Abstract 957: A Novel Interaction of Cardiac Troponin T with PKA Regulatory Subunits I and II
Increasing evidence suggests that cardiac myofilament activity is modulated by posttransla-tional modifications of cardiac troponin (cTn). In an attempt to identify novel proteins that associate with and modify cTn, we used the full-length human cardiac TnT as bait in the yeast two-hybrid system, and a human heart cDNA library as prey. Our screen of > 2×106 cDNA clones with hcTnT as bait, identified >1000 clones that grew on high-stringency plates, and 210 that turned the cells blue in the presence of α-gal, indicating positive interactions with hcTnT. DNA sequencing of the 210 clones led to the identification of 10 gene products: cTnI (identified in 180 clones), PKA-RIα, MLC2v, Dystrophin, PDZ and LIM domain 5, CGI-94, Muscle Creatine Kinase, ANF, Mitofusin-2 and an uncharacterized gene. HA-tagged clones (above) and myc-tagged cTnT were individually expressed using a rabbit reticulocyte lysate system and immunoprecipitated (IP) by agarose conjugated anti-myc and anti-HA antibodies. Therefore, the interaction of these proteins with cTnT was verified in a mammalian cellular milieu. Our finding suggests that cTnT, through its interaction with PKA regulatory domains (R), anchors PKA to the myofilament in close proximity to cTnI. This would explain the rapid, PKA dependent, phosphorylation of cTnI during increase intracellular cAMP levels following beta-adrenergic stimulation. While, PKA type I holoenzyme is predominantly cytoplasmic, the majority of PKA type II associates with cytoskeletal structures. Binding of PKA-RI and PKA-RII to intact and truncated forms of cTnT (amino acids 1–117; 118 –288; 1–180; 181–288) were determined by yeast two-hybrid and by IP assays. Our results demonstrate, for the first time, that both PKA-RI and RII associate with cTnT, and PKA-RI binds to a specific region of cTnT-1–180 fragment, whereas PKA-RII binds to a region of cTnT-181–288 fragment. PKA-dependent phosphorylation of cTnI-Ser 23/24 has profound effects on cardiac dynamics: modulating cross-bridge kinetics, energy consumption and calcium sensitivity. Therefore, it is important to have a mechanistic understanding of events that mediate and affect PKA-dependent cTnI phosphorylation.