Properties of cyclic AMP-independent catabolite gene activator proteins of Escherichia coli

The protein products of two crp alleles encoding mutationally altered catabolite gene activator proteins CAP and CAPc, which are functionally active in vivo in the absence of cAMP, were purified by an immunoaffinity purification procedure. These proteins bind cAMP with the same affinity as does the wild-type catabolite gene activator protein. From their susceptibility to the proteolytic enzyme subtilisin, we conclude that the two mutationally altered proteins adopt structural features adequate for biological activity and similar to the conformation that cAMP elicits or stabilizes in wild-type catabolite gene activator protein. We note, however, that their conformation is not unique and can be modulated by cAMP. The two altered proteins, CAP and CAPc, bind to the lactose promoter, giving rise to specific DNA-protein complexes in the absence of cAMP and promote initiation of specific lac messenger RNA synthesis.     

Structure of a complex of catabolite gene activator protein and cyclic AMP refined at 2.5 A resolution

The structure of a dimer of the Escherichia coli catabolite gene activator protein has been refined at 2.5 A resolution to a crystallographic R-factor of 20.7% starting with coordinates fitted to the map at 2.9 A resolution. The two subunits are in different conformations and each contains one bound molecule of the allosteric activator, cyclic AMP. The amino-terminal domain is linked to the smaller carboxy-terminal domain by a nine-residue hinge region that exists in different conformations in the two subunits, giving rise to approximately a 30 degree rotation between the positions of the small domains relative to the larger domains. The amino-terminal domain contains an antiparallel beta-roll structure in which the interstrand hydrogen bonding is well-determined. The beta-roll can be described as a long antiparallel beta-ribbon that folds into a right-handed supercoil and forms part of the cyclic AMP binding site. Each cyclic AMP molecule is in an anti conformation and has ionic and hydrogen bond interactions with both subunits.     

Phosphatidyl inositol inhibition of a sperm cyclic AMP-independent protein kinase

Phosphatidyl inositol has been found to inhibit strongly the activity of a cyclic AMP-independent protein kinase located on the external surface of goat epididymal intact spermatozoa. Phosphatidyl inositol at a concentration as low as 10 micrograms/ml inhibited nearly 50% of the ecto-kinase activity for the phosphorylation of the exogenous protein substrate: casein. Phosphatidyl ethanolamine at a relatively high concentration (125 micrograms/ml) inhibited slightly (approx 25%) the activity of the enzyme whereas other phospholipids: phosphatidyl serine and choline, diacyl glycerol, phosphatidic acid and myo-inositol-2-phosphate had no appreciable effect on the kinase activity. Phosphatidyl inositol has also served as a potent inhibitor of the phosphorylation of sperm ecto-phosphoproteins by the endogenous kinase activity of intact spermatozoa. By thin layer chromatography it has been shown that the observed inhibitory effect of the phospholipid was not due to any impurities or degraded products of phosphatidyl inositol. Phosphatidyl inositol inhibited the kinase activity noncompetitively with respect to casein and Mg2+ but uncompetitively with respect to ATP. The results raised the possibility that phosphatidyl inositol-mediated high affinity inhibition of protein kinase(s), may constitute a novel mechanism for the regulatory actins of the phospholipid in mammalian cells.     

A model for the non-specific binding of catabolite gene activator protein to DNA

The binding of E. coli catabolite gene activator protein (CAP) to non-specific sequences of DNA has been modelled as an electrostatic interaction between four basic side chains of the CAP dimer and the charged phosphates of DNA. Calculation of the electrostatic contribution to the binding free energy at various separations of the two molecules shows that complex formation is favored when CAP and DNA are separated by as much as 12 A. Thus, the long range electrostatic interactions may provide the initial energy for complex formation and also the correct relative orientation of CAP and DNA. The non-specific complex does not involve the penetration of amino acid side chains into the major grooves of DNA and permits 'sliding' of the protein along DNA, which would enhance the rate of association of CAP with the specific site as has been proposed previously for lac repressor. We propose that, as it 'slides', CAP is moving in and out of the major grooves in order to sample the DNA sequence. Recognition of the specific DNA site is achieved by a complementarity in structure and hydrogen bonding between amino acids and the edges of base pairs exposed in the major grooves of DNA.     

