Novel role for p90 ribosomal S6 kinase in the regulation of cardiac myofilament phosphorylation

Abstract

In myocardium, the 90-kDa ribosomal S6 kinase (RSK) is activated by diverse stimuli and regulates the sarcolemmal Na(+)/H(+) exchanger through direct phosphorylation. Only limited information is available on other cardiac RSK substrates and functions. We evaluated cardiac myosin-binding protein C (cMyBP-C), a sarcomeric regulatory phosphoprotein, as a potential RSK substrate. In rat ventricular myocytes, RSK activation by endothelin 1 (ET1) increased cMyBP-C phosphorylation at Ser(282), which was inhibited by the selective RSK inhibitor D1870. Neither ET1 nor D1870 affected the phosphorylation status of Ser(273) or Ser(302), cMyBP-C residues additionally targeted by cAMP-dependent protein kinase (PKA). Complementary genetic gain- and loss-of-function experiments, through the adenoviral expression of wild-type or kinase-inactive RSK isoforms, confirmed RSK-mediated phosphorylation of cMyBP-C at Ser(282). Kinase assays utilizing as substrate wild-type or mutated (S273A, S282A, S302A) recombinant cMyBP-C fragments revealed direct and selective Ser(282) phosphorylation by RSK. Immunolabeling with a Ser(P)(282) antibody and confocal fluorescence microscopy showed RSK-mediated phosphorylation of cMyBP-C across the C-zones of sarcomeric A-bands. In chemically permeabilized mouse ventricular muscles, active RSK again induced selective Ser(282) phosphorylation in cMyBP-C, accompanied by significant reduction in Ca(2+) sensitivity of force development and significant acceleration of cross-bridge cycle kinetics, independently of troponin I phosphorylation at Ser(22)/Ser(23). The magnitudes of these RSK-induced changes were comparable with those induced by PKA, which phosphorylated cMyBP-C additionally at Ser(273) and Ser(302). We conclude that Ser(282) in cMyBP-C is a novel cardiac RSK substrate and its selective phosphorylation appears to regulate cardiac myofilament function.

FIGURE 1.

Effects of the MEK inhibitor U0126 (A) or the RSK inhibitor D1870 (B) on ET1-induced phosphorylation of cMyBP-C at Ser²⁸², ERK at Thr²⁰²/Tyr²⁰⁴, RSK at Thr⁵⁷⁷, or RSK at Ser³⁸⁶ in ARVM. Cells were exposed to vehicle control or ET1 (50 nm for 10 min) following pretreatment with vehicle, U0126 (3 μm for 10 min) or D1870 (1 μm for 60 min). cMyBP-C, ERK, or RSK phosphorylation status was determined by immunoblot (IB) analysis using the indicated phosphospecific antibodies, with protein loading confirmed by immunoblot analysis using antibodies against total protein. Individual immunoblots illustrate representative experiments, and bar charts show quantitative data (n = 6–8). *, p < 0.05 versus corresponding control (con) group. Error bars, S.E.

FIGURE 2.

A, effects of D1870 on RSK NTK activity, as reflected by phosphorylation of a recombinant NHE1 fusion protein by endogenous RSK immunoprecipitated from ARVM, in an in vitro kinase (IVK) assay. Vehicle or D1870 (10 nm) was added to the reaction mixture after exposure of ARVM to ET1 (50 nm for 10 min) to activate RSK and subsequent RSK immunoprecipitation (top panel), or ARVM were pretreated with vehicle or D1870 (1 μm) for 60 min before exposure to ET1 to activate RSK and subsequent RSK immunoprecipitation (IP) (bottom panel). NHE1 phosphorylation status was determined by immunoblot (IB) analysis using a phosphospecific antibody and equal amounts of immunoprecipitated RSK in each sample confirmed by immunoblot analysis using an antibody against total RSK. Data are representative of three independent experiments. B, direct phosphorylation of cMyBP-C at Ser²⁸² by RSK and PKA in vitro. A recombinant protein comprising the c1c2 fragment of human cMyBP-C was used as substrate in an in vitro kinase assay, in the presence or absence of D1870 (10 nm) or H89 (100 nm), and phosphorylation by the PKA catalytic subunit or RSK2 was detected by immunoblot analysis using Ser(P)²⁸² phosphospecific cMyBP-C antibody. Equal protein loading was confirmed by Coomassie staining. C, effects of D1870 on PKA-mediated (left panel) or PKD-mediated (right panel) phosphorylation of cTnI at Ser²²/Ser²³, in ARVM exposed to isoproterenol (ISO; 10 nm for 10 min) or ET1 (50 nm for 10 min), respectively. cTnI phosphorylation status was determined by immunoblot analysis using Ser(P)²²/Ser(P)²³ phosphospecific cTnI antibody, with protein loading confirmed by immunoblot analysis using an antibody against total cTnI. Individual immunoblots illustrate representative experiments, and bar charts show quantitative data (n = 6). *, p < 0.05 versus corresponding control (con) group. Error bars, S.E.

FIGURE 3.

