Mst1 (mammalian sterile 20-like kinase 1) is a ubiquitously expressed serine/threonine kinase and its activation in the heart causes cardiomyocyte apoptosis and dilated cardiomyopathy. Its myocardial substrates, however, remain unknown. In a yeast two-hybrid screen of a human heart cDNA library with a dominant-negative Mst1 (K59R) mutant used as bait, cTn [cardiac Tn (troponin)] I was identified as an Mst1-interacting protein. The interaction of cTnI with Mst1 was confirmed by co-immunoprecipitation in both co-transfected HEK-293 cells (human embryonic kidney cells) and native cardiomyocytes, in which cTnI interacted with full-length Mst1, but not with its N-terminal kinase fragment. in vitro phosphorylation assays demonstrated that cTnI is a sensitive substrate for Mst1. In contrast, cTnT was phosphorylated by Mst1 only when it was incorporated into the Tn complex. MS analysis indicated that Mst1 phosphorylates cTnI at Thr31, Thr51, Thr129 and Thr143. Substitution of Thr31 with an alanine residue reduced Mst1-mediated cTnI phosphorylation by 90%, whereas replacement of Thr51, Thr129 or Thr143 with alanine residues reduced Mst1-catalysed cTnI phosphorylation by approx. 60%, suggesting that Thr31 is a preferential phosphorylation site for Mst1. Furthermore, treatment of cardiomyocytes with hydrogen peroxide rapidly induced Mst1-dependent phosphorylation of cTnI at Thr31. Protein epitope analysis and binding assays showed that Mst1-mediated phosphorylation modulates the molecular conformation of cTnI and its binding affinity to TnT and TnC, thus indicating functional significances. The results of the present study suggest that Mst1 is a novel mediator of cTnI phosphorylation in the heart and may contribute to the modulation of myofilament function under a variety of physiological and pathophysiological conditions.
Mst1 (mammalian sterile 20-like kinase 1) is an ubiquitously expressed serine/threonine kinase with a similarity to Ste20, an upstream activator of the mitogen-activated protein kinase pathway in budding yeast [1,2]. Mst1 contains an N-terminal catalytic domain and an autoinhibitory segment, followed by a dimerization domain and a nuclear localization motif in the non-catalytic C-terminal region . In addition, human Mst1 has two caspase cleavage sites situated between the catalytic and regulatory domains, which mediate the cleavage of the autoinhibitory domain [2,3]. In response to a variety of apoptotic stimuli, Mst1 is cleaved by caspases to produce a 34–36 kDa N-terminal constitutively active fragment and this cleavage markedly increases Mst1 kinase activity and translocates cleaved Mst1 to the nucleus, where it phosphorylates histone H2B on Ser14, resulting in apoptotic cell death [3–5]. In addition to caspase cleavage, Mst1 phosphorylation has been proposed previously to contribute to kinase activation . Several phosphorylation sites have been identified in Mst1, namely Thr175, Thr177, Thr183, Thr187, Ser327 and Thr387 [6,7], of which Thr183 and Thr187 appear to be essential for kinase activation [7,8]. The effect of phosphorylation at these sites on the activation of Mst1 may be amplified further by dimerization and eventually leads to the caspase cleavage, thereby constituting a powerful amplification loop of apoptotic responses .
The role of Mst1 in cardiac muscle function has previously been a novel focus of investigation. In cardiomyocytes, Mst1 is activated by pathological stimuli, such as hypoxia/reoxygenation in vitro and ischaemia/reperfusion in vivo . Cardiac-specific overexpression of Mst1 has been shown to cause dilated cardiomyopathy in mice . Inhibition of endogenous Mst1 prevents apoptosis of cardiomyocytes and cardiac dysfunction after myocardial infarction, without producing cardiac hypertrophy [9,10]. However, little is known about the cellular function of Mst1 besides apoptosis. Investigating its pathophysiological substrates in cardiac muscle will lead to a better understanding of myocardial adaptation to stress conditions. In the present study, we performed yeast two-hybrid screens of a human heart cDNA library using the dominant-negative form of Mst1 (K59R) as bait to identify myocardial proteins that represent potential Mst1 substrates. Structural and functional characterization provided evidence that cTn [cardiac Tn (troponin)] I is a novel substrate of Mst1 in the heart. Mst1 directly interacts with and phosphorylates cTnI. The pertinent phosphorylation sites were identified by MS analysis and confirmed by site-directed mutagenesis. Together with modulation of the molecular conformation and function of cTnI by Mst1 phosphorylation, our findings suggest that the Mst1 signalling pathway through cTnI phosphorylation may represent an important method of regulation of cardiac contractility under physiological and pathophysiological conditions.
