The oligopeptide DT‐2 is a specific PKG I inhibitor only in vitro, not in living cells

Abstract

cGMP is involved in the regulation of many cellular processes including cardiac and smooth muscle contractility, aldosterone synthesis and inhibition of platelet activation. Intracellular effects cGMP are mediated by cGMP-dependent PKs, cGMP-regulated PDEs and cGMP-gated ion channels. PKG inhibitors are widely used to discriminate PKG-specific effects. They can be divided into cyclic nucleotide-binding site inhibitors such as Rp-phosphorothioate analogues (Rp-cGMPS), ATP-binding site inhibitors such as KT5823, and substrate binding site inhibitors represented by the recently described DT-oligopeptides. As it has been shown that Rp-cGMPS and KT5823 have numerous non-specific effects, we analysed the pharmacological properties of the oligopeptide (D)-DT-2 described as a highly specific, membrane-permeable, PKG inhibitor. Specificity and potency of (D)-DT-2 to inhibit PKG activity was evaluated using biochemical assays in vitro and by substrate phosphorylation analysis in various cell types including human platelets, rat mesangial cells and rat neonatal cardiomyocytes. Despite potent inhibition of PKGI in vitro, (D)-DT-2 lost specificity for PKG in cell homogenates and particularly in living cells, as demonstrated by phosphorylation of different substrates. Instead, (D)-DT-2 modulated activity of other kinases including ERK, p38, PKB and PKC, thereby inducing unpredicted and often opposing functional effects. We conclude that DT-oligopeptides, as other inhibitors, cannot be used to specifically inhibit PKG in intact cells. Therefore, no specific pharmacological PKG inhibitors are available, and reliable studies of PKG signalling can only be made by using RNA knockdown or genetic deletion methods.