Abstract 1573: Phosphorylation of Cardiac Troponin I by Mammalian Sterile20-like Kinase Mst1
Mammalian sterile 20-like kinase 1 (Mst1) 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 human heart cDNA library with a dominant negative Mst1 (K59R) as bait, cardiac troponin I (cTnI) was identified as an Mst1-interacting protein. The interaction of cTnI with Mst1 was confirmed by co-immunoprecipitation in both co-transfected HEK293 cells and native cardiac myocytes, 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 its free form or in reconstituted troponin complex. In contrast, cardiac TnT was phosphorylated by Mst1 only when incorporated in the troponin complex. Mass spectrometric analysis indicated that Mst1 phosphorylates cTnI at Thr31, Thr51, Thr129, and Thr143. Substitution of Thr31 with Ala substantially reduced Mst1-mediated cTnI phosphorylation by approximately 90%, while replacement of either Thr51 orThr129, or Thr143 with Ala reduced Mst1-catalyzed cTnI phosphorylation by approximately 60%, suggesting that Thr31 is a preferential phosphorylation site for Mst1. 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. Our results suggest that Mst1 is a novel mediator of cTnI and/or cTnT phosphorylation in the heart and may contribute to the modulation of myofilament function under a variety of physiological and pathophysiological conditions.
This research has received full or partial funding support from the American Heart Association, AHA National Center.