Endogenous Type II cGMP-dependent Protein Kinase Exists as a Dimer in Membranes and Can Be Functionally Distinguished from the Type I Isoforms

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; ...

Section: Resultssupporting

confidence: 80%

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

Valtcheva

Nestorov

Beck

et al. 2009

“…Lastly, PET-cGMP is a less potent activator of PKG II because of the derivatization of the purine ring of cGMP into 1,N 2 -ethenoguanosine. This prevents the favorable hydrogen bonding between Asp-412 and the N1 and C2 positions of cGMP and may reduce the binding affinity (32).…”

Section: Discussionmentioning

confidence: 99%

Structural Basis of Cyclic Nucleotide Selectivity in cGMP-dependent Protein Kinase II

Campbell¹,

Kim

et al. 2016

Membrane-bound cGMP-dependent protein kinase (PKG) II is a key regulator of bone growth, renin secretion, and memory formation. Despite its crucial physiological roles, little is known about its cyclic nucleotide selectivity mechanism due to a lack of structural information. Here, we find that the C-terminal cyclic nucleotide binding (CNB-B) domain of PKG II binds cGMP with higher affinity and selectivity when compared with its N-terminal CNB (CNB-A) domain. To understand the structural basis of cGMP selectivity, we solved co-crystal structures of the CNB domains with cyclic nucleotides. Our structures combined with mutagenesis demonstrate that the guanine-specific contacts at Asp-412 and Arg-415 of the ␣C-helix of CNB-B are crucial for cGMP selectivity and activation of PKG II. Structural comparison with the cGMP selective CNB domains of human PKG I and Plasmodium falciparum PKG (PfPKG) shows different contacts with the guanine moiety, revealing a unique cGMP selectivity mechanism for PKG II.

“…Ref. 17) that are generally employed in autophosphorylation studies, because these require a high specific radioactivity of ATP. Under these conditions cGMP stimulated the autophosphorylation of rat recombinant cGK II expressed in COS-1 cells only 50%, because cGK II was already autophosphorylated to a considerable level in the absence of cGMP.…”

Section: Resultsmentioning

confidence: 99%

“…Reactions were stopped, and cyclic nucleotides were removed by a 250-fold dilution in the same buffer without ATP or cyclic nucleotides (resulting in a final 1000-fold dilution in the assay). Protein kinase activity was determined by incubation of the samples (4 ng of control or autophosphorylated His-cGK II or 4 -10 g of COS membrane protein in case of transiently expressed rat cGK II or mutants thereof) at 30°C for 4 min in 40 l of 20 mM Tris/HCl, pH 7.4, 0.15 M NaCl, 10 mM MgCl 2 , 5 mM ␤-mercaptoethanol, 0.1 mM 3-isobutyl-1-methylxanthine, 25 mM sodium ␤-glycerophosphate, 0.25% Triton X-100, 200 nM protein kinase A inhibitor, 0.1 mg/ml of a cGK substrate peptide 2A3 (RRKVSKQE (17) The amount of label incorporated into 2A3 was quantified as described previously (17).…”

Section: Construction and Expression Of Mutants Of Cgk Ii-thementioning

confidence: 99%

Autophosphorylation of cGMP-dependent Protein Kinase Type II

Vaandrager

Hogema

Edixhoven

et al. 2003

Self Cite

was found to be phosphorylated in a cGMP-dependent manner, whereas Ser 110 and Ser 97 were already prephosphorylated to a large extent in Sf9 cells. cGMP-dependent phosphorylation of cGK II was also demonstrated in intact COS-1 cells and intestinal epithelium. Substitution of most of the potentially autophosphorylated residues for alanines largely abolished the cGMP stimulation of the autophosphorylation. Prolonged autophosphorylation of purified recombinant cGK II in vitro resulted in a 40 -50% increase in basal kinase activity, but its maximal cGMP-stimulated activity and the EC 50 for cGMP remained unaltered. Mutation of the major phosphorylatable serines 110, 114, and 445 into "phosphorylation-mimicking" glutamates had no effect on the kinetic parameters of cGK II. However, replacing the slowly autophosphorylated residue Ser 126 by Glu rendered cGK II constitutively active. These results show that the fast phase of cyclic nucleotide-stimulated autophosphorylation of cGK II has a relatively small feed forward effect on its activity, whereas the secondary phase, presumably involving Ser 126 phosphorylation, may generate a constitutively active form of the enzyme.