Isolation and partial characterization of a carbohydrate-binding protein from a nematode-trapping fungus

A developmentally regulated carbohydrate-binding protein from the capture organs of Arthrobotrys oligospora, not present on hyphae, was isolated and partially characterized. Surface structures of A. oligospora were radiolabeled with [125I]iodosulfanilic acid. The fungus was homogenized, and the homogenate was passed over an affinity column containing N-acetyl-D-galactosamine immobilized to Sepharose 6B. The bound radiolabeled protein was eluted from the affinity column with a glycine-hydrochloride buffer (pH 3.0), concentrated, and chromatographed on a metal chelate affinity gel containing Ca2+. EDTA was used as an eluant for the radiolabeled protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis in combination with autoradiography revealed a molecular weight for the carbohydrate- and cation-binding polypeptide of ca. 20,000.     

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.     

cAMP-dependent protein kinase from Dictyostelium discoideum

The cAMP-dependent protein kinase (cAK) from Dictyostelium discoideum is an enzyme composed of one catalytic and one regulatory subunit. Upon binding of cAMP, the holoenzyme dissociates to liberate free active catalytic subunits. The cAK is developmentally regulated, ranging from very little activity in vegetative cells to maximal expression in postaggregative cells. Although there is no immunological cross-reaction between the subunits of cAKs from Dictyostelium and from other organisms, they share several biochemical properties. A complete cDNA for the regulatory subunit has been cloned and sequenced. Only one copy of the gene for the regulatory subunit is present per haploid genome. On the basis of the comparison of the structure of the cAK from Dictyostelium with its counterparts in yeast and higher eukaryotes, we propose a model for the evolution of cyclic-nucleotide-binding proteins.     

Biochemical characterization of extracellular cAMP-dependent protein kinase as a tumor marker

In a recent report (Cho et al., Proc. Natl. Acad. Sci. USA 97, 835-840, 2000), we showed that cancer cells of various cell types secrete cAMP-dependent protein kinase (PKA) into the conditioned medium and that in the serum of cancer patients this extracellular PKA (ECPKA) is upregulated 10-fold as compared with normal serum. Here, we characterized the enzymatic properties of ECPKA that is present in the conditioned medium of PC3M prostate cancer cells and in the serum of cancer patients, and we compared ECPKA with PKA found in the cell extracts of PC3M cells. ECPKA present in the conditioned medium and human serum was not activated by cAMP addition, but intracellular PKA activity was totally dependent on the addition of cAMP. This indicates that the ECPKA is present in active, free C subunit form, whereas intracellular PKA is present in inactive holoenzyme form. ECPKA activity increased in a substrate concentration- and time-dependent manner, as did intracellular PKA. Both ECPKA and intracellular PKA activities were specifically inhibited by the PKA inhibitor protein, PKI. However, ECPKA activity was more temperature-sensitive than intracellular PKA; after two cycles of freezing/thawing, only 20% of initial ECPKA activity was detected compared with over 40% of intracellular PKA activity. Western blot analysis revealed the presence of a 40 kDa C(alpha) subunit of PKA in both conditioned medium and in the serum of cancer patients. These results suggest that ECPKA, out of the context of cAMP regulation, may function as a growth factor promoting cell growth and transformation; thus, it may serve as a tumor biomarker.     

A natural non-protein low molecular weight cAMP-dependent protein kinase inhibitor from the insect Ceratitis capitata: Isolation and preliminary characterization

Three protein kinase inhibitors have been detected and isolated by gel filtration chromatography from the dipterous Ceratitis capitata. Two of them were proteinaceous; the third one was resistant to proteolytic treatments and its molecular weight ranged from 1000 and 6000. This inhibitor was purified to thin-layer electrophoretic homogeneity and a preliminary analysis carried out to determine its composition showed that it is a complex molecule with a probable peptidyl-purine structure. This new inhibitor acts competitively with ATP on the cAMP-dependent protein kinase activity and has been also found to inhibit the adenylate cyclase system. This metabolite may be important in regulating cAMP mediated responses in the insect.     

A structural model of the catalytic subunit-regulatory subunit dimeric complex of the cAMP-dependent protein kinase.

