Dephosphorylation specificities of protein phosphatase for cardiac troponin I, troponin T, and sites within troponin T
<p>Protein dephosphorylation by protein phosphatase 1 (PP1), acting in concert with protein kinase C (PKC) and protein kinase A (PKA), is a pivotal regulatory mechanism of protein phosphorylation. Isolated rat cardiac myofibrils phosphorylated by PKC/PKA and dephosphorylated by PP1 were used i...
| Format: | Article |
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| Language: | English |
| Published: |
Ivyspring International Publisher
2006-03-01
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| Series: | International Journal of Biological Sciences |
| Subjects: | |
| Online Access: | http://www.biolsci.org/v02p0001.htm |
| _version_ | 1811229073752457216 |
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| collection | DOAJ |
| description | <p>Protein dephosphorylation by protein phosphatase 1 (PP1), acting in concert with protein kinase C (PKC) and protein kinase A (PKA), is a pivotal regulatory mechanism of protein phosphorylation. Isolated rat cardiac myofibrils phosphorylated by PKC/PKA and dephosphorylated by PP1 were used in determining dephosphorylation specificities, Ca<sup>2+</sup>-stimulated Mg<sup>2+</sup>ATPase activities, and Ca<sup>2+</sup> sensitivities. In reconstituted troponin (Tn) complex, PP1 displayed distinct substrate specificity in dephosphorylation of TnT preferentially to TnI, <italic>in vitro</italic>. <italic>In situ</italic> phosphorylation of cardiomyocytes with calyculin A, a protein phosphatase inhibitor, resulted in an increase in the phosphorylation stiochiometry of TnT (0.3 to 0.5 (67%)), TnI (2.6 to 3.6 (38%)), and MLC2 (0.4 to 1.7 (325%)). These results further confirmed that though MLC2 is the preferred target substrate for protein phosphatase in the thick filament, the Tn complex (TnI and TnT) from thin filament and C-protein in the thick filament are also protein phosphatase substrates. Our <italic>in vitro</italic> dephosphorylation experiments revealed that while PP1 differentially dephosphorylated within TnT at multiple sites, TnI was uniformly dephosphorylated. Phosphopeptide maps from the <italic>in vitro</italic> experiments show that TnT phosphopeptides at spots 4A and 4B are much more resistant to PP1 dephosphorylation than other TnT phosphopeptides. Mg<sup>2+</sup>ATPase assays of myofibrils phosphorylated by PKC/PKA and dephosphorylated by PP1 delineated that while PKC and PKA phosphorylation decreased the Ca<sup>2+</sup>-stimulated Mg<sup>2+</sup>ATPase activities, dephosphorylation antagonistically restored it. PKC and PKA phosphorylation decreased Ca<sup>2+</sup> sensitivity to 3.6 µM and 5.0 µM respectively. However, dephosphorylation restored the Mg<sup>2+</sup>ATPase activity of PKC (99%) and PKA (95%), along with the Ca<sup>2+</sup> sensitivities (3.3 µM and 3.0 µM, respectively).</p> |
| first_indexed | 2024-04-12T10:08:25Z |
| format | Article |
| id | doaj.art-047939adb78a470c9aff107d83e8bfbb |
| institution | Directory Open Access Journal |
| issn | 1449-2288 |
| language | English |
| last_indexed | 2024-04-12T10:08:25Z |
| publishDate | 2006-03-01 |
| publisher | Ivyspring International Publisher |
| record_format | Article |
| series | International Journal of Biological Sciences |
| spelling | doaj.art-047939adb78a470c9aff107d83e8bfbb2022-12-22T03:37:22ZengIvyspring International PublisherInternational Journal of Biological Sciences1449-22882006-03-012119Dephosphorylation specificities of protein phosphatase for cardiac troponin I, troponin T, and sites within troponin T<p>Protein dephosphorylation by protein phosphatase 1 (PP1), acting in concert with protein kinase C (PKC) and protein kinase A (PKA), is a pivotal regulatory mechanism of protein phosphorylation. Isolated rat cardiac myofibrils phosphorylated by PKC/PKA and dephosphorylated by PP1 were used in determining dephosphorylation specificities, Ca<sup>2+</sup>-stimulated Mg<sup>2+</sup>ATPase activities, and Ca<sup>2+</sup> sensitivities. In reconstituted troponin (Tn) complex, PP1 displayed distinct substrate specificity in dephosphorylation of TnT preferentially to TnI, <italic>in vitro</italic>. <italic>In situ</italic> phosphorylation of cardiomyocytes with calyculin A, a protein phosphatase inhibitor, resulted in an increase in the phosphorylation stiochiometry of TnT (0.3 to 0.5 (67%)), TnI (2.6 to 3.6 (38%)), and MLC2 (0.4 to 1.7 (325%)). These results further confirmed that though MLC2 is the preferred target substrate for protein phosphatase in the thick filament, the Tn complex (TnI and TnT) from thin filament and C-protein in the thick filament are also protein phosphatase substrates. Our <italic>in vitro</italic> dephosphorylation experiments revealed that while PP1 differentially dephosphorylated within TnT at multiple sites, TnI was uniformly dephosphorylated. Phosphopeptide maps from the <italic>in vitro</italic> experiments show that TnT phosphopeptides at spots 4A and 4B are much more resistant to PP1 dephosphorylation than other TnT phosphopeptides. Mg<sup>2+</sup>ATPase assays of myofibrils phosphorylated by PKC/PKA and dephosphorylated by PP1 delineated that while PKC and PKA phosphorylation decreased the Ca<sup>2+</sup>-stimulated Mg<sup>2+</sup>ATPase activities, dephosphorylation antagonistically restored it. PKC and PKA phosphorylation decreased Ca<sup>2+</sup> sensitivity to 3.6 µM and 5.0 µM respectively. However, dephosphorylation restored the Mg<sup>2+</sup>ATPase activity of PKC (99%) and PKA (95%), along with the Ca<sup>2+</sup> sensitivities (3.3 µM and 3.0 µM, respectively).</p>http://www.biolsci.org/v02p0001.htmProtein phosphataseprotein kinase Cprotein kinase Atroponin complex |
| spellingShingle | Dephosphorylation specificities of protein phosphatase for cardiac troponin I, troponin T, and sites within troponin T International Journal of Biological Sciences Protein phosphatase protein kinase C protein kinase A troponin complex |
| title | Dephosphorylation specificities of protein phosphatase for cardiac troponin I, troponin T, and sites within troponin T |
| title_full | Dephosphorylation specificities of protein phosphatase for cardiac troponin I, troponin T, and sites within troponin T |
| title_fullStr | Dephosphorylation specificities of protein phosphatase for cardiac troponin I, troponin T, and sites within troponin T |
| title_full_unstemmed | Dephosphorylation specificities of protein phosphatase for cardiac troponin I, troponin T, and sites within troponin T |
| title_short | Dephosphorylation specificities of protein phosphatase for cardiac troponin I, troponin T, and sites within troponin T |
| title_sort | dephosphorylation specificities of protein phosphatase for cardiac troponin i troponin t and sites within troponin t |
| topic | Protein phosphatase protein kinase C protein kinase A troponin complex |
| url | http://www.biolsci.org/v02p0001.htm |