Restricted Protein Phosphatase 2A Targeting by Merkel Cell Polyomavirus Small T Antigen

Abstract

Merkel cell polyomavirus (MCV) is a newly discovered human cancer virus encoding a small T (sT) oncoprotein. We performed MCV sT FLAG-affinity purification followed by mass spectroscopy (MS) analysis, which identified several protein phosphatases (PP), including PP2A A and C subunits and PP4C, as potential cellular interacting proteins. PP2A targeting is critical for the transforming properties of nonhuman polyomaviruses, such as simian virus 40 (SV40), but is not required for MCV sT-induced rodent cell transformation. We compared similarities and differences in PP2A binding between MCV and SV40 sT. While SV40 sT coimmunopurified with subunits PP2A A␣ and PP2A C, MCV sT coimmunopurified with PP2A A␣, PP2A A␤, and PP2A C. Scanning alanine mutagenesis at 29 sites across the MCV sT protein revealed that PP2A-binding domains lie on the opposite molecular surface from a previously described large T stabilization domain (LSD) loop that binds E3 ligases, such as Fbw7. MCV sT-PP2A interactions can be functionally distinguished by mutagenesis from MCV sT LSD-dependent 4E-BP1 hyperphosphorylation and viral DNA replication enhancement. MCV sT has a restricted range for PP2A B subunit substitution, inhibiting only the assembly of B56␣ into the phosphatase holoenzyme. In contrast, SV40 sT inhibits the assembly of B55␣, B56␣ and B56 into PP2A. We conclude that MCV sT is required for Merkel cell carcinoma growth, but its in vitro transforming activity depends on LSD interactions rather than PP2A targeting. A pproximately 30 widely expressed serine/threonine (Ser/Thr) protein phosphatases (PPases) have been identified (1, 2). Protein phosphatase 2A (PP2A) is a combinatorial family of Ser/Thr phosphatases that are highly conserved in eukaryotes. The PP2A core enzyme structure is comprised of a 36-kDa catalytic C subunit (PP2A C) bound to a 65-kDa A regulatory subunit (PP2A A). This core heterodimer can be purified, but the PP2A holoenzyme generally contains a third, regulatory B subunit that provides substrate specificity and subcellular localization (3-7). Among the many combinatorial possibilities, there are two C catalytic subunits, at least two 〈 scaffolding subunits, and multiple B substrate recognition subunits (8), which allow for potentially hundreds of different phosphatase specificities and activities. Phosphatidylinositol 3-kinase (PI3K)/Akt, mitogen-activated protein kinase, c-Myc, and other cancer signaling pathways that impact cellular processes, including cell cycle progression and cellular tumorigenesis are regulated by specific PP2A isoforms (9). Aberrant expression and mutations in PP2A subunits have been implicated in human cancers (8,(10)(11)(12)(13)(14)(15)(16). IMPORTANCE Merkel cell polyomavirus is a newly discovered human cancer virus that promotes cancer, in part, through expression of itsPP2A activity is regulated in part by posttranslational modifications of subunits (17-23). Methylation of PP2A C at Leu309 activates the holoenzyme to allow binding of the B subunit (17), while phosphorylation o...

Emerging differential roles of the pRb tumor suppressor in trichodysplasia spinulosa-associated polyomavirus and Merkel cell polyomavirus pathogeneses

“…This negative finding was affirmed by our present study, in which MCPyV sT did not affect the phosphorylation status of pRb. Additionally, a report by Kwun et al confirms the limited MCPyV sT-PP2A binding capacity and suggests that the oncogenic capabilities of MCPyV sT may be attributed to its interactions with ubiquitin E3 ligases rather than PP2A [14]. Thus, the oncogenic action of MCPyV sT does not appear to rely on PP2A or its substrates [4,14].…”

Protein-mediated viral latency is a novel mechanism for Merkel cell polyomavirus persistence

“…MCV sT is generated from alternative splicing of the T antigen encoding gene to form an iron-sulfur protein with an unclear function for the virus (20,42). Similar to other polyomavirus sT proteins, MCV sT binds and inactivates some PP2A isoforms (43); however, it is transforming in rodent cells (44) and induces cell proliferation and tumorigenicity in transgenic mice (45)(46)(47). Its transforming activity has been mapped to a domain called the LT stabilization domain (LSD) that is separate from its PP2A-binding motif, and the LSD promiscuously binds SCF and anaphase-promoting complex E3 ligases (22,48).…”

Cutaneous Human Polyomavirus Small T Antigens and 4E-BP1 Targeting

“…The pathobiological effects and mechanisms of polyomavirus sT antigens are complex and dependent on the characteristics of different viruses as well as the specific tissue/cell context [7,8,18]. Computer-assisted genomic analysis of MCPyV, TSPyV, HPyV6, and HPyV7 demonstrated that all four sT antigens share an N-terminal DnaJ domain followed by protein phosphatase 2A-binding sequences and zinc-finger motifs [19,20].…”

“…Notably, polyomavirus small T (sT) antigens are strongly associated with cell proliferation pathways [6,7,8,9]. For example, in a previous report, we showed that TSPyV sT antigen activates the MEK-ERK-c-Jun pathway [7].…”