A Model for the Chemical Interactions of Adenosine 3':5'-Monophosphate with the R Subunit of Protein Kinase Type I. Refinement of the Cyclic Phosphate Binding moiety of Protein Kinase Type I

Jastorff, Bernd; Hoppe, Jürgen; Morr, Michael

doi:10.1111/j.1432-1033.1979.tb19750.x

Cited by 67 publications

(32 citation statements)

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“…A recent crystal structure of a large fragment containing the cAMP-binding domains of R subunit corroborated predictions of an earlier model based on the structure of the catabolite activator protein of Escherichia coli that most of the important contact residues for interaction with the ribose-cyclic phosphate of cAMP are in ␤-strands 6 and 7 of an antiparallel ␤-roll structure forming one face of the cAMP-binding pockets (5,6). It appears from this structure that Glu-201 2 in site A and Glu-325 in site B interact with the 2Ј-hydroxyl group of cAMP and that the guanidinium groups of Arg-210 in site A and Arg-334 in site B interact with the equatorial exocyclic oxygen of the cAMP phosphate group (5). Mutations at these residues or at the conserved Gly residues immediately upstream of the conserved Glu residues markedly reduced the affinities of the mutated sites for cAMP (7)(8)(9)(10).…”

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“…1). Studies with analogs of cAMP implicate the ribose-cyclic phosphate moiety of cAMP in the activation process (2,3). The adenine ring is thought to contribute to the specificity and high affinity of cAMP binding (2,4).…”

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“…Studies with analogs of cAMP implicate the ribose-cyclic phosphate moiety of cAMP in the activation process (2,3). The adenine ring is thought to contribute to the specificity and high affinity of cAMP binding (2,4). A recent crystal structure of a large fragment containing the cAMP-binding domains of R subunit corroborated predictions of an earlier model based on the structure of the catabolite activator protein of Escherichia coli that most of the important contact residues for interaction with the ribose-cyclic phosphate of cAMP are in ␤-strands 6 and 7 of an antiparallel ␤-roll structure forming one face of the cAMP-binding pockets (5,6).…”

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Arginine 210 Is Not a Critical Residue for the Allosteric Interactions Mediated by Binding of Cyclic AMP to Site A of Regulatory (RIα) Subunit of Cyclic AMP-dependent Protein Kinase

Steinberg

Symcox

Sollid

et al. 1996

Journal of Biological Chemistry

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The guanidinium groups of conserved arginines in the two intrachain cAMP-binding sites of regulatory (R) subunit of cAMP-dependent protein kinase have been implicated in the allosteric interactions by which cAMP binding leads to kinase activation. We have investigated the functional role of Arg-210, the conserved arginine in site A of murine type I␣ R subunit, by analyzing the effects of nine different substitutions at this residue on cAMP binding and allosteric properties of bacterially expressed RI␣ subunits. All substitutions reduced the cAMP binding affinity of site A, but the magnitude of reduction varied from several hundredfold to 10 6 -fold. The differential effects of the different substitutions could not easily be rationalized by interactions with cAMP and might, in part, reflect interactions with other residues in the unoccupied cAMP-binding pocket. None of the Arg-210 substitutions appeared to disrupt the allosteric interaction by which occupation of site A slows dissociation of cAMP from site B, although the effect was difficult to elicit in full with mutations that had strong effects on cAMP binding. The two weakest substitutions, Arg-210 3 Ile and Arg-210 3 Thr, could be shown to have essentially no effect on the allosteric interaction by which occupation of site A reduces the affinity of R subunit for the catalytic subunit. The weaker mutations had a smaller effect on kinase activation by the suboptimal activator R p -adenosine cyclic 3,5-phosphorothioate than by cAMP, suggesting that the analog largely bypasses interactions with the guanidinium group of Arg-210.Activation of cAMP-dependent protein kinase results from allosteric interactions by which cAMP binding to two intrachain cAMP-binding sites (sites A and B) in kinase regulatory (R) 1 subunit decreases by 5 orders of magnitude or more the affinity of R for the catalytic (C) subunit (reviewed in Ref. 1). Studies with analogs of cAMP implicate the ribose-cyclic phosphate moiety of cAMP in the activation process (2, 3). The adenine ring is thought to contribute to the specificity and high affinity of cAMP binding (2, 4). A recent crystal structure of a large fragment containing the cAMP-binding domains of R subunit corroborated predictions of an earlier model based on the structure of the catabolite activator protein of Escherichia coli that most of the important contact residues for interaction with the ribose-cyclic phosphate of cAMP are in ␤-strands 6 and 7 of an antiparallel ␤-roll structure forming one face of the cAMP-binding pockets (5, 6). It appears from this structure that 2 in site A and Glu-325 in site B interact with the 2Ј-hydroxyl group of cAMP and that the guanidinium groups of Arg-210 in site A and Arg-334 in site B interact with the equatorial exocyclic oxygen of the cAMP phosphate group (5). Mutations at these residues or at the conserved Gly residues immediately upstream of the conserved Glu residues markedly reduced the affinities of the mutated sites for cAMP (7-10). Arg-242, in the long ␣-helix perpendicular to the ␤-r...

