Localization of protein kinase A and vitronectin in resting platelets and their translocation onto fibrin fibers during clot formation.

Physiological stimulation of platelets with thrombin brings about the release of protein kinase A (PKA) into the plasma. In human blood, this kinase singles out and phosphorylates vitronectin (Vn), a multifunctional regulatory protein, which was proposed to play an important role in the control of fibrinolysis. Here we present immuno-cytochemical evidence to show: (i) that intact platelets possess on their surface an ecto-PKA which can preferentially phosphorylate Vn; (ii) that in the resting platelet, both the catalytic and the regulatory subunits of PKA are present on the platelet surface, in the surface-connected canalicular system, and within the alpha-granules of the platelets; (iii) that the process initiated upon platelet activation, which leads to the formation of fibrin fibers and consequently forms the fibrin net, is accompanied by a translocation of PKA, of Vn, and of PAI-1 onto the fibrin fibers. We propose that the localization and the translocation of these proteins in the fibrin net, together with our finding that PKA phosphorylation of Vn reduces its grip of PAI-1, can unleash PAI-1 in its free form. The free PAI-1 can then assume its latent (non inhibitory) conformation, allow plasminogen activators to trigger the formation of active plasmin, and to initiate fibrinolysis.

Phosphorylation of the retinoblastoma-related protein p130 in growth-arrested cells.

The retinoblastoma family of proteins including pRB, p107 and p130 undergoes cell cycle dependent phosphorylation during the mid-G1 to S phase transition. This phosphorylation is dependent upon the activity of cyclin D/cdk4. In contrast to pRB and p107, p130 is phosphorylated during G0 and the early G1 phase of the cell cycle. We observed that p130 is specifically phosphorylated on serine and threonine residues in T98G cells arrested in G0 by serum deprivation or density arrest. Identification of the phospho-serine and phospho-threonine residues revealed that most were clustered within a short co-linear region unique to p130, defined as the Loop. Deletion of the Loop region resulted in a change in the phosphorylation status of p130 under growth arrest conditions. Notably, deletion of the Loop did not affect the ability of p130 to bind to E2F-4 or SV40 Large T antigen, to induce growth arrest in Saos-2 cells, and to become hyperphosphorylated during the proliferative phase of the cell cycle. p130 undergoes specific G0 phosphorylation in a manner that distinguishes it from pRB and p107.

The carboxyl-terminal tail of kinase splitting membranal proteinase/meprin beta is involved in its intracellular trafficking.

The kinase splitting membranal proteinase (KSMP), was recently shown to be identical with the beta-subunit of meprin. Meprin is a metalloendoproteinase located in brush border membranes and composed of the two types of subunits, alpha and beta. Despite their high sequence homology and similar domain organization, meprin subunits are differently processed during maturation; meprin alpha is retained in the endoplasmic reticulum (ER), and undergoes a proteolytic removal of the transmembrane and cytoplasmic domains, prior to its export from this organelle. In contrast, meprin beta retains these domains even after reaching its final destination in the plasma membrane. Using truncated mutants of rat meprin beta expressed in Cos-7 and human embryonic kidney (HEK) 293 cells, we show here that the cytoplasmic tail is indispensable for its exit from the ER. A meprin beta mutant lacking the last 25 amino acids is shown to be transport-incompetent, although it does not contain any of the known ER retention signals. Systematic analysis of the rate of the ER to Golgi transport using a series of mutants with Ala or Pro substitutions in the tail, suggests that while no specific amino acid residue by itself is imperative for normal intracellular trafficking of meprin beta, the insertion of a bend at a distinct position in the tail (specifically by a Y685P mutation) suffices to retain this protein in the ER. We propose that the very length of the cytoplasmic tail, as well as its secondary structure are essential for the ER to Golgi transport of meprin beta, possibly by allowing an interaction with a cargo receptor.

Unveiling the substrate specificity of meprin beta on the basis of the site in protein kinase A cleaved by the kinase splitting membranal proteinase.

