Comparisons of the Polyomavirus Small T Antigens Demonstrate Novel Regulatory Pathways in Apoptosis and DIfferentiation.
Polyomaviruses have provided many insights into control of cell physiology. Studies of
their tumor antigens (Tags) have led to appreciation of the role of tyrosine
phosphorylation, PI3K and p53 in oncogenic transformation. This work explores signal
pathways regulated by polyomavirus small T antigen (PyST) that control differentiation
and regulate cell survival in fat, muscle ... read moreand bone models. Comparisons of murine
polyomavirus ST (PyST) to monkey polyomavirus SV40 (SV40 ST) have been especially useful
in parsing out the mechanisms involved. This work also makes use of PyST mutants
defective in specific interactions. Of the many PyST functions, we particularly
illustrate the importance of phosphatase 2A (PP2A) for PyST to regulate differentiation.
PP2A regulates almost all cell signaling pathways. The holoenzyme consists of a
catalytic C subunit and one of many regulatory B subunits bound to an A scaffolding
subunit. Of more than 80 PP2A isoforms, 10% use Abeta as a scaffold. Interaction with
PP2A plays a substantial role in known small T antigen functions. This work further
demonstrates the requirement of PP2A binding for PyST inhibition of differentiation in
muscle and fat as well as induction of apoptosis. Unlike PyST, SV40ST did not block
differentiation or induce apoptosis. This difference in ST biology can be explained by
the subset of PP2A A scaffolds the STs were targeting. Although both PyST and SV40ST
replace the B subunit of PP2A, PyST targets Abeta associated enzymes, while SV40ST only
bound A. Abeta proved to play a role in differentiation and apoptosis, as an
Abeta binding mutant SV40ST, 130AA, showed PyST like inhibition of differentiation. In
the absence of ST, significant knockdown of Abeta induced cell death, while a moderate
knockdown enhanced differentiation. These results point to the importance ofAbeta in
non-viral contexts. The protein kinase Akt is perhaps the principle effector of PI3
kinase signaling. Akt is an important signal regulator in differentiation. Blocking Akt
activity inhibited differentiation, while activating Akt promoted it. Small Ts regulate
differentiation through Akt. PyST inhibited Akt and its effect on differentiation was
reversed by the expression of activated Akt. SV40 ST activated Akt and promoted myoblast
differentiation; Akt inhibitor could block SV40ST activation of myoblast differentiation
in a dose dependent manner. Mechanistically, PyST likely inhibited Akt activity through
PP2A Abeta. PyST increased turnover of Akt S473 phosphate and enhanced PP2A Abeta/Akt
interaction. PP2A uses Abeta to negatively regulate Akt even in the absence of PyST.
Abeta complexed with Akt1 and Akt2 in immunoprecipitation experiments. During
differentiation, down-regulatedAbeta coincides with elevated Akt S473 phosphorylation.
Additionally, knockdown of Abeta promoted Akt activity and reduced turnover of phosphate
at Akt S473. These results imply that at least one role of Abeta as a tumor suppressor
may lie in its ability to regulate this all-important oncogenic kinase. Besides PP2A,
PyST also binds other putative regulators of differentiation: YAP/TAZ and lipin. While
mutants failing to bind lipin did not alter differentiation effects, PyST mutants
lacking the YAP/TAZ interaction were affected in bone differentiation and in
IBMX-dependent apoptosis. YAP is involved in transformation, apoptosis and cell
survival. It functions both in the cytoplasm and the nucleus. How PyST induces apoptosis
through YAP remains for future studies.
Thesis (Ph.D.)--Tufts University, 2013.
Submitted to the Dept. of Biochemistry.
Advisor: Brian Schaffhausen.
Committee: Alex Toker, Alexei Degterev, Amy Yee, and Andrew Bohm.
Keywords: Biology, and Cellular biology.read less