17 Structural order of the slow and fast intrasubunit cGMP-binding sites of type Iα cGMP-dependent protein kinase

Rb, Reed; Sandberg, M; Jahnsen, Tore; Sm, Lohmann; Sh, Francis; Jd, Corbin

doi:10.1016/s1040-7952(97)80020-1

Cited by 12 publications

(24 citation statements)

References 17 publications

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“…Interestingly, cNMP-BD or GAF domains are arranged in tandem pairs in cGKs or PDEs respectively. In cGKI, the Nterminal cNMP-BD, designated cNMP-BD A, has been described as a high-affinity binding site, whereas cNMP-BD B has a rather low cGMP affinity [25,26]. In PDE5, the N-terminal GAF domain, GAF-A, binds cGMP, leading to activation of the enzyme [27,28].…”

Section: Figure 1 Schematic Domain Structure Of Cgmp Indicatorsmentioning

confidence: 99%

Design of fluorescence resonance energy transfer (FRET)-based cGMP indicators: a systematic approach

Russwurm

Müllershausen

Friebe

et al. 2007

Biochemical Journal

131

150

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The intracellular signalling molecule cGMP regulates a variety of physiological processes, and so the ability to monitor cGMP dynamics in living cells is highly desirable. Here, we report a systematic approach to create FRET (fluorescence resonance energy transfer)-based cGMP indicators from two known types of cGMP-binding domains which are found in cGMP-dependent protein kinase and phosphodiesterase 5, cNMP-BD [cyclic nucleotide monophosphate-binding domain and GAF [cGMP-specific and -stimulated phosphodiesterases, Anabaena adenylate cyclases and Escherichia coli FhlA] respectively. Interestingly, only cGMP-binding domains arranged in tandem configuration as in their parent proteins were cGMP-responsive. However, the GAF-derived sensors were unable to be used to study cGMP dynamics because of slow response kinetics to cGMP. Out of 24 cGMP-responsive constructs derived from cNMP-BDs, three were selected to cover a range of cGMP affinities with an EC50 between 500 nM and 6 microM. These indicators possess excellent specifity for cGMP, fast binding kinetics and twice the dynamic range of existing cGMP sensors. The in vivo performance of these new indicators is demonstrated in living cells and validated by comparison with cGMP dynamics as measured by radioimmunoassays.

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Section: Figure 1 Schematic Domain Structure Of Cgmp Indicatorsmentioning

confidence: 99%

Design of fluorescence resonance energy transfer (FRET)-based cGMP indicators: a systematic approach

Russwurm

Müllershausen

Friebe

et al. 2007

Biochemical Journal

131

150

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“…Cyclic GMP-dependent protein kinase type I (PKG-I) 1 exists as a homodimer and is comprised of multiple functional domains. Each monomer contains a dimerization component, an autoinhibitory region, two cGMP-binding sites, and a catalytic domain (1,2).…”

mentioning

confidence: 99%

“…Each monomer contains a dimerization component, an autoinhibitory region, two cGMP-binding sites, and a catalytic domain (1,2). There are two splice variants of PKG-I, PKG-I␣ and PKG-I␤ (3-6), which show broad variation in tissue distribution (7)(8)(9)(10).…”

mentioning

confidence: 99%

A Conserved Serine Juxtaposed to the Pseudosubstrate Site of Type I cGMP-dependent Protein Kinase Contributes Strongly to Autoinhibition and Lower cGMP Affinity

