Interaction of the regulatory subunit (RII) of cAMP-dependent protein kinase with RII-anchoring proteins occurs through an amphipathic helix binding motif (original) (raw)
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European Journal of Biochemistry, 1994
Received January 19Eebruary 21, 1994) -EJB 94 005913 8-Piperidino-CAMP has been shown to bind with high affinity to site A of the regulatory subunit of CAMP-dependent protein kinase type I (AI) whereas it is partially excluded from the homologous site (AII) of isozyme I1 [Ggreid, D., Ekanger, R., Suva, R. H., Miller, J. P., and Dmkeland, S. 0. (1989), Eul: J. Biochem. 181,[28][29][30][31]. To further increase this selectivity, the (I?,)and (S,)diastereoisomers of 8-piperidino-CAMP[ S] were synthesized and analyzed for their potency to inhibit binding of 13H]cAMP to site A and site B from type I (rabbit skeletal muscle) and type I1 (bovine myocardium) CAMP-dependent protein kinases.
The Journal of biological chemistry, 1988
Each regulatory subunit of cAMP-dependent protein kinase has two tandem cAMP-binding sites, A and B, at the carboxyl terminus. Based on sequence homologies with the cAMP-binding domain of the Escherichia coli catabolite gene activator protein, a model has been constructed for each cAMP-binding domain. Two of the conserved features of each cAMP-binding site are an arginine and a glutamic acid which interact with the negatively charged phosphate and with the 2'-OH on the ribose ring, respectively. In the type I regulatory subunit, this arginine in cAMP binding site A is Arg-209. Recombinant DNA techniques have been used to change this arginine to a lysine. The resulting protein binds cAMP with a high affinity and associates with the catalytic subunit to form holoenzyme. The mutant holoenzyme also is activated by cAMP. However, the mutant R-subunit binds only 1 mol of cAMP/R-monomer. Photoaffinity labeling confirmed that the mutant R-subunit has only one functional cAMP-binding sit...
Journal of Biological Chemistry, 1990
Each protomer of the regulatory subunit dimer of CAMP-dependent protein kinase contains two tandem and homologous CAMP-binding domains, A and B, and cooperative CAMP binding to these two sites promotes holoenzyme dissociation. Several amino acid residues in the type I regulatory subunit, predicted to lie in close proximity to each bound cyclic nucleotide based on affinity labeling and model building, were replaced using recombinant techniques. The mutations included replacement of 1) Glu-200, predicted to hydrogen bond to the 2'-OH of CAMP bound to site A, with Asp, 2) Tyr-371, the site of affinity labeling with 8-N3-CAMP in site B, with Trp, and 3) Phe-247, the position in site A that is homologous to Tyr-371 in site B, with Tyr. Each mutation caused an approximate a-fold increase in both the K,(cAMP) and &(cAMP); however, the offrates for CAMP and the characteristic pattern of affinity labeling with S-N3-CAMP differed markedly for each mutant protein. Furthermore, these mutations affect the CAMP binding properties not only of the site containing the mutation, but of the adjacent nonmutated site as well, thus confirming that extensive crosscommunication occurs between the two CAMP-binding domains.
cAMP effector mechanisms. Novel twists for an ‘old’ signaling system
FEBS Letters, 2003
Cyclic AMP (cAMP) has traditionally been thought to act exclusively through cAMP-dependent protein kinase (cAPK, PKA), but a growing number of cAMP e¡ects are not attributable to general activation of cAPK. At present, cAMP is known also to directly regulate ion channels and the ubiquitous Rap guanine exchange factors Epac 1 and 2. Adding to the sophistication of cAMP signaling is the fact that (1) the cAPK holoenzyme is incompletely dissociated even at saturating cAMP, the level of free R subunit of cAPK being able to regulate the maximal activity of cAPK, (2) cAPK activity can be modulated by oxidative glutathionylation, and (3) cAPK is anchored close to relevant substrates, other signaling enzymes, and local compartments of cAMP. Finally, we will demonstrate an example of ¢ne-tuning of cAMP signaling through synergistic induction of neurite extensions by cAPK and Epac.
Journal of Biological Chemistry, 1999
The three G␣ i subunits were independently depleted from rat pituitary GH4C1 cells by stable transfection of each G␣ i antisense rat cDNA construct. Depletion of any G␣ i subunit eliminated receptor-induced inhibition of basal cAMP production, indicating that all G␣ i subunits are required for this response. By contrast, receptormediated inhibition of vasoactive intestinal peptide (VIP)-stimulated cAMP production was blocked by selective depletions for responses induced by the transfected serotonin 1A (5-HT1A) (G␣ i2 or G␣ i3 ) or endogenous muscarinic-M4 (G␣ i1 or G␣ i2 ) receptors. Strikingly, receptor activation in G␣ i1 -depleted clones (for the 5-HT1A receptor) or G␣ i3 -depleted clones (for the muscarinic receptor) induced a pertussis toxin-sensitive increase in basal cAMP production, whereas the inhibitory action on VIP-stimulated cAMP synthesis remained. Finally, in G␣ i2 -depleted clones, activation of 5-HT1A receptors increased VIP-stimulated cAMP synthesis. Thus, 5-HT1A and muscarinic M4 receptor may couple dominantly to G␣ i1 and G␣ i3 , respectively, to inhibit cAMP production. Upon removal of these G␣ i subunits to reduce inhibitory coupling, stimulatory receptor coupling is revealed that may involve G␥-induced activation of adenylyl cyclase II, a G i -stimulated cyclase that is predominantly expressed in GH4C1 cells. Thus G i -coupled receptor activation involves integration of both inhibitory and stimulatory outputs that can be modulated by specific changes in ␣ i subunit expression level.
