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

Dostmann, Wolfgang R.; Nickl, Christian K.; Thiel, Stefan; Tsigelny, Igor F.; Frank, Ronald; Tegge, Werner

doi:10.1016/s0163-7258(98)00063-1

Cited by 53 publications

(62 citation statements)

References 43 publications

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“…The electrostatic surface representation of this model displayed a different interaction site for peptide substrate when compared with the crystal structure of PKA in complex with PKI. In addition, molecular modeling of the docking of the TQAKRKKALA-amide inhibitor peptide onto this PKG model demonstrated that the peptide primarily binds to the small lobe (11). This in contrast to the PKI/PKA systems, in which the N-terminal part of PKI is largely associated with the large lobe of the catalytic domain and the C-terminal part of PKI is more outside the catalytic cleft.…”

Section: Discussionmentioning

confidence: 95%

“…Overall, this reiterates that the positioning of the photoactivatable amino acid with the peptide is important. To test the potential of cross-linking peptides to PKG via photoaffinity labeling we initially synthesized a photolabeled substrate peptide, based on the sequence TQAKRKKSLAMFLR, which is a high specificity peptide substrate of PKG (11). The phenylalanine was replaced by a photoactivable amino acid Phe(Tmd) at the p ϩ 4 position (P0 is the phosphate accepting serine).…”

Section: Discussionmentioning

confidence: 99%

“…The sites of the P0 cross-link for the PKA substrate (53) are highlighted in yellow, the equivalents of the p ϩ 4 cross-link site for PKG are highlighted in pink. Previously, a refined molecular model for the catalytic domain of PKG was presented (11). The electrostatic surface representation of this model displayed a different interaction site for peptide substrate when compared with the crystal structure of PKA in complex with PKI.…”

Section: Discussionmentioning

confidence: 99%

“…These two phenylalanine derivatives are commonly used as photoactivatable residues that allow for the covalent cross-linking between peptide ligands and the target protein (50,51). The substrate peptide TQAKRKKSLAMFLR (W64), in which the serine is the phosphate acceptor site (P0), was derived from a library screen directed to identify highly PKGspecific substrates (11,52). This peptide with a K m of 260 nM and a PKG/PKA specificity index of 19.2 is the most selective and specific synthetic substrate for PKG known today (Table 3).…”

Section: Strategy For Determining Peptide-binding Sites Using Photoacmentioning

confidence: 99%

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Mode of Action of cGMP-dependent Protein Kinase-specific Inhibitors Probed by Photoaffinity Cross-linking Mass Spectrometry

Pinkse

Rijkers

Dostmann

et al. 2009

Journal of Biological Chemistry

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The inhibitor peptide DT-2 (YGRKKRRQRRRPPLRKKK-KKH) is the most potent and selective inhibitor of the cGMPdependent protein kinase (PKG) known today. DT-2 is a construct of a PKG tight binding sequence (W45, LRKKKKKH, K I ‫؍‬ 0.8 M) and a membrane translocating sequence (DT-6, YGRKKRRQRRRPP, K I ‫؍‬ 1.1 M), that combined strongly inhibits PKG catalyzed phosphorylation (K I ‫؍‬ 12.5 nM) with ϳ1000-fold selectivity toward PKG over protein kinase A, the closest relative of PKG. However, the molecular mechanism behind this inhibition is not entirely understood. Using a combination of photoaffinity labeling, stable isotope labeling, and mass spectrometry, we have located the binding sites of PKGspecific substrate and inhibitor peptides. Covalent linkage of a PKG-specific substrate analogue was localized in the catalytic core on residues 356 -372, also known as the glycine-rich loop, essential for ATP binding. By analogy, the individual inhibitor peptides W45 and DT-6 were also found to cross-link near the glycine-rich loop, suggesting these are both substrate competitive inhibitors. A bifunctional photoreactive analogue of DT-2 was found to generate dimers of PKG. This cross-linking induced covalent PKG dimerization was not observed for an N-terminal deletion mutant of PKG, which lacks the dimerization domain. In addition, non-covalent mass spectrometry was used to determine binding stoichiometry and binding order of the inhibitor peptides. Dimeric PKG binds two W45 and DT-6 peptides, whereas only one DT-2 molecule was observed to bind to the dimeric PKG. Taken together, these findings imply that (i) the two individual components making up DT-2 are both targeted against the substrate-binding site and (ii) binding of a single DT-2 molecule inactivates both PKG monomers simultaneously, which is an indication that (iii) in cGMP-activated PKG the catalytic centers of both subunits may be in each other's proximity.Among the superfamily of protein kinases the two cyclic nucleotide-regulated protein kinases, cAMP-dependent protein kinase and cGMP-dependent protein kinase, form a closely related subfamily of serine/threonine protein kinases (1-4). Both proteins share several structural elements, such as the N-terminal dimerization domain, an autoinhibition site, two in-tandem cyclic nucleotide-binding sites, and a highly conserved catalytic core (Fig. 1, A and B). Despite these similarities, these two enzymes display differences, which account for their unique properties. Whereas PKA 2 is nearly ubiquitous, PKG is primarily found in the lung, cerebellum, and smooth muscles (5, 6). From a structural point of view these cyclic nucleotidedependent protein kinases differ as well. The holoenzyme of PKA is a tetramer composed of two regulatory and two catalytic subunits. The catalytic subunits are non-covalently attached to the regulatory subunit dimer. Upon interaction with cAMP, the catalytic subunits dissociate from the holoenzyme and are free to catalyze heterophosphorylation (Fig. 1C). The mammalian type I PKGs are homodimeri...

