Christian K. Nickl

Delineation of Selective Cyclic GMP-Dependent Protein Kinase Iα Substrate and Inhibitor Peptides Based on Combinatorial Peptide Libraries on Paper

Peptide libraries on cellulose paper have proven to be valuable tools for the a priori determination of substrate specificities of cyclic AMP- and cyclic GMP-dependent protein kinases (cAMP-kinase and cGMP-kinase) on the basis of octa-peptide sequences. Here, we report the extension of our peptide library screens to 12-mer and 14-mer peptide sequences, resulting in highly cGMP-kinase Ialpha selective peptides. The sequences TQAKRKKSLAMA-amide and TQAKRKKSLAMFLR-amide, with Km values for cGMP-kinase Ialpha of 0.7 and 0.26 microM and Vmax values of 11.5 and 10.9 micromol/min/mg, respectively, display a high specificity for this enzyme. Furthermore, replacing the phosphate acceptor residue serine with alanine in TQAKRKKSLAMA-amide resulted in the highly cGMP-kinase Ialpha selective inhibitor peptide TQAKRKKALAMA-amide, with inhibitor constants for cGMP-kinase Ialpha and cAMP-kinase of 7.5 microM and 750 microM, respectively. Selective cGMP-kinase inhibitors have the potential to play an important role in the elucidation of the distinct cellular functions of cGMP-kinase separate from those activated by cAMP-kinases, and, therefore, may play an important role as pharmaceutical targets. Molecular docking experiments of the most cGMP-kinase selective sequences on a molecular model of the catalytic domain of cGMP-kinase Ialpha suggest that they adopt unique conformations, which differ significantly from those observed for the cAMP-kinase-specific inhibitor PKI(5-24). Our results suggest that despite their structural similarities, cAMP-kinase and cGMP-kinase use distinct peptide substrate and inhibitor conformations, which could account for their unique substrate specificities. These findings are further supported by cAMP- and cGMP-kinase-selective inhibitor analogs with (D)-Ala residues at the inhibitory positions.

Exploring the mechanisms of vascular smooth muscle tone with highly specific, membrane-permeable inhibitors of cyclic GMP-dependent protein kinase Iα

The structural similarity of cyclic GMP-dependent protein kinase (cGPK) and cyclic AMP-dependent protein kinase (cAPK) has made it difficult to study cGPK pathways independent of those mediated by cAPK, primarily due to the lack of potent and selective cGPK inhibitors. We recently reported a novel peptide library screen specifically designed to select for tight-binding peptides that identified selective inhibitors of cGPK [Proc Natl Acad Sci USA, 97 (2000) 14772]. Iterative deconvolution of octameric library arrays on paper identified the sequence LRK(5)H (W45). 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 cAPK, respectively. Cellular internalization of highly charged W45 was accomplished by N-terminal fusion of membrane translocation sequences from either the human immunodeficiency virus tyrosine aminotransferase protein (47-59) DT-2 or from the Drosophila Antennapedia homeodomain (43-58) DT-3, respectively. For both fusion peptides, DT-2 and DT-3, we observed a potentiating effect with respect to the inhibitory potency, with K(i) values 40- to 80-fold lower than W45. Fluorescein-labeled DT-2 and DT-3 demonstrated rapid translocation through the cytosol and nuclei in a time-dependent manner using cultured cells and intact tissue samples (cerebral arteries). The physiological effects of DT-2 and DT-3 as selective cGPK inhibitors in smooth muscle were studied in small intact arteries. Nitric oxide, a cyclic GMP/cGPK activator, elicited a concentration-dependent dilation of isolated rat cerebral arteries, which was markedly inhibited by DT-2 and DT-3. Collectively, these results indicate that DT-2 and DT-3 effectively inhibit nitric oxide-induced vasodilation, further emphasizing the central role for cGPK in the modulation of vascular contractility.