MATERIALS AND METHODS
Screening of yeast two-hybrid library
The MATCHMAKER GAL4 yeast 2-hybrid system 3 was purchased from Clontech. The screening was performed on a human heart cDNA library using human dominant-negative Mst1(K59R) as bait as described previously . Positive colonies were subjected to multiple rounds of additional selection in the appropriate medium and β-galactosidase filter assays to verify specificity. The confirmed cDNA clones were sequenced to reveal their identities in comparison with the GenBank® databases.
Co-immunoprecipitation of cTnI and Mst1 in HEK-293 cells (human embryonic kidney cells) and cardiomyocytes
HEK-293 cells were transiently transfected with FLAG-tagged cTnI and Myc-tagged Mst1 expression plasmids using FuGENE™ 6 (Roche) following the manufacturer's instructions. HEK-293 cells or cardiomyocytes were lysed using a buffer containing 50 mM Tris/HCl (pH 8.0), 1% Nonidet P40, 150 mM NaCl and protease inhibitors. Co-immunoprecipitation of cTnI and Mst1 was performed as described previously . The following antibodies and beads were used for detection and immunoprecipitation: mouse monoclonal anti-Myc antibody (Invitrogen), mouse monoclonal anti-FLAG M2 antibody (Sigma), rabbit polyclonal anti-Mst1 antibody (Cell Signaling), mouse monoclonal anti-TnI antibody (Abcam), rabbit polyclonal anti-TnI antibody (Santa Cruz Biotechnology), anti-c-Myc–agarose affinity gel (Sigma), anti-FLAG M2–agarose (Sigma) and Protein G–Sepharose (Amersham Biosciences).
Western blotting analysis to identify the immunoprecipitated proteins was performed using horseradish peroxidase-conjugated donkey anti-rabbit and sheep anti-mouse (GE Healthcare) secondary antibodies, followed by detection using ECL® (enhanced chemiluminescence) reagents (Amersham Biosciences).
in vitro phosphorylation assays
To phosphorylate cTnI in vitro, recombinant cTnI, cTnC and cTnT (Lee Biosolutions), MBP (myelin basic protein) (Upstate) and reconstituted Tn complexes were incubated with recombinant active Mst1 (Upstate) and [γ-32P]ATP (PerkinElmer) in 2×kinase assay buffer [40 mM Hepes/NaOH pH 7.4, 20 mM MgCl2, 1 mM DTT (dithiothreitol), 1 mM ATP, 1 mM sodium orthovanadate, 50 mM sodium fluoride and complete protease inhibitor (Roche)] at 30 °C for 60 min. The reaction was terminated by the addition of 0.5 volumes of 3×Laemmli sample buffer and incubated at 95 °C for 5 min, and was then resolved by SDS/PAGE (12% gels) and autoradiography. For peptide MS analysis, recombinant human cTnI was phosphorylated by active Mst1 in the presence of non-radiolabelled ATP.
Reconstitution of the Tn complex
The Tn complex was reconstituted in vitro using recombinant wild-type or mutated cTnI, together with recombinant wild-type cTnC and cTnT, as described previously . The formation of the Tn complex was confirmed by non-denaturing SDS/PAGE.
Human cTnI was phosphorylated by Mst1 in vitro and then subjected to SDS/PAGE (10% gels), followed by Coomassie Blue R250 staining and tryptic in-gel digestion, and then analysed by MALDI–TOF–MS (matrix-assisted laser-desorption ionization–time of flight MS). The phosphorylated residues were identified further by MS/MS (tandem MS) analysis.