Biochemistry, 2004
Cyclic adenosine 5′-monophosphate (cAMP) is an ancient signaling molecule, and in vertebrates, a primary target for cAMP is cAMP-dependent protein kinase (PKA). (R p )-adenosine 3′,5′-cyclic monophosphothioate ((R p )-cAMPS) and its analogues are the only known competitive inhibitors and antagonists for cAMP activation of PKA, while (S p )-adenosine 3′,5′-cyclic monophosphothioate ((S p )-cAMPS) functions as an agonist. The crystal structures of a ∆(1-91) deletion mutant of the RIR regulatory subunit of PKA bound to (R p )-cAMPS and (S p )-cAMPS were determined at 2.4 and 2.3 Å resolution, respectively. While the structures are similar to each other and to the crystal structure of RIR bound to cAMP, differences in the dynamical properties of the protein when (R p )-cAMPS is bound are apparent. The structures highlight the critical importance of the exocyclic oxygen's interaction with the invariant arginine in the phosphate binding cassette (PBC) and the importance of this interaction for the dynamical properties of the interactions that radiate out from the PBC. The conformations of the phosphate binding cassettes containing two invariant arginine residues (Arg209 on domain A, and Arg333 on domain B) are somewhat different due to the sulfur interacting with this arginine. Furthermore, the B-site ligand together with the entire domain B show significant differences in their overall dynamic properties in the crystal structure of ∆(1-91) RIR complexed with (R p )-cAMPS phosphothioate analogue ((R p )-RIR) compared to the cAMP-and (S p )-cAMPS-bound type I and II regulatory subunits, based on the temperature factors. In all structures, two structural solvent molecules exist within the A-site ligand binding pocket; both mediate water-bridged interactions between the ligand and the protein. No structured waters are in the B-site pocket. Owing to the higher resolution data, the N-terminal segment (109-117) of the RIR subunit can also be traced. This strand forms an intermolecular antiparallel -sheet with the same strand in an adjacent molecule and implies that the RIR subunit can form a weak homodimer even in the absence of its dimerization domain.
Journal of Biological Chemistry, 1996
Two isoforms of the catalytic subunit of cAMPdependent protein kinase, C␣ and C1, are known to be widely expressed in mammals. Although much is known about the structure and function of C␣, few studies have addressed the possibility of a distinct role for the C proteins. The present study is a detailed comparison of the biochemical properties of these two isoforms, which were initially expressed in Escherichia coli and purified to homogeneity. C1 demonstrated higher K m values for some peptide substrates than did C␣, but C1 was insensitive to substrate inhibition, a phenomenon that was observed with C␣ at substrate concentrations above 100 M. C␣ and C1 displayed distinct IC 50 values for the ␣ and  isoforms of the protein kinase inhibitor, protein kinase inhibitor (5-24) peptide, and the type II␣ regulatory subunit (RII␣). Of particular interest, purified type II holoenzyme containing C1 exhibited a 5-fold lower K a value for cAMP (13 nM) than did type II holoenzyme containing C␣ (63 nM). This latter result was extended to in vivo conditions by employing a transcriptional activation assay. In these experiments, luciferase reporter activity in COS-1 cells expressing RII␣ 2 C1 2 holoenzyme was half-maximal at 12-fold lower concentrations of 8-(4-chlorophenylthio)-cAMP and 5-fold lower concentrations of forskolin than in COS-1 cells expressing RII␣ 2 C␣ 2 holoenzyme. These results provide evidence that type II holoenzyme formed with C1 is preferentially activated by cAMP in vivo and suggest that activation of the holoenzyme is determined in part by interactions between the regulatory and catalytic subunits that have not been described previously.
The cAMP binding domain: An ancient signaling module
Proceedings of the National Academy of Sciences, 2005
cAMP-binding domains from several different proteins were analyzed to determine the properties and interactions of this recognition motif. Systematic computational analyses, including structure-based sequence comparison, surface matching, affinity grid analysis, and analyses of the ligand protein interactions were carried out. These analyses show distinctive roles of the sugar phosphate and the adenine in the cAMP-binding module. We propose that the cAMPbinding regulatory proteins function by providing an allosteric system in which the presence or absence of cAMP produces a substantial structural change through the loss of hydrophobic interactions with the adenine ring and consequent repositioning of the C helix. The modified positioning of the helix in turn is recognized by a proteinbinding event, completing the allostery.