The amino acid sequence of a novel mammalian protein phosphatase, termed PPX (and designated PPP4 in the human genome nomenclature), has been deduced from the cDNA and shown to be 65% identical to PP2A alpha and PP2A beta and 45% identical to PPI isoforms, the predicted molecular mass being 35 kDa. PPX was expressed in the baculovirus system. Its substrate specificity and sensitivity to the inhibitors, okadaic acid and microcystin, were similar (but not identical) to the catalytic subunit of PP2A. However, PPX did not bind the 65 kDa regulatory subunit of PP2A. The intracellular localization of PPX was investigated by immunofluorescence using two different antibodies raised against bacterially expressed PPX and a PPX‐specific peptide. These showed that although PPX was distributed throughout the cytoplasm and the nucleus, intense staining occurred at centrosomes. The centrosomal staining was apparent in interphase and at all stages of mitosis, except telophase. In contrast, antibodies directed against bacterially expressed PP2A were not specifically localized to centrosomes. The human autoantibody #5051, which stains the pericentriolar material, colocalizes with PPX antibodies, suggesting that PPX may play a role in microtubule nucleation.

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 ...

Five protein serine,'threonine phosphatases (PP) have been identified by cloning cDNA from mammalian and Drosophila libraries. These novel enzymes. which have not yet been detected by the techniques of protein chemistry and enzymology.are termed PPV. PP2Bw, PPX, PPY and PPZ. The complete amino acid sequences of PPX, PPY and PPZ and an almost complete sequence of PPV are presented. In the catalytic domain PPV and PPX are more similar to PPZA (57-69~ identity) than PPI (45 49% identity), while PPY and PPZ are more similar to PPI (6&68% identity) than PP2A (44% identity). The cDNA for PPZB, encodes a novel CaZ+ /calmodulin-dependent protein phosphatase only 62% identical to PP2B in the catalytic domain. Approaches for determining the cellular functions of these protein phosphatases are discussed.

We have used phage display to isolate a range of human domain antibodies (dAbs) that bind to mouse, rat and/or human serum albumin (SA) and can be expressed at very high levels in bacterial, yeast or mammalian cell culture. In contrast to non-SA-binding dAbs, which have terminal half-lives of less than 45 min, the half-lives of these 12 kDa 'AlbudAbs' can match the half-life of SA itself. To demonstrate the use of AlbudAbs for extending the half-lives of therapeutic drugs, we created a fusion of the interleukin-1 receptor antagonist (IL-1ra) with an AlbudAb. Soluble IL-1ra is potent inhibitor of IL-1 signalling that is approved for the treatment of rheumatoid arthritis but has a relatively short in vivo half-life. Here we show that although the AlbudAb/IL-1ra fusion has a similar in vitro potency, its in vivo efficacy can be dramatically improved due to its extended serum half-life. AlbudAbs could potentially be used to generate a range of long half-life versions of many different drugs in order to improve their dosing regimen and/or clinical effect.