Determination of Cyclic Nucleotide-Dependent Protein Kinase Substrate Specificity by the Use of Peptide Libraries on Cellulose Paper

Dematin (band 4.9) is found in the junctional complex of the spectrin cytoskeleton that supports the erythrocyte cell membrane. Dematin is a member of the larger class of cytoskeleton-associated proteins that contain a modular "headpiece" domain at their extreme C termini. The dematin headpiece domain provides the second Factin-binding site required for in vitro F-actin bundling. The dematin headpiece is found in two forms in the cell, one of 68 residues (DHP) and one containing a 22-amino acid insert near its N terminus (DHP؉22). In addition, dematin contains the only headpiece domain that is phosphorylated, in vivo. The 22-amino acid insert in DHP؉22 appeared unstructured in NMR spectra; therefore, we have determined the three-dimensional structure of DHP by multidimensional NMR methods. Although the overall three-dimensional structure of DHP is similar to that of the villin headpiece, there are two novel characteristics revealed by this structure.

mentioning

confidence: 99%

The NMR Structure of Dematin Headpiece Reveals a Dynamic Loop That Is Conformationally Altered upon Phosphorylation at a Distal Site

Frank

Vardar

Chishti

et al. 2004

“…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%

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

Pinkse

Rijkers

Dostmann

et al. 2009

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...

“…Previous studies with peptide libraries defined optimal amino acid sequences (RKX(S/T), X is any amino acid) for phosphorylation by cGK (25). Although GKAP42 is preferentially phosphorylated by cGK-I in vitro and in vivo, a typical cGK phosphorylation site was not identified in GKAP42.…”

Section: Co-localization Of Cgk-i With Gkap42 In Mammalianmentioning

confidence: 99%

“…WT, wild type. defined optimal amino acid sequences for phosphorylation by cGK (RKX(S/T)) (25). Additionally, Glass and Smith (26) reported that the recognition site specific to cGK requires an arginine on the C-terminal side (underlined) of the phosphorylated residue, X(S/T)RX.…”

Section: Figmentioning

confidence: 99%

Binding and Phosphorylation of a Novel Male Germ Cell-specific cGMP-dependent Protein Kinase-anchoring Protein by cGMP-dependent Protein Kinase Iα

Yuasa¹,

Omori²,

Yanaka³

2000

cGMP-dependent protein kinase (cGK) is a major cellular receptor of cGMP and plays important roles in cGMP-dependent signal transduction pathways. To isolate the components of the cGMP/cGK signaling pathway such as substrates and regulatory proteins of cGK, we employed the yeast two-hybrid system using cGK-I␣ as a bait and isolated a novel male germ cell-specific 42-kDa protein, GKAP42 (42-kDa cGMP-dependent protein kinase anchoring protein). Although the N-terminal region (amino acids 1-66) of cGK-I␣ is sufficient for the association with GKAP42, GKAP42 could not interact with cGK-I␤, cGK-II, or cAMP-dependent protein kinase. GKAP42 mRNA is specifically expressed in testis, where it is restricted to the spermatocytes and early round spermatids. Endogenous cGK-I is co-immunoprecipitated with anti-GKAP42 antibody from mouse testis tissue, suggesting that cGK-I physiologically interacts with GKAP42. Immunocytochemical observations revealed that GKAP42 is localized to the Golgi complex and that cGK-I␣ is co-localized to the Golgi complex when coexpressed with GKAP42. Although both cGK-I␣ and -I␤, but not cAMP-dependent protein kinase, phosphorylated GKAP42 in vitro, GKAP42 was a good substrate only for cGK-I␣ in intact cells, suggesting that the association with kinase protein is required for the phosphorylation in vivo. Finally, we demonstrated that the kinase-deficient mutant of cGK-I␣ stably associates with GKAP42 and that binding of cGMP to cGK-I␣ facilitates their release from GKAP42. These findings suggest that GKAP42 functions as an anchoring protein for cGK-I␣ and that cGK-I␣ may participate in germ cell development through phosphorylation of Golgi-associated proteins such as GKAP42.