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Section: Discussionmentioning

confidence: 95%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Strategy For Determining Peptide-binding Sites Using Photoacmentioning

confidence: 99%

See 2 more Smart Citations

Mode of Action of cGMP-dependent Protein Kinase-specific Inhibitors Probed by Photoaffinity Cross-linking Mass Spectrometry

Pinkse

Rijkers

Dostmann

et al. 2009

Journal of Biological Chemistry

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“…cGKI, like its closest relative PKA, targets serine or threonine phosphorylation sites in its substrates within a basic consensus (38). Therefore, we used an adapted Ti 4ϩ -IMAC-based phosphopeptide enrichment strategy shown to be suited for basic kinase motifs (27).…”

Section: Alterations In the Cerebellar Proteome Of Cgki-ko Mice-mentioning

confidence: 99%

Alterations in the Cerebellar (Phospho)Proteome of a Cyclic Guanosine Monophosphate (cGMP)-dependent Protein Kinase Knockout Mouse

Corradini

Vallur

Raaijmakers

et al. 2014

Molecular & Cellular Proteomics

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The cyclic nucleotide cyclic guanosine monophosphate (cGMP) plays an important role in learning and memory, but its signaling mechanisms in the mammalian brain are not fully understood. Using mass-spectrometry-based proteomics, we evaluated how the cerebellum adapts its (phospho)proteome in a knockout mouse model of cGMPdependent protein kinase type I (cGKI). Our data reveal that a small subset of proteins in the cerebellum (ϳ3% of the quantified proteins) became substantially differentially expressed in the absence of cGKI. More changes were observed at the phosphoproteome level, with hundreds of sites being differentially phosphorylated between wildtype and knockout cerebellum. Most of these phosphorylated sites do not represent known cGKI substrates. An integrative computational network analysis of the data indicated that the differentially expressed proteins and proteins harboring differentially phosphorylated sites largely belong to a tight network in the Purkinje cells of the cerebellum involving important cGMP/cAMP signaling nodes (e.g. PDE5 and PKARII␤) and Ca 2؉ signaling (e.g. SERCA3). In this way, removal of cGKI could be linked to impaired cerebellar long-term depression at Purkinje cell synapses. In addition, we were able to identify a set of novel putative (phospho)proteins to be considered in this network. Overall, our data improve our understanding of cerebellar cGKI signaling and suggest novel players in cGKI-regulated synaptic plasticity. Molecular & Cellular

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Peptide Arrays on Solid Supports: A Tool for the Identification of Peptide Ligands

2009

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

Cited by 53 publications

References 43 publications

Mode of Action of cGMP-dependent Protein Kinase-specific Inhibitors Probed by Photoaffinity Cross-linking Mass Spectrometry

Mode of Action of cGMP-dependent Protein Kinase-specific Inhibitors Probed by Photoaffinity Cross-linking Mass Spectrometry

Alterations in the Cerebellar (Phospho)Proteome of a Cyclic Guanosine Monophosphate (cGMP)-dependent Protein Kinase Knockout Mouse

Peptide Arrays on Solid Supports: A Tool for the Identification of Peptide Ligands

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