Mst1 (mammalian sterile 20-like kinase 1) is a ubiquitously expressed serine/threonine kinase and its activation in the heart causes cardiomyocyte apoptosis and dilated cardiomyopathy. Its myocardial substrates, however, remain unknown. In a yeast two-hybrid screen of a human heart cDNA library with a dominant-negative Mst1 (K59R) mutant used as bait, cTn [cardiac Tn (troponin)] I was identified as an Mst1-interacting protein. The interaction of cTnI with Mst1 was confirmed by co-immunoprecipitation in both co-transfected HEK-293 cells (human embryonic kidney cells) and native cardiomyocytes, in which cTnI interacted with full-length Mst1, but not with its N-terminal kinase fragment. in vitro phosphorylation assays demonstrated that cTnI is a sensitive substrate for Mst1. In contrast, cTnT was phosphorylated by Mst1 only when it was incorporated into the Tn complex. MS analysis indicated that Mst1 phosphorylates cTnI at Thr31, Thr51, Thr129 and Thr143. Substitution of Thr31 with an alanine residue reduced Mst1-mediated cTnI phosphorylation by 90%, whereas replacement of Thr51, Thr129 or Thr143 with alanine residues reduced Mst1-catalysed cTnI phosphorylation by approx. 60%, suggesting that Thr31 is a preferential phosphorylation site for Mst1. Furthermore, treatment of cardiomyocytes with hydrogen peroxide rapidly induced Mst1-dependent phosphorylation of cTnI at Thr31. Protein epitope analysis and binding assays showed that Mst1-mediated phosphorylation modulates the molecular conformation of cTnI and its binding affinity to TnT and TnC, thus indicating functional significances. The results of the present study suggest that Mst1 is a novel mediator of cTnI phosphorylation in the heart and may contribute to the modulation of myofilament function under a variety of physiological and pathophysiological conditions.
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Research Article|
January 28 2009
Phosphorylation of cardiac troponin I by mammalian sterile 20-like kinase 1
Bei You;
Bei You
*Department of Cell Biology and Molecular Medicine, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Medical Science Building, Room G609, 185 South Orange Avenue, Newark, NJ 07103, U.S.A
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Guijun Yan;
Guijun Yan
*Department of Cell Biology and Molecular Medicine, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Medical Science Building, Room G609, 185 South Orange Avenue, Newark, NJ 07103, U.S.A
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Zhiling Zhang;
Zhiling Zhang
†Section of Molecular Cardiology, Evanston Northwestern Healthcare, Northwestern University Feinberg School of Medicine, 2650 Ridge Avenue, Evanston, IL 60201, U.S.A.
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Lin Yan;
Lin Yan
*Department of Cell Biology and Molecular Medicine, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Medical Science Building, Room G609, 185 South Orange Avenue, Newark, NJ 07103, U.S.A
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Jing Li;
Jing Li
‡Cell Signaling Technology, 3 Trask Lane, Danvers, MA 01923, U.S.A.
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Qingyuan Ge;
Qingyuan Ge
‡Cell Signaling Technology, 3 Trask Lane, Danvers, MA 01923, U.S.A.
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Jian-Ping Jin;
Jian-Ping Jin
1
†Section of Molecular Cardiology, Evanston Northwestern Healthcare, Northwestern University Feinberg School of Medicine, 2650 Ridge Avenue, Evanston, IL 60201, U.S.A.
1Correspondence may be addressed to either of these authors (email sunj1@umdnj.edu or jpjin@northwestern.edu).
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Jianxin Sun
Jianxin Sun
1
*Department of Cell Biology and Molecular Medicine, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Medical Science Building, Room G609, 185 South Orange Avenue, Newark, NJ 07103, U.S.A
1Correspondence may be addressed to either of these authors (email sunj1@umdnj.edu or jpjin@northwestern.edu).
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Publisher: Portland Press Ltd
Received:
July 01 2008
Revision Received:
October 22 2008
Accepted:
November 05 2008
Accepted Manuscript online:
November 05 2008
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2009 Biochemical Society
2009
Biochem J (2009) 418 (1): 93–101.
Article history
Received:
July 01 2008
Revision Received:
October 22 2008
Accepted:
November 05 2008
Accepted Manuscript online:
November 05 2008
Citation
Bei You, Guijun Yan, Zhiling Zhang, Lin Yan, Jing Li, Qingyuan Ge, Jian-Ping Jin, Jianxin Sun; Phosphorylation of cardiac troponin I by mammalian sterile 20-like kinase 1. Biochem J 15 February 2009; 418 (1): 93–101. doi: https://doi.org/10.1042/BJ20081340
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