摘要：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 such as AKT, RSK, AMPK and ROCK. We conclude that, in intact cells, H89 can affect activities of protein kinases other than PKA, and therefore responses to H89 should not be regarded as sufficient evidence for PKA involvement in a signaling process.
