Structure-Function Studies of the cAMP-Dependent Protein Kinase In Vitro and in Intact Cells (original) (raw)
Related papers
Full-length human cDNAs for all the different regulatory (R) and catalytic (C) subunits of CAMP-dependent protein kinases (PKA) were transcribed and translated in a cell-free in vitro system. The resulting proteins were characterized with respect to molecular size, isoelectric focusing, immunoreactivity, cAMP binding, and to what extent the RII protein subunits revealed mobility shifts upon phosphorylation by catalytic subunit of PKA. We were able to express cDNAs for all
CAMP-dependent protein kinase: prototype for a family of enzymes
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 1988
Protein kinases represent a diverse family of enzymes that play critical roles in regulation. The simplest and best-understood biochemically is the catalytic (C) subunit of cAMP-dependent protein kinase, which can serve as a framework for the entire family. The amino-terminal portion of the C subunit constitutes a nucleotide binding site based on affinity labeling, labeling of lysines, and a conserved triad of glycines. The region beyond this nucleotide fold also contains essential residues. Modification of Asp 184 with a hydrophobic carbodiimide leads to inactivation, and this residue may function as a general base in catalysis. Despite the diversity of the kinase family, all share a homologous catalytic core, and the residues essential for nucleotide binding or catalysis in the C subunit are invariant in every protein kinase. Affinity labeling and intersubunit cross-linking have localized a portion of the peptide binding site, and this region is variable in the kinase family. The ...
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.
Signaling through cAMP and cAMP-dependent protein kinase: Diverse strategies for drug design
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2008
The catalytic subunit of cAMP-dependent protein kinase has served as a prototype for the protein kinase superfamily for many years while structures of the cAMP-bound regulatory subunits have definded the conserved cyclic nucleotide binding (CNB) motif. It is only structures of the holoenzymes, however, that enable us to appreciate the molecular features of inhibition by the regulatory subunits as well as activation by cAMP. These structures reveal for the first time the remarkable malleability of the regulatory subunits and the CNB domains. At the same time, they allow us to appreciate that the catalytic subunit is not only a catalyst but also a scaffold that mediates a wide variety of protein:protein interactions. The holoenzyme structures also provide a new paradigm for designing isoform-specific activators and inhibitors of PKA. In addition to binding to the catalytic subunits, the regulatory subunits also use their N-terminal dimerization/docking domain to bind with high affinity to A Kinase Anchoring Proteins using an amphipathic helical motif. This targeting mechanism, which localizes PKA near to its protein substrates, is also a target for therapeutic intervention of PKA signaling.
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,
Journal of Biological Chemistry, 1997
Attempts to understand the physiological roles of the protein kinase inhibitor (PKI) proteins have been hampered by a lack of knowledge concerning the molecular heterogeneity of the PKI family. The PKI␥ cDNA sequence determined here predicted an open reading frame of 75 amino acids, showing 35% identity to PKI␣ and 30% identity to PKI1. Residues important for the high affinity of PKI␣ and PKI1 as well as nuclear export of the catalytic (C) subunit of cAMP-dependent protein kinase were found to be conserved in PKI␥. Northern blot analysis showed that a 1.3-kilobase PKI␥ message is widely expressed, with highest levels in heart, skeletal muscle, and testis. RNase protection analysis revealed that in most tissues examined PKI␥ is expressed at levels equal to or higher than the other known PKI isoforms and that in several mouse-derived cell lines, PKI␥ is the predominant PKI message. Partial purification of PKI activities from mouse heart by DEAE ion exchange chromatography resolved two major inhibitory peaks, and isoform-specific polyclonal antibodies raised against recombinant PKI␣ and PKI␥ identified these inhibitory activities to be PKI␣ and PKI␥. A comparison of inhibitory potencies of PKI␣ and PKI␥ expressed in Escherichia coli revealed that PKI␥ was a potent competitive inhibitor of C␣ phosphotransferase activity in vitro (K i ؍ 0.44 nM) but is 6-fold less potent than PKI␣ (K i ؍ 0.073 nM). Like PKI␣, PKI␥ was capable of blocking the nuclear accumulation of Flag-tagged C subunit in transiently transfected mammalian cells. Finally, the murine PKI␥ gene was found to overlap the murine adenosine deaminase gene on mouse chromosome 2. These results demonstrate that PKI␥ is a novel, functional PKI isoform that accounts for the previously observed discrepancy between PKI activity and PKI mRNA levels in several mammalian tissues.
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.
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.