Hydrophobic Derivatives of Glycopeptide Antibiotics as Inhibitors of Protein Kinases.

As key regulators of cell signaling, protein kinases (PKs) are attractive targets for therapeutic intervention in a variety of diseases. Herein, we report for the first time the inhibitory activity of polycyclic peptides, particularly, derivatives of glycopeptide antibiotics teicoplanin and eremomycin, against a panel of 12 recombinant human protein kinases and two protein kinases (CK1 and CK2) isolated from rat liver. Several of the investigated compounds inhibited various PKs with IC50 values below 10 µM and caused >90% suppression of the enzyme activity at 10 µM concentration. Kinetic analysis of the protein kinase CK2α inhibition by the teicoplanin aglycon analogue (7) demonstrated the non-competitive mechanism of inhibition (with regard to ATP). Interestingly, the inhibitory activity of some investigated compounds correlated with the earlier described antiviral activity against HIV, HCV, and other corona- and flaviviruses.

A "SYDE" effect of hierarchical phosphorylation: possible relevance to the cystic fibrosis basic defect.

The motif "SYDE", incorporating the protein kinase CK2 consensus sequence (S-x-x-E) has been found to be phosphorylated at both its serine and tyrosine residues in several proteins. Of special interest is the case of cystic fibrosis Transmembrane-conductance Regulator (CFTR), where this motif is close to the residue (F508), whose deletion is the by far commonest cause of cystic fibrosis. Intriguingly, however, CFTR S511 cannot be phosphorylated by CK2 to any appreciable extent. Using a number of peptide substrates encompassing the CFTR "SYDE" site we have recently shown that: (1) failure of CK2 to phosphorylate the S(511)YDE motif is due to the presence of Y512; (2) CK2 readily phosphorylates S511 if Y512 is replaced by a phospho-tyrosine; (3) the Src family protein tyrosine kinase Lyn phosphorylates Y512 in a manner that is enhanced by the deletion of F508. These data, in conjunction with the recent observation that by inhibiting CK2 the degradation of F508delCFTR is reduced, lead us to hypothesize that the hierarchical phosphorylation of the motif SYDE by the concerted action of protein tyrosine kinases and CK2 is one of the mechanisms that cooperate to the premature degradation of F508delCFTR.

Investigation on PLK2 and PLK3 substrate recognition.

Analyses of human phosphoproteome based on primary structure of the aminoacids surrounding the phosphor Ser/Thr suggest that a significant proportion of phosphosites is generated by a restricted number of acidophilic kinases, among which protein kinase CK2 plays a prominent role. Recently, new acidophilic kinases belonging to the Polo like kinase family have been characterized, with special reference to PLK1, PLK2, and PLK3 kinases. While some progress has been made in deciphering the PLK1-dependent phosphoproteome, very little is known about the targets of PLK2 and PLK3 kinases. In this report by using an in vitro approach, consisting of cell lysate phosphorylation, phosphoprotein separation by 2D gel electrophoresis and mass spectrometry, we describe the identification of new potential substrates of PLK2 and PLK3 kinases. We have identified and validated as in vitro PLK2 and PLK3 substrates HSP90, GRP-94, ß-tubulin, calumenin, and 14-3-3 epsilon. The phosphosites generated by PLK3 in these proteins have been identified by mass spectrometry analysis to get new insights about PLKs specificity determinants. These latter have been further corroborated by an in silico analysis of the PLKs substrate binding region.

[Observational study of adverse events in admissions in general hospital of Sassari about corporate planning of clinical risk management]. / Studio osservazionale degli eventi avversi relativi ai ricoveri del 2005, nell'ambito di un programma di gestione del rischio clinico della ASL di Sassari.

