Synthetic Phosphopeptides Enable Quantitation of the Content and Function of the Four Phosphorylation States of Phospholamban in Cardiac Muscle

Ablorh, Naa Adjeley; Dong, Xiaoqiong; James, Zachary M.; Xiong, Qiang; Zhang, Jianyi; Thomas, David D.; Karim, Christine B.

doi:10.1074/jbc.m114.556621

Cited by 16 publications

(27 citation statements)

References 69 publications

(48 reference statements)

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“…The four phosphorylation states of PLB are U-PLB (unphosphorylated), P16-PLB (phosphorylated at S16), P17-PLB (phosphorylated at T17), and 2P-PLB (phosphorylated at both sites). To quantitate these factors, we prepared purified synthetic standards for all 4 phosphorylation states of human phospholamban, enabling (a) calibration of western blots to account for imperfectly specific antibodies, and (b) control of SERCA2a/PLB stoichiometry and the isolation of each phosphorylation state in SERCA2a activity assays (Ablorh et al 2014). Human PLB sequences were chosen in anticipation of using them as standards to measure PLB phosphorylation in human tissue samples from the Sydney Heart Bank (Li et al 2013).…”

Section: Biochemical Analysismentioning

confidence: 99%

“…The first study (Ablorh et al 2012) measured X P as P16-PLB/Total-PLB, but a second study (Ablorh et al 2014) extended X P measurements to include all four PLB phosphorylation states (Fig. 4).…”

Section: Biochemical Analysismentioning

confidence: 99%

“…4A was used to construct and solve a system of four equations in four unknowns

{bold-italicI}_{normali} = Σ ε_{ij} c_{normalj}, i = 1, \dots, 4,

where I i is the intensity observed for one of the antibodies (row in Fig. 4A, j is the PLB phosphorylation state, ε ij is the slope of the standard curve (from synthetic standards) for phosphorylation state j blotted with antibody i, and c j is the unknown concentration of phosphorylation state j, determined by solving the system of equations (Ablorh et al 2014). These c j values are the concentrations of U-PLB, P16-PLB, P17-PLB, and 2P-PLB.…”

Section: Biochemical Analysismentioning

confidence: 99%

“…These c j values are the concentrations of U-PLB, P16-PLB, P17-PLB, and 2P-PLB. T-PLB was calculated as the sum of the 4 concentrations, and the mole fractions of each phosphorylated state (X P ) were then calculated from the ratio of the concentration of each phosphorylation state to T-PLB (Ablorh et al 2014). This method produces absolute and reproducible numbers, accounting for the imperfect specificities of commercial antibodies.…”

Section: Biochemical Analysismentioning

confidence: 99%

“…4B), a calculation made possible by including X 2P in our measurements where other groups had not. (Ablorh et al 2014). Quantitation of PLB phosphorylation states in both non-HF and HF hearts from the Sydney Heart Bank (Li et al 2013) are currently underway.…”

Section: Biochemical Analysismentioning

confidence: 99%

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Phospholamban phosphorylation, mutation, and structural dynamics: a biophysical approach to understanding and treating cardiomyopathy

Ablorh

Thomas

2015

Biophys Rev

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We review the recent development of novel biochemical and spectroscopic methods to determine the site-specific phosphorylation, expression, mutation, and structural dynamics of phospholamban (PLB), in relation to its function (inhibition of the cardiac calcium pump, SERCA2a), with specific focus on cardiac physiology, pathology, and therapy. In the cardiomyocyte, SERCA2a actively transports Ca2+ into the sarcoplasmic reticulum (SR) during relaxation (diastole) to create the concentration gradient that drives the passive efflux of Ca2+ required for cardiac contraction (systole). Unphosphorylated PLB (U-PLB) inhibits SERCA2a, but phosphorylation at S16 and/or T17 (producing P-PLB) changes the structure of PLB to relieve SERCA2a inhibition. Because insufficient SERCA2a activity is a hallmark of heart failure, SERCA2a activation (by gene therapy (Andino et al. 2008; Fish et al. 2013; Hoshijima et al. 2002; Jessup et al. 2011) or drug therapy (Ferrandi et al. 2013; Huang 2013; Khan et al. 2009; Rocchetti et al. 2008; Zhang et al. 2012)) is a widely sought goal for treatment of heart failure. This review describes rational approaches to this goal. Novel biophysical assays, using site-directed labeling and high-resolution spectroscopy, have been developed to resolve the structural states of SERCA2a-PLB complexes in vitro and in living cells. Novel biochemical assays, using synthetic standards and multidimensional immunofluorescence, have been developed to quantitate PLB expression and phosphorylation states in cells and human tissues. The biochemical and biophysical properties of U-PLB, P-PLB, and mutant PLB will ultimately resolve the mechanisms of loss of inhibition and gain of inhibition to guide therapeutic development. These assays will be powerful tools for investigating human tissue samples from the Sydney Heart Bank, for the purpose of analyzing and diagnosing specific disorders.

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Section: Biochemical Analysismentioning

confidence: 99%

Section: Biochemical Analysismentioning

confidence: 99%

“…4A was used to construct and solve a system of four equations in four unknowns