Biochemical evidence for the interaction of regulatory subunit of cAMP‐dependent protein kinase with IDA (Inter‐DFG‐APE) region of catalytic subunit

Sahara, Setsuko; Sato, Kenichi; Kaise, Hiroshi; Mori, Kotaro; Sato, Ayako; Aoto, Mamoru; Tokmakov, Alexander A.; Fukami, Yasuo

doi:10.1016/0014-5793(96)00302-x

Cited by 14 publications

(7 citation statements)

References 23 publications

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“…These residues are distributed about the surface of the C subunit, leaving only a narrow region of the C subunit to which the R subunit might bind that agrees strikingly with the region identified by the fitting of the crystal structures into our two-ellipsoid model of the RC dimer. Also supporting the proposed site of R-C interaction are the data of Sahara et al (39), who have shown that antipeptide antibodies to C subunit residues 187-199 or to residues 187-205 blocked the R-C interaction.…”

Section: -91supporting

confidence: 61%

Quaternary Structures of a Catalytic Subunit-Regulatory Subunit Dimeric Complex and the Holoenzyme of the cAMP-dependent Protein Kinase by Neutron Contrast Variation

Zhao

Hoye

Boylan

et al. 1998

Journal of Biological Chemistry

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Chimeric molecules of the cAMP-dependent protein kinase (PKA) holoenzyme (R 2 C 2 ) and of a ⌬ 1-91 RC dimer were reconstituted using deuterated regulatory (R) and protiated catalytic (C) subunits. Small angle scattering with contrast variation has revealed the shapes and dispositions of R and C in the reconstituted complexes, leading to low resolution models for both forms. The crystal structures of C and a truncation mutant of R fit well within the molecular boundaries of the RC dimer model. The area of interaction between R and C is small, seemingly poised for dissociation upon a conformational transition within R induced by cAMP binding. Within the RC dimer, C has a "closed" conformation similar to that seen for C with a bound pseudosubstrate peptide. The model for the PKA holoenzyme has an extended dumbbell shape. The interconnecting bar is formed from the dimerization domains of the R subunits, arranged in an antiparallel configuration, while each lobe contains the cAMP-binding domains of one R interacting with one C. Our studies suggest that the PKA structure may be flexible via a hinge movement of each dumbbell lobe with respect to the dimerization domain. Sequence comparisons suggest that this hinge might be a property of the R II PKA isoforms.Protein phosphorylation is one of the most important mechanisms for the regulation of biochemical function in eukaryotic cells. It is catalyzed by a family of enzymes, the protein kinases, of which several hundred have been identified. The cAMP-dependent protein kinase (PKA) 1 was one of the earliest kinases to be discovered, and it serves as a prototype for understanding kinase structure-function relationships and regulatory mechanisms (1, 2). In the absence of cAMP, PKA is an inactive tetramer (R 2 C 2 ) with two identical regulatory (R) and two identical catalytic (C) subunits. The two R subunits are homodimerized at their amino-terminal ends (3, 4), and, physiologically, the R subunit appears always to exist as a dimer. The R subunit also has two in tandem cAMP-binding sites and a pseudosubstrate autoinhibitory domain that binds to C and inhibits catalysis in the absence of cAMP. Upon binding cAMP, the PKA holoenzyme dissociates into an R 2 homodimer and two active C subunits (5-7). Whether dissociation is absolutely required for activation, however, remains in question (8, 9). Saturation of both cAMP-binding sites on each R is required for activation.There are three isoforms of C (C␣, C␤, and C␥) and two major isoforms of R (R I and R II ) that are further distinguished into subforms (␣ and ␤) (10). The physiological importance of these isozyme variations is not fully understood, but anchoring proteins (AKAPs) for R II give it a unique cellular distribution (11,12). R I and R II show sequence homology in their cAMP-binding and pseudosubstrate domains but differ extensively in their dimerization domains as well as in the sequence connecting the dimerization and pseudosubstrate domains.Structural data have been obtained for the individual PKA subunits, but info...

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Section: -91supporting

confidence: 61%

Quaternary Structures of a Catalytic Subunit-Regulatory Subunit Dimeric Complex and the Holoenzyme of the cAMP-dependent Protein Kinase by Neutron Contrast Variation

Zhao

Hoye

Boylan

et al. 1998

Journal of Biological Chemistry

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“…Evaluation of Models with Respect to Other Data-The proposed site for the R-C interaction in the R I␣ -based (and R II␤ -based) model is supported by the observation that that anti-peptide antibodies to C subunit fragments 187-199 and 187-205 blocked the R-C interaction (34). The proposed interface site is also compatible with the extensive set of mutation studies of yeast PKA C subunit homolog that identified a group of five residues involved in R subunit interaction and 31 C subunit surface residues unlikely to be at the R-C subunit interface (30, 31) (see "Materials and Methods").…”

Section: Resultsmentioning

confidence: 95%

A Structural Model of the Catalytic Subunit-regulatory Subunit Dimeric Complex of the cAMP-dependent Protein Kinase

