Highly specific, membrane-permeant peptide blockers
of cGMP-dependent protein kinase I? inhibit
NO-induced cerebral dilation
Wolfgang R. G. Dostmann*†, Mark S. Taylor*, Christian K. Nickl*, Joseph E. Brayden*, Ronald Frank‡,
and Werner J. Tegge‡
*Department of Pharmacology, Department of Molecular Physiology and Biophysics, University of Vermont, College of Medicine,
Burlington, VT 05405-0068; and‡AG Molecular Recognition, Gesellschaft fu ¨r Biotechnologische Forschung,
Mascheroder Weg 1, D-38124 Braunschweig, Germany
Communicated by Susan S. Taylor, University of California, San Diego, CA, October 3, 2000 (received for review September 5, 2000)
Arrays of octameric peptide libraries on cellulose paper were
screened by using32P-autophosphorylated cGMP-dependent pro-
tein kinase I? (cGPK) to identify peptide sequences with high
binding affinity for cGPK. Iterative deconvolution of every amino
acid position in the peptides identified the sequence LRK5H (W45)
as having the highest binding affinity. Binding of W45 to cGPK
resulted in selective inhibition of the kinase with Kivalues of 0.8
?M and 560 ?M for cGPK and cAMP-dependent protein kinase
(cAPK), respectively. Fusion of W45 to membrane translocation
signals from HIV-1 tat protein (YGRKKRRQRRRPP-LRK5H, DT-2) or
LRK5H, 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 Ki
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 substan-
tially decreased NO-induced dilation. This study provides func-
tional characterization of a class of selective cGPK inhibitor pep-
tides in vascular smooth muscle and reveals a central role for cGPK
in the modulation of vascular contractility.
protein kinase inhibitor ? combinatorial libraries ? SPOT method ?
membrane translocation signal ? smooth muscle
way, controlling a variety of cellular responses, ranging from
smooth muscle cell relaxation to neuronal synaptic plasticity (1,
2). The structural similarity of cGPK and its closest relative, the
cAMP-dependent protein kinase (cAPK), has made it difficult
to study cGPK pathways independent of those mediated by
cAPK, primarily because of the lack of potent and selective
cGPK inhibitors. Because recent studies have suggested that
cAMP and cGMP are each able to cross-activate either cGPK or
cAPK under physiological conditions, the specific role for cGPK
within the NO?cGMP-mediated signaling pathway remains ob-
scure (for a review see ref. 1). However, recent advances have
clearly identified specific intracellular targets for the cGPK
isozymes I? and I? (3, 4). Also, inactivation of the genes for
cGPK I??I? and cGPK II showed that the cGPK isozymes
regulate distinct cellular functions by pathways separate from
those mediated by cAPK (5, 6).
Attempts to identify cGPK-selective inhibitor peptides based
on the autoinhibitory domain of the enzyme or in vivo substrates
have been tedious at best, because of the lack of a well defined
surrounding the phosphate acceptor site has been established
(7). Various synthetic peptides have been used to analyze the
he cGMP-dependent protein kinases type I? and I? (cGPK)
act directly downstream in the NO-mediated signaling path-
sequence requirements for cGPK substrates (8–11). Recently,
we developed an iterative approach using phosphorylation of
peptide libraries on cellulose paper to determine a priori the
substrate specificity of cGPK versus cAPK. Consequently, we
identified the cGPK substrate sequence TQAKRKKSLAM-
FLR, in which the serine represents the phosphate-acceptor site
(12, 13). Substitution of this serine by alanine yielded cGPK
inhibitors with Ki values of 7.5–22 ?M (13) and improved
cGPK?cAPK selectivity, as has been reported with other syn-
thetic peptide derivatives (14, 15). However, all cGPK peptide
inhibitors known so far lack satisfactory potency and selectivity.
Here we report a peptide library screen specifically designed
First, we took advantage of the autophosphorylation properties
of cGPK, which provides the means to study the transient
enzyme–peptide interactions. Second, we used peptide libraries
that lack the phosphate acceptor residues serine and threonine
to select for peptide binding over phosphorylation. Linking the
best sequence from this screen to membrane translocation
signals (MTS) for intracellular delivery resulted in the highly
effective cGPK I? inhibitors DT-2 and DT-3. Finally, we have
demonstrated that both peptides are powerful tools for studying
the specific functional roles of cGPK in smooth muscle.
Enzyme Preparations. cAPK-C? was expressed and purified from
Escherichia coli (16), and cGPK type I? was expressed and
purified from SF9-insect cells as described in ref. 13.32P-labeling
of cGPK was accomplished by incubating the enzyme in buffer
A (50 mM Mops, pH 6.9?0.4 mM EDTA?1 mM Mg-
acetate?200 mM NaCl?1 mg/ml BSA?10 mM DTT) and
[?-32P]-ATP (0.1 mM; specific activity 1,600 cpm?pmol) for 2 h
at room temperature. Excess label was removed by using G-25
Sepharose chromatography, and SDS?PAGE and Western blot-
ting were performed (17).
