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Frnk Regulatory Complex Formation With Fak Is Regulated By Erk Mediated Serine 217 Phosphorylation, Taylor J. Zak

Dissertations

Focal adhesion kinase related non-kinase (FRNK) is an endogenous inhibitor of focal adhesion kinase (FAK) that has traditionally been used to inhibit FAK signaling in a variety of experiments and is also an important endogenous regulator of FAK signaling. More recently, FRNK has been shown to be of increasing importance in some pathologic conditions. Despite the increasing importance of FRNK, the molecular mechanism by which it functions remains unclear. In addition, FRNK contains several phosphorylation sites with unknown importance and function. Here I hypothesize that FRNK can inhibit FAK by binding directly to FAK within focal adhesions. Furthermore, I propose …

Cardiac Calcium Atpase Dimerization Measured By Fluorescence Resonance Energy Transfer And Chemical Cross-Linking, Daniel Blackwell

Dissertations

The cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA) establishes the intracellular calcium gradient across the sarcoplasmic reticulum membrane. It has been proposed that SERCA forms homo-oligomers that increase the catalytic rate of calcium transport. We investigated SERCA oligomerization in rabbit left ventricular myocytes using a photoactivatable cross-linker. Western blotting of cross-linked SERCA revealed higher molecular weight species consistent with SERCA oligomerization. Fluorescence resonance energy transfer (FRET) measurements in cells transiently transfected with fluorescently-labeled SERCA2a revealed that SERCA readily forms homo-dimers. These dimers formed in the absence or presence of the SERCA regulatory partner, phospholamban (PLB) and were unaltered by PLB phosphorylation …

L30a Mutation Of Phospholemman Mimics Effects Of Cardiac Glycosides On Isolated Cardiomyocytes, Ryan David Himes

Dissertations

In order to determine if mutations made to phospholemman (PLM) could increase PLM binding to the Na/K-ATPase (NKA) and cause positive inotropy we performed scanning mutagenesis of the transmembrane domain of PLM and measured FRET between each mutant and NKA. We observed increased binding to NKA for several PLM mutants compared to WT, including L27A, L30A, and I32A. In isolated cardiomyocytes, overexpression of WT PLM increased the amplitude of the Ca2+ transient compared to GFP control. Ca2+ transient amplitude was further increased by L30A PLM overexpression. L30A mutation also delayed Ca2+ extrusion and increased the duration of the cardiomyocyte contraction. …

The Binding Properties And Functional Consequences Of Ryr2-Cam Interaction, Yi Yang

Dissertations

The aim of my dissertation is to understand the regulation of RyR2. The whole dissertation is composed of two parts. The first part focused on RyR2-CaM interaction. The second focused on synthetic RyR2 domain peptide (DPc10), which worked as a powerful molecular tool for RyR2 functional and structural studies.

CaM has been long identified as an important cardiac RyR regulator. Broad studies suggest CaM is a critical RyR2 stabilizer and CaM-RyR2 interaction is a critical molecular substrate for arrhythmias and HF pathogenesis, but the in situ binding properties for CaM-RyR2 are still unknown. Here we, Using FRET detection and permeabilized …

An Investigation Of The Phospholamban-Serca Regulatory Interaction With Fluorescence Resonance Energy Transfer, Philip Adam Bidwell

Dissertations

With Fluorescence Resonance Energy Transfer (FRET), we are able to detect changes in the structure and affinity of the PLB-SERCA regulatory complex in live cells. Using this approach, we have detected a high level of PLB-SERCA interaction even at Ca2+ concentrations known to fully relieve PLB inhibition of SERCA, suggesting that dissociation is not required for relief of inhibition. We also detect no real-time change in PLB-SERCA binding over the course of a single Ca2+ transient in paced myocytes. The effect of Ca2+ on the PLB-SERCA interaction is best described as a reduced affinity with no change in the structure …