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Dostmann, W.R. et al. Highly specific, membrane-permeant peptide blockers of cGMP-dependent protein kinase I inhibit NO-induced cerebral dilation. Proc. Natl. Acad. Sci. USA 97, 14772-14777

Department of Pharmacology, Department of Molecular Physiology and Biophysics, University of Vermont, College of Medicine, Burlington, VT 05405-0068, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 01/2001; 97(26):14772-7. DOI: 10.1073/pnas.97.26.14772
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ABSTRACT Arrays of octameric peptide libraries on cellulose paper were screened by using (32)P-autophosphorylated cGMP-dependent protein kinase Ialpha (cGPK) to identify peptide sequences with high binding affinity for cGPK. Iterative deconvolution of every amino acid position in the peptides identified the sequence LRK(5)H (W45) as having the highest binding affinity. Binding of W45 to cGPK resulted in selective inhibition of the kinase with K(i) values of 0.8 microM and 560 microM for cGPK and cAMP-dependent protein kinase (cAPK), respectively. Fusion of W45 to membrane translocation signals from HIV-1 tat protein (YGRKKRRQRRRPP-LRK(5)H, DT-2) or Drosophila Antennapedia homeo-domain (RQIKIWFQNRRMKWKK-LRK(5)H, DT-3) proved to be an efficient method for intracellular delivery of these highly charged peptides. Rapid translocation of the peptides into intact cerebral arteries was demonstrated by using fluorescein-labeled DT-2 and DT-3. The inhibitory potency of the fusion peptides was even greater than that for W45, with K(i) values of 12.5 nM and 25 nM for DT-2 and DT-3, respectively. Both peptides were still poor inhibitors of cAPK. Selective inhibition of cGPK by DT-2 or DT-3 in the presence of cAPK was demonstrated in vitro. In pressurized cerebral arteries, DT-2 and DT-3 substantially decreased NO-induced dilation. This study provides functional characterization of a class of selective cGPK inhibitor peptides in vascular smooth muscle and reveals a central role for cGPK in the modulation of vascular contractility.

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    • "These results suggested that the binding of MYPT1 to PKG1α is not mediated by a LZ-LZ interaction. Because PKG1α prefers binding to RR and RK motifs (Dostmann et al., 2000) and there is an RK motif in the aa 888–928 sequence of MYPT1, the relevance of this sequence was investigated (Given et al., 2007). Mutants were generated, which lack or contain this sequence and which also lack or contain the leucine zipper. "
    Affinity Chromatography, 03/2012; , ISBN: 978-953-51-0325-7
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    • "Recently, peptide inhibitors that are highly selective for PKG-I have been discovered using a phage display library approach (Dostmann et al., 2000). DT-2 and DT-3 are 1000-fold more selective for PKG vs. PKA and exhibit a 100-fold selectivity for PKG-I vs. PKG-II (Dostmann et al., 2000). These inhibitors, thus, represent useful pharmacological tools when the need to determine the contribution of PKG-I in a biological response arises. "
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    ABSTRACT: Vascular endothelial growth factor (VEGF) stimulates nitric oxide (NO) production, which mediates many of its angiogenic actions. However, the angiogenic pathways that operate downstream of NO following VEGF treatment are not well characterized. Herein, we used DT-2 and DT-3, two highly selective cGMP-dependent protein kinase I peptide inhibitors to determine the contribution of PKG-I in VEGF-stimulated angiogenesis. Incubation of chicken chorioallantoic membranes (CAM) with PKG-I peptide inhibitors decreased vascular length in a dose-dependent manner, with DT-3 being more effective than DT-2. Moreover, inhibition of PKG-I with DT-3 abolished the angiogenic response elicited by VEGF in the rabbit eye cornea. PKG-I inhibition also blocked VEGF-stimulated vascular leakage. In vitro, treatment of cells with VEGF stimulated phosphorylation of the PKG substrate VASP through VEGFR2 activation; the VEGF-stimulated VASP phosphorylation was reduced by DT-2. Pre-treatment of cells with DT-2 or DT-3 inhibited VEGF-stimulated mitogen-activated protein kinase cascades (ERK1/2 and p38), growth, migration and sprouting of endothelial cells. The above observations taken together identify PKG-I as a downstream effector of VEGFR2 in EC and provide a rational basis for the use of PKG-I inhibitors in disease states characterized by excessive neovascularization.
    Vascular Pharmacology 11/2010; 53(5-6):215-22. DOI:10.1016/j.vph.2010.08.004 · 4.62 Impact Factor
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    • "To determine the role of basal PKG activity in regulating the rates of apoptosis, DNA synthesis, and proliferation, the PKG inhibitors DT-2 and DT-3 were used. DT-2 and DT-3 are unique among the various PKG inhibitors because of their high-level selectivity for PKG and the capability to inhibit not only stimulated PKG activity but also basal PKG activity [29] [30] [31]. "
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    ABSTRACT: Protein kinase G (PKG), a recognized downstream mediator of nitric oxide, is a key regulator of cardiovascular physiology and pathology. High-level stimulation of cyclic guanosine monophosphate/PKG signaling using high concentrations of nitric oxide donors, mimicking pathological conditions, induces apoptosis in vascular smooth muscle cells. In contrast, we have found that PKG at basal and moderately elevated activity prevents both spontaneous and toxin-induced apoptosis in many other cells. We hypothesized that PKG's apoptosis-regulatory role in vascular smooth muscle cells depends on PKG activation levels [low/basal-level activation prevents apoptosis, whereas high-level activation (hyperactivation) causes apoptosis]. Furthermore, we hypothesized that, although PKG hyperactivation inhibits vascular smooth muscle cell proliferation (potentially causing anti-atherogenic effects), basal PKG activity may promote vascular smooth muscle cell proliferation/atherogenesis. Involvement of PKG in apoptosis and proliferation was determined in unpassaged vascular smooth muscle cells from mouse aorta. Western blot analysis was used to determine PKG expression, and activators/inhibitors of PKG activity were used to determine involvement in apoptosis (Hoechst staining and DNA-fragmentation ELISAs) and proliferation (cell count, MTT assay, and BrdU incorporation). Both PKG-Iα and PKG-Iβ isoforms were expressed. Lower-level stimulation of PKG using the nitric oxide donor S-nitroso-acetylpenacillamine (10, 50 μM) significantly (P<.05) lowered spontaneous apoptosis, whereas S-nitroso-acetylpenacillamine at higher concentrations (500, 1000 μM) elevated apoptosis. Twenty-four-hour pretreatment with atrial natriuretic peptide, a PKG activator, completely prevented high-concentration, nitric oxide-induced apoptosis. Inhibition of basal PKG activity using highly selective PKG inhibitors, DT-2 and DT-3, significantly (P<.001) increased apoptosis and inhibited DNA synthesis/proliferation. The data suggest that basal/moderately elevated PKG activity protects against high/pathological-level nitric oxide-induced apoptosis and promotes DNA synthesis/proliferation in vascular smooth muscle cells, potentially important for atherogenesis.
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