Previous neutron scattering studies elaborated the topographical relationship of the regulatory (R(IIalpha)) and catalytic (C(alpha)) subunits of the cAMP-dependent protein kinase. We present here the results of a set of computations that lead to an atomic model of the cAMP-dependent protein kinase heterodimer, Delta(1-91)R(IIalpha)-C(alpha). The first step in the modeling utilized the crystal structures for the porcine C(alpha) and bovine Delta(1-90)R(Ialpha) or rat Delta(1-111)R(IIbeta), to homology-model structures of the species and isoforms that had been used in the neutron scattering experiments (bovine C(alpha) subunit and murine Delta(1-91)R(IIalpha) subunit, respectively). A docking procedure, constrained by the dimensions and positions of the ellipsoids in the neutron-derived R-C model as well as mutagenesis data, was used to develop "best fit" models for the heterodimer. Simulated annealing, molecular dynamics, and energy minimization were then used to refine the side chain packing at the heterodimer interface. For comparison, the calculations were done using the homology models derived from both the R(Ialpha) and R(IIbeta) crystal structures. Both resultant models had many similarities. Each predicted similar interfaces. The R(Ialpha)-based model has 25% more hydrogen bonds than that based on R(IIbeta), with seven of these potential bonds in common. The distribution of hydrophobic, polar, and charged residues at the interface was similar for both models, with a distribution more characteristic of the exposed surface residues than those in the protein interior. The calculated interface area in each is relatively small (<2000 A(2)). The R(Ialpha)-based model, however, has a significantly better fit with the scattering data and is therefore the one of distinctly higher probability. With its small interface area that has a high proportion of charged and polar residues, the complex appears poised for dissociation, and each subunit existing as a stable entity. This result is consistent with the known physiological events required for cAMP-dependent activation of the kinase.     

Isolation and study of the properties of the regulator subunit of cAMP-dependent protein kinase

The regulatory subunit of type II cAMP-dependent proteinkinase was isolated from cytosol of the rabbit small intestinal mucosa by affinity chromatography. The preparation contained 3 proteolytic enzymes and occurred in two forms differing as regards cAMP affinity. The cAMP-binding capacity of the preparation was equal to 17 nmol cAMP/mg protein. To study the topography of the cAMP-binding center, use was made of cAMP analogs. It was demonstrated that introduction of the substituents into the 8th position of the purine ring and substitution with respect to the N6-exoaminogroup affected insignificantly the analog affinity for the cAMP-binding center. At the same time the substituents introduced into the first position of the adenine base, into the area of the 2'-hydroxyl group of ribose and into the cyclophosphate part of the cAMP molecule considerably decreased the analog affinity for the regulatory center of type II cAMP-dependent proteinkinase.     

Isolation and partial characterization of a protein kinase NII from wheat germ chromatin

A protein kinase, type NII, has been purified from wheat germ chromatin. The enzyme, which uses both ATP and GTP as phosphoryl donors, catalyzes the phosphorylation of casein, phosvitin and E. coli RNA polymerase, but not of histone proteins. Polypeptide bands at 46 kDa, 37 kDa and 25 kDa were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autophosphorylation of the 25 kDa subunit was observed following incubation of the purified kinase with (-³²P)ATP and (-³²P)GTP.     

Molecular cloning and characterization of a putative protein kinase gene from the thermophilic fungus Thermomyces lanuginosus.

Based on the conserved amino acid sequence (DLKPEN) of serine-threonine protein kinase from several fungi, a degenerate primer was designed and synthesized. Total RNA was isolated from the thermophilic fungus Thermomyces lanuginosus. Using RACE-PCR, full-length cDNA of a putative serine-threonine protein kinase gene was cloned from T. lanuginosus. The full-length cDNA of T. lanuginosus protein kinase was 2551 bp and contained an 1806 bp open reading frame encoding a putative protein kinase precursor of 601 amino acid residues. Sequencing analysis showed that the cloned cDNA of T. lanuginosus had consensus protein kinase sequences. Conservative amino acid subdomains which most serine-threonine kinases contain can be found in the deduced amino acid sequence of T. lanuginosus putative protein kinase. Comparison results showed that the deduced amino acid sequence of T. lanuginosus putative protein kinase was highly homologous to that of Neurospora crassa dis1-suppressing protein kinase Dsk1. The putative protein kinase contained three arginine/serine-rich (SR) regions and two transmembrane domains. These showed that it might be a novel putative serine-threonine protein kinase.     