Synthesis of Peptide Libraries and cGPK Screening. The peptide
arrays on paper (SPOTs) were generated as described in refs. 12,
13, and 18. Ser and Thr were omitted from the defined and the
randomized positions resulting in arrays of 18 ? 18 sublibrary
spots. The following arrays were generated and used successively
in the iterative approach: 1, XXX12XXX; 2, XXXRK12X;
3, XRKKK12X; 4, 1RKKKKK2; 5, LRKKKKKH12; 6,
Abbreviations: cAPK, cAMP-dependent protein kinase; cGPK, cGMP-dependent protein
kinase; MTS, membrane translocation signal; PKI, protein kinase inhibitor.
†To whom reprint requests should be addressed. E-mail: firstname.lastname@example.org.
The publication costs of this article were defrayed in part by page charge payment. This
article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.
§1734 solely to indicate this fact.
December 19, 2000 ?
vol. 97 ?
To deliver the highly charged peptide W45 into living cells, we
synthesized two fusion peptides, DT-2 and DT-3, with MTS
sequences from the tat protein and from the antennapedia
homeodomain. Cellular internalization of the fusion peptides
was extensive (Fig. 2). Interestingly, the inhibitory potencies of
DT-2 and DT-3 were profoundly enhanced compared with the
inhibitory potency of W45 alone. However, the explanation for
this synergism is presently unclear. The MTS sequences (DT-5
and DT-6), perhaps because they are positively charged them-
selves, inhibited cGPK and cAPK (Table 1). The nature of the
cGPK inhibition by these peptides was of a linear mixed com-
petitive?noncompetitive type (Dixon plot analysis, data not
shown), involving two different and mutually exclusive binding
sites. This finding suggests that the membrane translocation
peptides can bind and inhibit cGPK at a different site from the
catalytic cleft. Thus, fusion of the MTS sequences to W45 may
yield the observed synergistic and competitive inhibition by
linking the MTS and W45 affinity binding motifs in a single
peptide sequence. Irrespective of the mechanism, the observed
synergism improves the utility of these compounds in biological
Inhibitors of cGPK with nanomolar inhibition constants have
not been reported, and the observed selectivities over cAPK of
20,000 (DT-3) and 1,300 (DT-2) are exceptional among protein
kinase inhibitors. We also observed highly selective cGPK
inhibition by the fusion peptides in mixtures of recombinant
cGPK?cAPK, as well as in intact cells. Clearly more work is
needed to investigate the mechanism of uptake, cellular distri-
bution, temporal dynamics, and proteolytic stability of these
peptides. We are currently addressing these issues by the use of
retro-inverse sequences of DT-2 and DT-3.
NO has a central role in vascular biology, but its mechanisms
of action have not yet been fully elucidated (1). NO-induced
relaxation of vascular smooth muscle does seem to involve
activation of cGPK, leading to alterations in [Ca2?]iand effects
on myosin light chain kinase and phosphatase activities (1, 3). It
was reasonable, therefore, to look for functional effects of the
cGPK-inhibitor peptides in intact arteries by using a NO donor
as a vasodilator agent. DT-2 and DT-3 decreased the dilator
potency of NONOate by 50- to 100-fold when applied to intact
of functionally active inhibitors into the vascular smooth muscle
The parallel shift in dose–response curves suggests that a
competitive inhibition of cGPK by the inhibitor peptides, as
observed for the purified enzyme, also occurs in vascular smooth
muscle cells in situ. In contrast to the inhibitory effects of the
carrier peptides DT-5 and DT-6 on purified cGPK in vitro, we
observed no appreciable effects of the carriers alone on the
NO-induced dilations of intact arteries. We infer from this
observation that, at the extracellular concentrations used in the
intact artery experiments, the carrier sequences do not reach
We did note some direct contractile action of DT-2 or DT-3
when applied in concentrations greater than 2 ?M or 500 nM,
respectively. This observation underscores the need for careful
study. A possible explanation for the contractile activity of the
inhibitors is that basally active cGPK may be important in
regulating cerebral arterial diameter, but further studies are
required to verify this action.
The present study has resulted in the discovery of selective
inhibitors of cGPK and has defined an effective means for
intracellular delivery of these compounds. Further, we have
demonstrated the ability of the inhibitors to alter NO-induced
cerebral vasodilation and substantiated a central role of cGPK as
a mediator of this response. The development of these mem-
brane-permeable, selective cGPK inhibitors should allow much
clearer dissection of the roles of cGPK in living cells than has
heretofore been possible.
We thank B. Kornak and S. Daenicke for excellent technical assistance
with the peptide synthesis and Drs. Mark Nelson and Karen Lounsbury
for their helpful reviews. This work was supported by Deutsche For-
schungsgemeinschaft Grants Do329?3-3 and Do329?4-1, the Lake
Champlain Cancer Research Organization and the Totman Medical
Research Trust (to W.R.G.D.), and National Institutes of Health Grants
HL44455 (to M.S.T.) and HL58231 (to J.E.B.).
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Table 2. Summary of peptide effects on NONOate mediated
0.43 ? 0.15
47 ? 9*†
34 ? 18*‡
0.92 ? 0.67
0.13 ? 0.09
*, Indicates significant difference (P ? 0.05) from the untreated group,
whereas † and ‡ indicate significant difference (P ? 0.05) from DT-6 and
Dostmann et al.
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