Expression and purification of cTnI mutants
In order to achieve high expression of cTnI in bacteria, two bases in the second and the fourth codons of cTnI cDNA (Ala2, GCG→GCC; and Gly4, GGG→GGT) were mutated before cloning . in vitro site-directed mutagenesis was performed to obtain the different mutated cTnI cDNA constructs using the QuikChange® multi-site-directed mutagenesis kit (Stratagene). The modified and mutated cTnI cDNA constructs were cloned into the NdeI and BamHI sites of the pET-21b vector and verified by DNA sequencing. BL21(DE3) competent cells (Stratagene) were transformed with the pET-21b constructs and induced with 1 mM IPTG (isopropyl β-D-thiogalactoside) at 37 °C for 3 h. Recombinant cTnI was purified as described previously .
Monoclonal antibody epitope analysis
The binding affinity between an antibody and its antigenic epitope depends on the three-dimensional structural fit. ELISA epitope analysis  was employed to examine the conformational differences between non-phosphorylated and Mst1-phosphorylated cTnI. A monoclonal TnI-1 antibody against an epitope in the C-terminal domain of cTnI , a monoclonal 4H6 antibody against the central region of TnI, and a polyclonal rabbit anti-TnI antibody (RATnI)  were used to monitor the conformational changes that alter the antibody-binding affinity.
An ELISA-based solid-phase protein-binding assay  was used to investigate the interactions of non-phosphorylated and Mst1-phosphorylated cTnI with TnT and TnC. All of the experiments were performed in triplicate.
The results for the epitope analysis and protein-binding assays are means±S.D. Statistical analyses were performed using ANOVA or Student's t test, with P<0.05 considered significant.
Interaction of cTnI with Mst1
Since Mst1 plays an essential role in initiating apoptosis in cardiomyocytes and the development of dilated cardiomyopathy [9,10], the present study aimed to identify the substrates of Mst1 in cardiomyocytes using yeast two-hybrid screening of a human heart cDNA library with the dominant negative Mst1 (K59R) as bait. After screening 2×106 clones, a total of 40 positive clones were identified, nine of which contained a full-length cDNA encoding cTnI.
To confirm that the interaction of Mst1 with cTnI does occur in mammalian cells, co-immunoprecipitation was performed in HEK-293 cells co-transfectively expressing Myc-tagged Mst1 and FLAG-tagged cTnI. Immunoprecipitation of Myc-tagged Mst1 led to co-precipitation of FLAG-tagged cTnI. As a control, the anti-Myc antibody did not immunoprecipitate FLAG-tagged cTnI in the absence of Myc-tagged Mst1. Similarly, immunoprecipitation of FLAG-tagged cTnI resulted in the co-immunoprecipitation of Myc-tagged Mst1, whereas the anti-FLAG antibody did not immunoprecipitate Myc-tagged Mst1 in the absence of FLAG-tagged cTnI (Figure 1A). These findings indicate that cTnI and Mst1 can form a tightly bound complex in mammalian cells.
Interaction of cTnI with Mst1
To determine whether there is an endogenous interaction of cTnI and Mst1 in cardiomyocytes, we performed immunoprecipitation with an anti-Mst1 antibody using cell lysates obtained from neonatal rat cardiac myocytes. Indeed, cTnI co-immunoprecipitated with the anti-Mst1 antibody, but not with the non-immune IgG (Figure 1B). The result indicates that cTnI interacts with Mst1 in cardiomyocytes under physiological conditions.
Mst1 contains an N-terminal catalytic domain (amino acids 1–326) and a C-terminal regulatory domain (amino acids 327–487) . To further map the cTnI-interaction domain of Mst1, we constructed the kinase domain and regulatory domain of Mst1 in pCS26MT vector with a 6×Myc tag and transfected these constructs into HEK-293 cells along with the FLAG–cTnI expression plasmid. Cell lysates from transfected HEK-293 cells were immunoprecipitated using an anti-FLAG antibody and analysed by Western blotting. We found that FLAG–cTnI bound only to full-length Mst1, but not to the N-terminal or C-terminal fragments (Figure 1C). As a negative control, the anti-FLAG antibody did not immunoprecipitate Myc–Mst1 in cells co-transfected with Myc–Mst1 and the empty FLAG vector. These results suggest that full-length Mst1 is required for the interaction with cTnI.
