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O Marin

Publications and source records attributed to O Marin.

At least 55 records · Page 3Linked to original sources
PubMed1996

Mapping the residues of protein kinase CK2 alpha subunit responsible for responsiveness to polyanionic inhibitors.

The quadruple mutation of the whole basic cluster, K74KKK77 conserved in the catalytic subunits of protein kinase CK2 and implicated in substrate recognition, not only abolishes inhibition by heparin but even induces with some peptide substrates an up to 5-fold stimulation by heparin in the 0.5-5 micrograms/ml concentration range. Two other mutants defective in substrate recognition, R191, 195K198A and K79R80K83A, display either a 100-fold reduction or no alteration at all in heparin inhibition, respectively. In contrast sensitivity to heparin inhibition is increased 30-fold by a single mutation affecting Arg-228 while it is not altered by a triple mutation in the small insert of subdomain XI (mutant R278K279R280A). The effect of the same mutations on inhibition by pseudosubstrate EEEEEYEEEEEEE is different, the mutant displaying the most reduced sensitivity being R191,195K198A, followed by K74-77A and K79R80K83A; the other mutants are almost indistinguishable from CK2 wild type. Substantial reduction of inhibition by poly(Glu,Tyr)4:1 is only observable with mutant R191,195K198A, whereas R228A is significantly more sensitive to inhibition. These data show that the mode of inhibition of CK2 by polyanionic compounds occurs through substantially different mechanisms involving residues that are variably concerned with substrate recognition.

Amino Acid Sequence↗
PubMed1996

Isolation from spleen of a 57-kDa protein substrate of the tyrosine kinase Lyn. Identification as a protein related to protein disulfide-isomerase and localisation of the phosphorylation sites.

A 57-kDa protein (p57) has been purified to homogeneity from a microsomal fraction of rat spleen. It is specifically and efficiently phosphorylated by the Src-like tyrosine kinase Lyn purified from the same source with a Km of 0.34 microM. The tyrosine kinases c-Fgr, Fyn, C-terminal Src kinase and p72syk, as well as the Ser/Thr-specific cAMP-dependent protein kinase and protein kinases CK1 and CK2 do not phosphorylate p57. C-terminal Src kinase, which acts to down-regulate the Src-like protein-tyrosine kinases, almost completely prevents the protein phosphorylation catalysed by Lyn. Protein mass fingerprinting with tryptic fragments identified p57 as a protein related to protein disulfide-isomerase which belongs to the superfamily of Cys-Gly-His-Cys-containing sequences. Lyn phosphorylates tyrosine residues Y444, Y453 and Y466 which are located in a highly acidic region of the protein at the C-terminus. Upon phosphorylation, p57 forms a complex with Lyn which can be immunoprecipitated with anti-Lyn IgG. The association which occurs between the phosphorylated substrate and the SH2 domain of the kinase is consistent with the suggested 'processive phosphorylation' model, which implies that a primary phosphorylation site of the substrate binds to the SH2 domain of the enzyme and triggers the phosphorylation at secondary site(s).

Amino Acid Sequence↗
PubMed1996

Activation of protein phosphatase-1 isoforms and glycogen synthase kinase-3 beta in muscle from mdx mice.

Three Protein Phosphatase-1 (PP1) isoforms (PP1 alpha, PP1 gamma-1 and PP1 delta) are found in skeletal muscle. These are bound to regulatory subunits, such as inhibitor 2 (I2) in the cytosol and G in the glycogen and microsomal fractions. In vitro, the PP1-12 complex is activated by Glycogen Synthase Kinase-3 (GSK-3 or FA). We investigated the activities and protein levels of the three PP1 isoforms and of GSK-3 in muscle of mdx dystrophic mice. PP1 was assayed as phosphorylase phosphatase, in the presence of 5 nM okadaic acid (which inhibits PP2A). Peptide antibodies were produced and used to investigate PP1 alpha, PP1 gamma-1 and PP1 delta. GSK-3 was assayed using a previously described peptide. This was synthesized in a pre-phosphorylated from, which avoids the additional use of Casein Kinase II. Higher PP1 activity was assayed in the cytosol from mdx rather than from control muscles. Immunoprecipitation indicated that only PP1 alpha and PP1 gamma-1 were more active. This was most likely due to enzyme activation, since the immunodetected proteins were unchanged. On the other hand, the immunodetected PP1 delta was lower in the glycogen and microsomal fractions from mdx muscle. GSK-3 was more active in the mdx extract Selective immunoprecipitation of GSK-3 alpha and GSK-3 beta indicated that both isoforms were activated. In the case of GSK-3 beta, the immunodetected protein was also increased. The changes described herein may be related to the pathological events occurring in the mdx muscle. These include increased protein degradation and turnover, and fibre regeneration. In fact, the decreased PP1 delta may be due to protein degradation and the increased GSK-3 may be the consequence of increased protein turnover or regeneration. The apparent correlation between the increased PP1 alpha and PP1 gamma-1 activities and the increased GSK-3 may agree with the hypothesis that GSK-3 activates the newly synthesized PP1.

