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
| Autor: | J.F. Kuo, Kazanietz Mg, Robert L. Raynor, Solaro Rj, Blumberg Pm, Jideama Nm, Thomas A. Noland, Guo X |
|---|---|
| Rok vydání: | 1996 |
| Předmět: |
Protein Kinase C-alpha
macromolecular substances In Vitro Techniques Mitogen-activated protein kinase kinase Biochemistry Isozyme Substrate Specificity Mice Substrate-level phosphorylation Troponin complex Animals Phosphorylation Protein kinase A Protein Kinase C Protein kinase C Binding Sites Chemistry Myocardium Troponin I Actomyosin Rats Isoenzymes Protein Kinase C-delta Mutation Calcium Cattle Ca(2+) Mg(2+)-ATPase PRKCE |
| Zdroj: | Biochemistry. 35:14923-14931 |
| ISSN: | 1520-4995 0006-2960 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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