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

Abstract

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...

cAMP-dependent protein kinase (PKA) complexes probed by complementary differential scanning fluorimetry and ion mobility–mass spectrometry

“…Even though PKA is used as a benchmark for studying other protein kinases, MS-based structural evaluation of the PKA complexes has not previously been reported to the best of our knowledge. MS studies previously evaluated ligand and inhibitor sensitivity of type I cGMP-dependent protein kinase (PKG) subunits [73,74]. However, PKG differs from PKA in that its regulation involves sensing of the cyclic GMP nucleotide within a single polypeptide.…”

cGMP-Dependent Protein Kinase Inhibitors in Health and Disease

“…In contrast PKG-II is bound to the plasma membrane by myristoylation of the N-terminal Gly2 residue. All three PKGs act as homodimers [1,2,3]. Generally, cGMP-dependent protein kinases consist of three domains:

amino-terminus with a leucine-zipper (important e.g., for homodimerization and targeting) and the autoinhibitory domain.

regulatory domain with a high- and low-affinity binding site for cGMP (important for the activation of the enzyme).

catalytic domain for ATP binding, which catalyses the transfer of the phosphate residue to the serine/threonine motif.

…”

17β-Estradiol Elevates cGMP and, via Plasma Membrane Recruitment of Protein Kinase GIα, Stimulates Ca2+ Efflux from Rat Hepatocytes

“…Rp-8-pCPT-cGMPS inhibits all three PKG isoforms (75) by blocking cGMP binding to the regulatory domain, thereby preventing exposure of the catalytic domain. DT-3 acts through a distinct mechanism to that of Rp-8-pCPTcGMPS; the PKGI-inhibitor peptide portion of DT-3, W45, prevents binding and, hence, phosphorylation of substrates at the PKGI catalytic domain (76). DT-3 inhibits isoforms I␣ and the I␤ at low M concentrations, whereas PKGII is relatively (ϳ100-fold less) insensitive to this inhibitor (77).…”