Amino Acid Sequence↗
PubMed1996

Phosphotyrosine specifies the phosphorylation by protein kinase CK2 of a peptide reproducing the activation loop of the insulin receptor protein tyrosine kinase.

Protein kinase CK2 is a ubiquitous Ser/Thr-specific protein kinase responsible for the phosphorylation of many proteins implicated in signal transduction. It phosphorylates both threonyl and seryl residue(s) of the insulin receptor beta-subunit. In this study, a series of peptides, reproducing all the threonyl sites of the intracellular domain of the insulin receptor that display the consensus sequence for CK2, has been synthesized and used as substrate for purified rat liver CK2. The only peptide readily phosphorylated is the one reproducing the activation loop of the insulin receptor (EIYET1160DYYA), including three tyrosines (Y1158, Y1162 and Y1163) whose phosphorylation through an intermolecular autocatalytic process promotes the activation of the receptor kinase. The phosphorylation efficiency of T1160 is increased almost 20-fold if these three tyrosines are previously phosphorylated. By using variably phosphorylated peptides, the tyrosine mainly responsible for such a hierarchical phosphorylation process has been identified as Y1163. It can be concluded, from these data, that T1160 situated in the activation loop of the insulin receptor, represents an excellent target for CK2, its phosphorylation being triggered by the previous autophosphorylation of the three tyrosyl residues surrounding it, with special reference to Y1163. These data are consistent with the implication of CK2 in the regulation of the activation process of the insulin receptor protein tyrosine kinase.

Animals↗
PubMed1995

Effect of polycation peptides on mitochondrial permeability transition.

Synthetic polycation peptides obtained with the basic aminoacids lysine, arginine and ornithine are able to inhibit the permeability transition induced in mitochondria by calcium ions and inorganic phosphate. At least three basic aminoacid residues must be present in the peptide in order to elicit the inhibitory effect. In the presence of synthetic polycations and similarly to spermine, a lack of correlation between inhibition of swelling and glutathione release is apparent, since glutathione release occurs before the onset of a large amplitude swelling. The same lack of correlation is observed in the presence of cyclosporin. From the results obtained with the above reported polycations, different in both aminoacid composition and length, it appears that the effect is not to be referred to the individual properties of the molecules examined but rather to their cationic character; in addition, a critical number of positive charges is necessary to elicit the effect.

Animals↗
PubMed1995

Site specificity of p72syk protein tyrosine kinase: efficient phosphorylation of motifs recognized by Src homology 2 domains of the Src family.

Protein tyrosine kinase p72syk purified from rat spleen has been assayed for its ability to phosphorylate a number of peptide substrates derived from naturally occurring phospho-acceptor sites. The phosphorylation efficiency is extremely variable, depending on the peptide sequence, with Km values in the 3-1500 microM range. The by far best peptide substrates, with Km values of 3 and 4 microM are those reproducing the phospho-acceptor sites of Vav and HS1 proteins, respectively. These sites include multiple acidic residues flanking tyrosine on both sides and they also display the consensus sequences (YEDL and YEEV) preferred by the SH2 domains of the Src family. Alteration of this consensus in the HS1 peptide, by replacing either the glutamic acid or valine, also reduces the phosphorylation efficiency by p72syk. Also the replacement of acidic residues at position -1 and, to a lesser extent at positions -3 and -4 (but not at positions +3 and +5) are detrimental. These observations may suggest a role of p72syk in the recruitment of ligands/substrates for the Src family enzymes. We also show that the HS1 peptide can be used for the specific monitoring of p72syk since neither the two Src-related c-Fgr and Lyn kinases (needing a hydrophobic instead of acidic residue at position -1) nor CSK appreciably phosphorylate it.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Phosphorylation and activation of protein kinase CK2 by p34cdc2 are independent events.