Two different intrachain cAMP sites in the cAMP-dependent protein kinase of the dimorphic fungus Mucor rouxii

cAMP sites of the cAMP-dependent protein kinase from the fungus Mucor rouxii have been characterized through the study of the effects of cAMP and of cAMP analogs on the phosphotransferase activity and through binding kinetics. The tetrameric holoenzyme, which contains two regulatory (R) and two catalytic (C) subunits, exhibited positive cooperativity in activation by cAMP, suggesting multiple cAMP-binding sites. Several other results indicated that the Mucor kinase contained two different cooperative cAMP-binding sites on each R subunit, with properties similar to those of the mammalian cAMP-dependent protein kinase. Under optimum binding conditions, the [3H]cAMP dissociation behavior indicated equal amounts of two components which had dissociation rate constants of 0.09 min-1 (site 1) and 0.90 min-1 (site 2) at 30 degrees C. Two cAMP-binding sites could also be distinguished by C-8 cAMP analogs (site-1-selective) and C-6 cAMP analogs (site-2-selective); combinations of site-1- and site-2-selective analogs were synergistic in protein kinase activation. The two different cooperative binding sites were probably located on the same R subunit, since the proteolytically derived dimeric form of the enzyme, which contained one R and one C component, retained the salient properties of the untreated tetrameric enzyme. Unlike any of the mammalian cyclic-nucleotide-dependent isozymes described thus far, the Mucor kinase was much more potently activated by C-6 cAMP analogs than by C-8 cAMP analogs. In the ternary complex formed by the native Mucor tetramer and cAMP, only the two sites 1 contained bound cAMP, a feature which has also not yet been demonstrated for the mammalian cAMP-dependent protein kinase.     

The regulatory subunit monomer of cAMP-dependent protein kinase retains the salient kinetic properties of the native dimeric subunit

Monomeric regulatory subunit (R) fragments of type II cAMP-dependent protein kinase were compared with the parent dimeric R. The monomeric fragments were generated by either endogenous proteolysis of rabbit muscle R or by trypsin treatment of bovine heart R in the holoenzyme form. During isolation of pure R from rabbit muscle, carboxyl-terminal fragments of Mr = 42,000 (42 K) and Mr = 37,000 by denaturing gels are generated by endogenous proteolysis. Although the autophosphorylation site is retained, the 42 K is not dimeric (as is its native 56 K precursor) but, in contrast to the monomeric 37 K product, actively reassociates with purified catalytic subunit (C). Several lines of evidence indicate a type II R origin of the 42 K. N-terminal sequence analysis of the 42 K shows some homology with known bovine RI, RII, and cGMP-dependent protein kinase sequences. Both cyclic nucleotide-binding sites (two/42 K or 37 K) and the site selectivity of cAMP analogs are retained in the monomeric fragments. When purified bovine heart holoenzyme, which contains a dimeric Mr = 56,000 R (denaturing gel analysis) and two C subunits, is treated with trypsin followed by separation procedures, the product is a fully recovered active enzyme with an unaltered ratio of cAMP binding to catalytic activity. From Mr considerations, the product is a dimer containing one intact C and a proteolyzed R of Mr = 48,000 on denaturing gels. This dimeric enzyme is not significantly different from the parent tetramer in cAMP concentration dependence (Hill constant = 1.63), [3H]cAMP dissociation behavior (both intrasubunit cAMP-binding sites are present), stimulation of [3H]cIMP binding by site-selective cAMP analogs, and synergism between two analogs in kinase activation. The data indicate that 1) proteolytic cleavage of the native R dimer can cause monomerization without appreciably affecting the inhibition of C and 2) essentially all of the cAMP binding cooperativity is an intrasubunit interaction.     