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Arginine 210 Is Not a Critical Residue for the Allosteric Interactions Mediated by Binding of Cyclic AMP to Site A of Regulatory (RIα) Subunit of Cyclic AMP-dependent Protein Kinase

Steinberg

Symcox

Sollid

et al. 1996

Journal of Biological Chemistry

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“…The derivatives 19-25 are not cyclic nucleotides. Some of them (19)(20)(21) can mimic the ribose-cyclic phosphate moiety of cAMP and bind to their receptor proteins [14,39] (Fig. 1 d) The polarity of all derivatives has been measured by highperformance liquid reversed phase chromatography.…”

Section: Selection Of Camp Derivativesmentioning

confidence: 99%

Substrate Specificity of Cyclic Nucleotide Phosphodiesterase from Beef Heart and from Dictyostelium discoideum

et al. 1983

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The substrate specificity of beef heart phosphodiesterase activity and of the phosphodiesterase activity at the cell surface of the cellular slime mold Dicfyostelium discoideum has been investigated by measuring the apparent K, and maximal velocity (V) of 24 derivatives of adenosine 3',5'-nionophosphate (CAMP). Several analogs have increased K,,, values, but unaltered V values if compared to CAMP; also the contrary (unaltered X, and reduced V) has been observed, indicating that binding of the substrate to the enzyme and ring opening are two separate steps in the hydrolysis of CAMP.cAMP is bound to the beef heart phosphodiesterase by dipole-induced dipole interactions between the adenine moiety and an aromatic amino acid, and possibly by a hydrogen bond between the enzyme and one of the exocyclic oxygen atoms; a cyclic phosphate ring is not required to obtain binding. cAMP is bound to the slime mold enzyme via a hydrogen bond at the 3'-oxygen atom, and probably via a hydrogen bond with one of the exocyclic oxygen atoms. A cyclic phosphate ring is necessary to obtain binding to the enzyme. A specific interaction (polar or hydrophobic) between the base moiety and the enzyme has not been demonstrated. A negative charge on the phosphate moiety is not required for binding of cAMP to either enzyme.The catalytic reaction in both enzymes is restricted to the phosphorus atom and to the exocyclic oxygen atoms. Substitution of the negatively charged oxygen atom by an uncharged dimethylamino group in axial or equatorial position renders the compound non-hydrolyzable. Substitution of an exocyclic oxygen by a sulphur atom reduces the rate of the catalytic reaction about 100-fold if sulphur is placed in axial position and more than 10000-fold if sulphur is placed in equatorial position. A reaction mechanism for the enzymatic hydrolysis of cAMP is proposed.cAMP is a key regulator of metabolism, function and growth of many cell types [l]. In mammalian cells CAMP is the second messenger of many hormones; it achieves its function via binding to an intracellular receptor, which is a protein kinase. In the cellular slime mold Dicfyostelium discoideum cAMP acts as the first messenger [2]; it is excreted by the cells and achieves its function via binding to a cell surface receptor. In D. discoideum cAMP induces chemotaxis and cell aggregation (for reviews see [3,4]).The level of cAMP is controlled by the rate of synthesis catalyzed by adenylate cyclase, the rate of excretion, and the rate of degradation by cyclic nucleotide phosphodiesterase.In mammalian tissue at least three types of phosphodiesterase have been demonstrated : a calmodulin-dependent enzyme, a CAMP-specific enzyme, and a third form characterized by cGMP activation (for reviews see [5,6]). In the cellular slime molds at least two enzymes have been demonstrated: one enzyme is localized on the cell surface and in the extracellular medium [7,8] and hydrolyzes cAMP and cGMP with about equal rates, and one cGMP-specific enzyme is localized intracellularly [9-111. cAMP and c...

