Signaling through cAMP and cAMP-dependent protein kinase: Diverse strategies for drug design (original) (raw)
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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.
Regulation of cAMP-dependent Protein Kinases
Journal of Biological Chemistry, 2010
cAMP-dependent protein kinases are reversibly complexed with any of the four isoforms of regulatory (R) subunits, which contain either a substrate or a pseudosubstrate autoinhibitory domain. The human protein kinase X (PrKX) is an exemption as it is inhibited only by pseudosubstrate inhibitors, i.e. RI␣ or RI but not by substrate inhibitors RII␣ or RII. Detailed examination of the capacity of five PrKX-like kinases ranging from human to protozoa (Trypanosoma brucei) to form holoenzymes with human R subunits in living cells shows that this preference for pseudosubstrate inhibitors is evolutionarily conserved. To elucidate the molecular basis of this inhibitory pattern, we applied bioluminescence resonance energy transfer and surface plasmon resonance in combination with site-directed mutagenesis. We observed that the conserved ␣H-␣I loop residue Arg-283 in PrKX is crucial for its RI over RII preference, as a R283L mutant was able to form a holoenzyme complex with wild type RII subunits. Changing the corresponding ␣H-␣I loop residue in PKA C␣ (L277R), significantly destabilized holoenzyme complexes in vitro, as cAMP-mediated holoenzyme activation was facilitated by a factor of 2-4, and lead to a decreased affinity of the mutant C subunit for R subunits, significantly affecting RII containing holoenzymes.
Structure-Function Studies of the cAMP-Dependent Protein Kinase In Vitro and in Intact Cells
There are 518 protein kinase genes in the human genome; this constitutes about 1.7% of all human genes. The cAMP-dependent protein kinase (PKA) serves as the prototypic model for the study of kinases because it contains a conserved catalytic core shared with all eukaryotic kinases, it is the simplest kinase, and it is one of the best-characterized serine/threonine kinases. PKA is ubiquitous in mammals and regulates multiple physiological mechanisms such as the cell cycle, apoptosis, cell motility, energy metabolism, and gene transcription through a well-defined intracellular signaling pathway. While PKA clearly has a central physiological role it is still unclear how PKA mediates multiple physiological mechanisms at the cellular level. Four approaches were used to explore this question using two PKA catalytic subunits, Cα and Cγ, which share 83% identity in primary structure but differ in function. The first approach sought to identify differences in primary structure between Cγ and...
Journal of molecular biology, 2004
The catalytic subunit of cAMP-dependent protein kinase has served as a paradigm for the entire kinase family. In the course of studying the structure -function relationship of the P þ 1 loop (Leu198 -Leu205) of the kinase, we have solved the crystal structure of the Tyr204 to Ala mutant in complexes with Mg·ATP and an inhibitory peptide at 1.26 Å , with overall structure very similar to that of the wild-type protein. However, at the nucleotide binding site, ATP was found largely hydrolyzed, with the products ADP-PO 4 retained in the structure. High-resolution refinement suggests that 26% of the molecules contain the intact ATP, whereas 74% have the hydrolyzed products. The observation of the substrate and product states in the same structure adds significant information to our understanding of the phosphoryl transfer process. Structural examination of the mutation site substantiates and extends the emerging concept that the hydrophobic core in the large lobe of the kinase might serve as a stable platform for anchoring key segments involved in catalysis. We propose that Tyr204 is critical for anchoring the P þ 1 loop to the core. Further analysis has highlighted two major connections between the P þ 1 loop and the catalytic loop (Arg165-Asn171). One emphasizes the hydrophobic packing of Tyr204 and Leu167 mediated through residues from the aF-helix, recently recognized as a signal integration motif, which together with the aE-helix forms the center of the hydrophobic core network. The other connection is mediated by the hydrogen bond interaction between Thr201 and Asp166, in a substrate-dependent manner. We speculate that the latter interaction may be important for the kinase to sense the presence of substrate and prepare itself for the catalytic reaction. Thus, the P þ 1 loop is not merely involved in substrate binding; it mediates the communication between substrate and catalytic residues.
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 ...
Progress in Biophysics & Molecular Biology, 1999
The protein kinase catalytic core in essence comprises an extended network of interactions that link distal parts of the molecule to the active site where they facilitate phosphoryl transfer from ATP to protein substrate. This review de®nes key sequence and structural elements, describes what is currently known about the molecular interactions, and how they are involved in catalysis. #
Folding and activity of cAMP-dependent protein kinase mutants
FEBS Letters, 2005
The catalytic subunit of cAMP-dependent protein kinase (PKA) can easily be expressed in Escherichia coli and is catalytically active. Four phosphorylation sites are known in PKA (S10, S139, T197 and S338), and the isolated recombinant protein is a mixture of different phosphorylated forms. Obtaining uniformly phosphorylated protein requires separation of the protein preparation leading to significant loss in protein yield. It is found that the mutant S10A/S139D/S338D has similar properties as the wild-type protein, whereas additional replacement of T197 with either E or D reduces protein expression yield as well as folding propensity of the protein. Due to its high sequence homology to Akt/PKB, which cannot easily be expressed in E. coli, PKA has been used as a surrogate kinase for drug design. Several mutations within the ATP binding site have been described to make PKA even more similar to Akt/PKB. Two proteins with Akt/PKB-like mutations in the ATP binding site were made (PKAB6 and PKAB8), and in addition S10, S139 and S338 phosphorylation sites have been removed. These proteins can be expressed in high yields but have reduced activity compared to the wild-type. Proper folding of all proteins was analyzed by 2D 1 H, 15 N-TROSY NMR experiments.
Journal of Biological Chemistry, 2006
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 280fold 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 cAMP-dependent 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 because the N-terminal site (A) is functionally deficient in Epac2 and completely lost in Epac1 (11). Despite overall amino acid sequence similarity, important differences exist between the cAMP domains of PKA and Epac. Most strikingly, the Glu interacting with the 2Ј-OH group of cAMP and conserved in the cAMP domains of all R subunits is replaced by Gln in Epac1 and by Lys in Epac2 (6). The effect of such a switch in PKA has not been studied. Cyclic nucleotide analogs are able to rapidly and reversibly activate cAMP receptors in intact cells. The first generation of cAMP analogs able to discriminate between Epac and PKA (20) has already been used successfully to dissect the contribution of each receptor to physiological cAMP responses (12-15). We wanted to understand the structural basis of the discriminative ability of these analogs and to probe Epac and PKA for useful *
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...