Li, Director), NHLBI Light Microscopy Core Facility (C. covalently attaching phosphate organizations to proteins. To comprehensively determine PKA substrates, we used genome editing (CRISPR-Cas9) to delete PKA from kidney epithelial cells followed by large-scale mass spectrometry to measure phosphorylation changes throughout the proteome; 229 PKA target sites were identified, many previously unrecognized. Surprisingly, PKA deletion caused seemingly paradoxical phosphorylation raises at many sites, indicating secondary activation of one or more mitogen-activated kinases. The data, coupled PF-04457845 with transcriptomics and standard proteomics, recognized a signaling network that clarifies the PF-04457845 effects of PKA that regulate cellular functions. in PKA knockout cells. SILAC-based quantitative phosphoproteomics recognized 229 PKA phosphorylation sites. Most of these PKA focuses on are thus far unannotated in public databases. Remarkably, 1,915 phosphorylation sites with the motif x-(S/T)-P showed improved phosphooccupancy, pointing to improved activity of one or more MAP kinases in PKA knockout cells. Indeed, phosphorylation changes associated with activation of ERK2 were seen in PKA knockout cells. The ERK2 site is definitely downstream of a direct PKA site in the Rap1Space, Sipa1l1, that indirectly inhibits Raf1. In addition, a direct PKA site that inhibits the MAP kinase kinase kinase Map3k5 (ASK1) is definitely upstream of JNK1 activation. The datasets were integrated to identify a causal network describing PKA signaling that clarifies vasopressin-mediated rules of membrane trafficking and gene transcription. The model predicts that, through PKA activation, vasopressin stimulates AQP2 exocytosis by inhibiting MAP kinase signaling. The PF-04457845 model also predicts that, through PKA activation, vasopressin stimulates transcription through induction of nuclear translocation of the acetyltransferase EP300, which raises histone H3K27 acetylation of vasopressin-responsive genes (confirmed by ChIP-seq). Heptahelical receptors that couple to the G protein stimulatory -subunit (Gs) regulate cell processes mainly through activation of protein kinase A (PKA). Inside a subset of G protein-coupled receptors (GPCRs), ligand binding results in activation of the heterotrimeric Gs, which activates adenylyl cyclases and raises intracellular cyclic AMP (cAMP). These Gs-coupled receptors include those that regulate glycogenolysis in the liver (glucagon and epinephrine), hydrolysis of triglycerides in adipose cells (epinephrine), PF-04457845 secretion of PF-04457845 thyroid hormone (thyroid-stimulating hormone), synthesis of steroid hormones in the adrenal cortex (adrenocorticotropic hormone), resorption of bone (parathyroid hormone), contractility and rate of contraction in the heart (epinephrine), and water excretion from the kidney (vasopressin) (2). Foremost among effectors of cAMP is definitely PKA, also known as cAMP-dependent protein kinase (3, 4). PKA is definitely a basophilic S/T kinase in the AGC family (5) that phosphorylates serines and threonines in target proteins that possess fundamental amino acids (R K) at positions ?3 and ?2 relative to the phosphorylation site [PKA target motif: (R/K)-(R/K)-x-(pS/pT), where x is any amino acid] (6C8). Lists of protein focuses on of PKA, recognized in reductionist studies, have been curated in databases such as Phospho.ELM (9), the Human being Protein Reference Database (10), PhosphoNET (11), and PhosphoSitePlus (12), although it is likely that many direct PKA focuses on are as yet unidentified. Some of the known PKA focuses on are additional protein kinases and phosphatases, meaning that PKA activation is likely to result in indirect changes in protein phosphorylation manifest like a signaling network, the details of which remain unresolved. To identify both direct and indirect focuses on of PKA in mammalian cells, we used CRISPR-Cas9 genome editing to expose frame-shifting indel mutations in both PKA catalytic subunit genes (and gene (16, 18). The studies recognized 229 phosphorylation sites in 197 proteins that showed decreased phosphooccupancy in cells with CRISPR-Cas9 deletion of PKA-C and PKA-C, including 47 sites in which phosphorylation was ablated by more than 90%. Many of these PKA ITGB2 target sites are previously unidentified as PKA substrates. Furthermore, there were many phosphorylation sites with increased phosphooccupancy that possessed a proline at position +1 relative to the phosphorylated amino acid. This indicates the PKA deletion secondarily activates one or more MAP kinases.