PR48, a Novel Regulatory Subunit of Protein Phosphatase 2A, Interacts with Cdc6 and Modulates DNA Replication in Human Cells

Abstract

Initiation of DNA replication in eukaryotes is dependent on the activity of protein phosphatase 2A (PP2A), but specific phosphoprotein substrates pertinent to this requirement have not been identified. A novel regulatory subunit of PP2A, termed PR48, was identified by a yeast two-hybrid screen of a human placental cDNA library, using human Cdc6, an essential component of prereplicative complexes, as bait. PR48 binds specifically to an amino-terminal segment of Cdc6 and forms functional holoenzyme complexes with A and C subunits of PP2A. PR48 localizes to the nucleus of mammalian cells, and its forced overexpression perturbs cell cycle progression, causing a G 1 arrest. These results suggest that dephosphorylation of Cdc6 by PP2A, mediated by a specific interaction with PR48, is a regulatory event controlling initiation of DNA replication in mammalian cells.Initiation of chromosomal DNA replication in eukaryotes is a multistep process that requires assembly of prereplicative complexes on chromosomal DNA, followed by unwinding of replication origins and loading of replicative polymerases and accessory proteins that catalyze DNA synthesis (reviewed in references 6, 34, and 35). Stringent control mechanisms, directed by the competing activities of multiple protein kinases and protein phosphatases, ensure that each segment of the genome is replicated once and only once in each cell cycle and that DNA replication is properly timed with respect to cell growth and mitosis.The serine/threonine protein phosphatase 2A (PP2A) exerts regulatory control over the initiation of DNA replication in yeast, viral, and vertebrate systems (19, 20, 33). This relationship was demonstrated first for replication of simian virus 40 (SV40) DNA, where dephosphorylation of specific serine residues by PP2A within large T antigen promotes DNA binding of T-antigen hexamers and unwinding of SV40 replication origins (33). More recently, immunodepletion of PP2A holoenzyme was observed to prevent chromosomal DNA replication in a Xenopus cell-free replication system (20). Neither assembly of prereplicative complexes nor elongation of replication forks is dependent on PP2A, but firing of replication origins is suppressed in the absence of this enzyme. This requirement for PP2A activity in promoting DNA replication presumably is based on direct or indirect modification of the proteins responsible for priming and/or firing replication origins. However, with the exception of SV40 large T antigen, specific phosphoprotein substrates of PP2A that are pertinent to its role in promoting DNA replication have not been identified.Cdc6 proteins are highly conserved in organisms as diverse as Saccharomyces cerevisiae, Xenopus laevis, and Homo sapiens (1,4,8,22,31,41) and exert unique functions at each of several steps of replication initiation. Cdc6 is required for formation of prereplicative complexes and is rate limiting for initiation of DNA replication in all species in which the function of Cdc6 has been examined (4, 9, 18, 36, 42). In yeast, forced overexpr...

Identification of the first PAR1 deletion encompassing upstream SHOX enhancers in a family with idiopathic short stature

“…This gene encodes a subunit of a serine/threonine phosphatase, which exerts regulatory control over DNA replication initiation. 13 The deletion of this gene was thought unlikely to be the cause of the observed short stature and no further analysis was undertaken at this time point.…”

Reduced expression of the A? subunit of protein phosphatase 2A in human gliomas in the absence of mutations in the A? and A? subunit genes

“…22 Importantly, we have shown that in every case the mutation renders the A␣ subunit defective in binding B or C subunits, depending on whether the mutation is located in the N-terminal B subunit binding domain (repeats 1-10) or in the C-terminal C subunit binding region (repeats [11][12][13][14][15] (Fig. 1).…”

“…The A and C subunits both exist as 2 isoforms (␣ and ␤ ), whereas the B subunits fall into 3 families designated B, [2][3][4][5] , BЈ (also called B56) 6 -10 and BЉ. [11][12][13][14] A model demonstrating how the A, B and C subunits interact in the holoenzyme is shown in Figure 1. 15,16 The existence of so many regulatory subunits suggests that the various forms of PP2A have distinct functions.…”

Disruption of protein phosphatase 2A subunit interaction in human cancers with mutations in the Aα subunit gene

“…The B' family consists of numerous isoforms and splice variants whose molecular masses range from 54 ± 68 kDa (Csortos et al, 1996;McCright et al, 1996;McCright and Virshup, 1995;Tanabe et al, 1996;Tehrani et al, 1996). The B'' family has at least four members, designated PR48, PR59, PR72 and PR130 (Hendrix et al, 1993;Nagase et al, 1997;Voorhoeve et al, 1999;Yan et al, 2000). PR72 and PR130 are splice variants of the same gene.…”