In the corporate planning of clinical risk management, we performed an observational retrospective study based on random sampling of admission in General Hospital of Sassari, in 2005. We examined 400 patient clinical documentations in order to find the most frequent adverse events (AE), according to the international literature. We looked for 9 different adverse events; for each of these we elaborated a form personal data and detailed information for each event. During the analysis of the clinical documentations we have found also adverse events not previously classified: they were recorded and classified. We classified the events as explicit, if declared in clinical documentation, and implicit if not declared but clearly present in the records. 47 EA included in the initial 9 categories were found; while other 26 were not included the defined categories, global frequency of AE in our sample resulted: 18.3%. The study is an initial approach to the survey of AE and needs to be refined by determination of liability, severity, predictability, preventability.

Dislocation resistance of ProRoot Endo Sealer, a calcium silicate-based root canal sealer, from radicular dentine.

AIM: To examine the dislocation resistance of three root canal sealers from radicular dentine with and without immersion in a simulated body fluid (SBF), using a modified push-out test design that produced simulated canal spaces of uniform dimensions under identical cleaning and shaping conditions. METHODOLOGY: Sixty single-rooted caries-free human canine teeth were used. Standardized simulated canal spaces were created using 0.04 taper ProFile instruments along the coronal, middle and apical thirds of longitudinal tooth slabs. Following NaOCl/ethylenediamine tetra-acetic acid cleaning, the cavities were filled with ProRoot Endo Sealer, AH Plus Jet or Pulp Canal Sealer. After setting, half of the cavities were tested with a fibre-optic light-illuminated push-out testing device. The rest were immersed in SBF for 4 weeks before push-out evaluation. Failure modes were examined with stereomicroscopy and field emission (FE)-scanning electron microscopy. RESULTS: Location of the sealer-filled cavities did not affect push-out strengths. ProRoot Endo Sealer exhibited higher push-out strengths than the other two sealers particularly after SBF storage (P < 0.001). Failure modes were predominantly adhesive and mixed for Pulp Canal Sealer and AH Plus Jet, and predominantly cohesive for ProRoot Endo Sealer. Spherical amorphous calcium phosphate-like phases that spontaneously transformed into apatite-like phases were seen in the fractured specimens of ProRoot Endo Sealer after SBF storage. CONCLUSIONS: When tested in bulk without a main core, both 'sealer type' and 'SBF storage' were significant in affecting push-out results. The ProRoot Endo Sealer demonstrated the presence of spherical amorphous calcium phosphate-like phases and apatite-like phases (i.e. ex vivo bioactivity) after SBF storage.

Pharmacological inhibition of protein kinase CK2 reverts the multidrug resistance phenotype of a CEM cell line characterized by high CK2 level.

Protein kinase CK2 is an ubiquitous and constitutively active kinase, which phosphorylates many cellular proteins and is implicated in the regulation of cell survival, proliferation and transformation. We investigated its possible involvement in the multidrug resistance phenotype (MDR) by analysing its level in two variants of CEM cells, namely S-CEM and R-CEM, normally sensitive or resistant to chemical apoptosis, respectively. We found that, while the CK2 regulatory subunit beta was equally expressed in the two cell variants, CK2alpha catalytic subunit was higher in R-CEM and this was accompanied by a higher phosphorylation of endogenous protein substrates. Pharmacological downregulation of CK2 activity by a panel of specific inhibitors, or knockdown of CK2alpha expression by RNA interference, were able to induce cell death in R-CEM. CK2 inhibitors could promote an increased uptake of chemotherapeutic drugs inside the cells and sensitize them to drug-induced apoptosis in a co-operative manner. CK2 blockade was also effective in inducing cell death of a different MDR line (U2OS). We therefore conclude that inhibition of CK2 can be considered as a promising tool to revert the MDR phenotype.

Prothrombin complex concentrates for urgent anticoagulation reversal in patients with intracranial haemorrhage.

