The transforming proteins of DNA tumor viruses interact with endogenous cellular factors that can play broad roles in the regulation of cell growth. The best-characterized example of an endogenous cell cycle regulator protein that interacts with a DNA virus transforming protein is pRB. However, there are other examples. A very high molecular weight (600 kDa) cellular protein (hereafter termed """"""""p600"""""""") forms a tight protein:protein complex with the E7 transforming factor of human and bovine papillomaviruses (HPV). In preliminary studies, my collaborators and I have shown that p600 plays a generalized role in anchorage independent growth and malignant transformation of many tumor cell types. The broad objective of work proposed here is to understand anchorage dependency and malignant transformation through purification and enzymatic characterization of E7-displacible, p600 binding proteins. I have two specific aims:
Aim 1 is to identify cellular factors that bind to p600 in an E7-dependent fashion. The p600 protein is too large for genetic manipulation or ectopic expression using conventional approaches. In preliminary studies, I have used a """"""""knock in"""""""" strategy to create ES cells that express tandem affinity epitope-tagged p600. In antibody pull down experiments with these cells, I have several p600- binding cellular proteins that are displaced by HPV E7. The study plan for aim 1 describes how I will purify and identify these E7-displacible factors using biochemical protocols and mass spectroscopy.
Aim 2 is to understand how E7 and E7-displacible cellular proteins might regulate p600 transforming functions. In preliminary studies, I have shown that p600 has ubiquitin ligase activity. I hypothesize that the ubuquitin ligase activity of p600 is regulated by interactions with E7 and E7-displacibile cellular factors. To test this hypothesis, I will characterize the ubiquitin ligase domain of p600 and identify E7-specific ubiquitin ligase substrates. In proof-of-concept studies for these two specific aims, I have already identified one p600-binding, E7-displacible protein as PKM2 - a protein kinase that has recently been shown to play a broad role in anabolic growth of tumors. I believe that these experiments will provide new insights into the molecular mechanisms of the malignant phenotype including anoikis and anchorage-dependent growth. In the fullness of time, the work could lead to novel new targets for anti cancer drug development.
p600 is a multifunctional protein that regulates cell motility, anchorage dependency, protein degradation, and apoptosis in response to Ca2+ signals. We believe that biochemical characterization of the p600 complexes would reveal molecular functions of anchorage-dependency, which is a major problem in understanding mechanisms of metastasis. This application would be significant not only for basic sciences, but also for drug discovery. If specific proteins bind to p600 in carcinoma cells to support anchorage-independent growth, these interactions could be potential targets for anti-cancer drugs.
|Nakaya, Takeo; Ishiguro, Kei-Ichiro; Belzil, Camille et al. (2013) p600 Plays Essential Roles in Fetal Development. PLoS One 8:e66269|
|Adelmant, Guillaume; Calkins, Anne S; Garg, Brijesh K et al. (2012) DNA ends alter the molecular composition and localization of Ku multicomponent complexes. Mol Cell Proteomics 11:411-21|