Overproduction of the membrane-bound components of the histidine permease from Salmonella typhimurium: identification of the M protein

The periplasmic histidine permease of Salmonella typhimurium is composed of a soluble histidine-binding protein and three membrane-bound components. These latter are produced in very small amounts and only two, the Q and the P protein, have been previously identified. This paper describes the construction of a plasmid carrying the hisQ, hisM, and hisP genes under the control of the lambda PL promoter, thus allowing great overproduction of those gene products. The M protein has been identified in such overproducing strains and its nature confirmed by constructing in vitro hisM deletions within the plasmid. With these results the identification of all components of the histidine permease has been completed.     

Kinetic analysis of inhibition of cAMP-dependent protein kinase catalytic subunit by the peptide-nucleoside conjugate AdcAhxArg6

Kinetic analysis of the inhibition of the phosphorylation of Kemptide, (LRRASLG), catalyzed by the catalytic subunit of cAMP-dependent protein kinase, by a peptide-nucleoside conjugate inhibitor AdcAhxArg6 was carried out over a wide range of ATP and peptide concentrations. A simple procedure was proposed for characterization of the interaction of this inhibitor with the free enzyme, and with the enzyme-ATP and enzyme-peptide complexes. The second-order rate constants, calculated from the steady-state reaction kinetics, were used for this analysis to avoid the complications related to the complex catalytic mechanism of the protein kinase catalyzed reaction.     

Metabolic oscillations in biochemical systems controlled by covalent enzyme modification

We analyze the conditions under which sustained oscillations develop in a biochemical system regulated autocatalytically by reversible, covalent enzyme modification. The analysis applies, for example, to the situation where adenylate cyclase (or guanylate cyclase) is activated through phosphorylation by a cAMP (or cGMP)-dependent protein kinase. The model then provides a non-allosteric mechanism for the periodic generation of cAMP or cGMP pulses. For certain parameter values close to those that produce oscillations, the system is excitable since it can amplify in a pulsatory manner suprathreshold perturbations. The results on excitable and oscillatory behavior are discussed in relation with the mechanism of cAMP relay and oscillation in the slime mold Dictyostelium discoideum.     

The preparation of cyclic nucleotide-depleted regulatory subunits from type II adenosine 3':5'-monophosphate-dependent protein kinase by a nondenaturing method

A nondenaturing method for the preparation of R subunits from type II cyclic AMP-dependent protein kinase is described. The procedure is based on the exchange of cyclic AMP, which is tightly bound to the R subunit, for more weakly bound cyclic GMP, which can be removed by washing and dialysis. Less than 5% of the available cyclic nucleotide-binding sites of R subunit prepared by this method contained cyclic AMP and less than 3% contained cyclic GMP. The C-subunit contamination (mol of C/mol of R monomer) was approximately 0.2%. These levels of contamination did not affect the properties of the R subunit as judged by (a) the ability of the R subunit to inhibit the activity of the C subunit and (b) the rate of exchange of cAMP into R2 . etheno-cAMP. The advantages of our method are that the protein is not subjected to denaturing conditions and that large amounts of material can be processed relatively rapidly.     

Cyclic AMP-dependent phosphorylation of pea proteins induced by forskolin

An increase in the level of phosphorylation of low-molecular-weight polypeptides in pea (Pisum sativum L.) leaves and changes in the content of some polypeptides after 10-min forskolin action in situ were demonstrated. A total level of protein phosphorylation in the homogenate of forskolin-treated leaves diminished after 25 min of its incubation in vitro. Using a highly specific inhibitor of cAMP-dependent protein kinases and cAMP treatments, we established that forskolin-induced change in the phosphorylation level of some polypeptides was cAMP-dependent.Translated from Fiziologiya Rastenii, Vol. 52, No. 1, 2005, pp. 27–35.Original Russian Text Copyright © 2005 by Karimova, Tyrykina, Zakharova.     

