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 subunit, C, and two regulatory subunits, A and B. The A subunit is rod shaped and consists of 15 nonidentical repeats. According to our previous model, the B subunit binds to repeats 1 through 10 and the C subunit binds to repeats 11 through 15 of the A subunit. Another form of PP2A, 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 of enzyme purification. To study the structure and relative abundance of different forms of PP2A, we generated monoclonal antibodies against the native A subunit. Two antibodies, 5H4 and 1A12, recognized epitopes in repeat 1 near the N terminus and immunoprecipitated free A subunit and core enzyme but not holoenzyme. Another antibody, 6G3, recognized 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 A alpha subunit (designated 6F9) precipitated 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 families all bind to repeat 1 of the A subunit, and the results confirm and extend our model of the holoenzyme. By sequential immunoprecipitations with 5H4 or 1A12 followed by 6F9, core enzyme and holoenzyme in cytoplasmic extracts from 10T1/2 cells were completely separated and they exhibited the expected specificities towards phosphorylase a and retinoblastoma peptide as substrates. Quantitative analysis showed that under conditions which minimized proteolysis and dissociation of holoenzyme, 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.
Kremmer
E
E
GSF-Forschungszentrum, Institut für Immunologie, Munich, Germany.
Ohst
K
K
Kiefer
J
J
Brewis
N
N
Walter
G
G
D000818:Animals; D000911:Antibodies, Monoclonal; D002460:Cell Line; D003067:Coenzymes / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D018604:Epitope Mapping; D010749:Phosphoprotein Phosphatases / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D011233:Precipitin Tests; D011487:Protein Conformation; D054648:Protein Phosphatase 2; D051381:Rats; D012091:Repetitive Sequences, Nucleic Acid
D000911:Antibodies, Monoclonal; D003067:Coenzymes; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2