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“…Animals have two major classes of cAMP-dependent protein kinase, designated as types I and II, that differ primarily in R (12,17,41). Kinase activation proceeds by a concerted reaction in which cAMP binds to the inactive holoenzyme and promotes dissociation of the catalytic subunit from R (4,14,17). Each R monomer has two binding sites for cAMP (3,7,40).…”

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Fine-structure mapping of charge-shift mutations in regulatory subunit of type I cyclic AMP-dependent protein kinase.

Steinberg¹

1984

Mol. Cell. Biol.

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A variety of structural mutations that alter functional properties of regulatory subunit (R) of type I cyclic AMP-dependent protein kinase are available in the cultured S49 mouse lymphoma cell system. Many of these mutations also alter the electrostatic charge of R by about 1 or 2 units. By a novel peptide mapping procedure, a number of these "charge-shift" structural mutations were localized to small regions within the R polypeptide. The procedure employed two-dimensional polyacrylamide gel electrophoresis to separate large overlapping fragments generated from denatured, affinity-purified R by limited digestion with papain. Mutations were mapped to intervals between the endpoints of these fragments. The position of one mutation was confirmed by mapping a new site for cleavage by Staphylococcus aureus V8 protease. Six different Ka mutations, which increase the concentrations of cyclic AMP required for kinase activation, mapped to three clusters in the carboxy-terminal half of R. Second-site mutations that cause phenotypic reversion of a single Ka mutant strain mapped to either side of the original mutation. By using charge-shift mutations for calibration, a map of charge density distribution was constructed for the R polypeptide. This map allowed tentative assignment of mutational lesions to portions of the R amino acid sequence implicated in cyclic AMP binding.Cyclic AMP (cAMP)-dependent protein kinases (EC 2.7.1.37), the intracellular transducers for responses involving cAMP elevation, have tetrameric structures consisting of two catalytic subunits and a regulatory subunit (R) dimer (16, 17). Animals have two major classes of cAMP-dependent protein kinase, designated as types I and II, that differ primarily in R (12, 17, 41). Kinase activation proceeds by a concerted reaction in which cAMP binds to the inactive holoenzyme and promotes dissociation of the catalytic subunit from R (4, 14, 17). Each R monomer has two binding sites for cAMP (3,7,40). These sites are distinguishable by differences in dissociation rates of bound cAMP and by differences in relative affinities for analogs of cAMP (15,24,25). Site 1 prefers cAMP derivatives modified in the C-8 position, and site 2 prefers derivatives modified in the N6 position. Both sites are thought to be important for kinase activation (20,26). Binding of cAMP induces conformational changes in R that can be detected by changes in reactivity of cysteine residues (1) or by changes in circular dichroism (27). These structural changes presumably contribute to cooperativity in cAMP binding (25)

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A Model for the Chemical Interactions of Adenosine 3':5'-Monophosphate with the R Subunit of Protein Kinase Type I. Refinement of the Cyclic Phosphate Binding moiety of Protein Kinase Type I

Cited by 67 publications

References 39 publications

Arginine 210 Is Not a Critical Residue for the Allosteric Interactions Mediated by Binding of Cyclic AMP to Site A of Regulatory (RIα) Subunit of Cyclic AMP-dependent Protein Kinase

Arginine 210 Is Not a Critical Residue for the Allosteric Interactions Mediated by Binding of Cyclic AMP to Site A of Regulatory (RIα) Subunit of Cyclic AMP-dependent Protein Kinase

Substrate Specificity of Cyclic Nucleotide Phosphodiesterase from Beef Heart and from Dictyostelium discoideum

Fine-structure mapping of charge-shift mutations in regulatory subunit of type I cyclic AMP-dependent protein kinase.

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