Journal
JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY
Volume 38, Issue 1, Pages 213-218Publisher
ELSEVIER SCI LTD
DOI: 10.1016/j.yjmcc.2004.10.014
Keywords
cardiac myofilament; troponin; phosphorylation; protein kinase C; phosphopeptide; electrophoresis; MALDI-TOF mass spectrometry
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Funding
- NCRR NIH HHS [S10 RR14686] Funding Source: Medline
- NHLBI NIH HHS [R01 HL 64035, HL 22231, P01 HL 62426] Funding Source: Medline
- NATIONAL CENTER FOR RESEARCH RESOURCES [S10RR014686] Funding Source: NIH RePORTER
- NATIONAL HEART, LUNG, AND BLOOD INSTITUTE [P01HL062426, R37HL022231, R01HL064035, R01HL022231] Funding Source: NIH RePORTER
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Phosphorylation of cardiac troponin I (cTnI) by cAMP-dependent kinase (PKA), protein kinase C (PKC) and potentially other kinases modulates the activity of myofilaments. To elucidate the signaling mechanisms involving this modulation, it is important to determine the phosphorylation states of cTnI and its phosphorylation sites in a simple and efficient manner. In this report., we describe a method to determine the phosphorylation states of cTnI with non-equilibrium isoelectric focusin gel electrophoresis (NEIEF). Our method easilyseparates cTnI species with a single-charge difference. To further establish a role of PKC-dependent phosphorylation of cTnI, we have applied this approach to analysis of cTnI phosphorylation in the Tn complex following treatment with recombinant PKC and in heart samples treated with a phorbol ester. Using mass spectrometry analysis of Tn and thin filaments, we identified Ser-23 and Ser-24 (normally considered to be PKA-dependent sites) as substrates for phosphorylation by PKC-beta and PKC-epsilon. (C) 2004 Elsevier Ltd. All rights reserved.
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