NOVEL INSIGHTS INTO TRANSFORMATION FROM A POLYOMAVIRUS MIDDLE TUMOR ANTIGEN CONDITIONAL EXPRESSION SYSTEM.
Rouleau, Cecile.
2016
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Abstract: The broad
range of tumors caused by murine Polyomavirus has led to the use of polyomaviruses as
model systems of transformation. These models have proven powerful discovery engines,
leading to breakthroughs, including tyrosine phosphorylation, phosphatidylinositol-3
(PI3) kinase, Src regulation, p53 and pRb function, that have advanced, beyond our
understanding of disease, knowledge... read moreof how we are alive. Middle tumor antigen (MT) is
the major viral oncoprotein. MT is always necessary and often sufficient to cause
tumors. It has no enzymatic activity and functions in membranes by engaging host cell
mechanisms. Because of its membrane localization and signaling properties, MT has been
likened to an activated tyrosine kinase receptor. The most significant finding of this
thesis is the identification of Yes-Associated Protein (YAP) as a novel interactor of MT
and contributor to MT transformation. The interaction was detected by proteomic analysis
of the MT complex. YAP is a known effector of the Hippo pathway, which controls organ
size and proliferation, and is reported to function either as a tumor suppressor or as
an oncogene depending on context. The basis for the oncogenic function of YAP is
understood to be its transcriptional activity. We show here that YAP makes a positive
contribution to MT transformation. MT affects YAP both indirectly and directly. YAP is
an indirect target of MT signaling, which stimulates its phosphorylation and
degradation. YAP also directly binds to MT. Binding counteracts the effect of MT
signaling: MT-bound YAP is dephosphorylated and stabilized compared to total cellular
YAP. Surprisingly, MT promotes the localization of YAP to membranes. These results
suggest that non-canonical oncogenic YAP functions exist that are independent of
YAP-directed transcription. Extension of these studies will certainly add to our
understanding of tumorigenesis. Other findings are presented beyond the physical and
functional characterization of the MT-YAP interaction. Our proteomic analysis of the MT
complex identified several novel interactors. These may shed light on MT mutants such as
E349K that have as yet unexplained phenotypes. Some, such as protein phosphatase PP4 and
the lipins, were previously suspected. Others, such as the nucleoporin Pom121 and
components in nucleocytoplasmic trafficking, including RanGAP and RanBP1/2, were
unexpected considering MT is anchored in the plasma membrane. These associations should
be explored further because a direct connection between the nuclear membrane and the
plasma membrane could open a new avenue of research. Other biological questions arose
from our use of a conditional MT expression system, generated to circumvent problems of
constitutive expression. Low-level MT could activate Ras and PI3K/Akt signaling, thought
sufficient for MT transformation, yet failed to transform. This suggested that Ras and
PI3K/Akt, although necessary, may not be sufficient. The new system also revealed a
novel paracrine activity whereby MT-conditioned medium promotes the survival of
non-transformed cells. This pointed to a possible mechanism of microenvironmental
regulation by MT. To understand this activity, analysis of the MT secretome by antibody
array and RNA-Seq was undertaken. MT dramatically up-regulated expression and secretion
of many growth factors and cytokines. A MT mutant defective in PI3K/Akt signaling
displayed paracrine survival activity, focusing attention on molecules not dependent
upon PI3K/Akt signaling. Several possible candidate survival factors emerged, among them
the chemokine CXCL10.
Thesis (Ph.D.)--Tufts University, 2016.
Submitted to the Dept. of Biochemistry.
Advisor: Brian Schaffhausen.
Committee: Alexei Degterev, Philip Hinds, and Larry Feig.
Keyword: Biochemistry.read less - ID:
- dv1406087
- Component ID:
- tufts:20532
- To Cite:
- DCA Citation Guide EndNote
