Fig 1: GDF6 Prodomain-CD99 Signaling Inhibits Src through Recruitment of CSK to the YQKKK Motif in the Intracellular Domain of CD99(A) GDF6 silencing or CD99 silencing activates Src in A673 cells. Activation of Src was assessed by phosphorylation of tyrosine 419.(B) Silencing of CD99 and CD99L2 activates Src in A673 cells.(C) Inhibition of Src by GDF6 requires CD99/CD99L2 and CSK. Transfection of 293 cells with GDF6 inhibited Src (lanes 1 and 2), which was abolished by CD99 and CD99L2 silencing (lanes 3 and 4) or by CSK silencing (lanes 5 and 6).(D) Inhibition of Src by CD99 requires CSK. Transfection of 293 cells with CD99 inhibited Src (lanes 1 and 2), which was abolished by CSK silencing (lanes 3 and 4).(E) CSK co-immunoprecipitates with CD99. C-terminally HA-tagged CSK was stably expressed in A673 cells and was immunoprecipitated with anti-HA antibody. The co-precipitation of endogenous CD99 was assessed by immunoblotting.(F) Human CD99, mouse CD99, and human CD99L2 co-immunoprecipitate with CSK. C-terminally FLAG-tagged human CD99, mouse CD99, and human CD99L2 were transfected in 293T cells, and the interaction with endogenous CSK was assessed by anti-FLAG immunoprecipitation followed by anti-CSK immunoblotting. Immunoprecipitation of each FLAG-tagged protein was verified by anti-FLAG immunoblotting.(G) Conserved YQKKKLCF-like motifs in the juxtamembrane regions of human CD99, mouse CD99, and human CD99L2.(H) The YQKKK motif interacts with CSK. Human CD99 intracellular domain (148–185), human CD99 intracellular domain lacking the YQKKKLCF sequence, YQKKKLCF, or YQKKK (all N-terminally fused to GFP) was transfected in 293T cells, and the interaction with endogenous CSK was assessed by anti-GFP immunoprecipitation followed by anti-CSK immunoblotting. Immunoprecipitation of each GFP-fusion protein was verified by anti-GFP immunoblotting (bottom).(I) The deletion of the YQKKK motif abrogates CD99-CSK interaction. C-terminally FLAG-tagged CD99 or CD99 lacking the YQKKK motif was co-transfected with GFP-tagged CSK or empty GFP vector in 293T cells. The CD99-CSK interaction was assessed by anti-GFP immunoprecipitation followed by anti-FLAG immunoblotting.(J) Endogenous GDF6 stimulates CD99-CSK interaction in Ewing sarcoma. The proximity ligation assays (PLAs) were used to assess the recruitment of CSK to CD99 in A673 cells stably expressing C-terminally HA-tagged CSK. The robust PLA signals were detected between CD99 and CSK-HA, which were abolished by GDF6 silencing. Scale bars: 20 µm. The quantification of PLA signals is shown at the bottom. *p < 0.05 (n = 5).(K) GDF6 stimulates the CSK recruitment to the YQKKK motif in CD99. HeLa cells, which do not endogenously express GDF6, were transfected with CD99-FLAG and CSK-HA and then treated with 293T cell conditioned medium expressing vector or GDF6, and the interaction between CD99-FLAG and CSK-HA was assessed by the PLA (top). GDF6 conditioned medium induced the recruitment of CSK-HA to CD99 (top). The same assay was performed using CD99 lacking the YQKKK motif, which showed that GDF6 conditioned medium was unable to induce the recruitment of CSK-HA to CD99 lacking the YQKKK motif (middle). Scale bars: 20 µm. The quantification of PLA signals is shown at the bottom. *p < 0.05 (n = 5).(L) Dominant-negative Src abrogates the proliferation inhibition by GDF6 silencing in A673 cells. A673 cells stably expressing dominant-negative Src or empty vector were transfected with GDF6 siRNA or control siRNA, and cell proliferation was assessed by IncuCyte (left). Dominant-negative Src reduced the phosphorylation of Src substrates, cortactin, p130 Cas, and STAT3 (right).(M) Src mutant lacking the CSK phosphorylation site (Y530F) inhibits A673 cell proliferation. Proliferation of A673 cells stably expressing Src Y530F or empty vector was assessed by IncuCyte (left). Src Y530F increased the phosphorylation of Src substrates, cortactin, p130 Cas, and STAT3 (right).(N) CSK abrogates the proliferation inhibition by GDF6 silencing in A673 cells. A673 cells stably expressing CSK or empty vector were transfected with GDF6 siRNA or control siRNA, and cell proliferation was assessed by IncuCyte.(O) A Src inhibitor, dasatinib, abrogates the proliferation inhibition by GDF6 silencing in A673 cells. Dasatinib was used at 50 nM.
