The transcription factor hypoxia-inducible factor 1 (HIF-1) is the primary sensor and regulator of intracellular O2-homeostasis in mammalian cells; inducing transcription of hundreds of genes that encode proteins critical for adaptive responses to hypoxia, or pathophysiological processes that deplete intracellular O2. cAMP-dependent protein kinase A (PKA) is one of the primary effectors of cAMP-responsive cellular processes, phosphorylating a multitude of proteins critical for cell physiology, cardiomyocyte contractility, and adaptive responses to acute and chronic stress and inflammation. Both HIF-1 and PKA play central roles in dozens of pathologies associated with cancer and diseases of the heart, including angiogenesis, cell migration, tissue and vasculature remodeling, immunosuppression and cancer cell immune evasion. Compared to what is known about regulation of HIF-1 in ischemic diseases and cancer, very little is known about the upstream intracellular signaling mechanisms that regulate HIF-1 in cardiomyocytes during the progression of chronic heart failure. To identify novel upstream mechanisms that regulate HIF-1 and are of particular relevance to HIF signaling in the heart, we used a proteomic approach to identify interacting protein partners of HIF-1? in primary rat neonatal cardiomyocytes (RNCMs). The R1a regulatory subunit of PKA was one protein identified of particular interest. Preliminary experiments have validated endogenous PKA as an interacting protein of HIF-1?, that pharmacological manipulation of pathways that increase intracellular cAMP increase HIF-1? transcriptional activity and protein levels in multiple cell lines, and that HIF-1a is a direct substrate of recombinant PKA in vitro. Based on these findings, we are proposing the following central hypothesis: PKA is a critical regulator of HIF-1 activity. To test this hypothesis we are proposing three Specific Aims.
Specific Aim 1 will conclusively determine if PKA is a critical mediator of HIF-1 activity by characterizing the effect of pharmacological and genetic gain- and loss-of-function of PKA on HIF-1? transcriptional activity and protein levels in transformed cell lines an primary RNCMs.
Specific Aims 2 and 3 will subsequently characterize the detailed mechanism by which PKA affects HIF-1 in cell lines and RNCMs.
For Aim 2, mass spectrometry and biochemical and molecular biological tools will be used to assess the affect of pharmacological and genetic manipulation of PKA on well characterized or potentially novel co-regulatory signaling axis' that mediate the protein stability and/or transactivation of HIF-1a.
For Aim 3, using the same tools we will identify and characterize specific amino acid residues phosphorylated by PKA on HIF-1? and determine which is functionally relevant for mediating PKA-dependent effects on HIF-1? in cells. Together these aims could provide a vital mechanistic link between a plethora of upstream regulators of cAMP and critical downstream HIF-dependent effects that orchestrate adaptive physiological responses, as well as maladaptive pathologies in diseases associated with hypoxia, inflammation and chronic stress (e.g. heart disease and cancer).
This application seeks funding to characterize the molecular mechanisms by which the master regulator of stress-responsive cAMP signaling, PKA, mediates the master regulator of O2-homeostasis, HIF-1, in mammalian cells. Both HIF-1 and PKA play central roles in mediating a multitude of pathologies associated with cancer and diseases of the heart. Interrogation of PKA as a critical mediator of HIF-1 activity could provide a vital link between a plethora of upstream regulators of cAMP and critical downstream HIF-dependent effects that orchestrate physiological adaptive responses, as well as maladaptive pathologies in diseases associated with tissue remodeling, inflammation, and/or chronic stress (e.g. cancer, cardiovascular disease and chronic heart failure).
| Bullen, John W; Tchernyshyov, Irina; Holewinski, Ronald J et al. (2016) Protein kinase A-dependent phosphorylation stimulates the transcriptional activity of hypoxia-inducible factor 1. Sci Signal 9:ra56 |