(D)-amino acid analogues of DT-2 as highly selective and superior inhibitors of cGMP-dependent protein kinase Iα

The cGMP-dependent protein kinase type I (PKG I) is an essential regulator of cellular function in blood vessels throughout the body. DT-2, a peptidic inhibitor of PKG, has played a central role in determining the molecular mechanisms of vascular control involving PKG and its signaling partners. Here, we report the development of (d)-amino acid DT-2 derivatives, namely the retro-inverso ri-(d)-DT-2 and the all (d)-amino acid analog, (d)-DT-2. Both peptide analogs were potent PKG Ialpha inhibitors with K(i) values of 5.5 nM (ri-(d)-DT-2) and 0.8 nM ((d)-DT-2) as determined using a hyperbolic mixed-type inhibition model. Also, both analogs were proteolytically stable in vivo, showed elevated selectivity, and displayed enhanced membrane translocation properties. Studies on isolated arteries from the resistance vasculature demonstrated that intraluminally perfused (d)-DT-2 significantly inhibited vasodilation induced by 8-Br-cGMP. Furthermore, in vivo application of (d)-DT-2 established a uniform translocation pattern in the resistance vasculature, with exception of the brain. Thus, (d)-DT-2 caused significant increases in mean arterial blood pressure in unrestrained, awake mice. Further, mesenteric arteries isolated from (d)-DT-2 treated animals showed a markedly reduced dilator response to 8-Br-cGMP in vitro. Our results clearly demonstrate that (d)-DT-2 is a superior inhibitor of PKG Ialpha and its application in vivo leads to sustained inhibition of PKG in vascular smooth muscle cells. The discovery of (d)-DT-2 may help our understanding of how blood vessels constrict and dilate and may also aid the development of new strategies and therapeutic agents targeted to the prevention and treatment of vascular disorders such as hypertension, stroke and coronary artery disease.

Direct modulation of the protein kinase a catalytic subunit α by growth factor receptor tyrosine kinases

The cyclic‐AMP‐dependent protein kinase A (PKA) regulates processes such as cell proliferation and migration following activation of growth factor receptor tyrosine kinases (RTKs), yet the signaling mechanisms that link PKA with growth factor receptors remain largely undefined. Here we report that RTKs can directly modulate the function of the catalytic subunit of PKA (PKA‐C) through post‐translational modification. In vitro kinase assays revealed that both the epidermal growth factor and platelet derived growth factor receptors (EGFR and PDGFR, respectively) tyrosine phosphorylate PKA‐C. Mass spectrometry identified tyrosine 330 (Y330) as a receptor‐mediated phosphorylation site and mutation of Y330 to phenylalanine (Y330F) all but abolished the RTK‐mediated phosphorylation of PKA‐C in vitro. Y330 resides within a conserved region at the C‐terminal tail of PKA‐C that allosterically regulates enzymatic activity. Therefore, the effect of phosphorylation at Y330 on the activity of PKA‐C was investigated. The Km for a peptide substrate was markedly decreased when PKA‐C subunits were tyrosine phosphorylated by the receptors as compared to un‐phosphorylated controls. Importantly, tyrosine‐phosphorylated PKA‐C subunits were detected in cells stimulated with EGF, PDGF, and Fibroblast growth factor 2 (FGF2) and in fibroblasts undergoing PDGF‐mediated chemotaxis. These results demonstrate a direct, functional interaction between RTKs and PKA‐C and identify tyrosine phosphorylation as a novel mechanism for regulating PKA activity. J. Cell. Biochem. 113: 39–48, 2012.

