ABSTRACT: Identification of PKA-dependent signaling network using CRISPR-Cas9 coupled with quantitative transcriptomics, proteomics and phosphoproteomics
Project description:Classically, connections within a complex network can be identified through systematically removing its components one at a time and observing the resulting functional changes throughout the network. We applied this concept to identify the signaling network downstream from protein kinase A (PKA) in epithelial cells expressing the Gs-coupled V2 vasopressin receptor. Both genes coding for PKA catalytic subunits (Prkaca and Prkacb) were modified using CRISPR-Cas9 to introduce indels resulting in the absence of detectable PKA catalytic subunit protein (immunoblotting and SILAC-based quantitative protein mass spectrometry). Analysis of multiple PKA double knockout (dKO) lines using SILAC-based quantitative phosphoproteomics showed that phosphorylation sites with decreased phospho-occupancy were dominated by the basophilic motif (R/K)-(R/K)-x-S, consistent with that seen for previously identified PKA targets. Overall, 233 PKA target sites were identified, the majority of which are not annotated as PKA sites in public databases. In addition, we identified a large number of sites with increased phospho-occupancy and the motif x-(S/T)-P, consistent with activation of one or more CMGC family kinases in response to PKA deletion. An unexpected finding was a complete, selective loss of expression of the Aqp2 gene (coding for a kidney-specific water channel) with PKA deletion observed both with quantitative proteomics and RNA-Seq based transcriptomics. Using large-scale data integration techniques, the quantitative proteomic, phosphoproteomic, and RNA-Seq datasets were integrated with prior data from the literature to identify a PKA signaling network that explains most of the cellular physiological responses to vasopressin in the target cells, including the regulation of Aqp2 gene transcription.
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:Small cell lung cancer (SCLC) is an aggressive subtype of lung cancer whose biology is still poorly understood. Using a multiplexed inhibitor beads assay, we identified active kinases in SCLC. Among those, we found that PKA is critical for the expansion of SCLC in culture and in vivo. PKA promotes the neuroendocrine epithelial state associated with SCLC tumor-initiating cells. Phosphoproteomics analyses identify ~200 PKA substrates and show that PKA controls multiple facets of SCLC growth. Notably, the PP2A phosphatase counteracts the oncogenic effects of PKA, and PP2A activators inhibit SCLC as single agents and with chemotherapy. Our data uncover key signaling networks in SCLC and indicate that targeting the PKA/PP2A pathway may help inhibit this lethal neuroendocrine cancer.
Project description:An effective combination of multi-omic datasets can enhance our understanding of complex biological phenomena. To build a context-dependent network with multiple omic layers, i.e., a trans-omic network, we performed phosphoproteomics, transcriptomics, proteomics, and metabolomics of murine liver for 4 h after insulin administration and integrated the time series. Structural characteristics and dynamic nature of the network were analyzed to elucidate the impact of insulin. Early and prominent changes in protein phosphorylation and persistent and asynchronous changes in mRNA and protein levels through non-transcriptional mechanisms indicate enhanced crosstalk between phosphorylation-mediated signaling and protein expression regulation. Metabolic response shows different temporal regulation with transient increases at early time points across categories and enhanced response in the amino acid and nucleotide categories at later time points due to process convergence. This extensive and dynamic view of the trans-omic network elucidates prominent regulatory mechanisms that drive insulin responses through intricate interlayer coordination.
Project description:The Ca2+/CaM-dependent protein kinase 2-delta (CAMK2D) has been proposed to be involved in vasopressin signaling in the renal collecting duct, which controls water and salt excretion by the kidney. RNA sequancing and quantitative proteomics analyses identified the expression of multiple CAMK2 isoforms, with CAMK2D being the most abundant in collecting duct cells. To investigate the role of CAMK2D in regulating RNA expression in response to vasopressin signaling, the transcriptome of CRISPR/Cas9-mediated Camk2d knock-out mpkCCD cells was profiled using RNA-Seq in the presence of vasopressin analogue dDAVP.
Project description:Osteoporosis and bone fractures affect millions of men and women worldwide and are often due to increased bone resorption (bone loss) mediated by osteoclasts. Here, we identify a novel role for the cytoplasmic protein ELMO1 as an important ‘signaling node’ controlling the bone resorption function of osteoclasts. Initially, we noted association of ELMO1 SNPs with bone abnormalities and altered bone density in humans. Experimentally, ELMO1 emerged as a promoter of bone loss wherein deletion of ELMO1 reversed osteoporosis / bone erosions in four in vivo mouse models: osteoprotegerin deficiency, ovariectomy, and two types of inflammatory arthritis. However, ELMO1 did not promote bone loss under homeostatic conditions. Mechanistic studies pointed to a larger ELMO1 signaling network that regulates osteoclast activity at several levels. First, transcriptomics coupled with CRISPR/Cas9 genetic deletion approaches identified new regulators of osteoclast function associated with Elmo1, including cathepsin G and myeloperoxidase. Second, defining the ‘ELMO1 interactome’ in osteoclasts via proteomics revealed membrane proteins and v-ATPases required for bone degradation. Third, ELMO1 affects the formation of the actin ring /sealing zone on bone-like surfaces and the distribution of osteoclast-specific proteases. Finally, a 3D structure-based inhibitory peptide targeting a highly conserved region of ELMO1 reduced bone resorption in wild type osteoclasts. Collectively, these data identify ELMO1 as a signaling hub that regulates osteoclast function and bone loss, with relevance to diseases such as osteoporosis and arthritis.
