Evidence that cyclic nucleotides activating rabbit muscle protein kinase I interact with both types of cAMP binding sites associated with the enzyme (original) (raw)
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European Journal of Biochemistry, 1989
cAMP analogs, all 96 of which were modified in the adenine moiety, were examined quantitatively for their ability to inhibit the binding of [3H]cAMP to each of the two classes (A and B) of cAMP-binding sites of type I (rabbit skeletal muscle) and type II (bovine heart) cAMP-dependent protein kinase. The study showed that analogs can be constructed that have a higher affinity than cAMP for a binding site. N6-phenyl-cAMP had 18-fold increased affinity for site A of RI (AI) and 40-fold increased affinity for site AII. 2-chloro-8-methylamino-cAMP had a 7-fold increased affinity for BI, and 8-(4-chlorophenylthio)-cAMP had 17-fold increased affinity for BII. Analogs could discriminate between the two classes of binding sites by more than two orders of magnitude in binding affinity: 2-chloro-8-methylamino-cAMP had 170-fold higher affinity for BI than for AI, and 2-n-butyl-8-thiobenzyl-cAMP had 700-fold higher affinity for BII than for AII. Analogs could also discriminate between the homologous binding sites of the isozymes: 2-n-butyl-8-bromo-cAMP had 260-fold higher affinity for AI than for AII (22-fold higher for BII than BI), and 8-piperidino-cAMP had 50-fold higher affinity for BII than for BI (and 50-fold higher for AI than for AII). The data suggest the following conclusions. (a) Stacking interactions are important for the binding of cAMP to all the binding sites. (b) Subtle differences exist between the sites as to the optimal electron distribution in the adenine ring since modifications that withdraw electrons at C2 and donate at C8 favour binding to BI, and disfavour binding to AI and AII. (c) There are no hydrogen bonds between the adenine ring of cAMP and any of the binding sites. (d) All sites bind cAMP in the syn conformation. (e) The subsites adjacent to the N6 and C8 positions may have nonpolar neighbouring regions since hydrophobic substituents at N6 could increase the affinity for AI and AII and similar substituents at C8 could increase the affinity for BII. Finally, (f) the sites differed in their ability to accomodate bulky substituents at C2 and C8. For all compounds tested, their potency as activators of protein kinases I and II was found to correlate, in a predictable fashion, to their mean affinity for the two classes of binding sites, rather than to the affinity for only one of the sites.
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.
Biochemistry, 1982
5'-[p-(Fluorosulfonyl)benzoyl] adenosine (FSBA) inactivates the catalytic subunit of the adenosine cyclic 3',5'-monophosphate dependent protein kinase isolated from bovine cardiac muscle by covalent modification of lysine-7 1, whereas 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) react with cysteines-199 and-343 to inactivate the enzyme. All three of these reagents have been postulated to modify residues at or near the active site of the catalytic subunit. ATP (2 mM) in the presence of excess Mg2+ (10 mM) protects the enzyme against inactivation by these reagents. AMP did not afford any protection, but adenosine slightly decreased the rate of inactivation. The specific effects of covalent modification of lysine-71 and cysteines-I99 and-343 on nucleotide binding were characterized by fluorescence-polarization titrations with lin-benzoadenine nucleotides as fluorescent ligands. lin-Benzoadenosine is a competitive inhibitor of the catalytic subunit with respect to ATP with a Ki (38 pM) similar to the Ki for adenosine (35 pM). This value agrees well with the Kd (32 pM) for adenosine determined by fluorescence-polarization titrations. lin-Benzoadenosine 5'-diphosphate (lin-benzo-ADP) has been shown to be a competitive inhibitor with respect to
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 Biochemical journal, 1977
The known amino acid sequences at the two sites on phosphorylase kinase that are phosphorylated by cyclic AMP-dependent protein kinase were extended. The sequences of 42 amino acids around the phosphorylation site on the alpha-subunit and of 14 amino acids around the phosphorylation site on the beta-subunit were shown to be: alpha-subunit Phe-Arg-Arg-Leu-Ser(P)-Ile-Ser-Thr-Glu-Ser-Glx-Pro-Asx-Gly-Gly-His-Ser-Leu-Gly-Ala-Asp-Leu-Met-Ser-Pro-Ser-Phe-Leu-Ser-Pro-Gly-Thr-Ser-Val-Phe(Ser,Pro,Gly)His-Thr-Ser-Lys; beta-subunit, Ala-Arg-Thr-Lys-Arg-Ser-Gly-Ser(P)-VALIle-Tyr-Glu-Pro-Leu-Lys. The sites on histone H2B which are phosphorylated by cyclic AMP-dependent protein kinase in vitro were identified as serine-36 and serine-32. The amino acid sequence in this region is: Lys-Lys-Arg-Lys-Arg-Ser32(P)-Arg-Lys-Glu-Ser36(P)-Tyr-Ser-Val-Tyr-Val- [Iwai, K., Ishikawa, K. & Hayashi, H. (1970) Nature (London) 226, 1056-1058]. Serine-36 was phosphorylated at 50% of the rate at which the beta-subunit...
