Defense Date

2008

Document Type

Thesis

Degree Name

Master of Science

Department

Biology

First Advisor

Robert Tombes

Abstract

Transient intracellular elevations of Ca2+ are common signaling mechanisms used to allosterically regulate proteins. One potential target of Ca2+ is Ca2+/calmodulin dependent protein kinase type II (CaMK-II). CaMK-II is a multi-functional protein kinase known to influence cellular pathways such as cell motility and cell cycle progression. Within the cell cycle, CaMK-II promotes the expression of the regulator protein cyclin D1, which is necessary for cell cycle progression. To further understand CaMK-II’s role in cyclin D1 expression, the binding partners of cytosolic CaMK-II were studied using mass spectrometry. Several proteins were identified including β actin, β tubulins, tropomodulin- 3 and Flightless-I. Flightless-I was of the most interest because of its role as a transcriptional co-activator of β-catenin-dependent genes such as cyclin D1 and its ability to translocate out of the nucleus following serum stimulation or CaMK-II activation. For this study, I sought to determine whether the interaction between CaMK-II and Flightless-I mediates transcription of cyclin D1. First, Flightless-I was linked to green fluorescent protein (GFP) and live cell imaging was performed. Under serum stimulation or constitutive CaMK-II expression, GFP-Flightless-I was cytosolic. Serum starvation or inhibition of CaMK-II expression resulted in the localization of GFP-Flightless-I to the nucleus. Next, a luciferase based reporter gene assay was used to evaluate the effect of Flightless-I and CaMK-II on β-catenin-dependent transcription of cyclin D1. Over-expression of Flightless-I or inhibition of CaMK-II both resulted in decreased β-catenin-dependent transcription; whereas suppression of Flightless-I by siRNA enhanced transcription. Taken together, these results suggest a novel mechanism whereby the interaction between CaMK-II and Flightless-I influences gene transcription necessary for cell cycle progression.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

8-11-2008

Included in

Biology Commons

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