Project description:When exploring the role of Gαs and β-arrestin in nuclear transcriptional programs downstream from β2-adrenergic receptor (β2AR), we noticed a dearth of information on PKA-regulated genes sets. Thus, we first used the tetracycline-regulated expression of wild type PKA Cα subunit in HEK293 cells to develop a PKA signature. This approach revealed multiple PKA-regulated genes, including well known PKA downstream transcriptional targets, such as PCK1 and FOS, and many new PKA transcriptional targets whose underlying mechanism can now be explored.
Project description:Phosphoproteomics studies of PKA catalytic subunit or its DNAJB1-fused chimeric form overexpressed in HEK293 cells, under various conditions including inhibitors. Also includes in vitro rephosphorylation of cell lysates by each construct.
Project description:Protein Kinase A (PKA) is a widely studied protein that has been viewed by most investigators as a single entity, although its catalytic subunits are coded in the genome by two separate genes, PKA catalytic alpha (Gene symbol: Prkaca) and PKA catalytic beta Gene symbol: Prkacb). At an amino-acid level, the two are 91.5 percent identical and the catalytic domains are virtually identical. (Footnote: A third entity PKA catalytic gamma, is not widely expressed and will not be considered here.) We have recently succeeded in using CRISPR-Cas9 to create disruptive mutations in both PKA genes (PKA double KO, or PKA dKO) in vasopressin-responsive kidney epithelial cells (mpkCCD cells). Here we carry out mass spectrometry based quantitative proteomics and phosphoproteomics separately in PKA catalytic-alpha and PKA catalytic-beta single knockouts address the issue of function difference between these two PKA catalytic subunits.
Project description:Temperature preference behavior in Drosophila depends on the level of PKA signaling in the mushroom bodies. To identify new components downstream to PKA, we carried out a genome-wide screen for genes regulated by PKA signaling in the mushroom bodies.
Project description:Temperature preference behavior in Drosophila depends on the level of PKA signaling in the mushroom bodies. To identify new components downstream to PKA, we carried out a genome-wide screen for genes regulated by PKA signaling in the mushroom bodies. Using the Gal4-UAS system, we increased or decreased PKA activity in the mushroom bodies by expressing dominant-negative (UAS-PKADN) or constitutively active PKA (UAS-PKACA), respectively. Expression of PKA transgenes was targeted to the mushroom bodies using the mushroom body-specific MB247-Gal4 driver. PKA expression was induced for 12-16 hours in three-day-old adults by inactivating the temperature-sensitive Gal80 at the restrictive temperature. We then analyzed gene-expression profiles to identify the genes showing altered expression levels in response to the high or low PKA activity.
