Tyrosine-phosphorylated SKAP55 interacts with CD45-D819V. (A) Full-length SKAP55 was subcloned in frame with the C-terminal Myc-His tag into the pcDNA3.1 vector. Truncated CD45-D819V was subcloned in frame with an N-terminal triple-HA tag into pAcTAG2 vector. (B) CD45-D819V interacts with SKAP55 expressed in serum-stimulated cells. 293T cells were transiently cotransfected with CD45-D819V and SKAP55. The transfected cells were either left untreated (−) or treated with serum (+). The cells (5 × 10⁶) were lysed and immunoprecipitated (IP) by anti-HA antibody. The immunoprecipitates were subjected to SDS-7% PAGE, and blotted by anti-Myc antibody (1:2,000). The bottom panel is a Western blot of total SKAP55 from whole-cell lysates from 2 × 10⁴ cells to ensure equal SKAP55 expression in samples. (C) Only the tyrosine phosphorylated form of SKAP55 interacts with CD45-D819V. The same membrane shown in panel B was stripped and reprobed with anti-phosphotyrosine antibody. The bottom panel is a Western blot of total CD45 from whole-cell lysates of 2 × 10⁴ cells, indicating equal CD45 expression of samples. (D) Chemical cross-linking verifies the direct interaction of CD45 with SKAP55. The serum-stimulated cells were either treated with 10 mM EDAC or left untreated. The cell lysates of 5 × 10⁶ cells were immunoprecipitated either by anti-Myc antibody and blotted by anti-HA antibody (left) or precipitated by anti-HA antibody and blotted by anti-Myc antibody (right) as done for panel B. Molecular mass markers (in kilodaltons) are on the left. (E) The endogenous CD45 interacts with the native SKAP55 in Jurkat cells in response to anti-CD3 antibody stimulation. Volumes of 5 × 10⁸ Jurkat cells were equally divided into two portions, one of which was treated with anti-CD3 antibody for 5 min, and the other portion of cells was left untreated. The cell lysates were immunoprecipated either with anti-CD45 antibody or with anti-SKAP55 antibody and subjected to SDS-7% PAGE. The anti-CD45 antibody immunocomplexes were blotted by anti-SKAP55 antibody, and total CD45 protein in cell lysates was determined by anti-CD45 antibody blotting analysis (left). Reciprocally, anti-SKAP55 immunocomplexes were subjected to anti-CD45 antibody probing, and total SKAP55 protein in cell lysates was assayed by anti-SKAP55 antibody to ensure equal amounts of SKAP55 protein were used in the experiments (right).

SKAP55 as substrate interacts with CD45-D819V through its binding to active site of phosphatase. (A) Phosphatase inhibitor pervanadate abolishes the interaction of SKAP55 with CD45-D819V in vivo. 293T cells were cotransfected with tagged CD45-D819V and SKAP55 as described for Fig. 2A. The transfected cells were stimulated with serum as in Fig. 2. The treated cells were lysed with lysis buffer with (+) or without (−) sodium pervanadate (2 mM). The cell lysates were precipitated either by anti-HA antibody or by immunoglobulin G (IgG) antibody. Immunoprecipitated (IP) complexes were subjected to SDS-7% PAGE and immunoblotted by anti-Myc antibody. (B) Association of SKAP55 with wild-type and mutant CD45-D819V in immunoprecipitates. As described for panel A, the tagged SKAP55 was cotransfected with tagged CD45 or its mutant CD45-D819V in 293T cells. The transfected cells were stimulated with serum as before. Cell lysates were immunoprecipitated by anti-HA antibody and immunoblotted by anti-Myc antibody (left) or the cells were immunoprecipitated by anti-Myc antibody and reprobed by anti-Myc antibody for the control of SKAP55 precipitation (right). The same membrane was stripped and reprobed by antiphosphotyrosine antibody (middle). The bottom panel is a Western blot of total CD45 from whole-cell lysates. (C) As described for panel B, CD45 and CD45-D918V were coprecipitated by anti-Myc antibody and immunoblotted by anti-HA antibody. The bottom panel is a Western blot of total SKAP55 from whole-cell lysates. (D) SKAP55 is a potential substrate for CD45 in T cells. CD45-deficient Jurkat cells (7 × 10⁶) were cotransfected with the constitutively active mutant Fyn-Y531F (FynYF), wild-type CD45, mutant CD45-D819V, and SKAP55 as indicated. The cell lysates were precipitated by anti-HA antibody and probed either by anti-Myc antibody (top), by antiphosphotyrosine antibody (upper middle), or by anti-HA antibody again (lower middle). The bottom panel is a Western blot of total Fyn from whole-cell lysates. Molecular mass markers (in kilodaltons) are on the left.