Recombinant isolated beta-subunit of protein kinase CK2 is readily phosphorylated by p34cdc2/cyclin B kinase at Ser209 with favourable kinetic constants (Km = 1.7 microM, Vmax = 20 nmol.min-1.mg-1). Two synthetic peptides reproducing the 170-215 and the 206-215 C-terminal fragments of the beta-subunit are also phosphorylated though with tenfold higher Km values (19.5 and 28.0 microM, respectively). In contrast, both the beta-subunit associated with the alpha-subunit to give the heterotetrameric holoenzyme and the native CK2 are not appreciably phosphorylated by p34cdc2. These data suggest that the Ser209 beta-subunit phosphorylation observed in intact cells occurs prior to beta-subunit incorporation into the holoenzyme. The isolated CK2 alpha-subunit is not phosphorylated to any appreciable extent by p34cdc2 kinase. Its catalytic activity is nevertheless increased up to fivefold upon incubation with p34cdc2/cyclin B kinase complex. Such a stimulation of activity is comparable to that induced by the beta-subunit and it is paralleled by a 40% decrease of p34cdc2/cyclin B catalytic activity. Similar to beta-subunit, p34cdc2/cyclin B also protects the alpha-subunit against thermal inactivation. CK2 holoenzyme is also stimulated by p34cdc2/cyclin B, albeit less dramatically than the isolated alpha-subunit. Such an effect is also evident with CK2 holoenzyme reconstituted with a mutated beta-subunit lacking the p34cdc2 phosphorylation site and it is not accompanied by any appreciable phosphorylation of either the beta or the alpha-subunit. These data indicate that in vitro CK2 alpha-subunit interacts with and is activated by p34cdc2/cyclin B kinase by a mechanism that does not imply the phosphorylation of CK2.

Amino Acid Sequence↗
PubMed1995

Dissection of the dual function of the beta-subunit of protein kinase CK2 ('casein kinase-2'): a synthetic peptide reproducing the carboxyl-terminal domain mimicks the positive but not the negative effects of the whole protein.

The dual function of the regulatory beta-subunit of protein kinase CK2 is highlighted by its ability to abolish calmodulin phosphorylation in contrast to its stimulatory effect on the phosphorylation of peptide substrates. Here we show that a synthetic peptide reproducing the C-terminal region of the beta-subunit (beta[170-215]) stimulates to a similar extent the phosphorylation of either the peptide substrate or calmodulin and also protects the catalytic alpha-subunit against thermal inactivation as efficiently as full-length beta-subunit. These data show that the positive and negative functions of the beta-subunit reside in physically separated domains and that the elements responsible for positive regulation are located in the C-terminal region.

Amino Acid Sequence↗
PubMed1995

Mapping the residues of protein kinase CK2 implicated in substrate recognition: mutagenesis of conserved basic residues in the alpha-subunit.

Six mutants of protein kinase CK2 alpha subunit in which basic residues have been mutated into alanines were assayed for their capability to phosphorylate the peptide RRRADDSDDDDD. Two mutants (R228A and R278K279R280A) behaved more or less as alpha wild type and one (H160,166A) was nearly inactive, hampering the calculation of kinetic parameters. In contrast 3 mutants (K74-77A, K79R80K83A and R191,195K198A) phosphorylated the peptide with reduced efficiency accounted for by increased Km and decreased Vmax values. By using derivatives of the RRRADDSDDDDD peptide in which individual aspartyl residues were variably replaced by alanine(s) and two peptide substrates derived from I-2 (KYRIREQESSGEEDSDL and RRKDLHDDEEDEEMSETADGE) it was shown that mutations in the 191-198, 74-77 and 79-83 regions were the least detrimental whenever the acidic determinants were lacking at positions +1, +4/+5 and +3, respectively. These data support the conclusion that the basic residues present in the p+1 loop of CK2 alpha specifically recognize the acidic determinant adjacent to the C-terminal side of serine, while the specificity determinants located more down-stream are variably recognized by different residues of the unique basic cluster spanning between Lys74 and Lys83.

Amino Acid Sequence↗
PubMed1995

Androgenic regulation of protein kinase CK1 in the prostatic cell.

We examined the androgenic regulation of protein kinase CK1 in rat ventral prostate cytosolic and chromatin fractions. On androgen deprivation, CK1 in both the nuclear and cytoplasmic compartments decreases slowly at a similar rate. Stimulation of regrowth in the prostate does not evoke an early differential modulation in the CK1 because it increased in both the cytosol and chromatin only by 24 h after androgenic stimulus. These changes in CK1 relate to its proposed role in cell regulation at mitosis and differ from those in protein kinase CK2 that demonstrates rapid modulations in the nuclear compartment under similar conditions.

Amino Acid Sequence↗
PubMed1994

Substrate recognition by casein kinase-II: the role of histidine-160.

