Cantharidin effects on protein phosphatases and the phosphorylation state of phosphoproteins in mice

Our earlier studies indicated that the action of cantharidin (CA) in mice is associated with binding to protein phosphatase 2A in liver cytosol and inhibition of its phosphorylase a phosphatase activity. In this investigation, we find that CA totally inhibits the phosphorylase a phosphatase activity in mouse liver, muscle, and skin cytosol at 5000 nM, with IC50s of 110-250 nM. About 50% of the phosphorylase a phosphatase activity of brain cytosol is sensitive to CA with an IC50 of approximately 80 nM and the remaining half is not inhibited even at 5000 nM. Intraperitoneal treatment of mice with CA leads to a dose-dependent decrease in phosphorylase a phosphatase activity with the aforementioned tissues displaying differential CA sensitivity. At 60 min after a 10 mg/kg CA dose, there is 90-95% inhibition of phosphorylase a phosphatase activity in liver and skin cytosol, 50% in muscle cytosol, and almost no inhibition in brain cytosol. The phosphorylation state of several phosphoproteins examined with tissue cytosol and [gamma-32P]ATP is increased by CA, in a concentration-dependent manner, as follows: endogenous glycogen phosphorylase a in muscle both in vitro and in vivo, and unidentified phosphoproteins in brain (approximately 34 and approximately 75 kDa) and skin (approximately 34 kDa) in vitro. These findings confirm the importance of protein phosphatases as primary targets of CA action in a variety of mouse tissues and, more generally, the possible use of CA and its analogs to investigate and potentially control some processes modulated by the reversible phosphorylation of proteins.