Stimulation of cGMP‐dependent protein kinase Iα by a peptide from its own sequence

Abstract

The structure of cGMP-dependent protein kinase Ia-(546 -576)-peptide amide (peptide-546) and its effects on cGMP-dependent protein kinase Ia (G-kinase) have been studied. By primary sequence analysis and analogy to a peptide that stimulates protein kinase C, peptide-546 was predicted to form part of the proteidpeptide binding site of G-kinase, and it was proposed that it would stimulate the enzyme by interaction with an autoinhibitory site. The portion of CAMP-dependent protein kinase analogous to peptide-546 forms part of the peptide substrate binding site, interacting with the peptide inhibitor residues ArgP-' and PheP-" (where p is the pseudophosphorylation site), through residues at positions corresponding to Glu4, ProlO and Serl3 in peptide-546. Peptide-546 is a reasonably potent G-kinase activator, increasing the turnover number with the peptide substrate ArgLys-Arg-Ser-Arg-Lys-Glu by about threefold with an activation constant that is about fivefold lower than the K,,, value of this peptide substrate.Peptide-546 does not appear to change the affinity of the enzyme for the above substrate, ATP or cGMP and does not affect the binding of ['HIcGMP to G-kinase. The activation does not seem to result from an interaction between peptide-546 and peptide substrates, and a kinetic scheme is proposed which is compatible with an action of peptide-546 on G-kinase independent of substrates. The activation is additive with that given by cGMP and causes the enzyme to enter a hitherto unrecognised superactive state. Peptide conformation has been monitored in mixed 2,2,2-trifluoroethanol/H,O solvents by circular dichroism: helical structure is observed in these mixtures when the 2,2,2-trifluoroethanol content is above 25%. The structure is lost only gradually on raising the temperature to 80°C with no clear melting transition.Assignment of the resonances in the 'H-NMR spectrum has allowed the identification of elements of secondary structure from detected nuclear Overhauser effects. In particular, a helical segment from Met18 to Arg26 is observed. The four proline residues (ProlO, Proll, Pro15 and Prol7) are all seen to be in the trans conformation, although additional, weaker peaks in the spectra may correspond to a minor conformer in which one or more of the prolines is in a cis conformation. The N-terminal residues are less structured but show some helical character.Calculation of the structure of the peptide using nuclear Overhauser effects (NOES) as distance constraints in distance geometry and molecular dynamics simulations revealed a well defined helical structure in the C-terminal region preceded by a less well defined proline-rich segment and Nterminal region. On the basis of these results, peptide-546 may adopt a conformation similar to that expected by analogy with CAMP-dependent protein kinase, and we propose that residues 546-576 of G-kinase are involved in enzyme autoinhibition.cGMP-dependent protein kinase is an enzyme involved in cellular signalling as a receptor protein for cGMP in Correspondence to J. P...

Functional Analysis of Type 1α cGMP-dependent Protein Kinase Using Green Fluorescent Fusion Proteins

“…Since this activity was not apparent in the PKG-deficient A549 cells, it is likely that the mutant catalytic domains were able to partially activate endogenous PKG. This can be explained if the T516A mutant was able to compete with endogenous wild-type catalytic regions for binding to the pseudosubstrate domains of the holoenzyme, as has been suggested to occur with isolated peptides corresponding to amino acids 546 -576 of the enzyme (40). This idea is supported by the observed interaction between the catalytic half of PKG (either FLAG-G1C or GFP-G1C) with the regulatory half of PKG in immunoprecipitation studies.…”

Cell cycle-dependent regulation of cellular ATP concentration, and depolymerization of the interphase microtubular network induced by elevated cellular ATP concentration in whole fibroblasts

“…3B), e.g., the K, value with respect to ATP of Ca2+/calmodulin-dependent protein kinase 2 is about 19 pM using MAP-2 as substrate [Gupta et al, 19921. Rabbit pancreas protein kinase C, casein kinase 2, cGMPdependent protein kinase 1 alpha, p4.5 casein kinase, have K, values in the same range [Ederveen et al, 1992;Jakoby and Traugh, 1992;Huggins et al, 1994;Angelov , 19941. As demonstrated in this work, the cellular ATP concentration varies through the cell cycle, being high during G,+M-phase (about 3.5 mM) and decreasing during GI-phase of the cell cycle, reaching its minimum at late G,/early S-phase (corresponding to about 2.2 mM). However, these changes are somewhat masked by the incomplete synchronization of our cell population, so the cellular ATP concentration during G, + M-phase may be considerably higher than 3.5 mM, and thereby exceed the ATP concentration needed to depolymerize microtubules ( Figs.…”