In vivo and in vitro binding of microcystin to protein phosphatases 1 and 2A.

The hepatotoxic microcystins (Mcyst) are potent inhibitors of the ser/thr protein phosphatases (PP1 and PP2A) with IC50's of 0.1-1.0 nM. Mcyst and other PP inhibitors like okadaic acid or calyculin A interact with the C-terminal region of PP1 and PP2A. Using [125I]-Mcyst and antibodies specific for PP1 and PP2A, we show by immunoprecipitation and autoradiography, that in hepatocytes Mcyst forms secondary covalent bonds with both PP1 and PP2A catalytic subunits. We demonstrate that the bond resulted from the reaction between the electrophilic alpha, beta unsaturated carbonyl of the methyldehydroalanine residue of Mcyst and the thiol of Cys 273 located in the C-terminal of PP1 (Cys 266 in PP2A), since site-directed mutagenesis of Cys 273 to Ala in PP1 alpha led to complete loss of ability for the formation of a covalent Mcyst-PP1 adduct.