Differential regulation of cardiac actomyosin S-1 MgATPase by protein kinase C isozyme-specific phosphorylation of specific sites in cardiac troponin I and its phosphorylation site mutants.
The significance of site-specific phosphorylation by protein kinase C (PKC) isozymes alpha and delta and protein kinase A (PKA) of troponin I (TnI) and its phosphorylation site mutants in the regulation of Ca(2+)-stimulated MgATPase activity of reconstituted actomyosin S-1 was investigated. The genetically defined TnI mutants used were T144A, S43A/S45A, S43A/S45A/T144A (in which the PKC phosphorylation sites Thr-144 and Ser-43/Ser-45 were respectively substituted by Ala) and N32 (in which the first 32 amino acids in the NH2-terminal sequence containing Ser-23/Ser-24 were deleted). Although the PKC isozymes displayed different substrate phosphorylation kinetics, PKC-alpha phosphorylated equally well TnI wild type and all mutants, whereas N32 was a much poorer substrate for PKC-delta. Furthermore, the two PKC isozymes exhibited discrete specificities in phosphorylating distinct sites in TnI and its mutants, either as individual subunits or as components of the reconstituted troponin complex. Unlike PKC-alpha, PKC-delta favorably phosphorylated the PKA-preferred site Ser-23/Ser-24 and hence, like PKA, reduced the Ca2+ sensitivity of the reconstituted actomyosin S-1 MgATPase. In contrast, PKC-alpha preferred to phosphorylate Ser-43/Ser-45 (common sites for all isozymes) and thus reduced the maximal Ca(2+)-stimulated activity of the MgATPase. In this respect, PKC-delta, by cross-phosphorylating the PKA sites, functioned as a hybrid of PKC-alpha and PKA. The site specificities and hence functional differences between PKC-alpha and -delta were most evident at low phosphorylation (1 mol of phosphate/mol) of TnI wild type and were magnified when S43A/S45A and N32 were used as substrates. The present study has demonstrated, for the first time, that distinct functional consequences could arise from the site-selective preferences of PKC-alpha and -delta for phosphorylating a single substrate in the myocardium, i.e., TnI.
Noland
T A
TA
Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Raynor
R L
RL
Jideama
N M
NM
Guo
X
X
Kazanietz
M G
MG
Blumberg
P M
PM
Solaro
R J
RJ
Kuo
J F
JF
D000205:Actomyosin / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000378:metabolism*; D051379:Mice; D009154:Mutation; D009206:Myocardium / Q000201:enzymology / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D051381:Rats; D013379:Substrate Specificity; D019210:Troponin I / Q000235:genetics / Q000378:metabolism*
D007527:Isoenzymes; D019210:Troponin I; D000205:Actomyosin; C497259:Prkcd protein, mouse; C497260:Prkcd protein, rat; C496630:Prkca protein, mouse; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D017301:Ca(2+) Mg(2+)-ATPase; D002118:Calcium