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Molecular insights into the disease-causing mechanisms of human phospholamban mutations Open Access


Other title
phosphorylation by protein kinase A
hereditary mutations in phospholamban
SERCA dysregulation
Type of item
Degree grantor
University of Alberta
Author or creator
Ceholski, Delaine K
Supervisor and department
Young, Howard (Biochemistry)
Examining committee member and department
Holmes, Charles (Biochemistry)
Fliegel, Larry (Biochemistry)
Alexander, Todd (Physiology and Pediatrics)
Rao, Rajini (Physiology, Johns Hopkins School of Medicine)
Department of Biochemistry

Date accepted
Graduation date
Doctor of Philosophy
Degree level
The movement of calcium across sarcoplasmic reticulum (SR) membranes is essential in the contraction-relaxation cycle of muscle. An influx of calcium into the muscle cell from the SR triggers muscle contraction and its removal by the sarco(endo)plasmic reticulum calcium ATPase (SERCA) causes muscle relaxation. Phospholamban (PLN) reversibly regulates SERCA by inhibiting its apparent calcium affinity and inhibition is reversed by phosphorylation of PLN by protein kinase A (PKA). The role of SERCA in heart disease has been underscored by the identification of hereditary mutations in the cytoplasmic domain of PLN that have been linked to heart disease: Arg9-to-Cys (R9C), Arg9-to-Leu (R9L), Arg9-to-His (R9H), and deletion of Arg14 (R14del). This thesis aims to provide functional insight into the disease-causing mechanisms of these mutations. We wanted to examine how can mutations in the cytoplasmic domain of PLN cause such severe phenotypes when it is well documented that most of the inhibitory capacity of PLN comes from the transmembrane domain. Using alanine-scanning followed by more selective mutagenesis of the cytoplasmic domain of PLN, we were able to reveal mechanistic insight into how these hereditary mutations alter regulation of SERCA. R14del PLN resulted in constitutive inhibition of SERCA caused by lack of response to β-adrenergic stimulation. While hydrophobic mutation of Arg9, including R9C and R9L, eliminated both SERCA inhibition and PLN phosphorylation, an aromatic substitution (R9H) selectively disrupted phosphorylation. It has been hypothesized that R9C PLN has an altered oligomeric state, which contributes to its disease phenotype. Using a monomeric variant of PLN, we examined the role of PLN oligomerization in disease-causing and -mimicking mutations. We can conclude that the role of Arg9 in PLN is complex – it is not only important for inhibition of SERCA and efficient phosphorylation of the PLN monomer, it is also critical for PKA recognition in the context of the PLN pentamer. Considering the multifaceted role of Arg9 in SERCA inhibition and PKA-mediated phosphorylation, it is not surprising that Arg9 is a hotspot for disease-associated mutations in PLN.
Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
Citation for previous publication
Ceholski DK, Trieber CA, and Young HS. Hydrophobic imbalance in the cytoplasmic domain of phospholamban is a determinant for lethal dilated cardiomyopathy. Journal of Biological Chemistry. 2012;287:16521-16529.Ceholski DK, Trieber CA, Holmes CFB, and Young HS. Lethal, hereditary mutants of phospholamban elude phosphorylation by protein kinase A. Journal of Biological Chemistry. 2012;287:26596-605.

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