SKAP55 is translocated from cytoplasm to cell membrane upon anti-CD3 antibody stimulation. GFP-SKAP55, GFP-SKAP55-Y232F, and GFP-SKAP55-Y271F were transfected into CD45-positive (CD45⁺) and CD45-negative (CD45⁻) Jurkat cells. At 24 h following transfection, the transfected cells were treated with anti-CD3 antibody (+) or were left untreated (−). The translocation was visualized by confocal microscopy in a single cell as the representatives from the GFP-positive cells. (a and b) GFP-SKAP55 was transfected into CD45-positive Jurkat cells; (c and d) GFP-SKAP55 was transfected into CD45-deficient Jurkat cells; (e and f) mutant GFP-SKAP55-Y232F was transfected into CD45-positive Jurkat cells; (g and h) mutant GFP-SKAP55-Y271F was transfected into CD45-positive Jurkat cells; (i and j) GFP vector alone was transfected into CD45-positive Jurkat cells as control; (k and l) both GFP-SKAP55 and the constitutively active Fyn were transfected in CD45-deficient Jurkat cells. The ratio of the two plasmid DNA, namely Fyn to SKAP55, was 10:1 to ensure that the green cells were also expressed with the constitutively active Fyn.

Overexpression of SKAP55 activates MAPK kinase and induces tyrosine phosphorylation of PLC-γ1. (A) SKAP55 functions as a positive regulator in MAPK kinase pathway. Jurkat cells (2 × 10⁷) either stably transfected with wild-type SKAP55, with mutant SKAP55-Y232F, or with vector were treated with anti-CD3 antibody for 5 min or were left untreated. Total-cell lysates were subjected to SDS-7% PAGE and blotted by anti-phospho-ERK or anti-total-ERK antibody. The total SKAP55 protein in cell lysates was determined by anti-Myc blottings. (B) SKAP55 promotes the tyrosine phosphorylation of PLC-γ1. Aliquots of 8 × 10⁷ Jurkat cells were stablely transfected either with wild-type SKAP55, mutant SKAP55-Y232F, or vector for 24 h. The transfected cells were treated with anti-CD3 antibody for 5 min or were left untreated. The cell lysates were immunoprecipitated by anti-PLC-γ1 antibody and blotted either by antiphosphotyrosine antibody (top) or by anti-PLC-γ1 antibody (middle). The expression of SKAP55 was measured by anti-Myc blotting (bottom). Molecular mass markers (in kilodaltons) are on the left.

Catalytic D1 of CD45 interacts with SK region of SKAP55, and interaction in yeast two-hybrid system requires Src kinase activity. (A) The D1 domain of CD45-D819V interacts with SKAP55 in the presence of Src kinase. Gal4-SKAP55 was cotransformed into yeast with LexA-CD45-D819V, LexA-CD45-D1 (residues 596 to 942), or LexA-CD45-D2 (residues 943 to 1304), with or without the expression of wild-type Src kinase or the kinase-dead mutant Src-K297R. The interactions were evaluated using the β-galactosidase filter assay. After 60 min, the level of blue color in transformed yeasts was used as an indicator: −, no interaction; ++, strong interaction; +++, very strong interaction. The data summarize results of three independent experiments. (B) The SK region of SKAP55 interacts with CD45-D819V. SKAP55 was dissected into three regions: PH domain (residues 1 to 205), SK domain (residues 206 to 300), and SH3 domain (residues 301 to 359). As described for panel A, the individual domain was cotransformed with CD45-D819V into yeast, and the β-galactosidase filter assay was used to evaluate the interactions as described for panel A.

Tyrosine residue 232 of SKAP55 plays critical role for its interaction with CD45. (A) Mutant SKAP55-Y232F completely abolished its interaction with CD45-D819V. CD45-D819V was cotransfected with vector alone and SKAP55, SKAP55-Y271F, SKAP55-Y219F, or SKAP55-Y232F in 293T cells. The transfected cells were stimulated with serum for tyrosine phosphorylation as described for Fig. 2. The cells (7 × 10⁶) were lysed and immunoprecipitated (IP) by anti-HA antibody, subjected to SDS-7% PAGE, and blotted with anti-Myc antibody. The middle panel is a blot made by precipitating SKAP55 and reprobing with anti-Myc antibody. The bottom panel is a Western blot of total CD45 from whole-cell lysates of 2 × 10⁴ cells. (B) The CD45-bound SKAP55 was tyrosine phosphorylated upon serum stimulation. The same membrane shown in panel A was stripped and reprobed with anti-phosphotyrosine antibody. The bottom panel is a Western blot of total SKAP55 from whole-cell lysates of 2 × 10⁴ cells. The molecular mass markers (in kilodaltons) are on the left.

