Little is known about the relative role of cAMP-dependent protein kinase (cAPK) and guanine exchange factor directly activated by cAMP (Epac) as mediators of cAMP action. We tested cAMP analogs for ability to selectively activate Epac1 or cAPK and discriminate between the binding sites of Epac and of cAPKI and cAP-KII. We found that commonly used cAMP analogs, like 8-Br-cAMP and 8-pCPT-cAMP, activate Epac and cAPK equally as well as cAMP, i.e. were full agonists. In contrast, 6-modified cAMP analogs, like N 6 -benzoyl-cAMP, were inefficient Epac activators and full cAPK activators. Analogs modified in the 2-position of the ribose induced stronger Epac1 activation than cAMP but were only partial agonists for cAPK. 2-O-Alkyl substitution of cAMP improved Epac/cAPK binding selectivity 10 -100-fold. Phenylthio substituents in position 8, particularly with MeO-or Cl-in p-position, enhanced the Epac/cAPK selectivity even more. The combination of 8-pCPT-and 2-O-methyl substitutions improved the Epac/cAPK binding selectivity about three orders of magnitude. The cAPK selectivity of 6-substituted cAMP analogs, the preferential inhibition of cAPK by moderate concentrations of Rp-cAMPS analogs, and the Epac selectivity of 8-pCPT-2-O-methyl-cAMP was also demonstrated in intact cells. Using these compounds to selectively modulate Epac and cAPK in PC-12 cells, we observed that analogs selectively activating Epac synergized strongly with cAPK specific analogs to induce neurite outgrowth. We therefore conclude that cAMP-induced neurite outgrowth is mediated by both Epac and cAPK.

The cAMP-dependent protein kinase (PKA I and II) and the cAMP-stimulated GDP exchange factors (Epac1 and -2) are major cAMP effectors. The cAMP affinity of the PKA holoenzyme has not been determined previously. We found that cAMP bound to PKA I with a K d value (2.9 M) similar to that of Epac1. In contrast, the free regulatory subunit of PKA type I (RI) had K d values in the low nanomolar range. The cAMP sites of RI therefore appear engineered to respond to physiological cAMP concentrations only when in the holoenzyme form, whereas Epac can respond in its free form. Epac is phylogenetically younger than PKA, and its functional cAMP site has presumably evolved from site B of PKA. A striking feature is the replacement of a conserved Glu in PKA by Gln (Epac1) or Lys (Epac2). We found that such a switch (E326Q) in site B of human RI␣ led to a 280-fold decreased cAMP affinity. A similar single switch early in Epac evolution could therefore have decreased the high cAMP affinity of the free regulatory subunit sufficiently to allow Epac to respond to physiologically relevant cAMP levels. Molecular dynamics simulations and cAMP analog mapping indicated that the E326Q switch led to flipping of Tyr-373, which normally stacks with the adenine ring of cAMP. Combined molecular dynamics simulation, GRID analysis, and cAMP analog mapping of wild-type and mutated BI and Epac1 revealed additional differences, independent of the Glu/Gln switch, between the binding sites, regarding space (roominess), hydrophobicity/polarity, and side chain flexibility. This helped explain the specificity of current cAMP analogs and, more importantly, lays a foundation for the generation of even more discriminative analogs.Lower eukaryotes like Saccharomyces cerevisiae have as sole receptor for the signaling molecule cAMP the two cAMP-binding sites (A and B) of the regulatory (R) 4 subunit of the cAMPdependent protein kinase (PKA). These tandem cAMP binding domains can be traced in all four isoforms (RI␣, RI␤, RII␣, and RII␤) of mammalian PKA (1), in the cGMP-dependent protein kinases (2, 3), the cyclic nucleotide gated ion channels (3-5), and the exchange proteins directly activated by cAMP, Epac1, and Epac2 (6). In PKA conformational changes induced by cAMP binding to both site A and B are required to dissociate the catalytic (C) subunit from the holoenzyme complex (7,8).In contrast, cAMP binding to a single site of Epac is sufficient to relieve the tonic intrachain inhibition of its GDP exchange activity toward the small GTPase Rap (6, 9). A major issue in cell signaling is how the second messenger cAMP uses the receptors PKA and Epac to coordinate biological effects (10). Comparison of the cAMP affinity of Epac1 and PKA holoenzyme would help predict which of the two cAMP receptors, if present in the same compartment, is likely to be preferentially activated by a slight increase of cAMP. For this the cAMP affinity of PKA holoenzyme, so far unknown, must be determined. The functional cAMP site in Epac is presumably derived from the B site of PKA b...