Adenosine analogue–oligo-arginine conjugates (ARCs) serve as high-affinity inhibitors and fluorescence probes of type I cGMP-dependent protein kinase (PKGIα)

INTRODUCTION Type I cGMP-dependent protein kinase (PKGIalpha) belongs to the family of cyclic nucleotide-dependent protein kinases and is one of the main effectors of cGMP. PKGIalpha is involved in regulation of cardiac contractility, vasorelaxation, and blood pressure; hence, the development of potent modulators of PKGIalpha would lead to advances in the treatment of a variety of cardiovascular diseases. AIM Representatives of ARC-type compounds previously characterized as potent inhibitors and high-affinity fluorescent probes of PKA catalytic subunit (PKAc) were tested towards PKGIalpha to determine that ARCs could serve as activity regulators and sensors for the latter protein kinase both in vitro and in complex biological systems. RESULTS Structure-activity profiling of ARCs with PKGIalpha in vitro demonstrated both similarities as well as differences to corresponding profiling with PKAc, whereas ARC-903 and ARC-668 revealed low nanomolar displacement constants and inhibition IC(50) values with both cyclic nucleotide-dependent kinases. The ability of ARC-based fluorescent probes to penetrate cell plasma membrane was demonstrated in the smooth muscle tissue of rat cerebellum isolated arteries, and the compound with the highest affinity in vitro (ARC-903) showed also potential for in vivo applications, fully abolishing the PKG1alpha-induced vasodilation.

Inhibitors of Cyclic AMP- and Cyclic GMP-Dependent Protein Kinases

Publisher Summary The cyclic nucleotide-dependent protein kinases PKA and PKG serve as primary targets for the second messengers cAMP and cGMP, respectively. The structural similarities of PKA and PKG have posed a formidable obstacle in the design of selective inhibitors that specifically target cyclic nucleotide-dependent protein kinases and show little inhibitory potency to other basophilic Ser/Thr-kinases. The domain structures of PKA and PKG dictate key target sites for putative inhibitors. It illustrates the three distinct classes of inhibitors and their various sites of actions. The regulatory components of cyclic nucleotide-dependent protein kinases each harbor two tandem cyclic nucleotide binding sites that allow allosteric and cooperative control of kinase activity. Rp-phosphorothioate derivatives of cAMP and cGMP competitively inhibit cyclic nucleotide-dependent protein kinases by stabilizing the enzymes in their inactive holoenzyme states. A large pool of derivatives, moderate selectivity, and cell membrane permeability are regarded as their advantages as tools in intact cell studies. However, partial antagonism and limited potencies restrict their versatility. The catalytic components of cyclic nucleotide-dependent protein kinases contain two target sites for inhibitors: the ATP-binding site, and the substrate binding site. ATP-analogs are a highly resourceful group of protein kinase inhibitors. Cell membrane permeability and limited selectivity highlight their advantages and disadvantages, respectively. Peptide-derived inhibitors present the most potent and selective group of PKA and PKG blockers. Low cell membrane permeability remains their main obstacle in cellular research.

cGMP-dependent protein kinase from Toxoplasma gondii: functional expression in E. coli and molecular characterization

Background The apicomplexan parasite Toxoplasma gondii is an obligate intracellular human pathogen causing toxoplasmosis predominantly in immune-compromised hosts such as cancer and transplant patients as well as patients with AIDS [1]. A specific cGMP-dependent protein kinase (TgPKG) which appears to be crucial for host invasion has been identified in T. gondii and related coccidial protozoa [2]. However, detailed structural and biochemical analyses have been hampered due to the inability to functionally express these kinases in high yields in systems other than their parasite host organisms.

Development of Highly Specific, Membrane Permeable Peptide Blockers of cGMP-Dependent Protein Kinase Utilizing Affinity Selection from Peptide Libraries

The cGMP-dependent protein kinases type Iα and Iβ (cGPK) act directly downstream in the nitric oxide (NO) mediated signaling pathway in the control of 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 due to the lack of potent and selective cGPK inhibitors. Here we report a novel peptide library screen specifically designed to select for tight binding peptides that identified selective inhibitors of the kinases. The most potent sequences were made cell permeable by the addition of membrane translocation sequences from HIV and Drosophila and physiological effects of the constructs were studied in vivo and in vitro.