Tung

Walsh

Trewhella

2002

Journal of Biological Chemistry

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Previous neutron scattering studies elaborated the topographical relationship of the regulatory (R II␣ ) and catalytic (C ␣ ) subunits of the cAMP-dependent protein kinase. We present here the results of a set of computations that lead to an atomic model of the cAMP-dependent protein kinase heterodimer, ⌬ 1-91 R II␣ -C ␣ . The first step in the modeling utilized the crystal structures for the porcine C ␣ and bovine ⌬ 1-90 R I␣ or rat ⌬ 1-111 R II␤ , to homology-model structures of the species and isoforms that had been used in the neutron scattering experiments (bovine C ␣ subunit and murine ⌬ 1-91 R II␣ subunit, respectively). A docking procedure, constrained by the dimensions and positions of the ellipsoids in the neutron-derived R-C model as well as mutagenesis data, was used to develop "best fit" models for the heterodimer. Simulated annealing, molecular dynamics, and energy minimization were then used to refine the side chain packing at the heterodimer interface. For comparison, the calculations were done using the homology models derived from both the R I␣ and R II␤ crystal structures. Both resultant models had many similarities. Each predicted similar interfaces. The R I␣ -based model has 25% more hydrogen bonds than that based on R II␤ , with seven of these potential bonds in common. The distribution of hydrophobic, polar, and charged residues at the interface was similar for both models, with a distribution more characteristic of the exposed surface residues than those in the protein interior. The calculated interface area in each is relatively small (<2000 Å 2 ). The R I␣ -based model, however, has a significantly better fit with the scattering data and is therefore the one of distinctly higher probability. With its small interface area that has a high proportion of charged and polar residues, the complex appears poised for dissociation, and each subunit existing as a stable entity. This result is consistent with the known physiological events required for cAMP-dependent activation of the kinase.The cAMP-dependent protein kinase (PKA) 1 is a multifunctional kinase that serves as a prototype for understanding second messenger signaling and protein phosphorylation (1-3). In the absence of a cAMP signal, the enzyme is inactive and exists as a dimer of dimers, having two identical regulatory (R) and two identical catalytic (C) subunits. The C subunit has the conserved catalytic core structure that is common to the majority of protein kinases. The R subunit has a dimerization domain, followed by a pseudosubstrate sequence that inhibits C subunit activity and two in tandem cAMP-binding domains (in order from the N to C termini). When two cAMP molecules bind to each of the R subunits, the C subunit is activated, presumably via some sort of release of the pseudosubstrate sequence. The activation of the C subunit has been assumed to involve dissociation of the C subunits from the R 2 homodimer (4 -6). The strongest evidence for the dissociation of the C subunit is the fact that the C subunit, but not R 2 , is...

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“…Anti-phosphorylated MAP kinase antibody (#9101S) was from BioLabs (MA, USA). A synthetic tyrosine kinase substrate peptide, Cdc2 peptide (20 amino acids; VEKIGEGTYGVVYKARHKLS), and synthetic IDA (Inter-DFG-APE) peptides; IDA-Src (19 amino acids, LIEDNEYTARQGAKFPIKW), IDA-PKA (20 amino acids with a nonauthentic cysteine in the C-terminus, FAKRVKGRTWTLCGTPEYLC), IDA-MAPK (25 amino acids with a nonauthentic cysteine in the C-terminus, LARVA-DPDHDHTGFLTEYVATRWYRC), and IDA-EGFR (20 amino acids, LLGAEEKEYHAEGGKVPIKW) were synthesized and puri®ed as described (Fukami et al 1993;Sato et al 1995b;Sahara et al 1996;Tokmakov et al 1996). The tyrosinephosphorylated form of IDA-Src peptide (P-IDA-Src) was purchased from Sawadii Technology (Tokyo).…”

Section: Chemicals Antibodies and Peptidesmentioning

confidence: 99%

Adaptor protein Shc undergoes translocation and mediates up‐regulation of the tyrosine kinase c‐Src in EGF‐stimulated A431 cells

Sato

Kimoto²,

Kakumoto³

et al. 2000

Genes to Cells

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Biochemical evidence for the interaction of regulatory subunit of cAMP‐dependent protein kinase with IDA (Inter‐DFG‐APE) region of catalytic subunit

Cited by 14 publications

References 23 publications

Quaternary Structures of a Catalytic Subunit-Regulatory Subunit Dimeric Complex and the Holoenzyme of the cAMP-dependent Protein Kinase by Neutron Contrast Variation

Quaternary Structures of a Catalytic Subunit-Regulatory Subunit Dimeric Complex and the Holoenzyme of the cAMP-dependent Protein Kinase by Neutron Contrast Variation

A Structural Model of the Catalytic Subunit-regulatory Subunit Dimeric Complex of the cAMP-dependent Protein Kinase

Adaptor protein Shc undergoes translocation and mediates up‐regulation of the tyrosine kinase c‐Src in EGF‐stimulated A431 cells

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