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Phosphorylation in protein-protein binding: effect on stability and function.

ABSTRACT: Posttranslational modifications offer a dynamic way to regulate protein activity, subcellular localization, and stability. Here we estimate the effect of phosphorylation on protein binding and function for different types of complexes from human proteome. We find that phosphorylation sites tend to be located on binding interfaces in heterooligomeric and weak transient homooligomeric complexes. Analysis of molecular mechanisms of phosphorylation shows that phosphorylation may modulate the strength of interactions directly on interfaces and that binding hotspots tend to be phosphorylated in heterooligomers. Although the majority of complexes do not show significant estimated stability differences upon phosphorylation or dephosphorylation, for about one-third of all complexes it causes relatively large changes in binding energy. We discuss the cases where phosphorylation mediates the complex formation and regulates the function. We show that phosphorylation sites are more likely to be evolutionary conserved than other interfacial residues.

SUBMITTER: Nishi H

PROVIDER: S-EPMC3240861 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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Phosphorylation in protein-protein binding: effect on stability and function.

Nishi Hafumi H Hashimoto Kosuke K Panchenko Anna R AR

Structure (London, England : 1993) 20111201 12

Posttranslational modifications offer a dynamic way to regulate protein activity, subcellular localization, and stability. Here we estimate the effect of phosphorylation on protein binding and function for different types of complexes from human proteome. We find that phosphorylation sites tend to be located on binding interfaces in heterooligomeric and weak transient homooligomeric complexes. Analysis of molecular mechanisms of phosphorylation shows that phosphorylation may modulate the strengt ...[more]

PMID: 22153503

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Project description:RationaleCardiac myosin-binding protein-C (cMyBP-C) phosphorylation at Ser-273, Ser-282, and Ser-302 regulates myocardial contractility. In vitro and in vivo experiments suggest the nonequivalence of these sites and the potential importance of Ser-282 phosphorylation in modulating the protein's overall phosphorylation and myocardial function.ObjectiveTo determine whether complete cMyBP-C phosphorylation is dependent on Ser-282 phosphorylation and to define its role in myocardial function. We hypothesized that Ser-282 regulates Ser-302 phosphorylation and cardiac function during β-adrenergic stimulation.Methods and resultsUsing recombinant human C1-M-C2 peptides in vitro, we determined that protein kinase A can phosphorylate Ser-273, Ser-282, and Ser-302. Protein kinase C can also phosphorylate Ser-273 and Ser-302. In contrast, Ca(2+)-calmodulin-activated kinase II targets Ser-302 but can also target Ser-282 at nonphysiological calcium concentrations. Strikingly, Ser-302 phosphorylation by Ca(2+)-calmodulin-activated kinase II was abolished by ablating the ability of Ser-282 to be phosphorylated via alanine substitution. To determine the functional roles of the sites in vivo, three transgenic lines, which expressed cMyBP-C containing either Ser-273-Ala-282-Ser-302 (cMyBP-C(SAS)), Ala-273-Asp-282-Ala-302 (cMyBP-C(ADA)), or Asp-273-Ala-282-Asp-302 (cMyBP-C(DAD)), were generated. Mutant protein was completely substituted for endogenous cMyBP-C by breeding each mouse line into a cMyBP-C null (t/t) background. Serine-to-alanine substitutions were used to ablate the abilities of the residues to be phosphorylated, whereas serine-to-aspartate substitutions were used to mimic the charged state conferred by phosphorylation. Compared to control nontransgenic mice, as well as transgenic mice expressing wild-type cMyBP-C, the transgenic cMyBP-C(SAS(t/t)), cMyBP-C(ADA(t/t)), and cMyBP-C(DAD(t/t)) mice showed no increases in morbidity and mortality and partially rescued the cMyBP-C((t/t)) phenotype. The loss of cMyBP-C phosphorylation at Ser-282 led to an altered β-adrenergic response. In vivo hemodynamic studies revealed that contractility was unaffected but that cMyBP-C(SAS(t/t)) hearts showed decreased diastolic function at baseline. However, the normal increases in cardiac function (increased contractility/relaxation) as a result of infusion of β-agonist was significantly decreased in all of the mutants, suggesting that competency for phosphorylation at multiple sites in cMyBP-C is a prerequisite for normal β-adrenergic responsiveness.ConclusionsSer-282 has a unique regulatory role in that its phosphorylation is critical for the subsequent phosphorylation of Ser-302. However, each residue plays a role in regulating the contractile response to β-agonist stimulation.

Project description:BackgroundHeart failure (HF) with preserved ejection fraction (HFpEF) accounts for ≈50% of all cases of HF and currently has no effective treatment. Diastolic dysfunction underlies HFpEF; therefore, elucidation of the mechanisms that mediate relaxation can provide new potential targets for treatment. Cardiac myosin-binding protein-C (cMyBP-C) is a thick filament protein that modulates cross-bridge cycling rates via alterations in its phosphorylation status. Thus, we hypothesize that phosphorylated cMyBP-C accelerates the rate of cross-bridge detachment, thereby enhancing relaxation to mediate diastolic function.Methods and resultsWe compared mouse models expressing phosphorylation-deficient cMyBP-C(S273A/S282A/S302A)-cMyBP-C(t3SA), phosphomimetic cMyBP-C(S273D/S282D/S302D)-cMyBP-C(t3SD), and wild-type-control cMyBP-C(tWT) to elucidate the functional effects of cMyBP-C phosphorylation. Decreased voluntary running distances, increased lung/body weight ratios, and increased brain natriuretic peptide levels in cMyBP-C(t3SA) mice demonstrate that phosphorylation deficiency is associated with signs of HF. Echocardiography (ejection fraction and myocardial relaxation velocity) and pressure/volume measurements (-dP/dtmin, pressure decay time constant τ-Glantz, and passive filling stiffness) show that cMyBP-C phosphorylation enhances myocardial relaxation in cMyBP-C(t3SD) mice, whereas deficient cMyBP-C phosphorylation causes diastolic dysfunction with HFpEF in cMyBP-C(t3SA) mice. Simultaneous force and [Ca(2+)]i measurements on intact papillary muscles show that enhancement of relaxation in cMyBP-C(t3SD) mice and impairment of relaxation in cMyBP-C(t3SA) mice are not because of altered [Ca(2+)]i handling, implicating that altered cross-bridge detachment rates mediate these changes in relaxation rates.ConclusionscMyBP-C phosphorylation enhances relaxation, whereas deficient phosphorylation causes diastolic dysfunction and phenotypes resembling HFpEF. Thus, cMyBP-C is a potential target for treatment of HFpEF.

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Phosphorylation in protein-protein binding: effect on stability and function.

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Phosphorylation in protein-protein binding: effect on stability and function.

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