A, effects of heterologous expression of wild-type (wt) or kinase-inactive (ki) RSK isoforms on cMyBP-C phosphorylation at Ser²⁸² in ARVM. Cells infected with AdV:βGal (control), AdV:RSK1wt or AdV:RSK1ki (left panel) or AdV:EGFP (control), AdV:RSK2wt or AdV:RSK2ki (right panel) were exposed to vehicle or ET1 (50 nm for 10 min) following pretreatment with vehicle or D1870 (1 μm for 60 min). cMyBP-C phosphorylation status was determined by immunoblot (IB) analysis using Ser(P)²⁸² phosphospecific cMyBP-C antibody, with protein loading confirmed by immunoblot analysis using an antibody against total cMyBP-C. Immunoblot analysis using a pan-RSK antibody confirmed comparable overexpression of wt and ki forms of RSK1 and RSK2. Individual immunoblots illustrate representative experiments, and bar charts show quantitative data on cMyBP-C phosphorylation at Ser²⁸² (n = 7). *, p < 0.05 versus corresponding control (con) group; #, p < 0.05 versus corresponding ET1 group infected with AdV:βGal or AdV:EGFP. Error bars, S.E. B, effects of heterologous expression of wt or ki RSK isoforms on cTnI phosphorylation at Ser²²/Ser²³ in ARVM. Experimental details are identical to those described in A, except that cTnI phosphorylation was determined by immunoblot analysis using a Ser(P)²²/Ser(P)²³ phosphospecific cTnI antibody, with protein loading confirmed by immunoblot analysis using an antibody against total cTnI. Data are representative of four independent experiments.

FIGURE 4.

A, time course of RSK2- or PKA-mediated phosphorylation of wild-type (wt) and mutated cMyBP-C c1c2 domains. Recombinant proteins comprising the c1c2 fragment of human cMyBP-C, in wt form or carrying single Ser/Ala substitutions (S273A, S282A, or S302A), were used as substrate in an in vitro kinase (IVK) assay performed in the presence of [Y³²P]ATP, and phosphorylation by RSK2 or the PKA catalytic subunit was detected by autoradiography. B, time course of RSK2- or PKA-mediated Ser²⁸² phosphorylation in wt and mutated cMyBP-C c1c2 domains. Experimental details are identical to those described in A, except that the in vitro kinase assay was performed in the presence of nonradiolabeled ATP, and phosphorylation by RSK2 or the PKA catalytic subunit was detected by immunoblot (IB) analysis using Ser(P)²⁸² phosphospecific cMyBP-C antibody. In both A and B, protein loading was confirmed by Coomassie staining, and the data are representative of three independent experiments. C, characterization of Ser(P)²⁷³ and Ser(P)³⁰² phosphospecific cMyBP-C antibodies. Recombinant proteins comprising the c1c2 fragment of human cMyBP-C, in wt form or carrying single Ser/Ala substitutions (S273A, S282A, or S302A), were used as substrate in an in vitro kinase assay performed in the presence of nonradiolabeled ATP, and phosphorylation by the PKA catalytic subunit or RSK2 was detected by immunoblot analysis using Ser(P)²⁷³ or Ser(P)³⁰² phosphospecific antibodies. Equal protein loading was confirmed by Coomassie staining. D, ET1-induced phosphorylation of cMyBP-C at Ser²⁷³ and Ser³⁰² in ARVM. Cells were exposed to vehicle (con) or ET1 (50 nm for 10 min), following pretreatment with vehicle or D1870 (1 μm for 60 min). cMyBP-C phosphorylation status was determined by immunoblot analysis using Ser(P)²⁷³ or Ser(P)³⁰² phosphospecific cMyBP-C antibody, with protein loading confirmed by immunoblot analysis using an antibody against total cMyBP-C. Cell extract from ARVM exposed to isoproterenol (ISO; 10 nm for 10 min) was used as a positive control. Data are representative of three independent experiments.

FIGURE 5.

Confocal microscope images showing the localization of total cMyBP-C (A) or cMyBP-C phosphorylated at Ser²⁸² (B), in ARVM exposed to vehicle (con) or ET1 (50 nm for 10 min), following pretreatment with vehicle or D1870 (1 μm for 60 min). The cells were additionally immunolabeled with an α-actinin antibody, to demarcate the Z-discs, and nuclei were stained with DAPI. The images were obtained from perinuclear regions of each cell. In the merged images, red indicates α-actinin labeling, green indicates total or Ser(P)²⁸² cMyBP-C labeling, and the nuclei are stained blue. Scale bars, 10 μm.

FIGURE 6.

RSK2- and PKA-mediated phosphorylation of cMyBP-C (A) and cTnI in skinned ventricular myocytes (B) from cTnI-Ala₂ mice, as detected by immunoblot (IB) analysis using (A) Ser(P)²⁷³, Ser(P)²⁸², or Ser(P)³⁰² phosphospecific cMyBP-C antibody and (B) Ser(P)²²/Ser(P)²³ phosphospecific cTnI antibody. Protein loading was confirmed by immunoblot analysis using total cMyBP-C or cTnI antibody, with positive control sample (+ con) obtained from ARVM exposed to isoproterenol (10 nm for 10 min). Individual immunoblots illustrate representative experiments, and bar charts show quantitative data on cMyBP-C phosphorylation (n = 4). *, p < 0.05 versus corresponding no-kinase control (con) group. Error bars, S.E.

FIGURE 7.

Effects of RSK2- and PKA-mediated phosphorylation on (A and B) the Ca²⁺ sensitivity of force development and (C) cross-bridge cycle kinetics, in skinned ventricular trabeculae from cTnI-Ala₂ mice. A, mean force-pCa curves obtained before (pre; open circles) and after (post; filled circles) incubation of trabeculae with RSK2 (left panel) or PKA catalytic subunit (right panel). Force values were normalized to the maximum force, measured at pCa 4.5. B, mean pCa at 50% maximum force (pCa₅₀) obtained before (pre; open bars) and after (post; filled bars) incubation of trabeculae with RSK2 or PKA catalytic subunit. C, mean relative rate of force redevelopment (k_tr) at 50% maximum force obtained before (pre; open bars) and after (post; filled bars) incubation of trabeculae with RSK2 or PKA catalytic subunit. *, p < 0.05 versus corresponding pre-kinase value (n = 7; Student's paired t test). Error bars, S.E.