Separation of PP2A Core Enzyme and Holoenzyme with Monoclonal Antibodies against the Regulatory A Subunit: Abundant Expression of Both Forms in Cells

Abstract

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␣ 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.Protein phosphatase 2A (PP2A), the most abundant serine/ threonine-specific phosphatase in mammals, plays a role in many fundamental cellular processes, including cell division (7, 30), signal transduction (36), gene expression (52), and Drosophila development (34). The PP2A holoenzyme consists of a 36-kDa catalytic C subunit and a 65-kDa regulatory A subunit, which together form the core enzyme to which one of several B subunits is bound (8, 38). The A and C subunits both exist as two isoforms (␣ and ␤) (19, 63), whereas the B subunits fall into three families called B, BЈ, and BЉ, which are unrelated to each other by protein sequence. The B family has three members, B␣, B␤, and B␥, each with a molecular mass of around 55 kDa (18,33,41); the BЈ family consists of numerous isoforms and splice variants, whose molecular masses range from 54 to 68 kDa (10, 35, 57, 69); and the BЉ family has two members, which have molecular masses of 72 and 130 kDa and are splice variants of the same gene (20). The combination of these subunits can give rise to a large number of PP2A variants, which ...

HRX Leukemic Fusion Proteins Form a Heterocomplex with the Leukemia-associated Protein SET and Protein Phosphatase 2A

“…What forms of PP2A are bound to HRX is an important question because PP2A core enzyme (subunits A and C) has been shown to have different substrate specificities than PP2A holoenzyme (subunits A, B, and C) (39). At present we do not actually know how PP2A is bound to the HRX⅐SET⅐PP2A complex; however, our data suggest that SET must be present for maximal PP2A binding.…”

“…Bands were visualized with enhanced chemiluminescence (ECL) reagents (Bio-Rad). Western blotting of co-immunoprecipitation (coIP) for PP2A was done as described previously by Kremmer et al (39) using the 6G3 monoclonal antibody.…”

PP2A holoenzyme assembly: in cauda venenum (the sting is in the tail)

“…Therefore, the regulated binding and dynamic exchange of B-type subunits represent attractive mechanisms for controlling PP2A activity. In addition to the 'classical' PP2A trimers, the PP2A D core structure can exist independently in cells [11], and some dimers lack an A subunit, which is replaced by the a4 protein (Box 2). These PP2A C -a4 complexes are involved in the inhibition of apoptosis and in diseases such as Opitz syndrome [1][2][3].…”

Reduced expression of the A? subunit of protein phosphatase 2A in human gliomas in the absence of mutations in the A? and A? subunit genes

“…Equal amounts of protein were separated on 10% SDS polyacrylamide gels and transferred to PVDF membrane (Immobilon-P, Millipore). To detect the A, B and C subunits, the following primary antibodies were used: rat monoclonal anti-A subunit (6G3), 30 rabbit anti-B␣ subunit produced against the peptide KGAVDDDVAEADY, 31 mouse monoclonal anti-C subunit from Marc Mumby and rabbit anti-A␤ subunit produced against the peptide MAGASELGTGPGK coupled to keyhole limpet hemacyanin with glutaraldehyde. Appropriate secondary antibodies coupled to horseradish peroxidase (Jackson ImmunoResearch) were used as well as Renaissance chemiluminescence reagent (NEN Life Science Products).…”