The three aromatic amino acid hydroxylases (phenylalanine, tyrosine, and tryptophan hydroxylase) and nitric oxide synthase (NOS) all utilize (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)) as cofactor. The pterin binding site in the three hydroxylases is well conserved and different from the binding site in NOS. The structures of phenylalanine hydroxylase (PAH) and of NOS in complex with BH(4) are still the only crystal structures available for the reduced cofactor-enzyme complexes. We have studied the enzyme-bound and free conformations of BH(4) by NMR spectroscopy and molecular docking into the active site of the three hydroxylases, using endothelial NOS as a comparative probe. We have found that the dihydroxypropyl side chain of BH(4) adopts different conformations depending on which hydroxylase it interacts with. All the bound conformations are different from that of BH(4) free in solution at neutral pH. The different bound conformations appear to result from specific interactions with nonconserved amino acids at the BH(4) binding sites of the hydroxylases, notably the stretch 248-251 (numeration in PAH) and the residue corresponding to Ala322 in PAH, i.e., Ser in TH and Ala in TPH. On the basis of analysis of molecular interaction fields, we discuss the selectivity determinants for each hydroxylase and explain the high-affinity inhibitory effect of 7-tetrahydrobiopterin specifically for PAH.

The activation of protein kinase A involves the synergistic binding of cAMP to two cAMP binding sites on the inhibitory R subunit, causing release of the C subunit, which subsequently can carry out catalysis. We used NMR to structurally characterize in solution the RI␣-(98 -381) subunit, a construct comprising both cyclic nucleotide binding (CNB) domains, in the presence and absence of cAMP, and map the effects of cAMP binding at single residue resolution. Several conformationally disordered regions in free RI␣ become structured upon cAMP binding, including the interdomain ␣C:A and ␣C:A helices that connect CNB domains A and B and are primary recognition sites for the C subunit. NMR titration experiments with cAMP, B site-selective 2-Cl-8-hexylamino-cAMP, and A site-selective Protein kinase A (PKA)3 is a primary receptor for cyclic adenosine monophosphate (cAMP) in eukaryotic cells (1, 2). In the absence of cAMP, the enzyme is an inactive, tetrameric holoenzyme complex, composed of two regulatory (R) and two catalytic (C) subunits (R 2 C 2 ). The catalytic site of the C subunit is occluded by a short inhibitory sequence in the R subunit (residues 94 -99 in bovine RI␣) that connects the N-terminal dimerization domain to the two cyclic nucleotide binding (CNB) domains. Multiple contacts exist between the CNB domains and the C subunit. The enzyme is allosterically activated by cAMP (3, 4), whose binding to the R subunits causes dissociation of the C subunits from the holoenzyme complex, thereby rendering C catalytically active (5). Two CNB domains (A and B) are present in all four isoforms (RI␣, RI␤, RII␣, and RII␤) of mammalian PKA, and both need to be occupied by cAMP to achieve PKA dissociation under physiologically relevant conditions (for reviews, see Refs. 2 and 6). Newly transcribed R subunit (apoR) in the cell can complex with either cAMP or the C subunit of PKA. Binding of cAMP leads to a dramatically decreased affinity for the C subunit, whereas binding of the C subunit lowers the cAMP affinity by about 3 orders of magnitude (7), allowing the holoenzyme to respond to fluctuations in physiological cAMP concentrations (8, 9).Comparing the crystal structures of RI␣-(103-376) (numbering for bovine RI␣) with cAMP bound in both the A and B domains (10) with the structure of RI␣-(91-379) (R333K) complexed with the C subunit (4) revealed pronounced differences in the two CNB domains, in particular with respect to their relative positioning (Fig. 1). However, little is known about the structure of the ligand-free (apo) state of the R subunits. A truncated RI␣ (residues 119 -244), comprising most of the A domain, has been investigated by NMR (11-13). Note, however, that this truncated form lacks not only the B domain but also the C-terminal end of the A domain, in particular the ␣C:A and ␣CЈ:A helices. These helices are at the junction between domains A and B and are important elements for interaction with the C subunit (4). Moreover, this region is conserved in all CAP-related eukaryotic cAMP binding proteins, as...

