Spectrum of spontaneous missense mutations causing cyclic AMP-resistance phenotypes in cultured S49 mouse lymphoma cells differs markedly from those of mutations induced by alkylating mutagens

Gorman, K B; Steinberg, Robert A.

doi:10.1007/bf02254719

Cited by 13 publications

(9 citation statements)

References 48 publications

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“…Most attention in these cAMP binding domains has focused so far on a number of conserved residues that interact with the cyclic phosphate portion of cAMP and sequester it completely from solvent. Genetic and mutational analyses have confirmed the importance of these residues for cAMP binding (43,44). The results described here, however, focus on cAMP binding domain A of the R I ␣ subunit and dissect it into two subsites that are structurally and functionally distinct.…”

Section: Discussionmentioning

confidence: 73%

Dissecting cAMP Binding Domain A in the RIα Subunit of cAMP-dependent Protein Kinase

Huang

Taylor

1998

Journal of Biological Chemistry

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The two gene-duplicated cAMP binding domains in the regulatory subunits of cAMP dependent protein kinase are each comprised of an A helix, an eight-stranded ␤-barrel, and a B and C helix (1). The A domain is required for high affinity binding to C, while the B domain regulates access to the A domain. Using a combination of a yeast two-hybrid screen coupled with deletion analysis, cAMP binding domain A of R I was dissected into two structurally and functionally distinct subsites, one that binds cAMP and another that binds the C subunit. The minimum stable subdomain required for binding to C in the 1-3 micromolar range is composed of residues 94 -169, while residues 236 -244, mapped to the C helix of cAMP binding domain A, were defined as a second surface necessary for high affinity (5-10 nanomolar) binding to C. This portion of the C helix, due to its position directly between the two subsites, serves as a molecular switch for either a cAMP-bound conformation or a Cbound conformation and can thus modulate interactions of cAMP binding domain A with cAMP, with C, and with cAMP binding domain B.

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Section: Discussionmentioning

confidence: 73%

Dissecting cAMP Binding Domain A in the RIα Subunit of cAMP-dependent Protein Kinase

Huang

Taylor

1998

Journal of Biological Chemistry

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“…Glu-200, Ile-210, Ser-210, Thr-210, and Asp-324 mutations were introduced into the wild-type R subunit plasmid by cassette replacement using polymerase chain reaction-amplified cDNAs from S49 cell mutants (19). Asn-210, Asp-210, Gln-210, Glu-210, His-210, and Lys-210 mutations were generated by site-directed mutagenesis using the following degenerate oligonucleotide primer in a polymerase chain reaction-based mutagenesis protocol (22): 5Ј-GGAGCTTGGA(A/C/G)A(C/G)CTGGCTTTGA-3Ј.…”

Section: Methodsmentioning

confidence: 99%

“…Ile-210, Ser-210, and Thr-210 mutations had been identified as spontaneous or mutagen-induced substitutions found in "K a " mutant isolates of S49 mouse lymphoma cells (19) and were transferred from mutant cDNAs to the bacterial expression plasmid. Asn, Asp, Gln, Glu, His, and Lys substitutions were generated by site-directed mutagenesis to assess the role of ionic and/or hydrogen-bonding interactions in the functions of Arg-210.…”

mentioning

confidence: 99%

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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“…Previous analyses (8)(9)(10)(11)(12)(13)(14)(15)(16)) have led to the proposal of an initial allosteric model in which the ␣-and ␤-subdomains are directly coupled to each other through a salt bridge between E200 and R241 and also possibly through a hydrophobic hinge defined by the L203, I204, and Y229 side-chain cluster (9,11,12). However, mutations (17), sequence conservation analyses (1), structurebased comparisons (1), and genetic screening (18,19) indicate that several other sites, which are not accounted for by the existing model, are also likely to play an active role in the cAMP-mediated activation of PKA. To comprehensively understand this allosteric mechanism, it is therefore necessary to elucidate at high resolution how cAMP remodels the free energy landscape of CBD-A, which serves as the central controlling unit of PKA.…”

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confidence: 99%

cAMP activation of PKA defines an ancient signaling mechanism

Das

Esposito

Abu-Abed

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

115

106

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allostery ͉ NMR ͉ cyclic nucleotide binding domain T he cAMP binding domain (CBD) and cAMP are conserved from bacteria to humans as a ubiquitous signaling mechanism to translate extracellular stress signals into appropriate biological responses (1). The major receptor for cAMP in higher eukaryotes, cAMP-dependent PKA (2), is ubiquitous in mammalian cells where it exists in two forms: the inactive tetrameric holoenzyme and the active dissociated catalytic subunit (Csubunit). In the inactive holoenzyme, two C-subunits are bound to a dimeric regulatory subunit (R-subunit) (Fig. 1a). Upon binding cAMP, the R-subunits undergo a conformational change that unleashes the active C-subunits (3, 4). The Rsubunits are composed of an N-terminal dimerization/docking domain, a flexible linker that includes an autoinhibitory segment, and two tandem CBDs (CBD-A and CBD-B; Fig. 1b) (5). The CBD-A of the isoform I␣ of the R-subunit of PKA (RI␣) contains a noncontiguous ␣-subdomain, which mediates the interactions with the C-subunit and a contiguous ␤-subdomain that forms a ␤-sandwich and contains the cAMP binding pocket (i.e., the phosphate binding cassette or PBC) ( Fig. 1 c and d) (6).Crystal structures of CBD-A of RI␣ in its cAMP-(6) and C-bound (7) states have revealed two very different conformations, highlighting the conformational plasticity of this ancient domain. Although these static crystal structures define two stable end points, questions remain about the allosteric control of the reversible shuttling between the two states. How does the signal generated by cAMP binding to the PBC (Fig. 1 c and d) propagate through a long-range allosteric network that spans both ␣-and ␤-subdomains? Previous analyses (8-16) have led to the proposal of an initial allosteric model in which the ␣-and ␤-subdomains are directly coupled to each other through a salt bridge between E200 and R241 and also possibly through a hydrophobic hinge defined by the L203, I204, and Y229 side-chain cluster (9,11,12). However, mutations (17), sequence conservation analyses (1), structurebased comparisons (1), and genetic screening (18,19) indicate that several other sites, which are not accounted for by the existing model, are also likely to play an active role in the cAMP-mediated activation of PKA. To comprehensively understand this allosteric mechanism, it is therefore necessary to elucidate at high resolution how cAMP remodels the free energy landscape of CBD-A, which serves as the central controlling unit of PKA. For this purpose, we have investigated by NMR RI␣ (residues 119-244), a construct that spans both ␣-and ␤-subdomains of CBD-A and retains high-

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Spectrum of spontaneous missense mutations causing cyclic AMP-resistance phenotypes in cultured S49 mouse lymphoma cells differs markedly from those of mutations induced by alkylating mutagens

Cited by 13 publications

References 48 publications

Dissecting cAMP Binding Domain A in the RIα Subunit of cAMP-dependent Protein Kinase

Dissecting cAMP Binding Domain A in the RIα Subunit of cAMP-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

cAMP activation of PKA defines an ancient signaling mechanism

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