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. 2000 Dec 19;97(26):14772-7.
doi: 10.1073/pnas.97.26.14772.

Highly specific, membrane-permeant peptide blockers of cGMP-dependent protein kinase Ialpha inhibit NO-induced cerebral dilation

W R Dostmann  1 , M S Taylor, C K Nickl, J E Brayden, R Frank, W J Tegge
Affiliations

Highly specific, membrane-permeant peptide blockers of cGMP-dependent protein kinase Ialpha inhibit NO-induced cerebral dilation

W R Dostmann et al. Proc Natl Acad Sci U S A. .

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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Figures

Figure 1
Figure 1
Evolution of peptide libraries for the iterative screening of cGPK binding sequences. A, B, C, and F show phosphorescence images of consecutive generations of the library screens after binding of 32P-labeled cGPK. Each library membrane carries 18 × 18 spots, which resulted from substitutions of 18 amino acids (Ser and Thr were omitted) at positions 1 and 2 in the peptides, both positions being varied in alphabetical order according to the single letter code. The best combinations are indicated with arrows. (D) Western blot of material eluted from excised sublibrary spots (red numbers in C) by using a cGPK I-specific antibody. (E) PhosphorImage of the blot shown in D.
Figure 2
Figure 2
Translocation of fluorescein-labeled MTS fusion peptides (Fluo-DT-2 and Fluo-DT-3) in smooth muscle cells from cerebral arteries. The arteries were isolated from rat and incubated for 30 min with Fluo-DT-2 (A), Fluo-DT-3 (B), or Fluo-W45 (C). Confocal images (100×) of tissue samples were taken after washing arteries with physiological salt solution. Fluorescein excitation was imaged at 488 nm. Images show the smooth muscle layer of the arteries (optical thickness = 1.5 μm).
Figure 3
Figure 3
Differential inhibition of recombinant cGPK and cAPK by DT-3, DT-2, and PKI(5–24). The assays contained 1 nM enzyme, 16 μM substrate peptide TQAKRKKSLAMA (13), 1 μM cAMP/cGMP, 70 nM PKI(5–24), and 200 nM DT-2 or DT-3, as indicated. Kinase activity was determined for 1.5 min at 30°C in a final volume of 100 μl as described (13).
Figure 4
Figure 4
Inhibition of endogenous cGPK activity in human aortic smooth muscle cells by internalization of exogenous MTS-fusion peptides DT-2 and DT-3. Cells were preincubated with either W45, DT-5, DT-3, DT-6, or DT-2, and then cell extracts were assayed for kinase activity (n = 3–6). Endogenous cAPK activity was blocked with PKI(5–24). * and + indicate significant differences (P < 0.05, ANOVA followed by Bonferroni post hoc test) from the untreated and W45 treated control groups, respectively.
Figure 5
Figure 5
Inhibition of NO-mediated vasodilation of intact cerebral arteries by DT-2 and DT-3. Pressurized segments of rat posterior cerebral artery were dilated with the NO donor NONOate (1 nM to 1 mM, indicated by arrows) in the presence or absence of the fusion peptides DT-2 and DT-3 for 25 min. (A) Continuous diameter tracings are shown for untreated, DT-2 treated, and DT-3 treated vessels. (B) NONOate dose–response curves in untreated arteries (n = 11) or arteries exposed to DT-2 (n = 6) or DT-3 (n = 6).
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