Microsomal cAMP-independent histone H1 kinase activity in plasmacytoma, Morris hepatoma and normal liver

A protein kinase activity with high specificity for histone H1 was isolated from mouse plasmacytoma, Morris hepatoma and normal mouse liver and compared by ion exchange chromatography after DEAE-cellulose, hydroxylapatite and Sephadex G-200 chromatography. This cAMP-independent histone H1 kinase is not affected by the heat-stable cAMP-dependent protein kinase inhibitor. It has the following particular properties: it prefers GTP to ATP as substrate and was found to be present with a great activity only in neoplastic tissues. No phosphatase activity was detected in the partially purified histone H1 kinase fraction from normal and neoplastic cells. These results suggest either an increase amount of histone H1 kinase and/or of its activator in neoplastic cells, or the presence of a strong inhibitor in normal cells. This histone H1 kinase appears to be analogous to the chromatin bound kinase which phosphorylates histone H1 at the NH2 and COOH terminal regions. We might suggest an implication of this kinase in the regulation of cell division.     

Ontogenetic changes in protein dephosphorylating activity of the cytosolic compartment of rat erythrocytes

Total casein phosphatase activity of erythrocytes from one-month-old rats was separated by DEAE-cellulose chromatography into three peaks--E1, E2 and E3--and only into two peaks--E1 and E3--when the erythrocyte donors were six- and 12-month-old rats. The activity of E1 (Mr 330 K) decreased continuously in erythrocytes during the first year of postnatal life. E2 (Mr 230 K) also decreased and completely disappeared from the cells of 12-month-old rats. E3 (Mr 180 K) was the dominant molecular form in the cytosol of erythrocytes during the first year of life. It decreased only up to six months of life. In this form E3 seems to be cooperative with respect to the substrate and to inhibitor molecules. The decrease of its kinetic parameters (Vmax and K0.55) was also found during postnatal ontogenesis. E3 isolated from erythrocytes of older rats (6 and 12 months) was more susceptible to inhibitory effect of pyrophosphate and to the change of ionic strength of eluting buffer than the enzyme from one-month-old rats. 0.2 mol.1(-1) NaCl lowered Mr of E3 phosphatase from 180 K to 128 K only in older rats.     

Yeast cytochrome b2 gene: isolation with antibody probes

An efficient technique was used to clone the gene for yeast cytochrome b2, (a nuclear encoded mitochondrial protein) using the expression vector, lambda gt11 (lac 5 nin 5 c1857 S100). This enables the insertion of yeast DNA into the beta-galactosidase structural gene (lacZ) and promotes synthesis of hybrid proteins. Screening of antigen producing clones in the lambda gt11 recombinant genomic library was achieved using antiserum against cytochrome b2 according to Young and Davis (1983) Two recombinants containing part of the gene coding for cytochrome b2 were isolated and characterized as follows: by their expression in Escherichia coli cells, examined by immuno-blotting with antibodies to pure cytochrome b2. by DNA sequence analysis. One recombinant carries a 3 Kb yeast DNA insert which contains the whole nucleotide sequence encoding cytochrome b2 and a few amino acids of the amino terminal presequence.     

Purification and characterization of a dimer form of the cAMP-dependent protein kinase from mouse liver cytosol

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 R_II 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.     

Major 56,000-dalton, soluble phosphoprotein present in bovine sperm is the regulatory subunit of a type II cAMP-dependent protein kinase

It has been shown that cAMP-dependent phosphorylation of a soluble sperm protein is important for the initiation of flagellar motion. The suggestion has been made that this motility initiation protein, named axokinin, is the major 56,000-dalton phosphoprotein present in both dog sperm and in other cells containing axokinin-like activity. Since the regulatory subunit of a type II cAMP-dependent protein kinase is a ubiquitous cAMP-dependent phosphoprotein of similar subunit molecular weight as reported for axokinin, we have addressed the question of how many soluble 56,000-dalton cAMP-dependent phosphoproteins are present in mammalian sperm. We report that in bovine sperm cytosol, the ratio of the type I to type II cAMP-dependent protein kinase is approximately 1:1. The type II regulatory subunit is related to the non-neural form of the enzyme and undergoes a phosphorylation-dependent electrophoretic mobility shift. The apparent subunit molecular weights of the phospho and dephospho forms are 56,000 and 54,000 daltons, respectively. When bovine sperm cytosol or detergent extracts are phosphorylated in the presence of catalytic subunits, two major proteins are phosphorylated and have subunit molecular weights of 56,000 and 40,000 daltons. If, however, the type II regulatory subunit (RII) is quantitatively removed from these extracts using either immobilized cAMP or an anti-RII monoclonal affinity column, the ability to phosphorylate the 56,000- but not 40,000-dalton polypeptide is lost. These data suggest that the major 56,000 dalton cAMP-dependent phosphoprotein present in bovine sperm is the regulatory subunit of a type II cAMP-dependent protein kinase and not the motility initiator protein, axokinin.     

