Metals in the active site of native protein phosphatase-1

• Native PP1 from skeletal muscle is associated with iron and zinc.
• Treatment of native PP1 with inhibitor-2 reduces the level of associated iron.
• The activity and substrate specificity of PP1 is determined by the nature of the associated metals.

Protein phosphatase-1 (PP1) is a major protein Ser/Thr phosphatase in eukaryotic cells. Its activity depends on two metal ions in the catalytic site, which were identified as manganese in the bacterially expressed phosphatase. However, the identity of the metal ions in native PP1 is unknown. In this study, total reflection X-ray fluorescence (TXRF) was used to detect iron and zinc in PP1 that was purified from rabbit skeletal muscle. Metal exchange experiments confirmed that the distinct substrate specificity of recombinant and native PP1 is determined by the nature of their associated metals. We also found that the iron level associated with native PP1 is decreased by incubation with inhibitor-2, consistent with a function of inhibitor-2 as a PP1 chaperone.

TXRF analysis was used to detect iron and zinc in protein phosphatase-1 (PP1) purified from rabbit skeletal muscle. The distinct substrate specificity of recombinant and native PP1 is determined by the nature of their associated metals. We also found that the iron associated with native PP1 is removed by inhibitor-2.Figure optionsDownload as PowerPoint slide