STUDY 1: A Novel Interaction of PKA Regulatory Subunit I-A With Cardiac Troponin T

Congestive heart failure is the leading cause of death in the United States and the western world. Heart failure develops when the heart muscle becomes too weak to pump enough blood to meet the body\u27s demands. Intriguingly, very little is known about the mechanisms that underline the decline in heart function. My research efforts, therefore, were focused on increasing understanding of the mechanisms that govern the regulation of the heart. The ability of the heart to pump blood is largely determined by the association of two proteins, myosin and actin, which are regulated by the protein complex troponin. My research efforts focused on elucidating what cellular molecules interact with troponin and modify its function. I used the yeast two-hybrid system to prove the interaction between two proteins. First, I engineered yeast cells to carry the human troponin T (TnT) gene. I screened a human heart library for genes that associate with TnT; ten gene products were identified. This screen showed, for the first time, that protein kinase A (PKA) associates with TnT. TnT serves as an anchor for PKA that essentially switches troponin I (TnI) on. This activation of TnI leads to improved relaxation of the cardiac muscle. Because there are two different PKA proteins in the cell (PKA-RI and PKA-RII), I wanted to differentiate them in terms of association with TnT and to identify what portion of TnT interacts with each PKA. I produced four different fragments that cover the full length of TnT and tested for their association with PKA-RI and PKA-RII. My data showed that PKA-RII binds tighter than PKA-RI, and they seem to associate with the same fragment of TnT