In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 97, No. 26 ( 2000-12-19), p. 14772-14777
Abstract:
Arrays of octameric peptide libraries on cellulose paper were
screened by using 32 P-autophosphorylated cGMP-dependent
protein kinase Iα (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 μM and 560 μM 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.
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.97.26.14772
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
2000
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
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