Fig 2: CD99 Serves as the GDF6 Prodomain Receptor in Ewing Sarcoma(A) GDF6 prodomain co-immunoprecipitates with CD99 and CD99L2, a paralog of CD99, upon co-transfection in 293T cells. C-terminally FLAG-tagged CD99 and/or CD99L2 was co-transfected with GDF6 prodomain in 293T cells. The interaction between GDF6 prodomain and CD99/CD99L2 was assessed by anti-FLAG immunoprecipitation followed by immunoblotting for the indicated protein.(B) GDF6 prodomain interacts with GST-CD99(1–122) in vitro. GDF6 prodomain expressed in 293T cell conditioned medium was incubated with GST-CD99 extracellular domain (1–122) or GST, and the protein interaction was assessed by GST pull-down followed by immunoblotting. Asterisks denote GST-CD99(1–122) and GST.(C) GDF6 prodomain binds to Ewing sarcoma cells through CD99/CD99L2. C-terminally FLAG-tagged GDF6 prodomain secreted from transfected 293T cells was incubated for 8 h with A673 cells that were transfected with CD99 siRNA, CD99L2 siRNA, and control siRNA as indicated (“–” denotes controls without incubation). Cells were washed, and the whole-cell lysate was prepared to assess the binding of GDF6 prodomain-FLAG to the cells. GDF6 prodomain-FLAG bound to control siRNA-transfected cells, and the binding was reduced by CD99 and CD99L2 silencing. The silencing of CD99 and CD99L2 was verified by immunoblotting.(D) CD99/CD99L2 mediate the binding of GDF6 prodomain to Ewing sarcoma cells. GDF6 prodomain fused to alkaline phosphatase (AP), or AP alone was produced from transfected 293T cells and was incubated with A673 cells that were transfected with CD99 siRNA and/or CD99L2 siRNA or control siRNA as indicated. The binding of GDF6 prodomain-AP or AP to cells was visualized by AP reaction. GDF6 prodomain-AP bound to control siRNA-expressing A673 cells, and the binding was blocked by CD99 siRNA and CD99L2 siRNA. Scale bars: 20 µm.(E) GDF6 prodomain induces the internalization of CD99 expressed in HeLa cells. CD99 expressed in HeLa cells was located in the plasma membrane. GST-GDF6 prodomain caused the internalization of CD99. Scale bars: 20 µm.(F) GDF6 induces the internalization of endogenous CD99 in A673 cells. A673 cells were treated with 293T cell conditioned medium expressing vector or GDF6. The latter induced the internalization of endogenous CD99 detected by anti-CD99 immunofluorescence (left). Scale bars: 20 µm. The quantification of surface CD99 expression (right, *p < 0.05; n = 5).(G) GDF6 silencing results in accumulation of CD99 on A673 cell surface. A673 stably expressing C-terminally FLAG-tagged CD99 was transfected with control siRNA or GDF6 siRNA, and the location of CD99-FLAG was assessed by anti-FLAG immunostaining (left). Scale bars: 20 µm. GDF6 silencing caused the accumulation of CD99-FLAG on cell surface. CD99 siRNA abolished the immunostaining of CD99-FLAG. The quantification of surface CD99-FLAG expression (right, *p < 0.05; n = 5).(H) Microscale thermophoresis analysis of GDF6 prodomain-CD99 extracellular domain interaction. The interaction between C-terminally Fc-tagged CD99 extracellular domain (residues 23–122) and GDF6 prodomain (wild-type, A249E, or L289P) was analyzed by microscale thermophoresis. GDF6 prodomain mutants displayed lower KD values than wild-type GDF6 prodomain.
Fig 3: GDF6 Prodomain Mutants Associated with Klippel-Feil Syndrome Are Hyperactive in GDF6-CD99-Src Signaling(A) GDF6 A249E or L289P prodomain mutant displays enhanced co-immunoprecipitation with CD99 compared with wild-type GDF6 prodomain. GDF6 prodomain, GDF6 A249E prodomain, or GDF6 L289P prodomain was co-transfected with C-terminally FLAG-tagged CD99 or FLAG vector in 293T cells as indicated, and the interaction between each GDF6 prodomain and CD99-FLAG was assessed by anti-FLAG immunoprecipitation followed by immunoblotting.(B) GDF6 A249E or L289P prodomain mutant displays enhanced interaction with CD99 in vitro. Wild-type, A249E, or L289P GDF6 prodomain expressed in 293T cell conditioned medium was incubated with C-terminally Fc-tagged CD99 extracellular domain (residues 23–122), and protein interaction was assessed by protein A Sepharose pull-down followed by immunoblotting.(C) GDF6 A249E or L289P prodomain mutant displays enhanced binding to A673 cells. Ligand-binding assays were performed as in Figure 3C.(D) GDF6 A249E or L289P prodomain mutant more strongly inhibits Src than wild-type prodomain in 293 cells. Wild-type, A249E, or L289P GDF6 prodomain was stably expressed in 293 cells, and the levels of active Src (phosphorylated on Y419) were assessed by immunoblotting.(E) GDF6 A249E or L289P mutant more strongly inhibits Src than wild-type GDF6 in A673 cells. Wild-type, A249E, or L289P GDF6 was stably expressed in A673 cells, and the levels of active phosphorylated Src were assessed by immunoblotting.(F) GDF6 A249E or L289P prodomain mutant more strongly induces CSK recruitment to CD99.The PLA was performed in HeLa cells as in Figure 4K, using 293T cell conditioned medium expressing vector, GDF6, GDF6 prodomain, GDF6 A249E prodomain, or GDF6 L289P prodomain. Scale bars: 20 µm. The quantification of PLA signals is shown at the bottom. *p < 0.05 (n = 5).
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