The kinase splitting membranal proteinase (KSMP) is a metalloendopeptidase that inactivates the catalytic (C) subunit of protein kinase A (PKA) by clipping off its carboxyl terminal tail. Here we show that this cleavage occurs at Glu332-Glu333, within the cluster of acidic amino acids (Asp328-Glu334) of the kinase. The Km values of KSMP and of meprin beta (which reproduces KSMP activity) for the C-subunit are below 1 microM. The Km for peptides containing a stretch of four Glu residues are in the micromolar range, illustrating the significant contribution of this cluster to the substrate recognition of meprin beta. This conclusion is supported by a systematic study using a series of the C-subunit mutants with deletions and mutations in the cluster of acidics. Hydrophobic amino acids vicinal to the cleavage site increase the Kcat of the proteinase. These studies unveil a new specificity for meprin beta, suggesting new substrates that are 1-2 orders of magnitude better in their Km and Kcat than those commonly used for meprin assay. A search for substrates having such a cluster of acidics and hydrophobics, which are accessible to meprin under physiological conditions, point at gastrin as a potential target. Indeed, meprin beta is shown to cleave gastrin at its cluster of five glutamic acid residues and also at the M-D bond within its WMDF-NH2 sequence, which is indispensable for all the known biological activities of gastrins. The latter meprin cleavage will lead to the inactivation of gastrin and thus to the control of its activity.

The cleavage of protein kinase A by the kinase-splitting membranal proteinase is reproduced by meprin beta.

The Kinase-Splitting Membranal Proteinase (KSMP) is a metallo-endoproteinase that clips off the carboxyl terminus tail of the catalytic (C) subunit of protein kinase A to yield a truncated, catalytically inactive protein (C'). Here we report (a) a new procedure for the purification of KSMP, yielding a major protein band in SDS-polyacrylamide gel electrophoresis that correlates with the characteristic KSMP activity; (b) the sequence of tryptic peptides obtained from this band, suggesting an identity between this protein and meprin beta; (c) the immuno-detection by specific anti-peptide antibodies of both the alpha and the beta subunits of meprin in KSMP preparations; (d) the stable expression of meprin beta in a mammalian cell line (293) to establish a clone that constitutively expresses the full-length precursor of meprin beta; and (e) the optimalization of the proteolytic activation of this precursor to obtain an enzyme exhibiting the specific KSMP cleavage, suggesting that KSMP is either derived from, or identical with, the meprin beta gene product. It is hoped that these results will shed light on the possible physiological role of KSMP and the way it may affect protein kinase A-mediated processes.

Functional malleability of the carboxyl-terminal tail in protein kinase A.

The catalytic (C) subunit of protein kinase A (PKA) is regarded as a framework for the protein kinase family. Its sequence is composed of a conserved core (residues 40 300) between two segments at the amino and carboxyl termini of the protein. Since the various protein kinases differ in their specificity, it seems reasonable to assume that these nonhomologous segments may be involved in endowing each kinase with its individual specificity. Here we present data to show that the cluster of acidic amino acids (328DDYEEEE334) at the carboxyl-terminal "tail" of the C subunit, specifically Tyr330, contributes to its substrate recognition. This is based on three complementary lines of evidence: (i) on a conformation-sensitive cleavage of the C subunit by a kinase-splitting membranal proteinase that specifically recognizes this cluster, to demonstrate the occurrence in solution of "open" (cleavable) and "closed" (noncleavable) conformations of the C subunit; (ii) on analysis of the three-dimensional structures of the open and closed conformations of the C subunit, showing an approximately 7-A movement of the phenolic hydroxyl of Tyr330 to reach (in the closed conformation) an approximately 3-A distance from the nitrogen atoms of the Arg residue at position p-3 of the PKA consensus sequence; and (iii) on single-site mutations of the C subunit (e.g. Y330A) that show a significant contribution of Tyr330 to the Km of PKA for its substrates/inhibitors and to its catalytic efficacy (Vmax/Km).

