Protein phosphatase 2A (polycation-stimulated protein phosphatase L) was purified from porcine kidney and skeletal muscle. The 36-kDa catalytic and the 65-kDa putative regulatory (hereafter termed PR65) subunits of protein phosphatase 2A2 were separated by reverse-phase HPLC. Partial amino acid sequence data (300 residues) was obtained for PR65. Molecular cloning showed that two distinct mRNAs (termed alpha and beta) encoded the PR65 subunit. The cDNA encoding the alpha-isotype spanned 2.2 kilobases (kb) and contained an open reading frame of 1767 bases predicting a protein of 65 kDa, which was in good agreement with the size of the purified protein. The cDNAs encoding the beta-isotype contained an open reading frame of size similar to that of alpha-form but lacked an initiator ATG. Northern analysis, using RNA isolated from several human cell lines, indicated that the alpha-isotype was encoded by a mRNA of 2.4 kb that was much more abundant than the beta mRNA of 4.0 kb. Comparison of the predicted amino acid sequences of the two isotypes revealed 87% identity. The deduced protein sequences of the alpha- and beta-isotypes were found to be made up of 15 imperfect repeating units consisting of 39 amino acids. This repeating structure was conserved between species.

cDNA clones encoding the 65-kDa (PR65) and 55-kDa (PR55) regulatory subunits of protein phosphatase 2A from Xenopus laevis were isolated by homology screening with the corresponding human cDNAs, and used to analyze the developmental expression patterns of these genes. The PR65 subunit was found to be encoded by two genes, termed XPR65 alpha and XPR65 beta. The open reading frames of the alpha and beta cDNAs both span 1767 bp, and predict proteins of 64.4 kDa and 65.3 kDa, respectively, that are 87% identical. The predicted amino acid sequence of XPR65 alpha showed 95% and 84% identity with human PR65 alpha and PR65 beta proteins, respectively, whereas the identity of XPR65 beta with the same proteins was 87% and 86.5%, respectively. Only one type of Xenopus PR55 (XPR55) was isolated that showed 93% and 84% similarity to human PR55 alpha and PR55 beta, respectively. Analysis of the N-terminal region of XPR55 with the same regions of human PR55 alpha and PR55 beta, indicates that the XPR55 is the Xenopus homolog of the human PR55 alpha isoform. Despite the overall similarity with PR55 from other species, XPR55 has an N-terminal extention of at least 24 amino acids. In the ovary, a transcript of 2.8 kb, encoding the XPR65 beta, was predominantly expressed and these XPR65 beta mRNAs are present at a constant level during oogenesis until late embryogenesis. Expression of the 2.4-kb XPR65 alpha was low until the larval stage, then dramatically increased. In all adult tissues except ovary, the 2.4-kb alpha-specific mRNA was more abundant than the 2.8-kb beta transcript. Two transcripts of 2.4 kb and 2.5 kb, encoding the XPR55 subunit, were detected at a constant level throughout Xenopus oogenesis and during embryogenesis. Both transcripts were also expressed at similar levels in all adult tissues, but in a tissue-specific manner. Analysis of the XPR55 and XPR65 proteins using antibodies to recombinant proteins revealed that the overall levels of the two proteins were constant, in good agreement with mRNA data.