Biochemical and Biophysical Research Communications, 2003
The present study examined the existence of the adenosine A 1 ; A 2A ; and A 2B receptors and the effect of receptor activation on cAMP accumulation and protein phosphorylation in primary rat skeletal muscle cells. Presence of mRNA and protein for all three receptors was demonstrated in both cultured and adult rat skeletal muscle. NECA (10 À9-10 À4 M) increased the cAMP concentration in cultured muscle cells with an EC 50 of (95% confidence interval) ¼ 15 (5.9-25.1) lM, whereas CGS 21680 (10 À9-10 À4 M) had no effect on cAMP accumulation. Concentrations of [R]-PIA below 10 À6 M had no effect on cAMP accumulation induced by either isoproterenol or forskolin. NECA resulted in phosphorylation of CREB with an EC 50 of (95% confidence interval) ¼ 1.7 (0.40-7.02) lM, whereas ERK1/2 and p38 phosphorylation was unchanged. The results show that, although the A 1 ; A 2A ; and A 2B receptors are all present in skeletal muscle cells, the effect of adenosine on adenylyl cyclase activation and phosphorylation of CREB is mainly mediated via the adenosine A 2B receptor.
Molecular and Cellular Biochemistry, 1999
A protein kinase that phosphorylates histones and polysomal proteins was partially purified from mouse liver cytosol. The active enzyme has a molecular mass of 100 kDa and a phosphorylatable subunit of 54 kDa. Biochemical as well as immunological data suggest that the enzyme is a heterodimer composed of the catalytic subunit of cyclic AMP-dependent protein kinase and the RII regulatory subunit. This RC form does not seem to dissociate upon activation with 3′, 5′ cyclic AMP and exhibits identical specificity as the classical cAMP-dependent protein kinase (2.7.1.37). The enzyme is affected by the 3′, 5′ cyclic phosphates of adenosine mainly, but also of guanosine, uridine and cytidine in a substrate-dependent manner. Cyclic nucleotides slightly stimulate phosphate incorporation into histones, while phosphorylation of polysomal proteins in intact polysomes is dramatically increased. The substrate- specific stimulatory effects of 3′, 5′ cyclic nucleotides are due to repression of the inhibition exerted upon the reaction, by negatively charged macromolecules such as RNA, DNA and to a lesser extent heparin.
Phosphorylation Modulates Catalytic Function and Regulation in the cAMP-Dependent Protein Kinase
Biochemistry, 1995
Site-directed mutagenesis was used to remove a critical phosphorylation site, Thr-197, near the active site of the catalytic subunit of CAMP-dependent protein kinase. This residue is present in a number of protein kinases, and its phosphorylation largely influences catalytic activity. We changed Thr-197 to aspartic acid and alanine and measured the effects of these substitutions on the kinetic mechanism and inhibitor affinities. The mutants were expressed as the free catalytic subunit and as soluble fusion proteins of glutathione-S-transferase. The values for KAT^ and Kpeptide for all three mutants are raised by approximately 2 orders of magnitude relative to the wild-type enzyme. Viscosometric measurements indicate that elevations in Kpeptide are the result of reduced rates for phosphoryl transfer and not reduced substrate affinities. This implies that the loop that contains the phosphothreonine, the activation loop, does not reduce access to the substrate site as proposed for the inactive forms of cdk2 kinase [DeBont,