BackgroundThe regulatory subunit (R) of cAMP-dependent protein kinase (PKA) is a modular flexible protein that responds with large conformational changes to the binding of the effector cAMP. Considering its highly dynamic nature, the protein is rather stable. We studied the thermal denaturation of full-length RIα and a truncated RIα(92-381) that contains the tandem cyclic nucleotide binding (CNB) domains A and B.Methodology/Principal FindingsAs revealed by circular dichroism (CD) and differential scanning calorimetry, both RIα proteins contain significant residual structure in the heat-denatured state. As evidenced by CD, the predominantly α-helical spectrum at 25°C with double negative peaks at 209 and 222 nm changes to a spectrum with a single negative peak at 212–216 nm, characteristic of β-structure. A similar α→β transition occurs at higher temperature in the presence of cAMP. Thioflavin T fluorescence and atomic force microscopy studies support the notion that the structural transition is associated with cross-β-intermolecular aggregation and formation of non-fibrillar oligomers.Conclusions/SignificanceThermal denaturation of RIα leads to partial loss of native packing with exposure of aggregation-prone motifs, such as the B' helices in the phosphate-binding cassettes of both CNB domains. The topology of the β-sandwiches in these domains favors inter-molecular β-aggregation, which is suppressed in the ligand-bound states of RIα under physiological conditions. Moreover, our results reveal that the CNB domains persist as structural cores through heat-denaturation.

No abstract

No abstract

I161The cyclic AMP-dependent protein kinase (cA-PK) graphy of the cytosol fraction revealed shoulders of exists in two isoenzyme forms (cA-PKI and cA-PKII). The holoenzymes are tetramers of molecular mass about 170 kDa, composed of two regulatory (RI or MI) and two catalytic (C) subunits [ 1,2]. The intriguing question of whether the two isoforms have separate biological effects remains unanswered, and different subcellular localization of the isoenzymes may give clues.In fractured preparations of rat hepatocytes about 30% of cA-PK was tightly associated with the particulate fraction, such that release required 0.5% Triton X-100 for 30 min. Size-exclusion chromatoAbbreviation used: cA-PK, cyclic AMP-dependent protein kinase. TableRII eluting with apparent molecular weight 700-1200 kDa in addition to the expected holoenzyme peak, suggesting the presence of supramolecular complexes of RII in rat hepatocyte T o address the question of whether such complexes exist in the intact hepatocyte, cells were briefly permeabilized with digitonin, and the release of RI and MI into the medium determined. The functional pore size of the permeabilized surface membrane was assessed by h.p.1.c.-size-exclusion chromatography of the proteins leaked into the medium. For some of these experiments the hepatocyte proteins had been metabolically prelabelled with ["Slmethionine to allow a noncytosol. I Size distribution of digitonin-released hepatocyte proteins as judged by size-exclusion chomatography Rat hepatocytes were prepared and suspended in a synthetic medium [4], with a low (0.75 ,UM) methionine content. After pre-incubation under gyratory shaking (5% CO,/ 95% 0,) for 90 min with [3iS]methionine (75 pCi/ml), the cells were washed, resuspended in medium (pH 7.0) at 37°C containing 120 mM-KCI, 5 mM-K2HP0,, 5 rnnMgSO,, 5 mM-Na,HPO,, I5 mn-Hepes, soybean trypsin inhibitor (0.2 mg/ml), aprotinin (0.065 mg/ml), leupeptin (0.048 mg/ml), pepstatin (0.0 I 4 mg/ml), antipain (0.006 mg/ml), chymostatin (0.007 mg/ml), ATP (2.5 mM), and various concentrations of digitonin. After incubation for 30 s the cells were centrigued (500 gav; 10 s), and the supernatant and cell pellet separated. The supernatant was subjected t o ultracentrifugation (10 min, at lOOooOg) in a Beckman Airfuge, diluted 1.3 in h.p.1.c. buffer (150 mn-potassium phosphate), and 0.1 ml of sample subjected t o h.p.1.c. on two columns (30 cm TSKW400+60 cm TSKW3000) in tandem, with a flow rate of 0.1 ml/min. Fractions of 0.5 ml were collected and their radioactivity determined.The column was calibrated with serum albumin, transferrin, lactate dehydrogenase, purified rabbit muscle cA-PKI, purified bovine heart cA-PKII, apoferritin, thyroglobulin, and a2-macroglobulin. The radioactivity of the fractions eluting close to 3H,0, containing low-molecular-mass peptides like glutathione and methionine, was taken as unity, and amounts of 35S-labelled proteins related t o this.