Mst1-mediated phosphorylation of cTnI
Since Mst1 is a serine/threonine kinase , the interaction of Mst1 with cTnI prompted us to investigate whether cTnI is a substrate of Mst1. Therefore purified human cTnI was incubated with active Mst1 in the presence of [γ-32P]ATP in an in vitro phosphorylation assay. Equimolar quantities of MBP was included as a positive control. In order to study whether other Tn subunits were substrates of Mst1, human cTnC and cTnT were also tested in the in vitro kinase assay. As shown in Figure 2(A), incubation of recombinant Mst1 with cTnI resulted in a robust phosphorylation of cTnI. The phosphorylation activity of Mst1 on cTnI was even higher than that on the known substrate MBP. In contrast, free cTnT and cTnC were both barely phosphorylated by Mst1. In addition, Mst1-mediated phosphorylation of cTnI produced a change in gel mobility as shown by SDS/PAGE (Figure 2B).
Mst1-mediated phosphorylation of cTnI
To investigate further whether Mst1 phosphorylates cTnI in the Tn complex, the Tn complex was reconstituted in vitro and then subjected to the in vitro phosphorylation assay in the presence of active Mst1. The results showed that cTnI in the Tn complex was also strongly phosphorylated by Mst1 (Figure 2C). Interestingly, although free cTnT was barely phosphorylated by Mst1, it was markedly phosphorylated when incorporated into the Tn complex, albeit to a lesser extent when compared with cTnI (Figure 2C). Together, these results indicate that cTnI is a good substrate for Mst1, not only in the free form, but also when present in the Tn complex. Furthermore, cTnI may also serve as an Mst1-anchoring protein in the Tn complex and facilitate the phosphorylation of cTnT by the kinase.
To determine the kinetics of Mst1-mediated phosphorylation of cTnI in the Tn complex, the time course of Mst1 phosphorylation of the reconstituted Tn complex was determined in vitro. Mst1 phosphorylates cTnI in the Tn complex in a time-dependent manner and reached saturation at 90 min (Figure 2D).
Identification of the phosphorylation sites by MALDI–TOF-MS
To determine Mst1 phosphorylation sites on cTnI, we performed tryptic peptide mapping using MALDI–TOF-MS after Mst1 phosphorylation treatment. As shown in Table 1, computer-assisted proteomic analysis revealed four phosphorylated tryptic ions with m/z ratios of 1067.4532, 1589.9648, 1325.6299 and 997.527, corresponding to cTnI residues 28–36+1 phosphate group, residues 46–58+1 phosphate group, residues 121–131+1 phosphate group and residues 139–145+1 phosphate group respectively. The phosphorylated residues were further identified by MS/MS spectra (see Supplementary Figure S1 at http://www.BiochemJ.org/bj/418/bj4180093add.htm). The results suggest that Mst1 phosphorylates cTnI at Thr31, Thr51, Thr129 and Thr143 (amino acids numbered as in the human cTnI amino acid sequence) (Table 1). Alignments of cTnI amino acid sequences bearing Mst1-mediated phosphorylation sites indicated that they are conserved among multiple species (Figure 3).
|Phosphopeptide||Calculated mass||Observed mass|
|Phosphopeptide||Calculated mass||Observed mass|
Sequence alignments of cTnI from various species
To identify the cTnI site(s) that are accessible for Mst1-mediated phosphorylation within the Tn complex, we reconstituted the complex further in vitro using recombinant wild-type or mutated cTnI (T31A, T51A, T129A, T143A or T31A/T51A/T129A/T143A) and recombinant wild-type cTnC and TnT. Wild-type cTnI and each of the cTnI mutants were all incorporated into the Tn complex effectively (results not shown). Interestingly, the T31A mutation had a greater impact on cTnI phosphorylation by Mst1 than the other single mutant constructs. The T31A mutation substantially attenuated Mst1-catalysed cTnI phosphorylation by approx. 90%. In contrast, mutation of Thr51 or Thr129 or Thr143 to alanine residues resulted in the partial attenuation of Mst1-catalysed cTnI phosphorylation by approx. 60% (Figures 4A and 4B). Furthermore, the combined replacement of all four threonine residues with alanine residues abolished Mst1-mediated phosphorylation of cTnI completely. These results further suggest that Mst1 phosphorylates threonine residues at positions 31, 51, 129, and 143 in cTnI and that Thr31 appears to be a preferential phosphorylation site.
