Date

January 2008

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Biochemistry and Molecular Biology

Institution

Oregon Health & Science University

Abstract

p63 is a member of the p53 tumor suppressor family that is critical for epithelial differentiation and cancer progression. Unlike p53, p63 is not a classical tumor suppressor because of the various roles of its isoforms. The balance of these isoforms is critical to cell homeostasis and perturbation of this balance is a hallmark of cancer. Currently, the molecular mechanisms regulating p63’s function remain largely unclear. Therefore, studies of p63’s signaling pathways and post-translational modifications are important to discerning the mechanisms governing p63’s role and that of the larger p53 family in cellular homeostasis. This study is one of a few which examine a specific protein governing p63’s post-translational modification, identifying SCF?TrCP1 as an E3 ubiquitin ligase for p63. My study begins with the striking observation that ?TrCP1 raises the steady-state levels of endogenous TAp63?, but not TAp63?. These data are corroborated by studies demonstrating that the protein half-life of TAp63? increases with co-expression of ?TrCP1 endogenously and exogenously. Then, I determined that this increased stabilization is in fact due to direct interaction of SCF?TrCP1 with p63, as ?TrCP1 binds the TAp63?, ?Np63?, TAp63?, and ?Np63? isoforms exogenously with a higher affinity for the TAp63? isoform versus the ?Np63? isoform. Further, this interaction occurs endogenously, as ?TrCP1 binds TAp63? and ?Np63?, but not TAp63? in keratinocytes. Further, ?TrCP1 interacts with TAp63? through regions on TAp63?’s N- and C- termini which may allow for differential regulation of the various isoforms according to our protein binding assays. Then, to study the functional outcome of this direct effect of ?TrCP1 which increased p63’s stabilization, I performed several assays on downstream promoter function and also cell cycle regulation. For these functional assays, I chose to focus on TAp63?, since it is the most transcriptionally active p63 isoform. I found that the stabilization of TAp63? leads to upregulation of p21 at the mRNA and protein level that is associated with an enrichment of TAp63? at the p21 promoter at both canonical and a novel p63 binding sites. This overall increase of p21 causes an increase in G1/S phase cell cycle arrest. Next, since SCF?TrCP1 is an E3 ubiquitin ligase, I characterized the ubiquitylation of p63. My data showed that the TAp63 isoforms had a higher degree of global ubiquitylation than the ?Np63 isoforms. Further, SCF?TrCP1 ubiquitylated TAp63? in vitro and in cells. Using in vitro ubiquitylation assays, I found that ubiquitylation of p63 occurred on p63’s N-terminus region, and extended from TAp63? through a K48 poly-ubiquitin linkage. This ubiquitylation was lost in the presence of ?F?TrCP1, a mutant which can bind, but not ubiquitylate p63. In fact the stabilization and activation of TAp63? by ?TrCP1 is due to ubiquitylation, since TAp63? was not stabilized, could not upregulate p21, and had significantly decreased binding at an established p21 promoter site and a novel-p63 binding region when co-expressed with ?F?TrCP1. Also supporting the effect of ubiquitylation on the stability of TAp63? are data showing that two point mutants of a putative canonical ?TrCP1 interaction motif in the N-terminal domain, while not displaying any difference in binding, displayed differing ubiquitylation and stabilization patterns compared with TAp63?. Therefore, my study reveals SCF?TrCP1 as an E3 ligase which activates TAp63? through ubiquitylation, providing a new mechanism for differential activation of p63’s isoforms in development and cancer.

Identifier

doi:10.6083/M4513W4D

School

School of Medicine

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