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

Scientific Reports
Kavee LimbutaraMark A Knepper

Abstract

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 mouse cell lines derived from mpkCCD cells were treated with H89 over a range of concentrations commonly used in the literature, followed by mass spectrometry-based phosphoproteomics to globally assess changes in phosphorylation. From a total of 14,139 phosphorylation sites quantified, we found that 571 and 263 phosphorylation sites with significant changes in abundance in PKA-intact and PKA-null cells, respectively. Analyses of sequence logos generated from significantly decreased phosphorylation sites in PKA-intact and PKA-null cells both revealed a preference for basic amino acids at position -3 and -2. Thus, H89 appears to inhibit basophilic kinases even in the absence of PKA. Likely H89 targets include basophilic protein kinases su...Continue Reading

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Citations

Oct 6, 2020·American Journal of Physiology. Renal Physiology·Viswanathan RaghuramMark A Knepper
Dec 22, 2020·British Journal of Pharmacology·Arnab DattaMark A Knepper
Mar 7, 2021·Biomolecules·Mohammad Sarif MohiuddinHideki Kamiya
Apr 7, 2021·Infection and Immunity·Karissa J MuñozChristine Sütterlin
Jul 1, 2021·Cell Reports·Mohammad Abu-OdehAlan R Saltiel
Jul 3, 2021·Nature Reviews. Nephrology·Emma T B Olesen, Robert A Fenton

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Datasets Mentioned

BETA
E9Q616

Methods Mentioned

BETA
Protein Assay
Assay
reverse phase chromatography

Software Mentioned

Perseus
Excel
Linear Models for Microarray Data ( LIMMA ) R
R
MaxQuant
PhosphoLogo

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