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

Abstract

Arrays of octameric peptide libraries on cellulose paper were screened by using 32 protein kinase inhibitor ͉ combinatorial libraries ͉ SPOT method ͉ membrane translocation signal ͉ smooth muscle T he cGMP-dependent protein kinases type I␣ and I␤ (cGPK) act directly downstream in the NO-mediated signaling pathway, 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 NO͞cGMP-mediated signaling pathway remains obscure (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 consensus sequence. Only a relative preference for basic residues surrounding the phosphate acceptor site has been established (7). Various synthetic peptides have been used to analyze the 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 K i values of 7.5-22 M (13) and improved cGPK͞cAPK selectivity, as has been reported with other synthetic 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 to select for tight binding peptides rather than substrate peptides. 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. MethodsEnzyme Prepa...

Probing noncovalent protein—Ligand interactions of the cGMP-dependent protein kinase using electrospray ionization time of flight mass spectrometry

“…Recombinant PKG I␣ was expressed and purified from SF9-insect cells according to Dostmann et al [18]. Guanosine 3=,5=-cyclic monophosphate, Adenosine 5=-triphosphate sodium salt, ␤,␥-imidoadenosine 5=-triphosphate tetralithium salt, magnesium acetate tetrahydrate and manganese(II) chloride were purchased from Sigma (St. Louis, MO).…”

Possible Existence of Common Internalization Mechanisms among Arginine-rich Peptides

“…Basic peptides derived from the HIV-1 1 Tat protein (Tat-(48 -60)) and Drosophila Antennapedia protein (Antp-(43-58)) have been reported to have the ability to translocate through the cell membranes and to carry exogenous molecules into the cytoplasm and nucleus (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). A 119-kDa protein, ␤-galactosidase, genetically fused with the former peptide segment, was successfully carried into various tissues in mice including the brain via intraperitoneal injection (6).…”

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

“…Consequently, other classes of cGKI inhibitors are increasingly used for intact cell studies. These inhibitors are based on cell-permeable modified peptide substrates (18) or on the allosteric activator cGMP (see Fig. 1A) (19).…”