BACKGROUND: Intracranial haemorrhage (ICH) is a serious and potentially fatal complication of oral anticoagulant therapy (OAT). Prothrombin complex concentrates (PCCs) produce a rapid and effective reversal of OAT effects, but little evidence exists on their efficacy and safety in the management of ICH in patients on OAT. AIM: To evaluate the efficacy and safety of PCCs for the rapid reversal of OAT in patients with ICH. METHODS: Patients suffering from acute ICH while receiving OAT were eligible for this prospective cohort study if their international normalized ratio (INR) was > or = 2.0. Stratified 35-50 IU kg(-1) PCC doses were infused based on initial INR. RESULTS: A total of 92 patients (50 males; mean age 74 years, range 34-92 years) were included. The median INR at presentation was 3.3 (range 2-9). At 30 min after PCC administration the median INR was 1.4 (range 0.9-3.1), declining to < or = 1.5 in 75% of patients. The benefit of PCC was maintained for a long time, since in 98% of all post-infusion time points through 96 h the median INR remained < or = 1.5 (median 1.19; range 0.9-2.3). During hospitalization neither thrombotic complications nor significant adverse events were observed and 11 patients died (11.9%). None of the deaths was judged to be related to PCC administration. CONCLUSIONS: PCC administration is an effective, rapid and safe treatment for the urgent reversal of OAT in patients with ICH. Broader use of PCC in this clinical setting appears to be appropriate.

Protein kinase CK2 phosphorylates and upregulates Akt/PKB.

Treatment of Jurkat cells with specific inhibitors of protein kinase CK2 induces apoptosis. Here we provide evidence that the anti-apoptotic effect of CK2 can be at least partially mediated by upregulation of the Akt/PKB pathway. Such a conclusion is based on the following observations: (1) inhibition of CK2 by cell treatment with two structurally unrelated CK2 inhibitors induces downregulation of Akt/PKB, as judged from decreased phosphorylation of its physiological targets, and immunoprecipitate kinase assay; (2) similar results are observed upon reduction of CK2 catalytic subunit by the RNA-interference technique; (3) Akt/PKB Ser129 is phosphorylated by CK2 in vitro and in vivo; (4) such a phosphorylation of activated Akt/PKB correlates with a further increase in catalytic activity. These data disclose an unanticipated mechanism by which constitutive phosphorylation by CK2 may be required for maximal activation of Akt/PKB.

Phosphorylation of phosvitin by casein kinase-2 provides the evidence that phosphoserines can replace carboxylic amino acids as specificity determinants.

The consensus sequence of casein kinase-2 consists of a serine (threonine) followed by a cluster of glutamic and/or aspartic acids, the one at position +3 playing an especially crucial role (Marin et al., (1986) Eur. J. Biochem. 160, 239-244 and Kuenzel et al. (1987) J. Biol. Chem. 262, 9136-9140). None of the 123 serines of the main phosvitin component (34 kDa) fulfils such a requirement (Byrne et al. (1984) Biochemistry 23, 4275-4279), rather, most of them are clustered into stretches of up to 14 entirely phosphorylated residues. Three out of the four threonines lie close to the N-terminal side of such phosphoseryl blocks. Here we show that native 34 kDa phosvitin is a poor substrate of casein kinase-2, its radiolabeling occurring mostly at threonine residue(s); a very slight (1%) previous dephosphorylation with acid phosphatase converts phosvitin into an excellent substrate for casein kinase-2, its phosphorylation occurring almost exclusively at serine residues. Extensive dephosphorylation however (greater than 40%) reduces the phosphorylation efficiency of casein kinase-2. These results show that phosphoserine residues can replace carboxylic residues as specificity determinants for casein kinase-2.

Identification of pseudo 'phosphothreonyl-specific' protein phosphatase T with a fraction of polycation-stimulated protein phosphatase 2A.

