Expression in Escherichia coli and characterization of the heat-stable inhibitor of the cAMP-dependent protein kinase (original) (raw)
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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.
The Journal of biological chemistry, 1983
Eighty different adenine-modified cAMP analogs were tested as activators of rabbit muscle protein kinase I (cAKI) in an in vitro phosphotransferase assay. All the analogs tested were able to activate completely the kinase. The affinities of the cAMP derivatives for the two types (A and B) of binding sites associated with the regulatory moiety of cAKI were determined under conditions similar to those used in the phosphotransferase assay. The potency of the cAMP analogs as cAKI activators was found to correlate with the mean affinity for sites A and B, rather than to the affinity for only one of the sites. This was true whether cAKI was assayed at low or near physiological ionic strength, whether the concentration of cAKI binding sites was 0.2 or 400 nM, and whether the kinase substrate was mixed histones or homogeneous phenylalanine-4-monooxygenase. Furthermore, site A-selective and site B-selective cAMP analogs activated cAKI synergistically. Finally, it was shown that the degree of...
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.
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.
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 Biological Chemistry, 2002
The complex of the subunits (RI␣, C␣) of cAMP-dependent protein kinase I (cA-PKI) was much more stable (K d ؍ 0.25 M) in the presence of excess cAMP than previously thought. The ternary complex of C subunit with cAMP-saturated RI␣ or RII␣ was devoid of catalytic activity against either peptide or physiological protein substrates. The ternary complex was destabilized by protein kinase substrate. Extrapolation from the in vitro data suggested about one-fourth of the C subunit to be in ternary complex in maximally cAMP-stimulated cells. Cells overexpressing either RI␣ or RII␣ showed decreased CRE-dependent gene induction in response to maximal cAMP stimulation. This could be explained by enhanced ternary complex formation. Modulation of ternary complex formation by the level of R subunit may represent a novel way of regulating the cAMP kinase activity in maximally cAMP-stimulated cells. The cAMP-dependent protein kinase (cA-PK) 1 differs from other kinases in having the catalytic site and the autoinhibitory site on two different subunits. The inactive cA-PK holoenzyme, when studied at nanomolar concentrations, dissociates into catalytic (C) and regulatory (R) subunits in the presence of cAMP (1). There is sparse evidence about the behavior of cA-PK at higher, more physiologically relevant, concentrations. Apparently, it is tacitly assumed that both isozymes (cA-PKI and cA-PKII) are completely dissociated by cAMP in the intact cell. The cAMP-induced decrease of fluorescent resonance transfer between microinjected C␣-FITC and RI␣-TRITC (2), and between genetically encoded fluorescent C␣ and RII (3) has reinforced this notion, although such studies are not designed to tell whether the dissociation of cA-PK is complete or not (4). Recently, C/EBP null mice were shown to have increased liver RI and RII, and attenuated cAMP-stimulated hepatic gene induction (5). Protein kinase inhibitor null mice, having 50%
The Journal of Peptide Research, 2009
Peptides derived from the inhibitor of CAMP-dependent protein kinase, PKI, have been studied by 2D 'H NMR techniques. These include the inhibitor PKI(6-22), the substrate [Ala2"-Ser"]PKI(5-24), and a phosphorylated form of the latter [Ala'o-Ser"P]PKI(5-24). A homologous fold was found in the three peptides which consisted of an h'-terminal segment in helical conformation to residue 13 and a C-terminal segment poorly defined conformationally. A parallel study was carried out by molecular dynamics (MD) for the inhibitor peptide PKI(5-24). The N-terminal helix, as observed in the crystal structure of the catalytic subunit-PKI(5-24) complex. was conserved in the M D simulations with the enzyme-free inhibitor. Similarly the Gly'J-Glyl' turn was apparent in all M D structures, whereas the C-terminal region, residues 18-24, was directed towards the N-terminal helix in contrast to the extended conformation of this segment pointing away from the A'-terminal helix in the crystal structure. This is primarily due to ionic interaction between Asp' and Arg". Indeed. a detailed analysis of the NOE contacts by NOESY at low temperature (2-C) shows the occurrence of pH-dependent contacts with Phe'". We conclude that the binding of short inhibitors, such as PKI(5-24), to the enzyme involves a conformational rearrangement of the C-terminal region. The substrate [Ala'o-Ser"]PKI(5-24) and the product [Alazo-SerZ1P]PKI(5-24), give very similar structures with local rearrangements involving some of the side chains. 0 Munksgaard 1997. K r j itorris: CAMP-dependent protein kinase: P K I x NMR: molecular dlnamics The heat-and acid-stable protein kinase inhibitor (PKI) was initially purified from rabbit skeletal muscle (l) , and the entire 75-amino acid sequence of this isoform, PKIr, has been determined (2). PKI specifically inhibits the catalytic subunit of the CAMPdependent protein kinase (3). The inhibitory region of PKI was localized, using proteolytic cleavage. to be in the first third of the molecule (4). Inhibition by the peptide PKI(5-24), as well as by full length PKI, is competitive with respect to protein substrate (5). The C-terminal segment of PKI(5-24) contains the pseudo-substrate site required for inhibition of the catalytic subunit of CAMP-dependent protein kinase (5, 6). The sequence Arg' 5-Thr-Gly-Arg-Arg-Asn-Ala-Ile2' of PKI is similar to the typical-Arg-X-X-Arg-Arg-X-Ser-hyd-phosphorylation consensus sequence found for substrates of the CAMP-depend-en{ protein kinase (7). The N-terminal domiin of Abbreviations: PKI, the heat-stable inhibitor of CAMP-dependent p~1 (5~2 4) has been shown to confer tight binding to the CAMP-dependent protein kinase (5, 6).
The Journal of biological chemistry, 1993
The catalytic subunit of cAMP-dependent protein kinase expressed in Escherichia coli is a phosphoprotein. By in vivo labeling with [32Pi]orthophosphate, the sites of phosphorylation were identified as Ser-10, Ser-139, Thr-197, and Ser-338. Two of these sites, Thr-197 and Ser-338, are found in the mammalian enzyme (Shoji, S., Titani, K., Demaille, J. G., and Fischer, E. H. (1979) J. Biol. Chem. 254, 6211-6214). The predominant isoform is phosphorylated at Ser-10, Ser-338, and Thr-197. The isoforms cannot be readily interconverted by in vitro autophosphorylation, suggesting that the phosphates are relatively stable once the mature protein is assembled. Unlike the mammalian enzyme, the recombinant enzyme is not myristylated at its animo terminus. By coexpressing the catalytic subunit and N-myristyl transferase, the recombinant catalytic subunit is myristylated, and, under these conditions, phosphorylation at Ser-10 is reduced. The fact that recombinant catalytic subunit mutants that ar...
Recombinant type I regulatory subunit of the cAMP‐dependent protein kinase is biologically active
FEBS Letters, 1987
The cDNA for the porcine type I regulatory subunit (RI) of the cAMP‐dependent protein kinase (cAMP‐PK) was cloned into two different bacterial expression vectors: pKK223 and pUC18. Recombinant RI was produced by bacteria transformed with either construct, and purified by affinity chromatography. Both the native RI from the pKK223 construct and the RI with an amino terminal extension of eight amino acids from the pUC18 construct were found to be completely native with regard to inhibition of the catalytic subunit activity and cAMP binding.
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 ...