Methylation of the protein phosphatase 2A catalytic subunit is essential for association of B alpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen

Binding of different regulatory subunits and methylation of the catalytic ( C) subunit carboxy-terminal leucine 309 are two important mechanisms by whi ch protein phosphatase 2A (PP2A) can be regulated. In this study, both gene tic and biochemical approaches were used to investigate regulation of regul atory subunit binding by C subunit methylation. Monoclonal antibodies selec tively recognizing unmethylated C subunit were used to quantitate the methy lation status of wild-type and mutant C subunits. Analysis of 13 C subunit mutants showed that both carboxyterminal and active site residues are impor tant for maintaining methylation in vivo. Severe impairment of methylation invariably led to a dramatic decrease in B alpha subunit binding but not of striatin, SG2NA, or polyomavirus middle tumor antigen (MT) binding. In fac t, most unmethylated C subunit mutants showed enhanced binding to striatin and SG2NA. Certain carboxy-terminal mutations decreased B alpha subunit bin ding without greatly affecting methylation, indicating that B alpha subunit binding is not required for a high steady-state level of C subunit methyla tion. Demethylation of PP2A in cell lysates with recombinant PP2A methylest erase greatly decreased the amount of C subunit that could be coimmunopreci pitated via the B alpha subunit but not the amount that could be coimmunopr ecipitated with A alpha subunit or MT. When C subunit methylation levels we re greatly reduced in vivo, B alpha subunits were found complexed exclusive ly to methylated C subunits, whereas striatin and SG2NA in the same cells b ound both methylated and unmethylated C subunits. Thus, C subunit methylati on is critical for assembly of PP2A heterotrimers containing B alpha subuni t but not for formation of heterotrimers containing MT, striatin, or SG2NA. These findings suggest that methylation may be able to selectively regulat e the association of certain regulatory subunits with the A/C heterodimer.