Protein phosphatase T from rat liver, so termed due to its activity toward [32P-Thr]casein and its marked preference for the phosphopeptide Arg-Arg-Ala-Thr(P)-Val-Ala over its phosphoseryl derivative (Donella Deana, A., Marchiori, F., Meggio, F. and Pinna, L.A. (1982) J. Biol. Chem. 257, 8565-8568), is shown here to belong to the family of type 2A protein phosphatase according to Cohen's nomenclature (Ingebritsen, T.S. and Cohen, P. (1983) Eur. J. Biochem. 132, 255-261). In particular, protein phosphatase T is endowed with phosphorylase phosphatase activity that is stimulated by protamine, histone H1 and heparin, it is inhibited by spermine, it does not bind to heparin-Sepharose and it readily dephosphorylates the phosphopeptide Arg-Arg-Leu-Ser(P)-Ile-Ser-Thr-Glu-Ser reproducing the phosphorylation site of the alpha-subunit of phosphorylase kinase. The Mr of protein phosphatase T determined by gel filtration under non-denaturating conditions is about 150 kDa and its activity ratio toward histone H1 phosphorylated by protein kinase C versus histone H1 phosphorylated by cAMP-dependent protein kinase is unusually high. Some properties of protein phosphatase T, such as its weak binding to DEAE-cellulose and its high stimulation by protamine as compared to a relatively poor stimulation by histone H1, suggest that it may be similar to subtype 2Ao of protein phosphatase 2A.

Random tyrosine and glutamic acid-containing polymers are very powerful inhibitors of casein kinase-2.

The random heteropolymers Glu/Tyr(4:1) and Glu/Tyr(1:1) that are widely used as substrates for tyrosine protein kinases, are very powerful competitive inhibitors of casein kinase-2, but not of casein kinase-1, with respect to the protein substrate, their Ki values being one to two orders of magnitude lower than those of polyglutamates of similar size. The inhibitory power is reduced if tyrosine is partially replaced by alanine, as in the polymer Glu/Ala/Tyr(6:3:1) and it disappears upon inclusion of lysine, the polymer Glu/Ala/Lys/Tyr(2:6:5:1) actually behaving as a stimulator. These data indicate that non-phosphorylatable hydroxylic residues in addition to acidic ones are required in order to optimize the binding of pseudo-substrates to the catalytic site of casein kinase-2.

Comparative study of mitochondrial and cytosol protein kinase activities.

Both cytosol and mitochondria of rat liver display protein kinase activity, cyclic AMP-independent, which is resolved by Sepharose 6B filtration and P-cellulose chromatography into multiple forms phosphorylating, besides endogenous mitochondrial membrane-bound proteins, also exogenous phosphoproteins such as casein and phosvitin. However, the forms by far predominant in the cytosol phosphorylate both phosphorylserine and phosphorylthreonine residues of casein, while most of the activity associated to mitochondrial structures is due to the forms phosphorylating only phosphorylserine residues.

Casein kinases and their protein substrates in rat liver cytosol: evidence for their participation in multimolecular systems.

We have shown by gel filtration on Sepharose 4B at low ionic strength that casein kinases S (type 1), heparin-insensitive, and TS (type 2), heparin-inhibited, of rat liver cytosol participate in two distinct multimolecular systems, Ve/Vo = 1.25 and Ve/Vo = 1.90, respectively, both less retarded than the peak of cAMP-dependent protein kinase activity (Ve/Vo = 2.04). Both casein kinase I and casein kinase II complexes are unstable in 0.5 M NaCl, giving rise by gel filtration under these conditions to the free forms of casein kinase S (Ve/Vo = 2.37, Mr 34 000) and casein kinase TS (Ve/Vo = 2.10, Mr 130 000), respectively. In contrast, the elution volume of cAMP-dependent protein kinase activity is always the same irrespective of the ionic strength of the medium. Casein kinase I, accounting for the whole casein kinase S activity of cytosol, also contains a phosphorylatable 31-kDa protein (p31) which is a substrate of casein kinase S, since its phosphorylation is insensitive to heparin, the heat-stable inhibitor and trifluoperazine, but it is prevented by beryllium. Casein kinase II, on the other hand, apparently results from the association of the whole casein kinase TS (type 2) of rat liver cytosol with a 90-kDa protein substrate (p90) which is distinct from glycogen synthase according to their different peptide mappings. The radiolabelling of p90 is inhibited by heparin, unlabeled GTP and polyglutamates, while it is dramatically and specifically enhanced by polylysine. At least three more protein bands of Mr 58 000, 52 000 and 37 000 are phosphorylated by casein kinase TS in the casein kinase II fraction: their co-elution with casein kinase TS, however, seems to be accidental and their radiolabeling in the presence of polylysine is almost negligible compared to that of p90. It is concluded that p31 and p90 may represent specific targets of casein kinase S and casein kinase TS, respectively, whose intimate association with the enzymes could be functionally significant.