Anti-head and anti-tail antibodies against distinct epitopes in the catalytic subunit of protein kinase A. Use in the study of the kinase splitting membranal proteinase KSMP.

Protein kinases share a considerable sequence homology in their catalytic core (residues 40-300 in PKA). Each core is flanked by "head" and "tail" segments at its amino- and carboxy-termini, which are different in the various kinases. These end segments may play an important role in creating the preferential affinity of each kinase for its physiological substrates or regulatory ligands. Here we describe three anti-peptide antibodies (alpha P-1, alpha P-2, and alpha P-3) that specifically recognize the head and tail segments of the catalytic subunit (C) of PKA. (i) alpha P-1 (against 6A-K23) react with C when denatured but not when in its native structure; (ii) alpha P-2 (against 319K-I335), bind to the site in C cleaved by the kinase splitting membranal proteinase (KSMP) and inhibit this cleavage of C; (iii) alpha P-3 (against 338S-F350) react with C but not with the KSMP cleavage product C', useful for detecting a KSMP-like activity in different tissues and subcellular loci. The combined use of the antibodies described here provides a strict definition of C, and thus a high degree of fidelity in its biorecognition.

[High molecular weight forms of GTP-binding regulatory proteins from the bovine cerebellum]. / Vysokomolekuliarnye formy GTP-sviazyvaiushchikh reguliatornykh belkov iz mozzhechka byka.

A new form of a low Km GTPase belonging to the family of regulatory GTP-binding G-proteins has been identified in bovine cerebellum. The molecular weight of this G-protein is several times as high as that of other G-proteins known to be alpha beta gamma heterotrimers: i. e., Gs, Gi, Go, transducin and a new G-protein which had recently been isolated in our laboratory from bovine cerebellum. The high molecular weight G-protein is stable against dissociation; its molecular mass does not change after treatment with DTT, colchicine and NaF. Using antibodies against the alpha-subunit of the formerly isolated cerebellar G-protein and the transducin beta-subunit, it was demonstrated that the both immunoreactive subunits are present in the high molecular weight G-protein. The two forms of the cerebellar G-proteins, i. e., "high" and "low molecular weight" ones, differ drastically in terms of the Mg2+ effect on their GTPase activity. Whereas at submicromolar concentrations of Mg2+ the GTPase activity of the former is virtually absent, the GTPase activity of the latter is more elevated in the presence of EDTA than in the presence of Mg2+.

[Dependence of the activity of phi X 174 B-promoter in the expression of Escherichia coli gal-operon on the number of its copies and their orientation]. / Zavisimost' aktivnosti B-promotora bakteriofaga phiX174 v ékspressii gal-operona Escherichia coli ot chisla ego kopii i ikh orientatsii.

Promoter activities of different restriction fragments of the R8 DNA region of phage phi X 174 were compared. The studied DNA fragments included HindII fragment R8 (B-promoter), its left portion 49 nucleotide long, and the central segment containing 113 nucleotides generated by AluI. The promoter activity of these fragments was quantitated by the appearance of uridyltransferase and galactokinase activities in Escherichia coli clones carrying plasmids pHD68-17. The gal-promoters of these plasmids was substituted for the three aforementioned restriction fragments. The R8 region and its central part (BII-promoter) had comparable promoter activities while the left part containing the putative BI-promoter, did not induce clones with the expressed gal-operon. Clones containing 1, 2, 3 copies of the promoter fragment R8 were selected. No clones were revealed with more copies. All selected di- and tri-promoter clusters in plasmids had the same correct orientation of all inserted promoters with respect to the gal-operon. The expression of the gal-operon in E. coli was nearly directly proportional to the number of the phi X 174 B-promoters inserted before the operon.

A new procedure for the simultaneous large-scale purification of bacteriophage-T4-induced polynucleotide kinase, DNA ligase, RNA ligase and DNA polymerase.

