SEPARATION OF PP2A CORE ENZYME AND HOLOENZYME WITH MONOCLONAL-ANTIBODIES AGAINST THE REGULATORY A-SUBUNIT - ABUNDANT EXPRESSION OF BOTH FORMS IN CELLS

Protein phosphatase 2A (PP2A) holoenzyme is composed of a catalytic su bunit, C, and two regulatory subunits, A and B. The A subunit is rod s haped and consists of 15 nonidentical repeats. According to our previo us model, the B subunit binds to repeats 1 through 10 and the C subuni t binds to repeats 11 through 15 of the A subunit. Another form of PP2 A, core enzyme, is composed only of subunits A and C. It is generally believed that core enzyme does not exist in cells but is an artifact o f enzyme purification. To study the structure and relative abundance o f different forms of PP2A, we generated monoclonal antibodies against the native A subunit. Two antibodies, 5H4 and 1A12, recognized epitope s in repeat 1 near the N terminus and immuneprecipitated free A subuni t and core enzyme but not holoenzyme. Another antibody, 6G3, recognize d an epitope in repeat 15 at the C terminus and precipitated only the free A subunit. Monoclonal antibodies against a peptide corresponding to the N-terminal 11 amino acids of the Aa! subunit (designated 6F9) p recipitated free A subunit, core enzyme, and holoenzyme. 6F9, but not 5H4, recognized holoenzymes containing either B, B', or B'' subunits. These results demonstrate that B subunits from three unrelated gene fa milies all bind to repeat 1 of the A subunit, and the results confirm and extend our model of the holoenzyme. By sequential immuno-precipita tions with 5H4 or 1A12 followed by 6F9, core enzyme and holoenzyme in cytoplasmic extracts from 10T1/2 cells were completely separated and t hey exhibited the expected specificities towards phosphorylase a and r etinoblastoma peptide as substrates. Quantitative analysis showed that under conditions which minimized proteolysis and dissociation of holo enzyme, core enzyme represented at least one-third of the total PP2A. We conclude that core enzyme is an abundant form in cells rather than an artifact of isolation. The biological implications of this finding are discussed.