Structure of catabolite gene activator protein at 2.9-A resolution. Incorporation of amino acid sequence and interactions with cyclic AMP

The amino acid sequence of the Escherichia coli catabolite gene activator protein has been fit into a 2.9-A resolution electron density map. Each subunit of the dimer consists of two structurally distinct domains. The larger NH2-terminal domain is seen to bind cyclic AMP and forms all of the contacts between the subunits. The cyclic AMP is completely buried between the interior of the "beta roll" structure of the large domain and a long alpha helix; it makes important hydrogen-bonding interactions with residues from both subunits. The guanidinium group of a buried Arg makes an internal salt link with the phosphate of cyclic AMP. The 6-amino group of adenine interacts simultaneously with both subunits. This interaction with both subunits and the fact that cyclic GMP and cyclic IMP do not activate catabolite gene activator protein suggest that the binding of cyclic AMP may alter the relative orientation of the two subunits, which in turn would change the structure of a DNA binding site that is presumed to span the two smaller domains. The distribution and nature of side chains in the small domain do not rule out the possibility that catabolite gene activator protein binds to left-handed B-DNA.     

Beta-adrenergic regulation of a myocardial actin gene via a cyclic AMP-independent pathway.

The skeletal alpha-actin gene encodes a major component of the embryonic cardiac sarcomere that is strongly and selectively re-induced during beta-adrenoceptor-mediated hypertrophy in neonatal rat cardiac myocytes. We present evidence that beta-adrenergic induction of this gene is mediated, not by cAMP, but by a calcium-dependent pathway involving ryanodine-sensitive calcium stores. Nifedipine-induced blockade of the plasma membrane L-type calcium entry channel prevented induction of skeletal alpha-actin mRNA by isoproterenol. Activation of calcium entry by the dihydropyridine agonist Bay K8644 independently induced skeletal alpha-actin mRNA, as did cholera toxin-mediated activation of Gs. Induction of skeletal alpha-actin mRNA by compounds that directly elevate cAMP was weak relative to their effects on other cAMP-dependent phenomena and required calcium entry. In addition, selective inhibition of protein kinase A with KT5720 did not block beta-adrenergic induction of skeletal alpha-actin. Calcium ionophore A23187 did not induce skeletal actin, but prevented its induction by isoproterenol. Ryanodine had bimodal effects: 10(-10) M ryanodine induced skeletal alpha-actin mRNA, whereas 10(-6) M ryanodine prevented skeletal actin induction by beta-adrenergic stimuli. We postulate that beta-adrenergic stimulation of skeletal alpha-actin mRNA requires G-protein-coupled calcium channel activation and compartmentalized calcium release in a manner independent of the cAMP/protein kinase A signal pathway.     

A cyclic AMP-independent protein kinase from Candida albicans.

A cyclic AMP-independent protein kinase which phosphorylates casein was purified to homogeneity from Candida albicans by affinity and ion-exchange chromatography. This protein kinase exhibits maximal activity with casein as substrate and is not stimulated by cyclic AMP or cyclic GMP. The Mr of the purified enzyme is 115,000, as determined by h.p.l.c. It migrates as a single band on gel electrophoresis and has three non-identical subunits, of Mr 44,000, 28,500 and 26,000, as determined by SDS/polyacrylamide-gel electrophoresis. This enzyme is insensitive to heparin, but is inhibited by polyamines. Furthermore, it is sensitive to thermal denaturation and to thiol reagents.     

DNA sequence determinants for binding of the Escherichia coli catabolite gene activator protein.

