Phosphorylation Changes in Response to Kinase Inhibitor H89 in PKA-Null Cells

Limbutara, Kavee; Kelleher, Andrew; Yang, Chin‐Rang; Raghuram, Viswanathan; Knepper, Mark A.

doi:10.1038/s41598-019-39116-2

Cited by 30 publications

(27 citation statements)

References 25 publications

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“…Almost all vasopressin‐mediated phosphorylation changes in murine immortalized cortical collecting duct (mpkCCD) cells were ablated when both PKA catalytic genes were deleted (PKA‐null cell) with CRISPR‐Cas9 genome editing (Datta et al, 2020). In addition, an inhibitor of PKA and other basophilic kinases, H‐89, blocked a large array of phosphorylation events in PKA‐intact mpkCCD cells that were not blocked in PKA‐null cells (Limbutara, Kelleher, Yang, Raghuram, & Knepper, 2019). Understanding of vasopressin signalling beyond PKA activation by cAMP remains incomplete.…”

Section: Introductionmentioning

confidence: 99%

Phosphoproteomic identification of vasopressin‐regulated protein kinases in collecting duct cells

Datta

Yang

Salhadar

et al. 2021

British J Pharmacology

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Background and Purpose The peptide hormone vasopressin regulates water transport in the renal collecting duct largely via the V2 receptor, which triggers a cAMP‐mediated activation of a PKA‐dependent signalling network. The protein kinases downstream from PKA have not been fully identified or mapped to regulated phosphoproteins. Experimental Approach We carried out systems‐level analysis of large‐scale phosphoproteomic data quantifying vasopressin‐induced changes in phosphorylation in aquaporin‐2‐expressing cultured collecting duct (mpkCCD) cells. Quantification was done using stable isotope labelling (SILAC method). Key Results Six hundred forty phosphopeptides were quantified. Stringent statistical analysis identified significant changes in response to vasopressin in 429 of these phosphopeptides. The corresponding phosphoproteins were mapped to known vasopressin‐regulated cellular processes. The vasopressin‐regulated sites were classified according to the sequences surrounding the phosphorylated amino acids giving 11 groups. Among the vasopressin‐regulated phosphoproteins were 25 distinct protein kinases. Among these, six plus PKA appeared to account for phosphorylation of about 81% of the 313 vasopressin‐regulated phosphorylation sites. The six downstream kinases were salt‐inducible kinase 2 (Sik2), cyclin‐dependent kinase 18 (Cdk18), calmodulin‐dependent kinase kinase 2 (Camkk2), protein kinase D2 (Prkd2), mitogen‐activated kinase 3 (Mapk3) and myosin light chain kinase (Mylk). Conclusion and Implications In V2 receptor‐mediated signalling, PKA is at the head of a complex network that includes at least six downstream vasopressin‐regulated protein kinases that are prime targets for future study. The extensive phosphoproteomic data reported in this study are provided as a web‐based data resource for future studies of GPCRs.

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Section: Introductionmentioning

confidence: 99%

Phosphoproteomic identification of vasopressin‐regulated protein kinases in collecting duct cells

Datta

Yang

Salhadar

et al. 2021

British J Pharmacology

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“…The cells were seeded in a 6-well plate at a density of 5 × 10 5 cells/well in MM. After 24 h, the cells were treated with or without glucagon in the presence or absence of 10 µmol/L H89, PKA inhibitor [ 46 ], in MM. After 30 min, the cells were incubated with a cell lysis buffer supplied by the manufacturer for another 30 min on ice with occasional vortexing.…”

Section: Methodsmentioning

confidence: 99%

Glucagon Prevents Cytotoxicity Induced by Methylglyoxal in a Rat Neuronal Cell Line Model

et al. 2021

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Although diabetic polyneuropathy (DPN) is a frequent diabetic complication, no effective therapeutic approach has been established. Glucagon is a crucial hormone for glucose homeostasis but has pleiotropic effects, including neuroprotective effects in the central nervous system. However, the importance of glucagon in the peripheral nervous system (PNS) has not been clarified. Here, we hypothesized that glucagon might have a neuroprotective function in the PNS. The immortalized rat dorsal root ganglion (DRG) neuronal cell line 50B11 was treated with methylglyoxal (MG) to mimic an in vitro DPN model. The cells were cultured with or without glucagon or MG. Neurotoxicity, survival, apoptosis, neurite projection, cyclic adenosine monophosphate (cAMP), and protein kinase A (PKA) were examined. Glucagon had no cytotoxicity and rescued the cells from neurotoxicity. Cell survival was increased by glucagon. The ratio of apoptotic cells, which was increased by MG, was reduced by glucagon. Neurite outgrowth was accelerated in glucagon-treated cells. Cyclic AMP and PKA accumulated in the cells after glucagon stimulation. In conclusion, glucagon protected the DRG neuronal cells from MG-induced cellular stress. The cAMP/PKA pathway may have significant roles in those protective effects of glucagon. Glucagon may be a potential target for the treatment of DPN.

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“…However, evidence from cell-free experiments has suggested that H89 can also inhibit other protein kinases (Davies et al, 2000). Limbutara et al (Limbutara et al, 2019) used PKA-null and PKAintact mouse cell lines derived from mpkCCD cells (see Section V) and quantitative phosphoproteomics to investigate the specificity of H89 over the range of concentrations commonly used in the literature. From a total of 14,507 phosphorylation sites quantified, the authors found that 578 phosphorylation sites were significantly changed in abundance in PKAintact and 267 sites were significantly changed in PKA-null cells.…”

Section: Effect Of Kinase Inhibitor H89 On Protein Phosphorylatimentioning

confidence: 99%

Phosphoproteomic Identification of Vasopressin/cAMP/Protein Kinase A–Dependent Signaling in Kidney

Salhadar

Matthews

Raghuram

et al. 2021

Molecular Pharmacology

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Water excretion by the kidney is regulated by the neurohypophyseal peptide hormone vasopressin through actions in renal collecting duct cells to regulate the water channel protein, aquaporin-2. Vasopressin signalling is initiated by binding to a G-protein coupled receptor V2R, which signals through Gsα, adenylyl cyclase 6, and activation of the cAMP-regulated protein kinase (PKA). Signaling events coupling PKA activation and aquaporin-2 regulation were largely unknown until the advent of modern protein mass spectrometry techniques that allow proteomewide quantification of protein phosphorylation changes (phosphoproteomics). This short review documents phosphoproteomic findings in collecting duct cells describing the response to V2selective vasopressin agonists and antagonists, the response to CRISPR-mediated deletion of PKA, results from in vitro phosphorylation studies using recombinant PKA, the response to the broad spectrum kinase inhibitor H89, and the responses underlying lithium-induced nephrogenic diabetes insipidus. These phosphoproteomic datasets have been made available online for modelling vasopressin signalling and signalling downstream from other Gsα-coupled receptors.

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Phosphorylation Changes in Response to Kinase Inhibitor H89 in PKA-Null Cells

Cited by 30 publications

References 25 publications

Phosphoproteomic identification of vasopressin‐regulated protein kinases in collecting duct cells

Phosphoproteomic identification of vasopressin‐regulated protein kinases in collecting duct cells

Glucagon Prevents Cytotoxicity Induced by Methylglyoxal in a Rat Neuronal Cell Line Model

Phosphoproteomic Identification of Vasopressin/cAMP/Protein Kinase A–Dependent Signaling in Kidney

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