The Commonly Used cGMP-dependent Protein Kinase Type I (cGKI) Inhibitor Rp-8-Br-PET-cGMPS Can Activate cGKI in Vitro and in Intact Cells

Abstract

cGMP is a cyclic-nucleotide second messenger with multiple targets and functions. Small-molecule modulators of cGMP generators and effectors are important biochemical tools as well as established and prospective drugs for the treatment of human diseases, such as erectile dysfunction, pulmonary hypertension, and various cardiovascular disorders (1-3). cGMP is generated by nitric oxide-or natriuretic peptide-stimulated guanylyl cyclases and can bind to and modulate the activity of at least three classes of cGMP effector proteins: cyclic nucleotide-hydrolyzing phosphodiesterases (PDEs), 2 cyclic nucleotide-gated cation channels, and cGMP-dependent protein kinases (cGKs, also known as protein kinase G or PKG) (4).Based on pharmacological and genetic studies, the cGK type I (cGKI) is considered the major mediator of cGMP signaling in many tissues including the cardiovascular system (5-8). The mammalian cGKI is a cytosolic Ser/Thr protein kinase comprising an N-terminal regulatory domain with two cGMPbinding sites and a C-terminal catalytic domain. It exists in two isoforms termed cGKI␣ and cGKI␤. The isozymes have identical cGMP-binding sites and catalytic domains but differ in their N-terminal ϳ100 amino acids, which contribute to homodimerization, sensitivity to cGMP activation, and interaction with anchoring and substrate proteins. Recent in vivo studies with transgenic mice demonstrated that both isoforms can induce smooth muscle relaxation and vasodilation (9), but the respective molecular mechanisms behind these effects are controversial (6, 10). Similarly, opposing effects of cGMP/cGKI signaling have been reported on the growth and phenotype of vascular smooth muscle cells (VSMCs) (11,12). The inconsistency of the results concerning the function of cGKI might in part be related to unexpected effects of the pharmacological cGKI activators and inhibitors that are commonly used to distinguish between cGKI-dependent and cGKI-independent signaling. For instance, cGMP analogues can bind to multiple * This work was supported by grants from the Deutsche Forschungsgemeinschaft. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 2 The abbreviations used are: PDE, phosphodiesterase; cGKI, cGMP-dependent protein kinase type I; PET, 8-Br-PET-cGMP; Rp-PET, Rp-8-Br-PETcGMPS; 8-Br-PET-cGMP, ␤-phenyl-1,N 2 -etheno-8-bromoguanosine-3Ј,5Ј-cyclic monophosphate; Rp-8-pCPT-cGMPS, 8-(4-chlorophenylthio)-guanosine-3Ј, 5Ј-cyclic monophosphorothioate, Rp-isomer; Rp-8-Br-PET-cGMPS, ␤-phenyl-1,N 2 -etheno-8-bromoguanosine-3Ј,5Ј-cyclic monophosphorothioate, Rp-isomer; VASP, vasodilator-stimulated phosphoprotein; VSMC, vascular smooth muscle cell; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; ESI-MS, electrospray ionization-mass spectrometry; ...

The roles of protein kinases in learning and memory

“…This suggests that PKG signaling in the amygdala is required for LTM formation. Consistent results have been obtained with pharmacological inhibition in the amygdala, although there is concern about the specificity of PKG blockers (Valtcheva et al 2009). Post-training injection of the most specific PKG inhibitor into the lateral amygdala impairs cued fear LTM (Ota et al 2008).…”

Catalytic Activity of cGMP-Dependent Protein Kinase Type I in Intact Cells Is Independent of N-Terminal Autophosphorylation

“…Antibodies used were rabbit anti-cGKI common (DH) (1∶5000), a pan-specific (nonphospho-specific) antiserum detecting both cGKIα and cGKIβ [26], rabbit anti-VASP (1∶1000, Cell Signaling, 9A2, 3132), rabbit anti-GAPDH (1∶5000, Cell Signaling, 14C10, 2118), and rabbit anti-Akt (1∶1000; Cell Signaling, 9272). The polyclonal rabbit antisera against phospho-cGKI species that were generated and characterized in this study were AffPS3 (1∶100), PS6 (1∶2000), and PS7 (1∶2000).…”

cGMP-Dependent Protein Kinases and cGMP Phosphodiesterases in Nitric Oxide and cGMP Action

“…Some cN analogs (e.g., 1,N 2 -phenyletheno-cGMP) are more potent PKGI activators than cGMP, and others, (e.g., 8-bromo-␤-phenyl-1,N 2 -ethenoguanosine-3Ј,5Ј-cyclic monophosphorothioate, Rp-isomer) bind to the allosteric site but only partially activate catalysis; the latter compound is commonly used as a PKGI inhibitor because it blocks access of the more effective activator, cGMP, to the binding sites (Sekhar et al, 1992;Butt et al, 1994b;Taylor et al, 2004;Poppe et al, 2008;Valtcheva et al, 2009). Cyclic nucleotide analogs have proved to be highly useful tools in investigating biological effects mediated by PKAs, EPACs, or PKGs; the analogs freely traverse the cell membrane to directly act on the target proteins, thereby circumventing involvement of proteins that generate the signaling molecule, factors involved in delivery of the signal to the target cell, specific membrane receptors, or the adenylyl or guanylyl cyclases that produce the cNs (Beebe et al, 1988a,b;Francis et al, 1988;Christensen et al, 2003;Dao et al, 2006;Poppe et al, 2008).…”