRFP1): #, P < 0.05; ##, P < 0.01.

Figure 6.

The SKAP55 DM is required for microcluster entry, microcluster stabilization, and the pro-adhesive functions of SKAP55. (A) Domain structures of the ΔDM and DM alone SKAP55 chimeras. (B) Differentially tagged SKAP55 (S1) and SKAP-Hom (S2) chimeras were coexpressed in E6.1 Jurkat cells. Immunoprecipitations were performed with anti-YFP antibody. Total lysates and immunoprecipitates were blotted with anti-YFP or anti-Flag (n = 4). (C) J14.SY and JSKAP.SY cells transiently expressing the ΔDM and DM alone chimeras were stimulated, imaged, and presented as in Fig. 2 B. See Tables 1 and 2 for microcluster properties and experiment numbers. Bars: (MOT and conventional images) 10 µm; (kymographs) 5 µm × 60 s. (D) Cells were lysed and Western blotted to determine relative expression of the WT, ΔDM, and DM-only chimeras (n = 3). (E) Composite microcluster traces for the conditions in C; numbers in parentheses indicate the total number of cells examined. Line intensity corresponds to the fraction of microclusters surviving; arrowheads identify points of half-maximal microcluster dissociation. (F) Fraction of cells scored as displaying unstable contacts (n = 5 or more). (G and H) Fractional retention on anti-CD3–coated or fibronectin-coated coverslips, as in Fig. 4 (n = 3). (I) Proposed mechanism by which the SKAP55 dimer stabilizes SLP-76 microclusters downstream of TCR ligation. Error bars indicate mean ± SEM. From parental J14.SY cells (with or without mRFP1): *, P < 0.05; **, P < 0.01. From JSKAP.SY (with or without mRFP1): #, P < 0.05; ##, P < 0.01. From JSKAP.SY with SKAP55.WT.mRFP add-back: †, P < 0.05.

Figure 7.

An artificial SKAP55 TD is sufficient for recruitment to and stabilization of SLP-76 microclusters, and for adhesion to TCR but not integrin ligands. (A) Domain structure of the SKAP55 TD chimera. (B) J14.SY cells were transiently transfected with WT and TD chimeras and blotted to confirm comparable expression levels (n = 3). (C) J14.SY and JSKAP.SY cells expressing the TD chimera were stimulated, imaged, and presented as in Fig. 2 B. See Tables 1 and 2 for microcluster properties and experiment numbers. Bars: (conventional images) 10 µm; (kymographs) 5 µm × 60 s. (D) Composite microcluster traces for the conditions examined in C; numbers in parentheses indicate the total number of cells examined. Line intensity corresponds to the fraction of microclusters surviving; arrowheads identify points of half-maximal microcluster dissociation. (E) Fraction of cells scored as displaying unstable contacts (n = 6). (F and G) Fractional retention on anti-CD3–coated or fibronectin-coated coverslips, as described in Fig. 4 (n = 3). Error bars indicate mean ± SEM. From parental J14.SY cells (with or without mRFP1): *, P < 0.05; **, P < 0.01. From JSKAP.SY (with or without mRFP1): #, P < 0.05. From JSKAP.SY with SKAP55.WT.mRFP1 add-back: †, P < 0.05.

Figure 8.

The SKAP55 DM is required for binding to RapL and RIAM, and governs the recruitment of talin into TCR-induced contacts. (A and B) 3×Flag.TRT-tagged SKAP55 wild-type (WT), DM deleted (ΔDM), and TD chimeras were expressed in J14.SY cells and immunoprecipitated via the Flag epitope. Blots of total lysates and immunoprecipitates are shown (n = 3). (C and D) Images of SLP-76.YFP (blue) and endogenous talin immunofluorescence (green) in J14.SY or JSKAP.SY cells stimulated on anti-CD3–coated plates and fixed after 10 min. Boxed regions were magnified 4× (shown below) to show microclusters at higher resolution (n = 3 or more). (D) JSKAP.SY cells were transfected with wild-type (WT) or TD SKAP55.3×Flag.TRT chimeras (red) before stimulation. (E) The frequency of cells exhibiting talin clustering and the central accumulation of talin were scored manually, using the images acquired above. (F and G) Cells were prepared as in C and D, but after fixation and before immunofluorescent staining cell bodies were sheared away, leaving cellular material only within the areas of tight contact between the cell and stimulatory substrate. Bars: (main panels) 10 µm; (enlarged panels) 2.5 µm. Error bars indicate mean ± SEM. From parental J14.SY cells: **, P < 0.01. From JSKAP.SY cells: ##, P < 0.01. From JSKAP.SY reconstituted with SKAP55.WT.3×Flag.TRT: ††, P < 0.01.

Figure 9.

SKAP55 is dispensable for β₁ integrin clustering downstream of TCR ligation. (A) J14.SY and JSKAP.SY cells were stimulated on anti-CD3–coated plates and fixed after 10 min. Immunofluorescent images of endogenous β₁ integrin (blue) and endogenous talin (red), and direct images of SLP-76.YFP (green) are shown in greyscale and as a pseudocolored merged image (n = 3). (B and C) Higher-resolution merged images of representative J14.SY (B, left) or JSKAP.SY (C, left) cells prepared as in A. SLP-76, talin, and β₁ integrin microclusters were identified algorithmically and used to derive regions of adjacency (right panels). The pseudocoloring scheme is indicated on the right. (n = 3). Bars, 10 µm. (D) Enrichment of talin, SLP-76, and β₁ clustered areas in distinct domains of the contact (edge, middle, and center), relative to the entire contact. (E) Fraction of each domain of the contact occupied by the indicated regions of adjacency. For D and E, data are shown ±SD (error bars) for six cells acquired in three independent experiments. From parental J14.SY cells (with or without mRFP1): °, P < 0.10; *, P < 0.05; **, P < 0.01. (F) Diagram showing the binding sites of the fluorescent probes used in A–E.

Figure 10.

Distinct roles of SKAP55 domains in microcluster stabilization, adhesion via the TCR, and integrin regulation. (A) SKAP55-dependent multimers enable microcluster stabilization and movement, and are required for adhesion via the TCR and via integrins. Microcluster persistence requires interactions capable of joining LAT, Gads, SLP-76, and ADAP into a minimal signaling complex. Constituents of stable microclusters (e.g., ADAP), may interact directly or indirectly with the actin cytoskeleton to promote T cell adhesion and/or microcluster movement. (B) The SKAP55 DM motif couples SLP-76 microclusters to RIAM and talin, enabling the conformational regulation of integrins via their β chains. (C) Models depicting the maturation or termination of the integrin-activating complex in B. Domains are abbreviated as follows: 4HB, four helix bundle; CC, coiled-coil region; FERM, FAK, ezrin, radixin, and moesin domain; PRR, proline-rich region; RA, Ras-association domain; hSH3, helically extended Src-homology 3 domain; TBH, talin-binding helix.