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Formation and regulation of the Notchic transcription complex

Kaplan, Fred M.
http://rave.ohiolink.edu/etdc/view?acc_num=ucin1204685263

Abstract Details

2008, PhD, University of Cincinnati, Medicine : Molecular Genetics, Biochemistry, and Microbiology.
Numerous intricate cellular processes are implemented through direct cell-to-cell interactions. Depending on cell type, the Notch signal transduction pathway initiates a variety of cellular processes including proliferation, differentiation, and apoptosis through these cell-to-cell interactions. For proper development and cellular homeostasis, tight regulation of Notch signaling is essential. Inappropriate Notch signaling is observed in tumors of many tissue types, indicating that deregulation of Notch signaling is involved in initiating or maintaining the neoplastic phenotype. Therefore, understanding the mechanisms responsible for the tight regulation of Notch signaling is critical. In mammals, there are four known Notch genes (Notch1-4) that encode single transmembrane-spanning cell surface receptors. Current models of the Notch signal transduction pathway suggest that the extracellular domain of Notch interacts with the extracellular domain of ligands found on adjacent cells. Ligands from the DSL (Delta, Serrate and Lag-2) family of proteins interact with the Notch receptor and these interactions dictate a series of proteolytic events that release the intracellular domain of Notch (Notchic) from the plasma membrane. Notchic translocates into the nucleus where it interacts with Skip, the DNA binding protein CSL [CBF1, Su(H) and Lag-1]and transcriptional co-activators of the Mastermind-Like family to activate transcription. Although there is a general understanding about the proteolytic events involved in Notch activation, little is known regarding the events involved in active complex assembly. In both naturally occurring tumors and in model systems, cells transformed by Notch contain two distinct nuclear Notchic complexes. However, only one of these two complexes associates with Mastermind and CSL to form an activation complex. In contrast, the other Notchic complex does not appear to be associated with either Mastermind or CSL. Mutations in either Notchic or Mastermind that perturb the formation of the activation complex, but not the formation of the Notchic only complex, also inhibit the function of Notchic. This indicates the importance of the activation complex in Notchic transcriptional activity. However, what role the Notchic complex that does not contain either Mastermind or CSL plays in Notch signaling remains to be determined. One intriguing possibility is that formation of the activation complex is derived from the Notchic that is not associated with either Mastermind or CSL. Here, we report that the activation complex contains a Notchic monomer associated with Mastermind and CSL, while the Notchic complex without Mastermind and CSL consists of Notchic multimers. Furthermore, we demonstrate that the initial step in activation complex assembly is the formation of Notchic multimers and that formation of Notchic multimers is necessary for efficient Notchic transcriptional activity. Subsequently, Notchic multimers form a complex with Skip (ski interacting protein), which then recruits Mastermind to form the pre-activation complex. The interaction between the pre-activation complex and CSL results in the loading of Notchic and Mastermind onto CSL to form the activation complex. We further demonstrate that the Notchic transcription complex is regulated by multiple phosphorylation events. First, we demonstrate that the ankyrin domain of Notchic is phosphorylated by casein kinase 2 (CK2) at two sites. Secondly, we demonstrate that Notchic is hyperphosphorylated in a region between amino acids 2203-2240 as it forms an active transcription complex with Mastermind and CSL. Both of these phosphorylation events negatively regulate the activity of the Notchic transcription complex. The hierarchal phosphorylation event, by CK2, inhibits the formation of the Notchic transcription complex on DNA. How the phosphorylation event between amino acids 2203-2240 negatively regulates the Notchic transcription complex and the kinase(s) involved in this regulation remain unknown. These studies represent an initial characterization of the biochemical mechanisms involved in formation and regulation of the Notchic transcription complex.
Anthony Capobianco, PhD (Committee Chair)
Carolyn Price, PhD (Committee Member)
Joanna Groden, PhD (Committee Member)
Jerry Lingrel, PhD (Committee Member)
Kenji Fukasawa, PhD (Committee Member)
Rhett Kovall, PhD (Committee Member)
137 p.
Biochemistry
Notch; multimer; phosphorylation

Recommended Citations

Citations

  • Kaplan, F. M. (2008). Formation and regulation of the Notchic transcription complex [Doctoral dissertation, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1204685263

    APA Style (7th edition)

  • Kaplan, Fred. Formation and regulation of the Notchic transcription complex. 2008. University of Cincinnati, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin1204685263.

    MLA Style (8th edition)

  • Kaplan, Fred. "Formation and regulation of the Notchic transcription complex." Doctoral dissertation, University of Cincinnati, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1204685263

    Chicago Manual of Style (17th edition)

ucin1204685263
849
© 2008, all rights reserved.
This open access ETD is published by University of Cincinnati and OhioLINK.