The consensus DNA site for binding of the Escherichia coli catabolite gene activator protein (CAP) is 22 base pairs in length and is 2-fold symmetric: 5'-AAATGTGATCTAGATCACATTT-3'. Positions 4 to 8 of each half of the consensus DNA half-site are the most strongly conserved. In this report, we analyze the effects of substitution of DNA base pairs at positions 4 to 8, the effects of substitution of thymine by uracil and by 5-methylcytosine at positions 4, 6, and 8, and the effect of dam methylation of the 5'-GATC-3' sequence at positions 7 to 10. All DNA sites having substitutions of DNA base pairs at positions 4 to 8 exhibit lower affinities for CAP than does the consensus DNA site, consistent with the proposal that the consensus DNA site is the ideal DNA site for CAP. Specificity for T:A at position 4 appears to be determined solely by the thymine 5-methyl group. Specificity for T:A at position 6 and specificity for A:T at position 8 appear to be determined in part, but not solely, by the thymine 5-methyl group. dam methylation has little effect on CAP.DNA complex formation. The thermodynamically defined consensus DNA site spans 28 base pairs. All, or nearly all, DNA determinants required for maximal affinity for CAP and for maximal thermodynamically defined CAP.DNA ion pair formation are contained within a 28-base pair DNA fragment that has the 22-base pair consensus DNA site at its center. The quantitative data in this report provide base-line thermodynamic data required for detailed investigations of amino acid-base pair and amino acid-phosphate contacts in this protein-DNA complex.     

Purification and properties of a cyclic AMP-independent protein kinase from calf thymus nuclei

A phosphoprotein kinase (ATP : protein phosphotransferase, EC 2.7.1.37) from calf thymus nuclei was purified by DEAE-cellulose chromatography, hydroxyapatite, and Sepharose 6B gel filtration. The enzyme is a cyclic AMP-independent protein kinase by the following criteria: (a) the protein kinase did not bind cyclic AMP; (b) no inhibition of activity was obtained with the heat-stable protein kinase inhibitor from rabbit skeletal muscle; (c) the regulatory subunit of cyclic AMP-dependent protein kinase had no effect on activity; and (d) no inhibition was obtained with antibody to cyclic AMP-dependent protein kinase. The nuclear cyclic AMP-independent protein kinase readily phosphorylated protamine on serine and to a lesser extent on threonine. Homologous nucleoplasmic RNA polymerase (EC 2.7.7.6) is a better substrate than arginine-rich histone, phosvitin or casein. Physical characteristics of the enzyme are described.     

Purification and properties of a cyclic AMP-independent protein kinase from calf thymus nuclei.

A phosphoprotein kinase (ATP : protein phosphotransferase, EC 2.7.1.37) from calf thymus nuclei was purified by DEAE-cellulose chromatography, hydroxyapatite, and Sepharose 6B gel filtration. The enzyme is a cyclic AMP-independent protein kinase by the following criteria: (a) the protein kinase did not bind cyclic AMP; (b) no inhibition of activity was obtained with the heat-stable protein kinase inhibitor from rabbit skeletal muscle; (c) the regulatory subunit of cyclic AMP-dependent protein kinase had no effect on activity; and (d) no inhibition was obtained with antibody to cyclic AMP-dependent protein kinase. The nuclear cyclic AMP-independent protein kinase readily phosphorylated protamine on serine and to a lesser extent on threonine. Homologous nucleoplasmic RNA polymerase (EC 2.7.7.6) is a better substrate than arginine-rich histone, phosvitin or casein. Physical characteristics of the enzyme are described.     

Intracellular location of catabolite activator protein of Escherichia coli.

The catabolite activator protein was assayed in extracts from the minicell-producing Escherichia coli strain P678-54. The level of catabolite activator protein was found to be the same in both parent cells and purified minicells, regardless of whether the bacteria were grown on glucose (which leads to low intracellular cyclic adenosine monophosphate levels) or on glycerol-yeast extract or LB broth (which lead to high cyclic adenosine monophosphate concentrations in the cell). Thus, at any given time most catabolite activator protein molecules are found in the cytoplasm. The implications of this for the mechanism of catabolite activator protein action at catabolite-sensitive operons are discussed.