A preferred AMPK phosphorylation site adjacent to the inhibitory loop of cardiac and skeletal troponin I

Abstract

5 0 -AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that is activated when cellular AMP to ATP ratios rise, potentially serving as a key regulator of cellular energetics. Among the known targets of AMPK are catabolic and anabolic enzymes, but little is known about the ability of this kinase to phosphorylate myofilament proteins and thereby regulating the contractile apparatus of striated muscles. Here, we demonstrate that troponin I isoforms of cardiac (cTnI) and fast skeletal (fsTnI) muscles are readily phosphorylated by AMPK. For cTnI, two highly conserved serine residues were identified as AMPK sites using a combination of high-resolution top-down electron capture dissociation mass spectrometry, 32 P-incorporation, synthetic peptides, phospho-specific antibodies, and site-directed mutagenesis. These AMPK sites in cTnI were Ser149 adjacent to the inhibitory loop and Ser22 in the cardiac-specific N-terminal extension, at the level of cTnI peptides, the intact cTnI subunit, whole cardiac troponin complexes and skinned cardiomyocytes. Phosphorylation time-course experiments revealed that Ser149 was the preferred site, because it was phosphorylated 12-16-fold faster than Ser22 in cTnI. Ser117 in fsTnI, analogous to Ser149 in cTnI, was phosphorylated with similar kinetics as cTnI Ser149. Hence, the master energy-sensing protein AMPK emerges as a possibly important regulator of cardiac and skeletal contractility via phosphorylation of a preferred site adjacent to the inhibitory loop of the thin filament protein TnI.

TNNI1, TNNI2 and TNNI3: Evolution, regulation, and protein structure–function relationships

“…Ser 150 can be phosphorylated by P21-activated kinase (Pak) (Buscemi et al, 2002; Ke et al, 2004) and AMP-activated protein kinase (AMPK), resulting in increased Ca 2+ sensitivity of cardiac myofibrils, prolonged relaxation (Oliveira et al, 2012), and increased development of adrenergic-induced myocardial hypertrophy (Taglieri et al, 2011). Equivalent to Ser 150 in cTnI, Ser 118 in fsTnI was also reported to be phosphorylated by AMPK (Sancho Solis et al, 2011). As AMPK is a key regulator of cellular energetics, phosphorylation of Ser 150 may suggest an adaptive mechanism in energy deprivation of both cardiac and skeletal muscles.…”

Alpha1 catalytic subunit of AMPK modulates contractile function of cardiomyocytes through phosphorylation of troponin I

“…Phosphorylation of cTnI is a key regulatory mechanism in cardiac muscle contraction and relaxation. Recently, phosphorylation of cTnI at Ser149 has been identified as a substrate for AMPK activation (Oliveira et al, 2012; Sancho Solis et al, 2011). The results in our study displayed that cTnI (Ser 149 ) from WT cardiomyocytes has been up-regulated after A-769662 treatment compared to vehicle group, which supports our hypothesis that activation of AMPK by A-769662 could regulate the phosphorylation status of cTnI, leading to the result of increase contractility of cardiomyocytes.…”

“…Membranes were probed with anti-rabbit AMPK-α (1:1000, Cell Signaling), anti-rabbit phosphor-AMPK (Thr 172 , 1:1000, Cell Signaling), anti-rabbit ACC (1:1000, Cell Signaling), and anti-rabbit phosphor-ACC (Ser 79 , 1:1000, Cell Signaling), followed by incubation with horseradish peroxidase (HRP)-coupled anti-rabbit secondary antibody (Cell Signaling Technology Inc, Beverly, MA). For the detection of cTnI Ser 149 phosphorylation, a polyclonal antibody was generated against the phosphopeptide LRRVRIS(phos)ADAMMQA and purified with affinity cross-absorption with the nonphosphorylated peptide (Sancho Solis et al, 2011). Blue X-ray film (Phenix, Candler, NC) was used for photon detection and image development.…”

“…Phosphorylation at Ser 23/24 of cTnI by PKA or PKG results in promising effects on cardiac contractility (Layland et al, 2005). Recent studies also showed that AMPK changes the phosphorylation status of cTnI at Ser 149 , suggesting a promising mechanism of AMPK mediation in contractility of cardiomyocytes (Nixon et al, 2012; Oliveira et al, 2012; Sancho Solis et al, 2011). …”

Comprehensive Characterization of AMP-Activated Protein Kinase Catalytic Domain by Top-Down Mass Spectrometry

“…Purified recombinant AMPK Δ protein (10 μM) was incubated with the LKB1/Ste20-related adaptor (STRAD)/mouse protein 25(MO25) complex (100 nM) with a substrate-to-enzyme ratio of 100:1 in 100 μl kinase buffer (50 mM HEPES pH 7.4, 5 mM MgCl 2 , 1 mM CaCl 2, 2 mM DTT, 200 μM ATP and protease inhibitor cocktail) at 30 °C for 5, 10, and 30 min [40]. The reaction was stopped by freezing the samples at −80 °C.…”