Release of [3H]adenosine cyclic 3',5'-phosphate bound to the regulatory moiety of protein kinase I in the presence of magnesium(2+), ATP and the catalytic subunit of the kinase

Øgreid, D; Døskeland, Stein Ove

doi:10.1021/bi00276a026

Cited by 33 publications

(17 citation statements)

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“…Kinetic evidence [22] as well as results from cAMP binding site mutants [10] and cAMP analogs [25,26] have indicated that kinase activation is mediated via a sequential kinetic mechanism. First, cAMP The cAMP analogs are listed according to their R l A/B selectivity [5].…”

Section: Discussionmentioning

confidence: 99%

“…1 and Table 1 the two cAMP binding sites in the wild-type R I subunit are readily distinguished by their cAMP dissociation rates. The rapid dissociation (kon-= 1.0 min -1) represents the exchange of cAMP bound to site A while the slower dissociation (kofr = 0.0167 min -]) is the exchange from site B [22]. Dissociation rates were measured with high salt in the buffer, a condition known to emphasize biphasic exchange [23].…”

Section: Purification and Camp-bindingmentioning

confidence: 99%

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Ala³³⁵ is essential for high‐affinity cAMP‐binding of both sites A and B of cAMP‐dependent protein kinase type I

et al. 1995

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A single amino acid substitution (Ala33SAsp) in cAMP binding site B of the regulatory subunit of cAMP-dependent protein kinase type I was sufficient to abolish high affinity cAMP binding for both cAMP binding sites A and B. Furthermore, the Ala33SAsp mutation increased the activation constant for cAMP of the mutant holoenzyme 30-fold and also enhanced the rate of holoenzyme formation. Thus, the substitution was responsible for the dominant negative phenotype of the enzyme. Activation of mutant holoenzyme with site-selective cAMP analogs indicated that the enzyme dissociated through binding to site A only. Our results provide evidence that Ala 335 is an essential residue for high affinity cAMP binding of both sites as well as for the functional integrity of the enzyme.B has been shown to interact with the adenine moiety since alteration of this residue changed the affinity and specificity for cGMP [13]. Recently, a sub-clone of the rat myelocytic leukemia cell line (IPC-81) has been found to harbor a heterozygously expressed point mutation [14]. This mutated clone was selected by treatment with 8-CPT-cAMP and identified as Alato-Asp at the invariant position 335 [14]. In order to study the effects of this mutation on ligand binding and holoenzyme activation we used recombinant protein expression techniques to obtain large quantities of pure recombinant Ala335Asp mutant protein. Materials and methods

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Section: Discussionmentioning

confidence: 99%

Section: Purification and Camp-bindingmentioning

confidence: 99%

Ala³³⁵ is essential for high‐affinity cAMP‐binding of both sites A and B of cAMP‐dependent protein kinase type I

et al. 1995

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“…It is known that addition ofthe C subunit causes the release of cAMP from the R-cAMP complex with either RI and RII (22,29,30). Mutant RII provided a suitable means for testing whether or not the effect of the C subunit on cAMP dissociation is due to the interaction ofthe C subunit with the hinge region.…”

Section: Resultsmentioning

confidence: 99%

“…The cAMP binding capacity of RII was measured with a Millipore filtration method described by D0skeland and Ogreid (25). The reaction mixture contained 2 ,uM [3HJcAMP (25)(26)(27)(28)(29)(30) Ci/mmol; 1 Ci = GBq), 50 mM potassium phosphate (pH 6.8), 1 mM EDTA, type IIA histone (Sigma) at 0.5 mg/ml, 2 M NaCl, and 1 Ag of RII. The binding reaction was carried out at 30'C for 30 min and terminated by adding ice-cold 95% (wt/vol) ammonium sulfate solution.…”

mentioning

confidence: 99%

A constitutively active holoenzyme form of the cAMP-dependent protein kinase.

