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. 2013 Feb 15;112(4):633-9.
doi: 10.1161/CIRCRESAHA.112.275602. Epub 2012 Dec 31.

GSK3β phosphorylates newly identified site in the proline-alanine-rich region of cardiac myosin-binding protein C and alters cross-bridge cycling kinetics in human: short communication

Diederik W D Kuster  1 , Vasco Sequeira, Aref Najafi, Nicky M Boontje, Paul J M Wijnker, E Rosalie Witjas-Paalberends, Steven B Marston, Cristobal G Dos Remedios, Lucie Carrier, Jeroen A A Demmers, Charles Redwood, Sakthivel Sadayappan, Jolanda van der Velden
Affiliations

GSK3β phosphorylates newly identified site in the proline-alanine-rich region of cardiac myosin-binding protein C and alters cross-bridge cycling kinetics in human: short communication

Diederik W D Kuster et al. Circ Res. .

Abstract

Rationale: Cardiac myosin-binding protein C (cMyBP-C) regulates cross-bridge cycling kinetics and, thereby, fine-tunes the rate of cardiac muscle contraction and relaxation. Its effects on cardiac kinetics are modified by phosphorylation. Three phosphorylation sites (Ser275, Ser284, and Ser304) have been identified in vivo, all located in the cardiac-specific M-domain of cMyBP-C. However, recent work has shown that up to 4 phosphate groups are present in human cMyBP-C.

Objective: To identify and characterize additional phosphorylation sites in human cMyBP-C.

Methods and results: Cardiac MyBP-C was semipurified from human heart tissue. Tandem mass spectrometry analysis identified a novel phosphorylation site on serine 133 in the proline-alanine-rich linker sequence between the C0 and C1 domains of cMyBP-C. Unlike the known sites, Ser133 was not a target of protein kinase A. In silico kinase prediction revealed glycogen synthase kinase 3β (GSK3β) as the most likely kinase to phosphorylate Ser133. In vitro incubation of the C0C2 fragment of cMyBP-C with GSK3β showed phosphorylation on Ser133. In addition, GSK3β phosphorylated Ser304, although the degree of phosphorylation was less compared with protein kinase A-induced phosphorylation at Ser304. GSK3β treatment of single membrane-permeabilized human cardiomyocytes significantly enhanced the maximal rate of tension redevelopment.

Conclusions: GSK3β phosphorylates cMyBP-C on a novel site, which is positioned in the proline-alanine-rich region and increases kinetics of force development, suggesting a noncanonical role for GSK3β at the sarcomere level. Phosphorylation of Ser133 in the linker domain of cMyBP-C may be a novel mechanism to regulate sarcomere kinetics.

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Figures

Figure 1
Figure 1. Identified phosphorylated peptides from cMyBP-C
A. Schematic domain structure of cMyBP-C. Cardiac MyBP-C consists of 8 Ig (rounded rectangle) and 3 fibronectin (diamond) domains labeled C0 (N-terminus) through C10 (C-terminus). Two additional domains are present in the N-terminal part of the protein, the Proline-Alanine rich region (PA) and the M-domain (M). Four previously described phosphorylation sites, Ser275, Ser284, Ser304 and Ser311 (indicated by grey diamonds), are located in the M-domain. Cardiac MyBP-C was semi-purified from donor and failing human myocardium. After trypsin digestion, peptides were scanned by tandem mass spectrometry for phosphorylated peptides. B. Peptide coverage (bold with gray background) of cMyBP-C semi-purified from donor myocardium. 58% of the total amino acid sequence was covered by the peptides identified by the MS/MS analysis. Modified amino acids are indicated in dark gray. C. Fragmentation spectrum with b and y ions indicated of peptide spanning from amino acid 282–300 showed a phosphorylated serine at position 284 in cMyBP-C from donor myocardium. D. Fragmentation spectrum of peptide spanning from amino acids 100–135 revealed a novel phosphorylation site on serine 133 in cMyBP-C semi-purified from donor heart tissue. Serine 133 is located in the PA region (indicated by black diamond in Figure 1A).
Figure 2
Figure 2. Site specific phosphorylation of cMyBP-C in donor and end-stage failing hearts
Immunoblot analysis of tissue homogenates from donor (n=5), and end-stage failing hearts from patients with idiopathic (IDCM; n=6) or ischemic (ISHD; n=6) cardiomyopathy revealed lower phosphorylation of Ser133 in failing compared to donor hearts. Phosphorylation of previously identified sites, Ser275, Ser284 and Ser304, was also lower. *P<0.01 IDCM and ISHD versus donor in 1-way ANOVA followed by Bonferroni post-hoc test; #P<0.05 ISHD versus IDCM.
Figure 3
Figure 3. Ser133 is phosphorylated by GSK3β
Human recombinant C0C2 fragment was incubated with either PKA or GSK3β for 2 hours at 37°C. Phosphorylation at serines 275, 284, 304 and 133 was determined with phospho-specific antibodies. A. PKA phosphorylated Ser275, 284 and 304, but not Ser133. B. GSK3β phosphorylated Ser304 and 133. C. To directly compare the relative capability of PKA and GSK3β to phosphorylate Ser133 and Ser304, human recombinant C0C2 was incubated without kinase, with PKA (10 pmol/min/µg) or with GSK3β (168 pmol/min/µg) and loaded onto the same gel in two different amounts followed by immunoblotting with site-specific antibodies. Ser133 was only phosphorylated by GSK3β while Ser304 was predominately phosphorylated by PKA. D. Recombinant 40kDa cMyBP-C (amino acids 1–271 from the human sequence) was incubated with a rough cytosolic fraction isolated from donor heart tissue with (n=3) or without (n=3) 2µM CT99021 (GSK3β antagonist) to determine whether endogenous GSK3β is responsible for Ser133 phosphorylation. PSer133 signal was normalized to total cMyBP-C (with antibody against C0 domain) and 40kDa + soluble fraction was set at 1. CT99021 significantly decreased PSer133 phosphorylation. *P<0.05, with versus without CT99021 in unpaired Student’s t-test. E. GSK3β protein levels were not changed in the end-stage failing hearts compared with donor hearts. F. Phosphorylation of another GSK3β target protein β-catenin was studied by Phos-tag analysis. Reversible interaction between phosphorylated proteins and Phos-tag reagent slow down migration of phosphorylated proteins through the gel. End-stage failing hearts showed a greater proportion of unphosphorylated β-catenin compared with donor hearts suggesting a reduced GSK3β activity. *P<0.01 IDCM and ISHD versus donor in 1-way ANOVA followed by Bonferroni post-hoc test.
Figure 4
Figure 4. Effect of GSK3β on the rate of force redevelopment
A. Incubation of skinned human left ventricular tissue from end-stage failing hearts with GSK3β (n=3) led to an increase of Ser133 phosphorylation while Ser304 phosphorylation was unchanged. B The rate of force redevelopment was determined at maximal calcium activation (max ktr) in Triton-permeabilized single cardiomyocytes isolated from left ventricular myocardium from end-stage heart failure patients (4 hearts; 11 cells). GSK3β significantly increased max Ktr, while upon control treatments (in the absence of GSK3β) no effect was found. *P<0.05, before versus after GSK3β in paired Student’s t-test.
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