Different oligomeric forms of protein phosphatase 2A activate and inhibit simian virus 40 DNA replication.

Abstract

The ability of simian virus 40 (SV40) large T antigen to Cellular kinases and phosphatases regulate the initiation of simian virus 40 (SV40) DNA replication. Unphosphorylated large T antigen is unable to unwind the viral origin of replication until phosphorylated on threonine 124 by a cyclindependent kinase (24). T antigen purified from mammalian cells is heavily phosphorylated on a number of additional serines and threonines (reviewed in reference 11). Phosphorylation on serines 120 and 123 by a nuclear form of casein kinase I (CKI) blocks T antigen's origin-unwinding activity, apparently by preventing functional interactions between Tantigen hexamers bound to the minimal origin of replication (5,6,21,44). This phosphorylation-mediated inhibition of T-antigen activity can be reversed in vitro by treatment of T antigen with alkaline phosphatase (13,28) or with the isolated catalytic subunit of protein phosphatase 2A (PP2AJ) (42,43).Since the phosphoryl groups on serine 123 (and probably serine 120) of T antigen have a high turnover rate in vivo (31) and T-antigen mutants with alanines instead of serines at these positions are inviable (34), these phosphorylation-dephosphorylation cycles are likely to be physiologically relevant. Two lines of evidence suggest that PP2A is the cellular phosphatase that dephosphorylates serines 120 and 123. First, PP2A was purified from HeLa extracts as a factor required for efficient in vitro SV40 DNA replication (42, 43). Second, addition of okadaic acid but not inhibitor 2 inhibited in vitro SV40 DNA replication (21). Since okadaic acid inhibits both PP2A and protein phosphatase 1, while inhibitor 2 inhibits protein phosphatase 1 but not PP2A, this suggests that PP2A is the sole cellular phosphatase which activates SV40 DNA replication. intracellular serine/threonine protein phosphatases (9). All members of the family appear to share a -36-kDa catalytic C subunit. This PP2Ac polypeptide can be dissociated from PP2A regulatory subunits in vitro but does not appear to exist as a monomer in vivo. Rather, the PP2A holoenzymes isolated to date contain both a catalytic subunit and a -65-kDa regulatory A subunit. These A and C subunits are each encoded by a pair of highly homologous genes (17). In addition, several heterotrimeric PP2A holoenzymes have been purified in a number of laboratories (reviewed in references 29 and 36); these enzymes contain a third B subunit. At least three distinct B subunits of 72, 55, and 54 kDa have been purified to date from skeletal and cardiac muscle and erythrocytes. cDNAs for the 55-and 72-kDa forms (and a 130-kDa splice variant) have been cloned (16,22). Interestingly, small t antigen of SV40 and small and middle T antigens of polyomavirus can also bind to the A-C complex in place of endogenous B subunits, leading to alterations in phosphatase activity (references 4 and 38 and references therein). Disruption of the gene encoding the 55-kDa B subunit homolog in Drosophila melanogaster leads to lethal defects in mitosis and wing development (23,40), ...

Upregulation of miR-27a contributes to the malignant transformation of human bronchial epithelial cells induced by SV40 small T antigen

“…The alteration of the phosphorylation status of signaling molecules has been demonstrated to be the primary action of the ST antigen in cell transformation (Janssens and Goris, 2001; Moreno Sablina and Hahn, 2008). Indeed, PP2A complexes formed by various B subunits confer specificity of substrates dephosphorylation (Ferrigno et al, 1993;Cegielska et al, 1994). Among them, several members of the B56 family have been reported to have important roles in control of PP2A potential tumorsuppressive activity by dephosphorylation of its target oncogene (Arnold and Sears, 2006;Margolis et al, 2006;Grochola et al, 2009;Shouse et al, 2010).…”

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

“…Therefore, a consequence of a drop in A␣ subunit levels is an increase in the concentration of free unbound C and B subunits, which normally do not exist in cells, and a corresponding reduction in the levels of core and holoenzyme. Because the substrate specificity of the monomeric C subunit is markedly different from core and holoenzyme, 35 we anticipate that the phosphorylation state of many PP2A substrates will change dramatically as a result of A␣ subunit reduction. Another consequence is that the unbound B subunits are unable to target the phosphatase to specific subcellular locations 8,10,35 or to mediate interactions with other growth-regulatory proteins (for review see Virshup 20 ).…”

“…Because the substrate specificity of the monomeric C subunit is markedly different from core and holoenzyme, 35 we anticipate that the phosphorylation state of many PP2A substrates will change dramatically as a result of A␣ subunit reduction. Another consequence is that the unbound B subunits are unable to target the phosphatase to specific subcellular locations 8,10,35 or to mediate interactions with other growth-regulatory proteins (for review see Virshup 20 ). For example, PP2A has been proposed to function as a tumor suppressor in the Wnt signaling pathway by binding to the tumor suppressor APC through a regulatory BЈ subunit, resulting in downregulation of ␤-catenin.…”

Functional interaction between a novel protein phosphatase 2A regulatory subunit, PR59, and the retinoblastoma-related p107 protein

“…It is widely believed that the multitude of B subunits plays a role in regulation of phosphatase activity, control of substrate speci®city and targeting to certain cellular compartments of the PP2A core dimer. That substrate speci®city is indeed conferred by the subunit composition of PP2A is demonstrated by the ®nding that PR72-containing PP2A holo-enzyme puri®ed from rabbit skeletal muscle in vitro preferentially dephosphorylates SV40 large T antigen phosphorylated by Casein Kinase I, whereas the PP2A core dimer, or PR55 containing holo-enzyme, preferentially dephosphorylates SV40 large T antigen on a site phosphorylated by recombinant cdc2 kinase (Cegielska et al, 1994). The diversity of activities of the PP2A holoenzyme is thus re¯ected in a diversity of possible trimeric PP2A complexes.…”