The contribution of four phosphorylation sites to Mst1-catalysed cTnI phosphorylation in the reconstituted Tn complex
To confirm further that Mst1 preferentially phosphorylates cTnI at Thr31, the reconstituted Tn complexes containing cTnI mutants with or without the T31A substitution were subjected to in vitro phosphorylation by Mst1 to determine the kinetics of phosphorylation. In the Tn complex, wild-type cTnI and mutations that retained Thr31 were effectively phosphorylated by Mst1 in a time-dependent manner, with maximal phosphorylation observed at 90 min. However, when Thr31 is mutated to alanine, Mst1-mediated cTnI phosphorylation was markedly diminished (Figure 4C). These results further indicate that Mst1 preferentially phosphorylates cTnI at Thr31.
Hydrogen peroxide-induced cTnI (Thr31) phosphorylation via Mst1 activation in cardiomyocytes
Oxidative stress has been shown to activate Mst1 in neuronal cells and cardiomyocytes [9,18]. To determine whether oxidative stress activates both Mst1 and cTnI phosphorylation in cardiomyocytes, we used hydrogen peroxide (100 μM) to stimulate Mst1 activity and cTnI phosphorylation. Indeed, hydrogen peroxide markedly stimulated Mst1 activation, as determined by autophosphorylation of Mst1 (Figure 5A). Using an anti-[phospho-specific cTnI (Thr31)] antibody, we found that cTnI phosphorylation was significantly increased, paralleling the time course of hydrogen peroxide-induced Mst1 activation (Figure 5A). Furthermore, silencing of Mst1 expression by use of a specific siRNA (small interfering RNA) substantially attenuated hydrogen peroxide-induced cTnI phosphorylation at Thr31 (Figures 5B and 5C). These results suggest that Mst1 activation is critical for hydrogen peroxide-mediated cTnI phosphorylation.
Mst1 mediates hydrogen peroxide-mediated cTnI (Thr31) phosphorylation in cardiomyocytes
Mst1-mediated phosphorylation induces conformational changes in cTnI
To investigate the structural and functional effects of Mst1 phosphorylation of cTnI, we investigated changes in cTnI molecular conformation upon Mst1 treatment. ELISA epitope analysis was performed to examine the binding affinity of monoclonal and polyclonal anti-TnI antibodies for non-phosphorylated and Mst1-phosphorylated cTnI. The titration curves of a monoclonal TnI-1 antibody that recognizes a C-terminal epitope  showed that Mst1-catalysed phosphorylation of cTnI did not change the molecular conformation in the C-terminal domain (Figure 6A). In contrast, the titration curves of a monoclonal 4H6 antibody that recognizes an epitope in the central region of TnI demonstrated that Mst1-catalysed phosphorylation of cTnI resulted in an increase in the binding affinity in comparison with that of the non-phosphorylated cTnI, as shown by the increased antibody dilution resulting in 50% maximum binding (P<0.005) (Figure 6B). This result indicates a change in the molecular conformation of the central region of cTnI due to Mst1-catalysed phosphorylation. This observation was supported further by epitope analysis using the polyclonal anti-TnI antibody RATnI. The RATnI titration curves demonstrated an increased maximum binding to Mst1-phosphorylated cTnI in comparison with the non-phosphorylated control (P<0.005) (Figure 6C). The conformational analysis results suggest that Mst1-catalysed phosphorylation resulted in a significant change in the molecular conformation of cTnI, laying a foundation for functional significance.
