Differential association of regulatory B subunits with a core heterodimer, composed of a catalytic (C) and a structural (A) subunit, is an important mechanism that regulates protein phosphatase 2A (PP2A). We have isolated and characterized three novel cDNAs related to the B` subunit of bovine cardiac PP2A. Two human (B`1 and B`2) and a mouse (B`3) cDNA encode for alternatively spliced variants of the B` subunit. The deduced primary sequences of these clones contain 12 of 15 peptides derived from the purified bovine B` subunit. Differences between the deduced sequences of the B` splice variants and the cardiac peptide sequences suggest the existence of multiple isoforms of the B` subunit. Comparison of the protein and nucleotide sequences of the cloned cDNAs show that all three forms of B` diverge at a common splice site near the 3`-end of the coding regions. Northern blot and reverse transcription-polymerase chain reaction analyses revealed that the B` transcripts (4.3-4.4 kb) are widely expressed and very abundant in heart and skeletal muscle. The expressed human and mouse B` proteins readily associated with the PP2A core enzyme in both in vitro and in vivo complex formation assays. Immunofluorescence microscopy revealed that epitope-tagged B` was localized in both the cytosol and nuclei of transiently transfected cells. The efficiency of binding of all three expressed proteins to a glutathione S-transferase-A subunit fusion protein was greatly enhanced by the addition of the C subunit. Expression of the B` subunits in insect Sf9 cells resulted in formation of ACB` heterotrimers with the endogenous insect A and C subunits. These results show that the B` subunit, which is the predominant regulatory subunit in cardiac PP2A, is a novel protein whose sequence is unrelated to other PP2A regulatory subunits. The nuclear localization of expressed B` suggests that some variants of the B` subunit are involved in the nuclear functions of PP2A.

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