Wang

Scott

McKnight

et al. 1991

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

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The major function of the regulatory (R) subunit ofthe cAMP-dependent protein kinase is to bind tightly to the catalytic (C) subunit to form an inactive holoenzyme in the absence of cAMP. The hinge region of the R subunit resembles the substrate recognition site for the C subunit and is known to be involved in the R-C subunit interaction. Two arginine residues in this region, Arg-92 and Arg-93, are suggested to be essential for holoenzyme formation. In this study, a mutant in which Arg-92 and Arg-93 of type II regulatory subunit (RH) were replaced with alanine was constructed. Formation of the holoenzyme from mutant RH and C subunits was analyzed by gel-filtration and cation-exchange chromatography. Mutant RH in its cAMP-free form formed a stable holoenzyme with the C subunit, which dissociated in the presence of cAMP. Interestingly, the holoenzyme formed from mutant RU and C subunits retained full enzymatic activity even in the absence of cAMP. Although mutant RU could no longer be phosphorylated by the C subunit, the rate of [3H]cAMP release from mutant RUI cAMP was increased by addition ofthe C subunit, indicating that C-induced cAMP release is not the result of the interaction of the C subunit with the hinge region. These results demonstrate that Arg-92 and Arg-93 are not essential for holoenzyme formation but are critical for inhibiting kinase activity in the holoenzyme probably by occupying the substrate binding site. The results suggest that, in addition to the hinge region, a second site on the RH subunit may interact with the C subunit in a cAMP-dependent manner.The cAMP-dependent protein kinase (PKA) consists of regulatory (R) and catalytic (C) subunits that associate to form an inactive tetrameric holoenzyme (R2C2). The binding of cAMP to the R subunit leads to dissociation of the inactive holoenzyme and the release of the active C subunit (1).Two major types of PKA have been classified based on the isozymic identity of the R subunit, which exists in two forms, RI and RII (2,3). Both forms of the R subunit share the general domain structure, which has been defined by limited proteolysis and affinity labeling (4). The 50 residues at the amino terminus of the R subunit are essential for dimerization to R2 (5). Two tandem cAMP-binding domains are located at the carboxyl terminus of the R subunit (6, 7). The sequence of these cAMP binding domains is homologous to that of the catabolite gene activator protein (CAP) from Escherichia coli, and a p-barrel structural model of cAMP-binding sites has been proposed, based on the crystallographic coordinates of CAP (8-10). The two cAMP binding sites (sites A and B) of the R subunit have different preferences for cAMP analogs and display different dissociation rates for cAMP (11,12).A proteolytically sensitive "hinge" region (residues 90-100, see Fig. 1) has been emphasized thus far as the site of interaction with the C subunit. The hinge region in RII contains a substrate consensus sequence, Arg-Arg-Xaa-Ser, while that in RI has a pseudosubstrate sequence,...

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“…Consequently, PDE7A1 can play a dual role in promoting PKA holoenzyme re-association following hormonal stimulation-localization of C to the vicinity of R/cAMP dimers and elimination of cAMP as it is released from R. R dimers compete effectively for binding with C pre-bound to PKI, and potentially also with C pre-bound to PDE7A1 (44). The binding of C to R/cAMP dimers facilitates cAMP dissociation, a rate-limiting step in PKA re-association (45)(46)(47). Cyclic AMP release from R is inhibited by physiological concentrations of cAMP (1 M), and rapid cAMP hydrolysis upon its release facilitates the release of the additional three cAMP molecules bound to R/cAMP and the consequent re-association of PKA.…”

Section: Discussionmentioning

confidence: 99%

PDE7A1, a cAMP-specific Phosphodiesterase, Inhibits cAMP-dependent Protein Kinase by a Direct Interaction with C