Isolation and characterization of the calcium- and phospholipid-dependent protein kinase (protein kinase C) subtypes from bovine heart

Protein kinase C was isolated from bovine heart by chromatography on DEAE-Sephacel, phenyl-Sepharose, poly(L-lysine) agarose, and hydroxylapatite. Estimates based upon enzyme recovery indicate 10-20 nmol/min of protein kinase C activity per gram of bovine ventricular myocardium. Hydroxylapatite column chromatography resolved the preparation into two peaks of calcium- and phospholipid-dependent protein kinase activity. By Western blot analysis, peaks 1 and 2 contained subtypes II (beta 2) and III (alpha), respectively. No cross-reactivity was observed, indicating that separation was complete. Type III, the major subtype detected, was subsequently purified to apparent homogeneity by chromatography on phosphatidylserine (PS) acrylamide. Type II activity could not be recovered following phosphatidylserine affinity chromatography. Phospho amino acid analysis showed that type III autophosphorylated at serine residues, whereas type II autophosphorylated at both serine and threonine residues. Among the various phospholipids tested for activity, PS was the most effective. Both subtypes were activated by 1-stearoyl-2-arachidonylglycerol (SAG) in the presence of phosphatidylserine and calcium. Activation of both subtypes occurred at calcium concentrations of less than 1 microM. In addition to several similarities, these two subtypes showed differences in activation and kinetic properties: type II was activated by cardiolipin, 1,2-and 1,3-dioleoylglycerol, and both cis- and trans-unsaturated fatty acids. Type III was activated to a lesser degree by cardiolipin and showed no response to 1,3-dioleoylglycerol. Type III was activated to a greater extent by 1,2-diacylglycerols and by cis-unsaturated fatty acids. In the presence of PS and SAG, type II exhibited substantial activity in the presence of 1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) without added calcium. Activation of types II and III by unsaturated fatty acids was independent of phospholipid and showed a lower apparent calcium affinity than that observed for activation by phosphatidylserine. These results show that cardiac protein kinase C subtypes II and III were functionally distinguishable and may play unique roles in the regulation of cardiac function.     

Circular dichroism of cAMP-dependent protein kinase from pig brain

The circular dichroism spectra of phosphorylated and non-phosphorylated forms of cAMP-dependent protein kinase from pig brain and those of the catalytical and regulatory subunits of the enzyme were studied. The percentage of the secondary structure components of the subunits was calculated. cAMP was shown to cause conformational changes of the enzyme. The conformation of the cyclic nucleotide within the cAMP--regulatory subunit complex was established. It was assumed that the conformation of the cAMP molecule during enzyme activation is subjected to inversion.     

Probes of the mitochondrial cAMP-dependent protein kinase

The development of a fluorescent assay to detect activity of the mitochondrial cAMP-dependent protein kinase (PKA) is described. A peptide-based sensor was utilized to quantify the relative amount of PKA activity present in each compartment of the mitochondria (the outer membrane, the intermembrane space, and the matrix). In the process of validating this assay, we discovered that PKA activity is regulated by the protease calpain. Upon exposure of bovine heart mitochondria to digitonin, Ca^2 +, and a variety of electron transport chain inhibitors, the regulatory subunits of the PKA holoenzyme (R₂C₂) are digested, releasing active catalytic subunits. This proteolysis is attenuated by calpain inhibitor I (ALLN). This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).     

Expression in Escherichia coli and characterization of the heat-stable inhibitor of the cAMP-dependent protein kinase

Pure heat-stable inhibitor of the cAMP-dependent protein kinase (PKI) has been isolated in high yield by using a bacterial expression vector constructed to synthesize the complete sequence of the rabbit muscle protein kinase inhibitor, plus an amino-terminal initiator methionine and glycine. Bacterially expressed PKI has an inhibitory activity identical to that of the protein isolated from rabbit skeletal muscle and, by gel filtration and gel electrophoresis, has the same physicochemical characteristics as the native physiological form of PKI. Fourier transformed infrared spectroscopy and CD establish that PKI has unusually large amounts of random coil and turn structures, with significantly smaller amounts of alpha-helix and beta structures.