Identification of a Functionally Critical Protein Kinase C Phosphorylation Residue of Cardiac Troponin T

Abstract

Cardiac Troponin T (cTnT) is one prominent substrate through which protein kinase C (PKC) exerts its effect on cardiomyocyte function. To determine the specific functional effects of the cTnT PKC-dependent phosphorylation sites (Thr197, Ser201, Thr206, and Thr287) we first mutated these residues to glutamate (E) or alanine (A). cTnT was selectively mutated to generate single, double, triple, and quadruple mutants. Bacterially expressed mutants were evaluated in detergent-treated mouse left ventricular papillary muscle fiber bundles where the endogenous troponin was replaced with a recombinant troponin complex containing either cTnT phosphorylated by PKC-alpha or a mutant cTnT. We simultaneously determined isometric tension development and actomyosin Mg-ATPase activity of the exchanged fiber bundles as a function of Ca2+ concentration. Our systematic analysis of the functional role of the multiple PKC phosphorylation sites on cTnT identified a localized region that controls maximum tension, ATPase activity, and Ca2+ sensitivity of the myofilaments. An important and novel finding of our study was that Thr206 is a functionally critical cTnT PKC phosphorylation residue. Its exclusive phosphorylation by PKC-alpha or replacement by Glu (mimicking phosphorylation) significantly decreased maximum tension, actomyosin Mg-ATPase activity, myofilament Ca2+ sensitivity, and cooperativity. On the other hand the charge modification of the other three residues together (T197/S201/T287-E) had no functional effect. Fibers bundles containing phosphorylated cTnT-wt (but not the T197/S201/T206/T287-E) exhibited a significant decrease of tension cost as compared with cTnT-wt.

Phosphorylation of Cardiac Troponin I at Protein Kinase C Site Threonine 144 Depresses Cooperative Activation of Thin Filaments

“…Myosin-S1 was made by chymotryptic digestion of rabbit psoas muscle myosin. Recombinant mouse cTnI (wild-type and mutant) in pET3d, mouse cTnT containing an N-terminal myc tag in pSBET, and human cTnC in pET3d were expressed and purified as described previously (20,25). It was shown that myc tag at the N terminus of cTnT has no effect on myofilament activity (20).…”

The role of protein kinase C-mediated phosphorylation of sarcomeric proteins in the heart—detrimental or beneficial?

“…A more direct study in which transgenic mice lacking PKC phosphorylation sites expressed approximately 50% fast skeletal TnT (fsTnT) provided evidence for a potential role of cTnT phosphorylation in depressing/reducing myofilament force (Montgomery et al 2001). Sumandea et al (2003) studied the specific functional effects by charge mutating the cTnT sites (Thr197, Ser201, Thr206, Thr287, mouse sequence) into aspartic acid and replacing the recombinant cTn into detergent-treated mouse LV papillary muscle fibers. Phospho-mimicking of Thr206, but not the other sites, in the TG mice resulted in decreased maximal tension, actomyosin Mg-ATPase activity, Ca 2+ -sensitivity, and cooperativity (Table 1).…”

Cardiac thin filament regulation

“…However, it does contain up to four potential PKC-specific phosphorylation sites [73]. Sumandea et al, employing recombinant Tn exchange techniques in skinned myocardium, demonstrated that Thr-206 is the functionally important site [73].…”

“…However, it does contain up to four potential PKC-specific phosphorylation sites [73]. Sumandea et al, employing recombinant Tn exchange techniques in skinned myocardium, demonstrated that Thr-206 is the functionally important site [73]. That is, either phosphorylation or a charge mutation phospho-mimic (Thr206Glu) at this residue significantly reduces maximum Ca 2+ -saturated force, myofilament Ca 2+ sensitivity, and crossbridge cycling rate.…”

“…Although the underlying molecular mechanisms are still largely unknown, it was noted that Thr-206 is located close to the end of a N-cap residue of an alpha-helix. Phosphorylation or Glu-substitution of this residue, therefore, could conceivably extend the alpha-helix up to Gly-200, causing either local or global conformational alterations of the Tn complex [73]. Regardless of the underlying mechanisms, it is clear that PKC-mediated phosphorylation of cTnT may play a significant role in the regulation of the cardiac thin filament.…”