Han¹,

Sonati²,

Rubin

et al. 2006

Journal of Biological Chemistry

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The N-terminal regulatory region of the high affinity cAMP-specific phosphodiesterase, PDE7A1, contains two copies of the cAMPdependent kinase (PKA) pseudosubstrate site RRGAI. In ␤TC3 insulinoma cells, PDE7A1 co-localizes with PKA II in the Golgicentrosome region. The roles PDE7A1 and its regulatory region play in cAMP signaling were examined by studying interactions with PKA subunits. PDE7A1 associates with the dissociated C subunit of PKA (C), but does not bind tetrameric PKA holoenzyme. High affinity binding of C by PDE7A1 inhibits kinase activity in vitro (IC 50 ‫؍‬ 0.5 nM). The domain containing PKA pseudosubstrate sites at the N terminus of PDE7A1 mediates complex formation with C. The PDE7A1 N-terminal repeat region inhibits C activity in CHO-K1 cells and also suppresses C dependent, cAMP-independent, physiological responses in yeast. Thus, PDE7A1 possesses a non-catalytic activity that can contribute to the termination of cAMP signals via direct inhibition of C. This study identifies a novel inhibitor of PKA and a non-catalytic affect of a cyclic nucleotide phosphodiesterase.Cyclic AMP (cAMP) is a second messenger that mediates diverse cellular responses to hormones and neurotransmitters. Cyclic AMP levels are regulated by rates of adenylyl cyclase-catalyzed synthesis and phosphodiesterase (PDE) 2 -mediated hydrolysis. Several targets for cAMP are known: cAMP-dependent protein kinases (PKAs), cAMP-activated guanine nucleotide exchange factors, and ion channels (1-4). PKAs phosphorylate multiple substrates and thereby regulate a broad range of physiological processes including gene expression via CREB/ATF and co-stimulatory transcription factors, glycogenolysis, lipolysis, catecholamine biosynthesis, synaptic transmission, and ion channel/transporter activities (1, 2, 5-7). PKA activity is stimulated by binding of cAMP to the regulatory subunits of inactive, tetrameric PKA complexes, and the consequent dissociation of active catalytic subunits. Free catalytic subunit (C) phosphorylates PKA substrates including nuclear targets. During interphase, the vast majority of PKA regulatory subunits and tetrameric complexes are excluded from the nucleus. A substantial proportion (principally PKA II) can be tethered to specific subcellular sites via interactions with anchor proteins (8). Subcellular targeting of PKA is often required for efficient transduction of cAMP signals (9). Down-regulation of PKA activity is achieved via PDE-mediated degradation of cAMP, dissociation of ligands from receptors, receptor desensitization, inhibitory G proteins, GTPase-activating proteins, holoenzyme reassociation, C degradation, compensatory expression of PKA regulatory subunit, and direct inhibition by the PKA inhibitor, PKI (10 -15). In addition to directly inhibiting catalytic activity, PKI also promotes export of C from the nucleus (16 -18).PKA regulatory subunits and PKI bind to C with high affinity (1, 2). The hinge regions of the type I and type II regulatory subunits of PKA interact with the catalytic cleft of C via the...

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Release of [3H]adenosine cyclic 3',5'-phosphate bound to the regulatory moiety of protein kinase I in the presence of magnesium(2+), ATP and the catalytic subunit of the kinase

Cited by 33 publications

References 46 publications

Ala³³⁵ is essential for high‐affinity cAMP‐binding of both sites A and B of cAMP‐dependent protein kinase type I

Ala³³⁵ is essential for high‐affinity cAMP‐binding of both sites A and B of cAMP‐dependent protein kinase type I

A constitutively active holoenzyme form of the cAMP-dependent protein kinase.

PDE7A1, a cAMP-specific Phosphodiesterase, Inhibits cAMP-dependent Protein Kinase by a Direct Interaction with C

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Release of [3H]adenosine cyclic 3',5'-phosphate bound to the regulatory moiety of protein kinase I in the presence of magnesium(2+), ATP and the catalytic subunit of the kinase

Cited by 33 publications

References 46 publications

Ala335 is essential for high‐affinity cAMP‐binding of both sites A and B of cAMP‐dependent protein kinase type I

Ala335 is essential for high‐affinity cAMP‐binding of both sites A and B of cAMP‐dependent protein kinase type I

A constitutively active holoenzyme form of the cAMP-dependent protein kinase.

PDE7A1, a cAMP-specific Phosphodiesterase, Inhibits cAMP-dependent Protein Kinase by a Direct Interaction with C

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Ala³³⁵ is essential for high‐affinity cAMP‐binding of both sites A and B of cAMP‐dependent protein kinase type I

Ala³³⁵ is essential for high‐affinity cAMP‐binding of both sites A and B of cAMP‐dependent protein kinase type I