Dataset Information

H89 Phosphoproteomics in PKA-Null Cells

ABSTRACT: Protein phosphorylation plays a crucial role in propagating cellular responses to both internal and external cues. One of the most important kinases responsible for protein phosphorylation is protein kinase A (PKA). N-[2-p-bromocinnamylamino-ethyl]-5-isoquinolinesulphonamide (H89) is often used as a “PKA specific inhibitor” to study the involvement of PKA in signaling pathways. However, evidence from cell-free experiments suggested that H89 can also inhibit other kinases. In this experiment, previously-generated mouse collecting duct cell lines with and without PKA were treated with H89, followed by mass spectrometry-based phosphoproteomics to globally assess changes in phosphorylation.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Epithelial Cell, Cell Culture

SUBMITTER: Kavee Limbutara

LAB HEAD: Mark A. Knepper

PROVIDER: PXD011167 | Pride | 2019-03-28

REPOSITORIES: Pride

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Publications

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

Limbutara Kavee K Kelleher Andrew A Yang Chin-Rang CR Raghuram Viswanathan V Knepper Mark A MA

Scientific reports 20190226 1

Protein phosphorylation, mediated by protein kinases, plays a crucial role in cellular regulation. One of the most important protein kinases is protein kinase A (PKA). N-[2-p-bromocinnamylamino-ethyl]-5-isoquinolinesulphonamide (H89) is often used as a "PKA specific inhibitor" to study the involvement of PKA in signaling pathways. However, evidence from cell-free experiments has suggested that H89 can also inhibit other protein kinases. In this study, previously generated PKA-null and PKA-intact ...[more]

PMID: 30808967

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Project description:Purpose: Deregulated phosphatidylinositol 3-kinase pathway signaling through AGC kinases including AKT, p70S6 kinase, PKA, SGK and Rho kinase, is a key driver of multiple cancers. The simultaneous inhibition of multiple AGC kinases may increase antitumor activity and minimize clinical resistance compared with a single pathway component. Experimental Design: We investigated the detailed pharmacology and antitumor activity of the novel clinical drug candidate AT13148, an oral ATP-competitive multi-AGC kinase inhibitor. Gene expression microarray studies were undertaken to characterize the molecular mechanisms of action of AT13148. Results: AT13148 caused substantial blockade of AKT, p70S6K, PKA, ROCK and SGK substrate phosphorylation and induced apoptosis in a concentration and time-dependent manner in cancer cells with clinically relevant genetic defects in vitro and in vivo. Antitumor efficacy in HER2-positive, PIK3CA-mutant BT474 breast, PTEN-deficient PC3 human prostate cancer and PTEN-deficient MES-SA uterine tumor xenografts was demonstrated. We show for the first time that induction of AKT phosphorylation at serine 473 by AT13148, as reported for other ATP-competitive inhibitors of AKT, is not a therapeutically relevant reactivation step. Gene expression studies showed that AT13148 has a predominant effect on apoptosis genes, whereas the selective AKT inhibitor CCT128930 modulates cell cycle genes. Induction of upstream regulators including IRS2 and PIK3IP1 due to compensatory feedback loops was observed. Conclusions: The clinical candidate AT13148 is a novel oral multi-AGC kinase inhibitor with potent pharmacodynamic and antitumor activity, which demonstrates a distinct mechanism of action from other AKT inhibitors. AT13148 will now be assessed in a first-in-human Phase I trial. The PTEN-deficient U87MG glioblastoma cell line was treated for 6 hours with vehicle control (DMSO) or to different concentrations of AT13148 and CCT128930 (0.1uM, 1xGI50 and 3XGI50).

Dataset Information

H89 Phosphoproteomics in PKA-Null Cells

Publications

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

Similar Datasets

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