Mst1-catalysed phosphorylation induces conformational changes in cTnI
Altering binding affinity of cTnI to TnT and TnC by Mst1 phosphorylation
To investigate the effects of Mst1-catalysed cTnI phosphorylation on the interactions with cTnT and TnC, ELISA solid-phase protein-binding experiments were performed . The results showed that Mst1-phosphorylated cTnI had a lower binding affinity for TnT than that of unphosphorylated cTnI, as indicated by the right-hand shift of the TnT concentration required for 50% maximum binding (P<0.05) (Figure 7A). The binding curves for TnC demonstrated that Mst1-phosphorylated cTnI had an increased binding affinity for TnC compared with that of unphosphorylated cTnI, as indicated by the significant left-hand shift of the TnC concentration required for 50% maximum binding, despite the presence or absence of Ca2+ (P<0.01 and P<0.05 respectively) (Figures 7B and 7C).
Mst1 catalysed phosphorylation affects the binding affinity of cTnI to TnT and TnC
Phosphorylation of cTnI is an important post-translational mechanism in the regulation of thin filament function and thereby in cardiac muscle contractility . Altered phosphorylation of cTnI and other myofilament proteins may also contribute causally to cardiac dysfunction in the transition from compensated hypertrophy to heart failure [21,22]. In the present study, we demonstrated that Mst1 interacts with and directly phosphorylates cTnI, resulting in conformational changes of cTnI and an alteration in its interactions with the Tn complex.
The contraction of cardiac muscle is based on actin–myosin interactions regulated by intracellular Ca2+ via the thin filament-based Tn–tropomyosin system . The Tn complex contains three subunits: the Ca2+-binding subunit TnC, the tropomyosin-binding subunit TnT and the inhibitory subunit TnI [24,25]. During muscle contraction, the binding of Ca2+ to TnC releases the TnI inhibition of actomyosin ATPase through allosteric protein–protein interactions among the Tn complex, tropomyosin and actin, and leads to muscle contraction. A key step in this signalling mechanism is the release of inhibition of TnI on the actin–myosin interaction . Phosphorylation of specific serine and threonine residues in cTnI has been identified as a major physiological mechanism for the alteration of myofilament properties [22,26]. To date, much attention has been focused on the phosphorylation-mediated regulation of cTnI function by PKA (protein kinase A) and PKC (protein kinase C). PKA has been demonstrated to phosphorylate cTnI at Ser22 and Ser23 (numbered as in the human cTnI sequence) in vitro [27,28], thus leading to a reduction in myofilament Ca2+ sensitivity , an increase in cross-bridge cycling [30,31] and an increase in the binding of cTnI to thin filaments. PKC has been shown to phosphorylate cTnI mainly at Ser43, Ser45 and Thr144 and reduce the maximal activity of actomyosin Mg2+-ATPase . In addition, cTnI may also be modified specifically by protein kinase G and p21-activated kinases .
In the present study, our results demonstrate that Mst1 targets cTnI specifically and catalyses the phosphorylation of cTnI at Thr31, Thr51, Thr129 and Thr143. These phosphorylation sites are mostly conserved in cTnI from multiple species. It is important to note that the decreases in phosphorylation of the mutant cTnI lacking one of the four Mst1 phosphorylation sites is non-proportional in comparison with wild-type cTnI, suggesting that the phosphorylation of cTnI at the four different threonine residues by Mst1 may be dependent on each other. In the reconstituted Tn complex, Thr31, which lies within the N-terminal extension of cTnI, appears to be a preferential phosphorylation site by Mst1, since replacement of Thr31 with an alanine residue reduced Mst1-catalysed cTnI phosphorylation markedly.
Another interesting finding is that when free cTnT was examined, it was not significantly phosphorylated by Mst1. However, when it was incorporated into the Tn complex, cTnT was markedly phosphorylated by Mst1 (Figure 4). Moreover, our preliminary results suggest that cTnT did not interact directly with Mst1 both in vitro and in co-transfected HEK-293 cells (results not shown). This implies that cTnI may serve as an anchoring protein to mediate the phosphorylation of cTnT by Mst1 in native cardiac muscle. The phosphorylation sites in cTnT by Mst1 remain to be identified in future studies.
