Phosphorylation of cardiac troponin I by mammalian sterile 20-like kinase 1.
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 Thr(31), Thr(51), Thr(129) and Thr(143). Substitution of Thr(31) with an alanine residue reduced Mst1-mediated cTnI phosphorylation by 90%, whereas replacement of Thr(51), Thr(129) or Thr(143) with alanine residues reduced Mst1-catalysed cTnI phosphorylation by approx. 60%, suggesting that Thr(31) is a preferential phosphorylation site for Mst1. Furthermore, treatment of cardiomyocytes with hydrogen peroxide rapidly induced Mst1-dependent phosphorylation of cTnI at Thr(31). 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.
You
Bei
B
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, USA.
Yan
Guijun
G
Zhang
Zhiling
Z
Yan
Lin
L
Li
Jing
J
Ge
Qingyuan
Q
Jin
Jian-Ping
JP
Sun
Jianxin
J
D000595:Amino Acid Sequence; D000818:Animals; D055162:Biocatalysis; D002460:Cell Line; D017124:Conserved Sequence; D004789:Enzyme Activation / Q000187:drug effects; D006321:Heart / Q000187:drug effects; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D019209:Troponin C / Q000378:metabolism; D019210:Troponin I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020107:Troponin T / Q000378:metabolism; D000099317:Hippo Kinases
D006861:Hydrogen Peroxide; D047908:Intracellular Signaling Peptides and Proteins; D017346:Protein Serine-Threonine Kinases; D019209:Troponin C; D019210:Troponin I; D020107:Troponin T; C095566:STK4 protein, human; D000099317:Hippo Kinases