THE CATALYTIC SUBUNIT OF PROTEIN PHOSPHATASE 2A IS CARBOXYL-METHYLATED IN-VIVO

We have used polyclonal antibodies against an internal peptide (residu es 169 to 182; Ab(169/182)) and a peptide corresponding to the carboxy l terminus (residues 299 to 309; Ab(299/309)) to look for in vivo modi fications of protein phosphatase 2A catalytic (PP2Ac) subunit. Treatme nt of extracts from human breast cancer (MCF7) cells with either alkal i or ethanol increased immunoreactivity of PP2Ac subunit severalfold o n Western blots with Ab(299/309), but did not apparently change molecu lar weight or isoelectric point of the protein. In contrast, immunorea ctivity with Ab(169/182) was unchanged by these treatments. Subsequent ly, we demonstrated that the increase in PP2Ac subunit recognition by Ab(299/309) coincides with the demethylation of this protein at the ca rboxyl-terminal leucine (Leu(309)). Methylation of PP2Ac subunit, in v itro, increases its activity toward both phosphorylase a and a phospho peptide. The carboxyl-terminal sequence (TPDYJFL) of PP2Ac subunit is completely conserved between mammals, yeast, fruit fly, and plants whi ch suggests that regulation of this enzyme activity by carboxyl-termin al methylation has been conserved during evolution.