A procedure for simultaneous large-scale purification of the bacteriophage-T4-induced polynucleotide kinase, DNA ligase, RNA ligase and DNA polymerase has been developed. The method involves bacterial cell disruption by sonication, fractionation of cell extract with polymin P, salt elution from the polymin pellets, ammonium sulfate precipitation, and subsequent column chromatography purification of the enzymes. To enrich the enzyme content highly in the initial source non-permissive Escherichia coli B-23 cells infected with T4 amN82 phage were used. The procedure described is rapid, reproducible, high in yield, and able to handle preparations using from 1 g to 200 g cell paste. It can be easily scaled up. The method results in large amounts of the enzymes with very high specific activities, good stability essential lacking exonuclease and endonuclease contamination. The final enzyme preparations were efficiently used in DNA sequencing and in multiple experiments on construction of various recombinant DNAs for cloning and expression in vivo.

Leu-enkephalin purification from E. coli cells carrying the plasmid with fused synthetic leu-enkephalin gene.

Chemically synthesized leu-enkephalin gene was fused to a large Eco RI-Bam HI fragment of pBR322 along with a Eco RI fragment of Ch4A phage DNA carrying the promoter and most of the E.coli beta-galactosidase gene. The resulting recombinant DNA was used to transform E. coli cells. Transformants were screened for Tc-sensitivity, Am-resistance, and beta-galactosidase constitutional synthesis. Restriction endonuclease analysis combined with DNA sequencing of the plasmid DNAs revealed a complete nucleotide leu-enkephalin sequence and Eco RI lac-operon fragment in two possible orientations. Radioimmunoassay for leu-enkephalin activity in BrCN-treated bacterial extracts showed that in vivo leu-enkephalin is synthesized only in strains carrying plasmids with the proper lac-fragment orientation. About 5.10(4) molecules of the former are synthesized per single E. coli cell. One of the clones was used for leu-enkephalin purification. Using 100 g of cells it is possible to obtain about 2 mg of practically pure leu-enkephalin.Images

Properties of Bacillus subtilis ATP-dependent deoxyribonuclease.

A purification procedure described previously resulting in electrophoretically pure Bacillus subtilis ATP-dependent DNAse has now been modified by adding a fractionation stage with Polymin P to permit large-scale isolation of the enzyme. It has been found that the enzyme molecule (Mr = 300000) consists of two large subunits with Mr 155000 and 140000. The purified enzyme has three activities: (1) DNAse on linear single-stranded and double-stranded DNAs (2) DNA-unwinding and (3) ATPase. Circular DNAs were not affected by the enzyme. Study of the dependence of these activities on temperature, pH, and ATP and Mg2+ concentrations has revealed two different states of the enzyme. At low ATP concentrations and alkaline pH, it showed chiefly nuclease action, degrading considerable amounts of DNA to small fragments five residues long on average. At higher ATP concentrations and neutral pH (more physiological conditions) it predominantly unwound DNA. Simultaneously it cut preferentially one of the duplex strands to fragments more than 1000 residues in length. The results obtained suggest that the energy of the enzyme-cleaved ATP is mainly expended on unwinding rather than on degrading DNA molecules.

[Isolation and properties of B. subtilis DNA-binding proteins inhibiting ATP-dependent deoxyribonuclease]. / Vydelenie i svoistva DNK-sviazyvaiushchikh belkov Bactillus subtilis, ingibiruiushchikh ATP-zavisimuiu dezoksiribonukleazu.

The fraction inhibiting ATP-dependent DNAase and some other enzyme activities was found in B. subtilis cell extracts. Two methods of its isolation were elaborated. It is established that the inhibiting activity fraction represents a set of some positively charged thermostable proteins of low molecular weight (M 9000--25 000). The inhibiting effect of the proteins in question may be attributed to their ability to form a complex with DNA. The complex is formed in low ionic strength conditions. The elevation of NaCl concentration to 0,3 M removes some proteins from the complex and causes the complete loss of inhibiting activity. At 0,5 M NaCl DNA-protein complex is completely dissociated. The discovered proteins seems to be localized in DNA-membrane cell fraction. It is supposed that these proteins (or some of them) are the structural ones of the bacterial nucleoid.