In comparison with skeletal muscle TnI, cTnI has an additional 27–33 amino acids at its N-terminus, and phosphorylation of Ser22 and Ser23 by PKA in this cardiac-specific segment has been shown to reduce myofilament Ca2+ sensitivity . In the present study, we, for the first time, demonstrate that in this cardiac specific segment, Thr31 is a primary site phosphorylated by a novel kinase, Mst1. Since Thr31 is close to the TnC-binding domain in the N-terminal portion of cTnI, the phosphate introduced by Mst1 may possibly influence the interaction between the N-terminus of cTnI and the C-terminus of cTnC [33,34]. This hypothesis is supported by the Mst1 phosphorylation-induced changes in the conformation of the 4H6 epitope (Figure 5) and the binding affinity for TnC (Figure 6). In addition, Mst1 phosphorylation of Thr51 of cTnI in a site that binds to the C-terminus of cTnT and the C-lobe of cTnC [33,34] may also have an effect on the interaction of cTnI with cTnT and cTnC. Indeed, our results demonstrate that phosphorylation of cTnI by Mst1 significantly changed the global conformation of cTnI and its binding affinity to both cTnT and cTnC. The effect of Mst1 phosphorylation of cTnI on the interaction with TnT may also be attributed to the Thr129 site in the I-T arm of the Tn complex.
The phosphorylation of cTnI by Mst1 at Thr143 located in the inhibitory region  is worth investigating further. Thr143 was identified previously as being one of the PKC phosphorylation sites in cTnI , and PKC-catalysed Thr143 phosphorylation has been shown to result in a reduction in the Ca2+ sensitivity of filament sliding  and an increase in myofilament calcium sensitivity . In this regard, Mst1 may represent another kinase that is able to phosphorylate cTnI at Thr143, resulting in similar functional effects. Taken together, our results suggest that Mst1 is a novel kinase that phosphorylates cTnI at multiple threonine sites in several important structural and functional domains which are responsible for protein–protein interactions among the Tn complex, tropomyosin and actin. Thus Mst1 may play important roles in modifying cardiac contractility through the phosphorylation of TnI and/or cTnT.
The functional significance of Mst1-mediated phosphorylation of cTnI in the heart remains elusive. In response to apoptotic stimuli, Mst1 is cleaved by caspases to a 34–36 kDa N-terminal constitutive active fragment that subsequently translocates to the nucleus where it phosphorylates histone H2B on Ser14, resulting in apoptosis [3–5]. In the heart, Mst1 is activated by ischaemia/reperfusion, and targeted overexpression of Mst1 in the heart has been shown to cause dilated cardiomyopathy in mice . In Mst1 transgenic mice, increased Mst1 activity was found to be associated with the full-length form rather than its cleaved form . Our results also demonstrated that cTnI interacted only with full-length Mst1, but not the N-terminal or C-terminal fragment. Deleting either one of these domains would probably change the molecular conformation of Mst1, thus affecting the binding of Mst1 to cTnI. Importantly, in response to oxidative stress, cTnI is phosphorylated at Thr31 in an Mst1-dependent manner. This implies that cTnI may be a pathophysiologically relevant substrate of Mst1 in the cytoplasm of cardiac myocytes. Since post-translational modification of cTnI, such as proteolysis and phosphorylation by kinase(s), is of particular importance in the modulation of cardiac myofilament function under stress conditions, the identification of cTnI as a novel substrate of Mst1 in the heart may open a new area of research towards a better understanding of cardiac contractile dysfunction associated with ischaemic heart diseases and heart failure.
In summary, the present study provides the first evidence which indicates that Mst1 is a novel kinase that interacts with and phosphorylates cTnI directly, resulting in conformational changes in cTnI and alterations in the protein–protein interactions in the Tn complex. Therefore Mst1 may play an important role in the regulation of cardiac muscle contractility under physiological and pathophysiological conditions, which requires further investigation.
This work was supported by the National Institutes of Health [grant numberHL078773] (to J.-P. J.); and by a Scientist Development Grant from the American Heart Association [grant number 0630047N] (to J. S.). Z. Z. was supported by a Postdoctoral Fellowship from the American Heart Association, Great Midwest Affiliate.