Project description:Transforming growth factor β (TGFβ) signaling is essential in cell growth and differentiation. Yet, the role of the individual TGFβ signaling components in human tissue homeostasis and transformation is still incompletely understood. Here we dissected the importance of the core components in the TGFβ signaling pathway by CRISPR/Cas9 genome editing of human keratinocytes. The edited keratinocytes were used for human organotypic skin cultures and global quantitative proteomics and phosphoproteomics by mass spectrometry. Characterization of cells and human organotypic skin tissues showed control of epithelial differentiation by Smad4-dependent TGF signaling through cell cycle regulation and ECM expression. In contrast, we found that the combined Smad4 dependent and independent pathways, governed by TGFβRII, controls epithelial homeostasis and prevents invasive growth by blocking epithelial inflammation and activation of p38 and ERK signaling. The study provides a framework for exploration of signaling pathways in human 3D tissue models and with global phosphoproteomics.
Project description:Vasopressin regulates renal water excretion by binding to the Gs-coupled vasopressin receptor (V2R) in collecting duct cells, resulting in cyclic AMP-dependent increases in epithelial water permeability through regulation of the aquaporin-2 (AQP2) water channel. Our prior studies showed that CRISPR-mediated deletion of protein kinase A (PKA) in cultured mpkCCD cells largely eliminates these regulatory events. These PKA-null cells provide a means of identifying PKA-independent signaling downstream from the V2 receptor. We carried out large-scale quantitative protein mass spectrometry (SILAC) to identify PKA-independent phosphorylation changes in response to V2R-selective vasopressin analog, dDAVP. The results show that V2R-mediated vasopressin signaling is predominantly, but not entirely, PKA-dependent. Target motif analysis of the phosphopeptides increased in response to dDAVP in PKA-null cells indicates that the vasopressin activates of one or more members of the AMPK/SNF1 subfamily of basophilic protein kinases. Among the upregulated phosphorylation sites were three known targets of SNF1-subfamily kinases, namely Lipe (S559), Crtc1 (S151) and Arhgef2 (S151). One of the phosphorylation sites that increased in occupancy in PKA-null cells was Ser256 of AQP2, a site critical for vasopressin-mediated trafficking of AQP2 to the cell surface. Beyond this, PKA-independent active site phosphorylation changes were also seen for protein kinases Stk39 (SPAK) and Prkci (Protein kinase C iota). Cyclic AMP levels were ~10-fold higher in PKA-null than in PKA-intact cells in the presence of phosphodiesterase inhibitor IBMX, consistent with a marked acceleration of cAMP production in PKA-null cells. The findings are indicative of substantial PKA-independent signaling downstream from the Gs-coupled V2 receptor.
Project description:In this study, we coupled microarray-based transcriptomics and MS-based phosphoproteomics assay to determine mRNA, protein, and phosphopeptide expression levels from 71 autopsied temporal cortical samples, with varying degree of Alzheimer's Disease (AD)-related neurofibrillary pathology. With computational analysis, we identified disease-related transcript, protein and phosphopeptide expression patterns, associated with distinct biological processes and cell types.
Project description:A major goal in the discovery of signaling networks is to identify regulated phosphorylation sites and map them to the protein kinases responsible for their phosphorylation. The V2 vasopressin receptor is a Galphas-coupled GPCR that is responsible for regulation of renal water excretion through control of osmotic water transport in kidney collecting duct cells. Genome editing experiments have demonstrated that virtually all vasopressin-triggered phosphorylation changes are dependent on PKA, but events downstream from PKA are obscure. Here we used: 1) TMT-based quantitative phosphoproteomics to track phosphorylation changes over time in native collecting duct cells isolated from rats; 2) a clustering algorithm to classify time course data; and 3) Bayes’ Theorem to integrate the dynamic phosphorylation data with multiple prior “omic” data sets to identify a set of protein kinases that are regulated secondary to PKA activation. The data establish three PKA-dependent protein kinase modules whose regulation mediate the physiological effects of vasopressin at a cellular level. The three modules are 1) a pathway involving several Rho/Rac/Cdc42-dependent protein kinases that control actin cytoskeleton dynamics; 2) MAP kinase and cyclin-dependent kinase pathways that control cell proliferation; and 3) calcium/calmodulin-dependent signaling. The findings provide a template for investigating signaling via other Galphas-coupled GPCRs.