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 of h... read moreow 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
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