Figure 4.

UV- and calpain-dependent protein tyrosine phosphatase degradation requires previous PTP oxidation. (A) Antioxidant treatment attenuates UV-induced degradation. A431 cells were treated with NAC (30 mM) for 2 min before irradiation, and the lysates were immunoblotted. (B) Reversible oxidation converts PTP1B to a calpain substrate in vitro. Left panel: Cells were treated with H₂O₂ (5 mM) at 37°C for 15 min and PTP1B was immunoprecipitated. The precipitates were divided into two parts, one of which was digested with 0.4 U of calpain at 37°C for 30 min. Right panel: PTP1B was immunoprecipitated from an untreated cell lysate, and either reversibly or irreversibly oxidized by treatment with 50 μM of H₂O₂ for 4 or 16 h, respectively (Salmeen et al, 2003; supplementary Fig 3 online) and exposed to calpain as in the left panel. (C) Reversible oxidation of PTP1B increases the susceptibility to calpain but not to other proteinases. PTP1B was immunoprecipitated and reversibly oxidized (as in Fig 3B) or mock-treated. The immunoprecipitates were then left untreated or were treated with 0.4 U of the indicated proteinases at 37°C for different periods. PTP1B protein digestion was measured by quantification of the PTP1B band in immunoblots. The ratio of the amount of reversibly oxidized versus non-oxidized PTP1B remaining after digestion is depicted. (D) Combined treatment with H₂O₂ (as in (B)) and ionomycin (as in Fig 3A) leads to PTP degradation. Immunoblots of lysates are shown.