A study with model substrates of the structure of the sites phosphorylated by rat liver casein kinase TS.

Two new sites phosphorylated by rat liver cyclic AMP-independent casein kinase TS have been identified in denatured pepsin and soybean antiprotease C-II, exhibiting the sequences: Cys-Ser-Ser(P)-Ile-Asp-Ser and His-Ser3(P)-Asp-Asp-Glu, respectively. Their phosphorylation efficiency has been compared to that of previously identified sites and the effects of chemical modifications in the vicinity of the phosphorylatable residue have been studied. The results obtained support the following conclusions: 1. All sites affected by casein kinase TS conform to the sequence: Ser/Thr-X-Glu/Asp which is also believed to be required by the mammary gland casein kinase. Threonine appears to be less suitable for phosphorylation then serine. The presence of some additional residues on the C-terminal side also appears to be required. 2. X can be either an additional acidic residue or a natural one, but not a basic residue. The contiguity of an acidic cluster to the C-terminal side of the target greatly improves the phosphorylation efficiency. 3. The residues N-terminal to the target one do not seem to be relevant for determining the site recognition by the protein kinase. 4. The predicted secondary structure constantly occurring at the phosphorylation sites is the beta-turn: apparently the bend must include both the target residue and the acidic determinant at the n + 2 position.

Substitution of phosphotyrosine for sulphotyrosine in biologically active peptides. Enzymatic phosphorylation of a progastrin peptide confers immunoreactivity reminiscent of the sulphated derivative.

The peptide SAEEEDQYN, corresponding to the carboxyl-terminal tryptic fragment of rat progastrin, whose penultimate tyrosyl residue is sulphated in the native peptide, is phosphorylated with Km values of 120 and 180 microM by two spleen tyrosine protein kinases, termed TPK-IIB and TPK-III, respectively. Another spleen tyrosine protein kinase related to the src family (TPK-I/lyn) is poorly active toward this peptide, displaying a Km 6.5 mM. The Tyr-phosphorylated peptide is recognized by an antibody (L304), which reacts with both sulphated and unmodified peptides, while it is not recognized by a second antibody (L303), which reacts with unmodified peptide yet not with the sulphated derivative. These data, in conjunction with previous observations (Hofsteenge, J., Stone, S.R., Donella-Deana, A. and Pinna, L.A. (1990) Eur. J. Biochem. 188, 55-59) support the view that phosphotyrosine is an effective surrogate for sulphotyrosine in a wide spectrum of biological activities.

The use of phosphopeptides to distinguish between protein phosphatase and acid/alkaline phosphatase activities: opposite specificity toward phosphoseryl/phosphothreonyl substrates.

The four main classes of protein phosphatases (PP-1, 2A, 2B and 2C), although differing in their ability to dephosphorylate phosphopeptide substrates, invariably display a marked preference toward phosphothreonyl peptides over their phosphoseryl counterparts. Conversely, all the acidic and alkaline phosphatases tested so far dephosphorylate phosphoseryl derivatives far more readily than phosphothreonyl ones. This opposite behaviour provides a criterion for discriminating between protein dephosphorylating activity due to authentic protein phosphatases as compared to nonspecific acid and/or alkaline phosphatases. In particular the phosphothreonyl peptides RRATPVA and RRREEETPEEEAA appear to be especially suited for detecting the activity of PP-2C and PP-2A, since they are hardly dephosphorylated by acid and alkaline phosphatases. Conversely, the phosphoseryl peptides SPEEEEE and RRASPVA can provide a sensitive evaluation of the majority of acid and alkaline phosphatases, while being refractory to protein phosphatases.

An analysis of the substrate specificity of insulin-stimulated protein kinase-1, a mammalian homologue of S6 kinase-II.

