STK25 phosphorylates PRKAR1A to regulate PKA signaling

Project Summary The development of new therapies in heart failure is a critical need and current drug development in this field is not sufficient to keep pace with the increasing incidence and mortality of this disease. Novel therapeutic targets are required as are model systems that more closely resemble human cardiac physiology. Protein kinase A (PKA) is a cAMP sensitive kinase that is the central relay for beta-adrenergic stimulation of cardiomyocyte contraction and calcium flux. With a broad set of substrates, specificity of signaling relies on careful regulation of both cAMP metabolism and kinase activity. Regulatory subunits of PKA both inhibit kinase activity and help recruit effectors and macromolecular binding partners to orchestrate kinase signaling. Type I regulatory subunits are broadly expressed yet how their function is modulated is not well known. In recently published work, we demonstrate that the Type Iα regulatory subunit (PRKAR1A) is phosphorylated by the kinase STK25. In studies performed in human induced pluripotent stem cell derived cardiomyocytes (iPSC-CM), phosphorylation of PRKAR1A led to inhibition of PKA kinase activity and downstream signaling in response to cAMP through increased binding to the catalytic subunit. Knockout studies of Stk25 in mice confirmed its in vivo role of inhibiting PKA activity. In further preliminary data, the Stk25 knockout was associated with improved outcomes after myocardial infarction with decreased fibrosis and increased cardiac function. In a set of logical and feasible aims, we propose to tests the hypothesis that STK25 phosphorylation of PRKAR1A leads to inhibition of PKA activity and that this signaling between STK25 and PRKAR1A has therapeutic potential. In Aim 1, we use genetically modified iPSC-CM’s to investigate the mechanism of how phosphorylation leads to increased inhibition of PKA activity as well as characterize the changes to the macromolecular PKA complex in response to phosphorylation of PRKAR1A. In Aim 2, transgenic mice with knock-in mutations are utilized to investigate Prkar1a phosphorylation and its regulation of PKA activity in vivo. In the third aim, an inducible conditional knockout of Stk25 is generated in mice and is used to explore the mechanism underlying the improvement after myocardial infarction in response to loss of Stk25. We will examine if loss of Stk25 after a myocardial infarction imparts any benefit and whether a decrease in Prkar1a phosphorylation mediates this improvement. We also will investigate a novel inhibitor to explore the pharmacologic potential of targeting this pathway after myocardial infarction in vivo. We believe that this proposal will have significant impact on our understanding of PKA regulation in cardiomyocytes and establish the phosphorylation of PRKAR1A as a as a potential therapeutic modality in myocardial infarction and heart failure.