Busch

Bessay

Francis

et al. 2002

Journal of Biological Chemistry

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Serines 64 and 79 are homologous residues that are juxtaposed to the autoinhibitory pseudosubstrate site in cGMP-dependent protein kinase type I␣ and type I␤ (PKG-I␣ and PKG-I␤), respectively. Autophosphorylation of this residue is associated with activation of type I PKGs. To determine the role of this conserved serine, point mutations have been made in PKG-I␣ (S64A, S64T, S64D, and S64N) and PKG-I␤ (S79A). In wild-type PKG-I␣, basal kinase activity ratio (؊cGMP/؉cGMP) is 0.11, autophosphorylation increases this ratio 3-fold, and the K a and K D values for cGMP are 127 and 36 nM, respectively. S64A PKG-I␣ basal kinase activity ratio increases 2-fold, cGMP binding affinity increases ϳ10-fold in both K a and K D , and activation by autophosphorylation is slight. S64D and S64N mutants are nearly constitutively active in the absence of cGMP, cGMP binding affinity in each increases 18-fold, and autophosphorylation does not affect the kinase activity of these mutants. Mutation of the homologous site in PKG-I␤ (S79A) increases the basal kinase activity ratio 2-fold and cGMP binding affinity 5-fold over that of wild-type PKG-I␤. The combined results demonstrate that a conserved serine juxtaposed to the pseudosubstrate site in type I PKGs contributes importantly to enzyme function by increasing autoinhibition and decreasing cGMP binding affinity.Cyclic GMP-dependent protein kinase type I (PKG-I) 1 exists as a homodimer and is comprised of multiple functional domains. Each monomer contains a dimerization component, an autoinhibitory region, two cGMP-binding sites, and a catalytic domain (1, 2). There are two splice variants of PKG-I, PKG-I␣ and PKG-I␤ (3-6), which show broad variation in tissue distribution (7-10). Although the amino acid sequences of their cGMP-binding sites and catalytic domains are identical, the variants share only 36% homology in their 88 and 103 aminoterminal residues, respectively (1, 4 -6). This region of variable sequence includes the autoinhibitory domain and the autophosphorylation sites, which impart distinct functional characteristics to the kinases. The autoinhibitory domain contains a sequence that mimics the substrate sequence ((R/K)(R/K) X(S/T)X) except that an alanine instead of a phosphorylatable serine or threonine is located at P 0 , the position of phosphorylation. Thus, the sequence is referred to as a pseudosubstrate site. Interactions involving residues in the pseudosubstrate site have been demonstrated to be important in maintaining these kinases in an inactive state, and it has been proposed that this simulates interaction of this sequence with the substrate-binding site in the catalytic domain (11, 12). The interaction between the pseudosubstrate site and the catalytic domain maintains the enzyme in an autoinhibited state. Removal of the pseudosubstrate site by proteolysis relieves much of the autoinhibition (13,14), and perturbation of certain residues within the pseudosubstrate sequence of synthetic PKG peptides interferes with the inhibitory potencies of the peptides (15). ...

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“…In a fashion similar to cyclic nucleotide binding to PKA, there is a high-affinity and a low-affinity site. In PKG, these sites are the A and B-sites, respectively, unlike PKA, where the order is reversed [13, 14]. Recent crystallographic studies have discovered that the A-site in the regulatory subunit of PKG I isoforms can bind both cAMP and cGMP, although the implications for this are yet unexplored [16].…”

Section: The Cyclic Nucleotide Binding Sites In Pkg Are Discretementioning

confidence: 99%

“…First, the regulatory domains carry two in-tandem cGMP binding sites A and B (Figure 2b) [2, 4]. In contrast to PKA, the N-terminal A site is the high affinity binding site and the C-terminal B site the low affinity site in PKG [13, 14]. It has been a particular challenge to understand these compositional differences between PKA and PKG in terms of their contributions to the molecular mechanisms of kinase activation.…”

Section: Introductionmentioning

confidence: 99%

The switch helix: A putative combinatorial relay for interprotomer communication in cGMP-dependent protein kinase

Moon

Osborne

Dostmann

2013

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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For over three decades the isozymes of cGMP-dependent protein kinase (PKG) have been studied using an array of biochemical and biophysical techniques. When compared to its closest cousin, cAMP-dependent protein kinase (PKA), these studies revealed a set of identical domain types, yet containing distinct, sequence-specific features. The recently solved structure of the PKG regulatory domain showed the presence of the switch helix (SW), a novel motif that promotes the formation of a domain-swapped dimer in the asymmetric unit. This dimer is mediated by the interaction of a knob motif on the C-terminal locus of the SW, with a hydrophobic nest on the opposing protomer. This nest sits adjacent to the cGMP binding pocket of the B-site. Priming of this site by cGMP may influence the geometry of the hydrophobic nest. Moreover, this unique interaction may have wide implications for the architecture of the inactive and active forms of PKG.

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17 Structural order of the slow and fast intrasubunit cGMP-binding sites of type Iα cGMP-dependent protein kinase

Cited by 12 publications

References 17 publications

Design of fluorescence resonance energy transfer (FRET)-based cGMP indicators: a systematic approach

Design of fluorescence resonance energy transfer (FRET)-based cGMP indicators: a systematic approach

A Conserved Serine Juxtaposed to the Pseudosubstrate Site of Type I cGMP-dependent Protein Kinase Contributes Strongly to Autoinhibition and Lower cGMP Affinity

The switch helix: A putative combinatorial relay for interprotomer communication in cGMP-dependent protein kinase

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