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.

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A simple, improved procedure for the isolation of the phosphotyrosyl phosphatase activator (PTPA) from rabbit skeletal muscle has been developed. The majority of the protein phosphatase 2A (PP2A) was separated from PTPA at an early stage in the procedure. The procedure yields approximately 1 mg essentially pure PTPA/kg rabbit skeletal muscle; it was also applied to porcine brain and the yeast Saccharomyces cerevisiae. The physico-chemical properties of PTPA obtained from all sources are very similar. The pure rabbit skeletal muscle protein was used to raise polyclonal goat antibodies and to affinity purify these antibodies. Immunological studies revealed the presence of PTPA in all mammalian tissues and cell lines examined with differences in tissue distribution, brain showing the highest concentration. PTPA could only be detected in cytosolic fractions. Using a semi-quantitative immunological assay (Western blot), the in vivo concentration could be estimated to be micromolar, which is in the same range as the PP2A target. The purified Xenopus oocyte PTPA showed only a weak cross reactivity, whereas yeast PTPA was not recognised by the antibody indicating some evolutionary diversity of the protein. In a PTPA-affinity column chromatography, the weak interaction with PP2A was independent of the presence of ATP.Mg, a necessary cofactor in the activation process. Interaction of PTPA with PP2A in a 1:1 ratio induces a low (kcat = 3 min-1) ATPase activity that is inhibited by okadaic acid, ADP and non-hydrolysable ATP analogues.

-EJB 95 0106/1 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 XPR65a and XPR65p. The open reading frames of the a and p 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 XPR65a showed 95 % and 84% identity with human PR65a and PR65p proteins, respectively, whereas the identity of XPR65P 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 PR55a and PR55p, respectively. Analysis of the N-terminal region of XPR55 with the same regions of human PR55a and PR55/3, indicates that the XPR55 is the Xenopus homolog of the human PR55a 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 XPR65P, was predominantly expressed and these XPR65P mRNAs are present at a constant level during oogenesis until late embryogenesis. Expression of the 2.4-kb XPR65a was low until the larval stage, then dramatically increased. In all adult tissues except ovary, the 2.4-kb a-specific mRNA was more abundant than the 2.8-kb p 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.