Additional PKA phosphorylation sites in human cardiac troponin I.
We used mass spectrometry to monitor cAMP-dependent protein kinase catalysed phosphorylation of human cardiac troponin I in vitro. Phosphorylation of isolated troponin I by cAMP-dependent protein kinase resulted in the covalent incorporation of phosphate on at least five different sites on troponin I, and a S22/23A troponin I mutant incorporated phosphates on at least three sites. In addition to the established phosphorylation sites (S22 and S23) we found that S38 and S165 were the other two main sites of phosphorylation. These 'overphosphorylation' sites were not phosphorylated sufficiently slower than S22 and S23 that we could isolate pure S22/23 bisphosphorylated troponin I. Overphosphorylation of troponin I reduced its affinity for troponin C, as measured by isothermal titration microcalorimetry. Phosphorylation of S22/23A also decreased its affinity for troponin C indicating that phosphorylation of S38 and/or S165 impedes binding of troponin I to troponin C. Formation of a troponin I/troponin C complex prior to cAMP-dependent protein kinase treatment did not prevent overphosphorylation. When whole troponin was phosphorylated by cAMP-dependent protein kinase, however, [(32)P]phosphate was incorporated only into troponin I and only at S22 and S23. Mass spectrometry confirmed that overphosphorylation is abolished in the ternary complex. Troponin I bisphosphorylated exclusively at S22 and S23 troponin I showed reduced affinity for troponin C but the effect was diminished with respect to overphosphorylated troponin I. These results show that care should be exercised when interpreting data obtained with troponin I phosphorylated in vitro.
Ward
D G
DG
School of Biosciences, University of Birmingham, UK.
Ashton
P R
PR
Trayer
H R
HR
Trayer
I P
IP
D001665:Binding Sites; D002151:Calorimetry; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004926:Escherichia coli; D006801:Humans; D013058:Mass Spectrometry; D009206:Myocardium / Q000378:metabolism*; D010766:Phosphorylation; D014162:Transfection; D019209:Troponin C / Q000378:metabolism; D019210:Troponin I / Q000378:metabolism*
D019209:Troponin C; D019210:Troponin I; D017868:Cyclic AMP-Dependent Protein Kinases