Identification and characterization of B′′‐subunits of protein phosphatase 2 A in Xenopus laevis oocytes and adult tissues

Abstract

Protein phosphatase 2A is a phosphoserine/threonine phosphatase implicated in many cellular processes. The core enzyme comprises a catalytic and a PR65/A-subunit. The substrate specificity and subcellular localization are determined by a third regulatory B-subunit (PR55/B, PR61/B¢ and PR72/130/B¢¢). To identify the proteins of the B¢¢ family in Xenopus laevis oocytes, a prophase Xenopus oocyte cDNA library was screened using human PR130 cDNA as a probe. Three different classes of cDNAs were isolated. One class is very similar to human PR130 and is probably the Xenopus orthologue of PR130 (XPR130). A second class of clones (XN73) is identical to the N-terminal part of XPR130 but ends a few amino acids downstream of the putative splicing site of PR130. To investigate how this occurs, the genomic structure of the human PR130 gene was determined. This novel protein does not act as a PP2A subunit but might compete with the function of PR130. The third set of clones (XPR70) is very similar to human PR48 but has an N-terminal extension. Further analysis of the human EST-database and the human PR48 gene structure, revealed that the human PR48 clone published is incomplete. The Xenopus orthologue of PR48 encodes a protein of 70 kDa which like the XPR130, interacts with the A-subunit in GST pull-down assays. XPR70 is ubiquitously expressed in adult tissues and oocytes whereas expression of XPR130 is very low in brain and oocytes. Expression of XN73 mainly parallels XPR130 with the exception of the brain.Keywords: phosphatase; PP2A; Xenopus laevis; cell cycle.Reversible protein phosphorylation is a key mechanism that regulates a number of different cellular events. The phosphorylation state of a protein is dependent on the opposing activities of kinases and phosphatases. Among the phosphatases, protein phosphatase 2A is a major serine/threonine protein phosphatase involved in many cellular events, such as signal transduction, DNA replication, transcription, translation, apoptosis and the cell cycle [reviewed in 1,2]. The core enzyme of PP2A is a heterodimer consisting of a catalytic (C) and structural (PR65/A) subunit. Different mechanisms are known to regulate the phosphatase activity.The PR65/A subunit serves as a scaffolding protein that interacts with a third regulatory subunit (B-subunit). There is evidence that PP2A is an obligate trimer in vivo [3,4], although other evidence suggests that a substantial proportion of PP2A can also exist in vivo as a dimer [5]. The association of a B subunit with the AC core dimer can result in altered substrate specificity, catalytic activity and subcellular localization. To date, three different B-subunit families have been described in eukaryotes: PR55/B, PR61/B¢ and PR72/130/B¢¢. Each family of B-subunits harbours different genes, some giving rise to different splice variants. PP2A activity is also subject to regulation by post-translational modifications, such as methylation. Methylation occurs on the carboxyl group of the C-terminal Leu309 by a specific methyltransferase [6,7] a...

Selection of Protein Phosphatase 2A Regulatory Subunits Is Mediated by the C Terminus of the Catalytic Subunit

“…All constructs and mutants were verified by DNA sequencing. The following primary antibodies were used in this study: mouse monoclonal anti-PP2A C and anti-PR65 antibodies as described previously (26), mouse monoclonal anti-methyl-PP2A C antibodies (Upstate), monoclonal anti-HA (clone 12CA5), anti-GST and anti-␣-tubulin antibodies (Sigma), rabbit polyclonal anti-poly(ADP-ribose) polymerase antibodies (Cell Signaling Technology), and anti-PR55␣ antibodies (Calbiochem for Western blotting, Upstate for immunoprecipitations). The polyclonal rabbit anti-LCMT1 and anti-peptide antibodies against the C terminus of PP2A C (amino acids 299 -309), specific for demethylated PP2A C , were described previously (12).…”

“…Surprisingly PR61␦ 1 , PR72, and PR70, another member of the PR72/BЉ family (26), could still associate with this PP2A C mutant (Fig. 3C), suggesting that these B subunits do not require any contacts with the last six C-terminal residues of PP2A C .…”

Protein Phosphatase 2A Is Targeted to Cell Division Control Protein 6 by a Calcium-binding Regulatory Subunit

“…Cloning of Full-length PR70-A human expressed sequence tag encoding the PR70 start codon was identified in the human expressed sequence tag data base using the MegaBLAST tool (www.ncbi.nlm.nih.gov/BLAST/) with the assembled PR70 sequence (21). A PR70 cDNA was constructed using the PR48 cDNA and the IMAGE Human Clone ID 5728169 (GenBank TM accession number BM544432), purchased from Invitrogen, using an internal NcoI restriction site present in the common region of BM54432 and PR48.…”

Identification and Functional Analysis of Two Ca2+-binding EF-hand Motifs in the B“/PR72 Subunit of Protein Phosphatase 2A

“…Five different BЉ isoforms have been identified in mammals: human PR72 and PR130 (the founding members of the family) (15), mouse PR59 (16), human PR48 (17), and the recently identified human G5PR (18). Although BЉ homologues have been described in plants (19), Xenopus laevis oocytes (20), Drosophila melanogaster (4,5) and Caenorhabditis elegans (protein CO6G1.5, GenBank accession number AAK32946), they are manifestly absent in yeast. PR72 and PR130 are splice variants generated from a single gene and share an identical C terminus.…”

“…Cdc6 is an ATPase required for the initiation of DNA replication that primarily acts by recruiting the minichromosome maintenance complex to origins of replication (22). Recent results indicate, however, that PR48 is a partial clone that would better be renamed as "PR70," fitting with its real molecular mass (20). G5PR was identified as a germinal center-associated nuclear protein (GANP)-associated molecule by yeast two-hybrid screening (18).…”