Tyrosine phosphorylation of SKAP55 is enhanced with anti-CD3 antibody stimulation. (A) The tyrosine phosphorylation of endogenous SKAP55. Volumes of 10 × 10⁷ cells were untreated (−) or treated (+) by anti-CD3 antibody and lysed. The endogenous SKAP55 was precipitated with anti-SKAP55 specific antibody from wild-type Jurkat (right) or CD45-deficient Jurkat (CD45⁻) (left) cells. Immunoprecipitates (IP) were subjected to SDS-7% PAGE and blotted with antiphosphotyrosine antibody. The immunoprecipitated SKAP55 protein was also reprobed by anti-SKAP55 antibody to indicate that an equal amount of SKAP55 was precipitated in the samples (bottom). (B) The tyrosine phosphorylation of the transfected exogenous SKAP55. SKAP55 and mutant SKAP55-Y232F were transfected into the Jurkat cells by electroporation. The cells were treated with anti-CD3 antibody (+) or were left untreated (−) and were lysed. The cell lysates were immunoprecipitated by anti-Myc antibody and subjected to SDS-7% PAGE. The membrane was probed by antiphosphotyrosine antibody. To ensure equal immunoprecipitation of SKAP55, the membrane was reprobed by anti-Myc antibody (bottom). (C) SKAP55 was tyrosine phosphorylated by coexpression of mutant Fyn in CD45-negative cells. CD45-deficient Jurkat cells (10 × 10⁷) were cotransfected with SKAP55, wild-type Fyn, or mutant Fyn-Y531F (FynYF). The cell lysates were immunoprecipitated by anti-Myc antibody and blotted by antiphosphotyrosine antibody (top), again by anti-Myc antibody (middle), or by anti-Fyn antibody (bottom). Molecular mass markers (in kilodaltons) are on the left.

SKAP55 functions as a positive regulator in IL-2 gene activation. (A) Activation of the IL-2 promoter by SKAP55 in Jurkat cells. Jurkat cells (10⁷) were transiently transfected by electroporation with 10 μg of wild-type (WT) SKAP55, mutant SKAP55-Y219F, SKAP55-Y232F, or SKAP55-Y271F, or vector only, together with 10 μg of IL-2-Luc plasmid carrying the luciferase gene driven by the IL-2 promoter. For the internal control of transfection efficiency, pSV-β-galactosidase vector (1 μg) was cotransfected into Jurkat cells. Each transfected sample was divided into two portions that were either stimulated with anti-CD3 antibody plus PMA (50 ng/ml) and ionomycin (1 μM) or left unstimulated and then cultured for an additional 6 h to activate IL-2 gene transcription. Following incubation, luciferase activities were measured. Relative activity was determined after normalization, with unstimulation being defined as equal to one. The efficiency of transfections was normalized with the activity of β-galactosidase produced by a transfected reporter vector. The data represent the average and standard deviation from three independent experiments. The inset on top represents anti-SKAP55 immunoblot showing the expression of transfected SKAP55 and its mutants. (B) Overexpression of SKAP55-Y232F in Jurkat cells induces the tyrosine hyperphosphorylation of Fyn. Jurkat cells stably expressing the empty vector, SKAP55, and mutant SKAP55-Y232F were treated with anti-CD3 antibody stimulation (+) or were left untreated (−). Cell lysates were prepared and immunoprecipitated (IP) with anti-Fyn antibody. The immunoprecipitates were subjected to SDS-7% PAGE, and the membrane was probed by antiphosphotyrosine antibody. The equal amounts of precipitated Fyn protein in the samples were shown (middle) by stripping the same membrane and reprobing it with anti-Fyn antibody. In the bottom panel, Fyn kinase activity was examined in vitro by its autophosphorylation and the phosphorylation of the exogenous substrate enolase. The immunoprecipitates of Fyn were incubated with the substrate enolase in the presence of [γ-³²P]ATP. Incorporation of the label in Fyn and enolase was determined by resolution on SDS-7% PAGE and autoradiography. Molecular mass markers (in kilodaltons) are on the left.

SKAP55 as an adapter bridges CD45 with Fyn for interaction. (A) Fyn was either transfected only with CD45-D819V or was cotransfected with both CD45-D819V and SKAP55 in 293T cells. At 48 h following the transfection, cell lysates were immunoprecipitated (IP) by anti-CD45 specific antibody and subjected to SDS-7% PAGE and immunoblotted by anti-Fyn antibody. The bottom panel is a Western blot of total CD45 from whole-cell lysates, indicating the equal expression of CD45-D819V in the two samples. (B) As described for panel A, the cell lysates were immunoprecipitated with anti-Fyn specific antibody. The immunoprecipitates were subjected to SDS-7% PAGE and immunoblotted with anti-Fyn antibody again, demonstrating its equal precipitation. The bottom panel is a Western blot of SKAP55 from whole-cell lysates, indicating its appropriate expression in the sample. Molecular mass markers (in kilodaltons) are on the left.