MAP2 is required for dendrite elongation,PKA anchoring in dendrites,and proper PKA signal transduction

Microtubule-associated protein 2 (MAP2) is a major component of cross-bridges between microtubules in dendrites, and is known to stabilize microtubules. MAP2 also has a binding domain for the regulatory subunit II of cAMP-dependent protein kinase (PKA). We found that there is reduction in microtubule density in dendrites and a reduction of dendritic length in MAP2-deficient mice. Moreover, there is a significant reduction of various subunits of PKA in dendrites and total amounts of various PKA subunits in hippocampal tissue and cultured neurons. In MAP2-deficient cultured neurons, the induction rate of phosphorylated CREB after forskolin stimulation was much lower than in wild-type neurons. Therefore, MAP2 is an anchoring protein of PKA in dendrites, whose loss leads to reduced amount of dendritic and total PKA and reduced activation of CREB.     

Discovery of allostery in PKA signaling

Cyclic AMP (cAMP)-dependent protein kinase (PKA) was the second protein kinase to be identified, and the PKA catalytic (C)-subunit serves as a prototype for the large protein kinase superfamily that contains over 500 gene products. The protein kinases regulate many biological functions in eukaryotic cells and are now also a major therapeutic target. The discovery of PKA nearly 50 years ago was quickly followed by the identification of the regulatory subunits that bind cAMP and release the catalytic activity from the holoenzyme. Thus in PKA we see the convergence of two major signaling mechanisms—protein phosphorylation and second messenger signaling through cAMP. Crystallography provides a foundation for understanding function, and detailed knowledge of the structure of the isolated regulatory (R)- and catalytic (C)-subunits has been extremely informative. Yet it is the R₂C₂ holoenzyme that predominates in cells, and the allosteric features of PKA signaling can only be fully appreciated by seeing the full-length protein. The symmetry and the quaternary constraints that one R:C heterodimer exerts on the other in the holoenzyme simply are not present in the isolated subunits or even in the R:C heterodimer.     

Catalytic independent functions of a protein kinase as revealed by a kinase-dead mutant: study of the Lys72His mutant of cAMP-dependent kinase

A highly conserved lysine in subdomain II is required for high catalytic activity among the protein kinases. This lysine interacts directly with ATP and mutation of this residue leads to a classical "kinase-dead" mutant. This study describes the biophysical and functional properties of a kinase-dead mutant of cAMP-dependent kinase where Lys72 was replaced with His. Although the mutant protein is less stable than the wild-type catalytic subunit, it is fully capable of binding ATP. The results highlight the effect of the mutation on stability and overall organization of the protein, especially the small lobe. Phosphorylation of the activation loop by a heterologous kinase, 3-phosphoinositide-dependent protein kinase-1 (PDK-1) also contributes dramatically to the global organization of the entire active site region. Deuterium-exchange mass spectrometry (DXMS) indicates a concerted stabilization of the entire active site following the addition of this single phosphate to the activation loop. Furthermore the mutant C-subunit is capable of binding both the type I and II regulatory subunits, but only after phosphorylation of the activation loop. This highlights the role of the large lobe as a scaffold for the regulatory subunits independent of catalytic competency and suggests that kinase dead members of the protein kinase superfamily may still have other important biological roles although they lack catalytic activity.     

Diversity of Bisubstrate Binding Modes of Adenosine Analogue-Oligoarginine Conjugates in Protein Kinase A and Implications for Protein Substrate Interactions

Crystal structures of the catalytic subunit α of cAMP-dependent protein kinase (PKAc) with three adenosine analogue-oligoarginine conjugates (ARCs) are presented. The rationally designed ARCs include moieties that, in combination, target both the ATP- and the peptide-substrate-binding sites of PKAc, thereby taking advantage of high-affinity binding interactions offered by the ATP site while utilizing an additional mechanism for target specificity via binding to the peptide substrate site. The crystal structuresdemonstrate that, in accord with the previously reported bisubstrate character of ARCs, the inhibitors occupy both binding sites of PKAc. Further, they show new binding modes that may also apply to natural protein substrates of PKAc, which have not been revealed by previous crystallographic studies. The crystal structures described here contribute to the understanding of the substrate-binding patterns of PKAc and should also facilitate the design of inhibitors targeting PKAc and related protein kinases.     

