Date of Completion
2016
Document Type
Honors College Thesis
Department
Biology
Thesis Type
Honors College, College of Arts and Science Honors
First Advisor
Dr. Paula B. Deming
Second Advisor
Dr. Bryan Ballif
Keywords
Protein Kinase A, PKA, src, phosphorylation
Abstract
Abstract: Protein kinase A (PKA) is a cyclic-AMP (cAMP) dependent kinase and is known to regulate many processes, specifically proliferation and migration. PKA activity also plays an important role in the metastasis of ovarian cancer. PKA has been shown to localize to the leading edge of migrating ovarian cancer cells and is required for invasive potential (McKenzie, Campbell et al. 2011). Src family kinases (SFKs) are non-receptor tyrosine kinases that become activated after the stimulation of a variety of plasma membrane receptors. SFKs are proto-oncogenes, that play key roles in signal transduction pathways involved in cell division, motility, adhesion, and survival in both normal and cancer cells. In cancers, SFKs are particularly important in regulating the processes that promote invasion and metastasis. During chronic stress signaling, PKA activates Src through direct phosphorylation (Armaiz-Pena, Allen et al. 2013). The Deming Lab has also shown that active Src family kinases can regulate PKA activity through phosphorylation of the catalytic subunit of PKA at Tyrosine 69. The Deming lab has also made a mutant that cannot be phosphorylated. In the mutant the Tyrosine (Y) has been changed to a Phenylalanine (F) at site 69 (Y69F. Given that phosphorylation at Y69 enhances PKA activity and that PKA and SFKs have been linked in ovarian cancer migration and invasion, I hypothesize that hypothesize that activation of SFK’s induces PKA-C phosphorylation and regulation of downstream PKA signaling. The goal of my research was to investigate this interaction in wild type PKA catalytic subunit (PKA-C) and Y69F-PKA-C using cell culture and other molecular techniques. The second aim of my project was to design a system utilizing siRNA knockdown technology and rescuing with a C-terminally fluorescently tagged exogenous PKA-C so that later experiments can be aimed at charactering the Y69F-PKA-C.
To address the first aim, I investigated the 1) effect of Src inhibition on global PKA activity in response to epidermal growth factor (EGF) stimulation and 2) the formation lamellipodia response to EGF stimulation when wild type PKA-C is overexpressed or when the Y69 mutant is expressed. The results presented here suggest that 1) Src phosphorylates PKA in response to EGF signaling leading to increased activitiy and 2) the phosphorylation of Y69 seems to play an important role in the ability of cells to form lamellipodia. The siRNA system proposed shows optimal knockdown of endogenous PKA-C after 72 hours of exposure. Due to the affinity of the siRNA for both endogenous and exogenous PKA-C, silent point mutations were designed and tested to convey resistance to siRNA degradation. These mutations show promising resistance to siRNA degradation and will be used for later experiments to characterize Y69F-PKA-C.
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.
Recommended Citation
Todero, Jenna E., "Establishing an Ovarian Cancer cell culture model system in order to study the molecular interaction between Src Family Kinases and Protein Kinase A" (2016). UVM Honors College Senior Theses. 210.
https://scholarworks.uvm.edu/hcoltheses/210