The specificity determinants for insulin-stimulated protein kinase-I (ISPK-1) have been investigated with synthetic peptides based on naturally-occurring protein phosphoacceptor sequences. Peptides (Arg-Arg-Xaa-Ser-Xaa) that fulfill the consensus sequence for cyclic-AMP-dependent protein kinase (PK-A) are also phosphorylated readily by ISPK-1. The phosphorylation efficiency is improved by increasing the number of N-terminal arginine residues and by moving the arginyl cluster one residue further away from the serine, the nonapeptide (Arg)4-Ala-Ala-Ser-Val-Ala being the best substrate among all the short peptides tested (Km = 15 microM). Conversely, the substitution of either Thr for Ser or Lys for Arg is detrimental. Likewise, two flanking Pro residues and an Arg immediately N-terminal to the Ser act as negative specificity determinants. While the specificity of ISPK-1 shows several similarities to that of PK-A, including an absolute requirement for basic residues on the N-terminal side of the target Ser, it differs in several other respects including (1), the detrimental effect of a Lys for Arg substitution which is still compatible with some phosphorylation by ISPK-1, but not PK-A; (2), the presence of C-terminal acidic residues which are tolerated very well by ISPK-1, but are detrimental to PK-A; (3), the effect of substituting Phe for Val in the peptide Arg-Arg-Ala-Ser-Val-Ala, which improves the efficiency of phosphorylation by PK-A (lowering the Km 4-fold), but has no effect on phosphorylation by ISPK-1. These differences in peptide substrate specificity may account in part for the different rates of phosphorylation of physiological substrates for ISPK-1 and PK-A, such as the G subunit of protein phosphatase-1.

The autophosphorylation and p34cdc2 phosphorylation sites of casein kinase-2 beta-subunit are not essential for reconstituting the fully-active heterotetrameric holoenzyme.

Two mutants of human casein kinase-2 beta-subunit with short deletions at either their amino (delta 1-4) or carboxy (delta 209-215) terminal side have been created that have lost the capability to undergo autophosphorylation and p34cdc2 mediated phosphorylation, respectively. Both mutants give rise to reconstituted CK2 holoenzymes displaying basal catalytic activity, thermostability and responsiveness to polylysine, identical to those of wild-type holoenzyme, whose reconstitution, moreover, is not affected by previous phosphorylation of the beta-subunit at either its N-terminal or C-terminal sites. Unlike the wild-type beta and beta(delta 209-215), however, beta(delta 1-4) fails to confer to the reconstituted holoenzyme the typical responsiveness to NaCl stimulation. These results suggest that while neither the autophosphorylation nor the p34cdc2 phosphorylation sites are required for conferring a stable structure and full catalytic activity to CK2, the autophosphorylation site is implicated in the NaCl-dependent fine tuning of CK2 activity.

Phosphorylation of ornithine decarboxylase in intact erythroleukemia cells.

32P-labeled ornithine decarboxylase was isolated by immunoprecipitation from murine erythroleukemia cells incubated in a medium containing [32P]ortophosphoric acid. Analysis of immunoprecipitate by SDS-polyacrylamide gel electrophoresis and autoradiography revealed a radiolabeled band, which corresponded to the position of mouse ornithine decarboxylase, phosphorylated in vitro by casein kinase-2. A preparation of casein kinase-2 purified from nuclei of erythroleukemia cells could also phosphorylate mouse ornithine decarboxylase.

Location of the phosphorylation site for casein kinase-2 within the amino acid sequence of ornithine decarboxylase.

The sizes of the radiolabeled fragments obtained by CNBr and DMSO/HBr digestion of 32P-labeled ornithine decarboxylase phosphorylated by rat liver casein kinase TS (type-2) are consistent with the location of the phosphorylation site within the sequence(303-309) Ser-Asp-Asp-Glu-Asp-Glu-Ser. Parallel experiments with synthetic peptides rule out the suitability of Ser-309, as well as of other serines of ornithine decarboxylase having just two or three acidic residues close to their C terminal side. Ser-303 appears, therefore, to be the main if not the only target for casein kinase-2.