STAT5 is cleaved by calpain in vivo and in vitro.
(A) Dibucaine treatment of platelets leads to cleavage of STAT5. The platelets were incubated with either calpeptin (20 μM) or dimethyl sulfoxide (DMSO; vehicle of calpeptin; final, 0.1%) for 5 minutes. The platelets were then treated with dibucaine (1 mM) for 15 minutes. Whole-platelet lysates (1.5 × 107 cells/lane) were analyzed by 7.5% to 15% gradient SDS-PAGE. The separated proteins were electrophoretically transferred from the gel to nitrocellulose membranes. STAT5 was detected by immunoblotting with a pan anti-STAT5 MoAb (left panel), anti-STAT5A (middle panel), or anti-STAT5B (right panel). Arrows indicate the relative position of intact STAT5; arrowheads show the positions of cleaved products of STAT5. R indicates control resting platelets; D, dibucaine-treated cells after incubation in DMSO; D + C, dibucaine plus calpeptin; and IB, immunoblotting. (B) In vitro cleavage of STAT5. STAT5 was precipitated by either anti-STAT5A or anti-STAT5B. The immunoprecipitates were untreated or incubated with purified μ-calpain in the presence or absence of calcium ion. After denaturing in SDS, STAT5 was detected by immunoblotting with a pan anti-STAT5 MoAb. (C) Truncated STAT5 molecules cleaved in vitro were not recognized by the anticarboxy-terminal domain of STAT5. IP indicates immunoprecipitation. Methods were the same as those as described in the legend for Figure 1B except that STAT5 was recognized by an antibody against the carboxy-terminal domain of STAT5. (D) Cleavage of STAT5 during platelet aggregation. Platelets were stimulated with thrombin (1 U/mL) for 30 minutes with or without stirring. After addition of ethyleneglycotetraacetic acid (5 mM) and EDTA (5 mM), platelets were lysed by boiling in SDS sample buffer. STAT5 was detected by immunoblotting as described in the legend for Figure 1A by using the pan anti-STAT5 MoAb. Lane 1 shows resting platelets; lane 2, results after thrombin stimulation of platelets for 30 minutes, with stirring, in the absence of Arg-Gly-Asp-Ser; and lane 3, results after thrombin stimulation for 30 minutes without stirring. The arrow indicates full-length STAT5 and the arrowhead a cleaved form of STAT5.
