Classification and Phylogenetic Analysis of the cAMP-Dependent Protein Kinase Regulatory Subunit Family (original) (raw)
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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...
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.
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 ...
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.
European Journal of Biochemistry, 1994
We have isolated and characterized cDNA and genomic DNA clones encoding the catalytic subunit (C) of CAMP-dependent protein kinase in the aquatic fungus Blastocladiella emersonii. The C-subunit amino acid sequence derived from the nucleotide sequence predicts a basic polypeptide of 424 residues, excluding the initiator methionine, which by amino-terminal sequence analysis has been shown to be absent from the mature protein. The Blastocladiella C presents a 70-amino-acid extension at the amino terminus, when aligned to the mouse Ca subunit, being one of the largest C subunits already characterized. The B. emersonii C-gene-coding region is interrupted by three introns, ranging in size over 57-69 bp. The positions of the introns are quite different from those found in other species, suggesting a considerable amount of evolutionary drift in the gene structure. The 5'-flanking region lacks recognizable TATA or CCAAT sequences, is remarkably high in GC content (70%), and primer extension experiments indicate that transcription initiates from multiple sites. Several sequence motifs were identified in the promoter region which could be involved in the developmental control of this gene.
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, 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.