Two DNA fragments, AP-1 and AP-2, encoding amino acid sequences closely related to Ser/Thr protein phosphatases were amplified from Arabidopsis thaliana genomic DNA. Fragment AP-1 was used to screen A. thaliana cDNA libraries and several positive clones were isolated. Clones EP8a and EP14a were sequenced and found to encode almost identical proteins (97% identity). Both proteins are 306 amino acids in length and are very similar (79-80% identity) to the mammalian isotypes of the catalytic subunit of protein phosphatase 2A. Therefore, they have been designated PP2A-1 and PP2A-2. A third cDNA clone, EP7, was isolated and sequenced. The polypeptide encoded (308 amino acids, lacking the initial Met codon) is 80% identical with human phosphatases 2A and was named PP2A-3. The PP2A-3 protein is extremely similar (95% identity) to the predicted protein from a cDNA clone previously found in Brassica napus. Southern blot analysis of genomic DNA using AP-1 and AP-2 probes, as well as probes derived from clones EP7, EP8a and EP14a strongly indicates that at least 6 genes closely related to type 2A phosphatases are present in the genome of A. thaliana. Northern blot analysis using the same set of probes demonstrates that, at the seedling stage, the mRNA levels for PP2A-1, PP2A-3 and the gene containing the AP-1 sequence are much higher than those of PP2A-2 and AP-2. These results demonstrate that a multiplicity of type 2A phosphatases might be differentially expressed in higher plants.

We have recently reported the existence of multiple isoforms of the catalytic subunit of protein phosphatase 2A (PP2A) in Arabidopsis thaliana and the molecular cloning of cDNAs encoding three of these proteins (PP2A-1, PP2A-2, PP2A-3). The reported cDNA encoding PP2A-3 was truncated at the 5' terminus, lacking a short fragment of the N-terminal coding sequence. We have now isolated a near full-length cDNA encoding the entire PP2A-3 protein (313 residues). The clone includes 188 nucleotides of 5'-untranslated region, where a 44 bp long poly(GA) track is found. We also describe the cloning of a cDNA encoding a fourth isoform of PP2A (PP2A-4). The polypeptide contains 313 residues being 98% identical to PP2A-3 and only 80% identical to both PP2A-1 and PP2A-2. The mRNA for PP2A-4 is 1.4 kb in length and, although predominantly expressed in roots, it is also found in other organs. It is concluded that in A. thaliana the isoforms of PP2A can be grouped in two extremely conserved subfamilies.

Nuclear inhibitor of protein phosphatase-1 (NIPP-1) is one of two major regulatory subunits of protein phosphatase-1 in mammalian nuclei. We report here the cloning and structural characterization of the human NIPP-1 genes, designated PPP1R8P and PPP1R8 in human gene nomenclature. PPP1R8P (1.2 kb) is a processed pseudogene and was localized by in situ hybridization to chromosome 1p33±32. PPP1R8 is an authentic NIPP-1 gene and was localized to chromosome 1p35. PPP1R8 (25.2 kb) is composed of seven exons and encodes four different transcripts, as determined from cDNA library screening, reverse transcriptase-PCR (RT-PCR) and/or EST (expressed sequence tag) database search analysis. NIPP-1a mRNA represents the major transcript in human tissues and various cell lines, and encodes a polypeptide of 351 residues that only differs from the previously cloned calf thymus NIPP-1 by a single residue. The other transcripts, termed NIPP-1b, g and d, are generated by alternative 5 H -splice site usage, by exon skipping and/or by alternative polyadenylation. The NIPP-1b/d and NIPP-1g mRNAs are expected to encode fragments of NIPP-1a that differ from the latter by the absence of the first 142 and 224 residues, respectively. NIPP-1g corresponds to`activator of RNA decay-1' (Ard-1) which, unlike NIPP-1a, displays in vitro and endoribonuclease activity and lacks an RVXF consensus motif for interaction with protein phosphatase-1. While the NIPP-1a/b/d-transcripts were found to be present in various human tissues, the NIPP-1g transcript could only be detected in human transformed B-lymphocytes.Keywords: protein phosphatase-1; NIPP-1; gene structure; splice variants.The Ser/Thr protein phosphatases of type-1 (PP-1) 1 represent a widely distributed and conserved group of enzymes that dephosphorylate key proteins in various cellular processes [1,2]. They consist of a type-1 catalytic subunit (PP-1 C ) and one or two regulatory subunits that modulate the catalytic activity and bring the phosphatase in close proximity to its physiological substrates. In mammalian cells, a major fraction of the cytoplasmic PP-1 C is targeted to glycogen by a G-subunit (PP-1G) and to myosin by an M-subunit (PP-1M). Three PP-1 holoenzymes have been identified in mammalian nuclei, designated PP-1N NIPP-1 , PP-1N R111 and PP-1N sds22 [3,4]. PP-1N sds22 is a rare holoenzyme that contains the mitotic regulator sds22 as a noncatalytic subunit [4]. PP-1N R111 is a heterodimer between the catalytic subunit and an inhibitory polypeptide of 111 kDa [3], that is most likely identical to the recently cloned p99 [5] or PNUTS [6]. PP-1N NIPP-1 is a heterodimer between PP-1 C and the nuclear inhibitor of protein phosphatase-1 (NIPP-1) [3].NIPP-1 is a 38.5-kDa modular protein, with basic N-terminal and C-terminal domains and an acidic central domain [7]. The central domain of NIPP-1 contains an RVXF consensus-motif for interaction with PP-1 C [8,9]. PP-1N NIPP-1 is completely inactive but can be activated by phosphorylation of the central domain of NIPP-1 by protein kinases...

In a recent paper [Ariño et al., Plant Mol Biol 21: 475-485 (1993)] we reported the amplification of a DNA fragment (AP-2) from the genome of Arabidopsis thaliana encoding an amino acid sequence corresponding to a Ser/Thr protein phosphatase distantly related to type 2A protein phosphatases. In this paper we report the use of the AP-2 fragment to isolate several cDNA clones from a leaf cDNA library. Two of these (EP124 and EP129) largely overlap and contain the AP-2 sequence, whereas a third clone (EP128) is different although very related in sequence (86% of identity). Clones EP124/EP129 and EP128 were found to encode two highly related polypeptides (93% identity) of 305 residues, showing a very high identity (83%) to the catalytic subunit of protein phosphatase X (PPX) from rabbit. Therefore, they have been named PPX-1 (EP124/EP129) and PPX-2 (EP128). Southern blot analysis of genomic DNA indicates that only these two genes encoding phosphatases closely related to PPX are present in the genome of A. thaliana. Both PPX-1 and PPX-2 are expressed at very low levels in A. thaliana flowers, leaves, stems and roots. The expression levels of four previously identified type 2A phosphatases are higher than those of PPX genes. PP2A-1 appears to be the major mRNA species detected in all the tissues analyzed.

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