Phosphorylation of the alpha-subunit of Na,K-ATPase from duck salt glands by cAMP-dependent protein kinase inhibits the enzyme activity

Although it was shown earlier that phosphorylation of Na,K-ATPase by cAMP-dependent protein kinase (PKA) occurs in intact cells, the purified enzyme in vitro is phosphorylated by PKA only after treatment by detergent. This is accompanied by an unfortunate side effect of the detergent that results in complete loss of Na,K-ATPase activity. To reveal the effect of Na,K-ATPase phosphorylation by PKA on the enzyme activity in vitro, the effects of different detergents and ligands on the stoichiometry of the phosphorylation and activity of Na,K-ATPase from duck salt glands (11-isoenzyme) were comparatively studied. Chaps was shown to cause the least inhibition of the enzyme. In the presence of 0.4% Chaps at 1 : 10 protein/detergent ratio in medium containing 100 mM KCl and 0.3 mM ATP, PKA phosphorylates serine residue(s) of the Na,K-ATPase with stoichiometry 0.6 mol P_i/mol of -subunit. Phosphorylation of Na,K-ATPase by PKA in the presence of the detergent inhibits the Na,K-ATPase. A correlation was found between the inclusion of P_i into the -subunit and the loss of activity of the Na,K-ATPase.     

Distribution of (14C)MCA and its derivatives in mouse tissues (author's transl)

Distribution of [¹⁴C]MCA and its derivatives in mouse tissuesThe distribution of radioactivity due to 3-methylcholanthrene (MCA) and/or its metabolites was evaluated in different organs of mothers, foetuses and newborns at 6 and 60–66 h following a single intragastric administration of 1 mg of [¹⁴C]MCA to pregnant CF-1 mice. The extent of bound [¹⁴C]MCA nd/or its metabolites to nuclear and cytoplasmic proteins was evaluated in organs which were proven to be either susceptible (lung) or non-susceptible (kidney) to the carcinogenic effect of MCA. The present data indicate a slightly higher level of free and bound MCA in the subcellular fractions of the lung than in the kidney and concurrently a higher level of covalently bound MCA to the nuclear DNA in the lungs than in the kidney.     

Dynamic features of cAMP-dependent protein kinase revealed by apoenzyme crystal structure

To better understand the mechanism of ligand binding and ligand-induced conformational change, the crystal structure of apoenzyme catalytic (C) subunit of adenosine-3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) was solved. The apoenzyme structure (Apo) provides a snapshot of the enzyme in the first step of the catalytic cycle, and in this unliganded form the PKA C subunit adopts an open conformation. A hydrophobic junction is formed by residues from the small and large lobes that come into close contact. This "greasy" patch may lubricate the shearing motion associated with domain rotation, and the opening and closing of the active-site cleft. Although Apo appears to be quite dynamic, many important residues for MgATP binding and phosphoryl transfer in the active site are preformed. Residues around the adenine ring of ATP and residues involved in phosphoryl transfer from the large lobe are mostly preformed, whereas residues involved in ribose binding and in the Gly-rich loop are not. Prior to ligand binding, Lys72 and the C-terminal tail, two important ATP-binding elements are also disordered. The surface created in the active site is contoured to bind ATP, but not GTP, and appears to be held in place by a stable hydrophobic core, which includes helices C, E, and F, and beta strand 6. This core seems to provide a network for communicating from the active site, where nucleotide binds, to the peripheral peptide-binding F-to-G helix loop, exemplified by Phe239. Two potential lines of communication are the D helix and the F helix. The conserved Trp222-Phe238 network, which lies adjacent to the F-to-G helix loop, suggests that this network would exist in other protein kinases and may be a conserved means of communicating ATP binding from the active site to the distal peptide-binding ledge.