Casein kinase-II (CK-II) belongs to the protein kinases recognizing serine/threonine in proximity to acidic residues in protein substrates. Crystallography and mutagenesis studies on the cAMP-dependent protein kinase (PKA) disclosed that glutamic acid-170 (E170), is important for interaction of substrates with the enzyme. At a position corresponding to E170 in PKA most Ser/Thr kinases have an aspartic or glutamic acid, while CK-II has a histidine residue (H160). In order to examine the relevance of this substitution for CK-II substrate specificity, a mutant of the catalytic alpha subunit (H160D), in which H160 was changed to aspartic acid, was made. Our results show that H160 is not primarily involved in canonical substrate recognition, but does interact with an acidic residue located at position -2 with respect to the target Ser/Thr.

Amino Acid Sequence↗
PubMed1994

Inhibition of macroautophagy and proteolysis in the isolated rat hepatocyte by a nontransportable derivative of the multiple antigen peptide Leu8-Lys4-Lys2-Lys-beta Ala.

The multiple antigen peptide derivative, Leu8-Lys4-Lys2-Lys-beta Ala (Leu8-MAP), was synthesized by attaching the carboxyl of leucine to the NH2 termini of a branched lysine core, termed MAP, creating a molecule of about 1900 Da with 8 leucine residues. On a molar basis (independent of the number of leucine substitutions), Leu8-MAP was as effective as leucine in suppressing macroautophagy and proteolysis; moreover, it exhibited the same apparent Km (about 0.1 mM). The effect was specific for leucine since Ile8-MAP was inactive. It is of interest, though, that Leu8-MAP did not elicit the multiphasic response typical of leucine but instead evoked the single site inhibition normally seen with leucine plus the co-regulator alanine. Some free leucine was produced from Leu8-MAP during hepatocyte incubations, but the amounts were insufficient to account for the inhibition. Although this degradation created species of Leu-MAP that had lost 1-3 residues of leucine, their inhibitory effectiveness was not diminished. Because the extracellular/intracellular distribution ratio of [3H]-Leu8-MAP was 100:1 or greater, the direct transport of Leu8-MAP across the plasma membrane into the cytosolic compartment can be excluded. Hence, cytosolic concentrations of Leu8-MAP will be at least 100-fold smaller than those of leucine under conditions of comparable proteolytic inhibition. For these and related reasons, effects attributable to the recognition of Leu8-MAP cannot be explained by signals generated within the cytosol. They could, however, be mediated from site(s) on the plasma membrane or within associated vesicles.

Animals↗
PubMed1994

Identification and localization of the myotonic dystrophy gene product in skeletal and cardiac muscles.

We have raised a polyclonal antibody against a synthetic peptide chosen within the deduced sequence of the myotonic dystrophy gene product. This antibody binds to a protein whose molecular weight is in the range of 50-54 kDa in Western blotting of rat, rabbit and human muscles. Biochemical studies seem to indicate that this protein is a peripheral component of sarcoplasmic reticulum as well as of plasma membrane.

Adult↗
PubMed1994

Phosphorylation of synthetic fragments of inhibitor-2 of protein phosphatase-1 by casein kinase-1 and -2. Evidence that phosphorylated residues are not strictly required for efficient targeting by casein kinase-1.

The major phosphorylation site for both casein kinase-2 (CK2) and casein kinase-1 (CK1) in protein phosphatase-1 (PP-1) inhibitor-2 (I-2) is Ser86. Minor phosphorylation sites affected by either CK2 or CK1 are Ser120/Ser121 and Ser174, respectively. A synthetic peptide of 25 amino acids encompassing residues 67-93 of I-2 is phosphorylated by either CK2 or CK1 at its seryl residue corresponding to Ser86 with higher Vmax and Km values similar to those of the intact protein (9 vs 7.2 microM and 14.2 vs 5.3 microM with CK2 and CK1, respectively). No detectable phosphorylation of this peptide which also includes the glycogen synthase kinase-3 (GSK-3) site (Thr72), could be observed with either GSK-3 or p34cdc2 kinase whether or not its seryl residue equivalent to Ser86 had been previously phosphorylated by CK2. Shorter derivatives of I-2(67-93), encompassing residues 72-93 and 78-93, are also readily phosphorylated by both CK1 and CK2, with phosphorylation efficiencies similar to those of the parent peptide. A synthetic heptadecapeptide reproducing the phosphoacceptor site around Ser120/Ser121 is phosphorylated by CK2, but not to any detectable extent by CK1, with a Km value fivefold higher than that of the corresponding pentadecapeptide including Ser86 (78-93). A synthetic pentadecapeptide (166-180) reproducing the phosphoacceptor site around Ser174 is phosphorylated by CK1 less efficiently than the pentadecapeptide including its main phosphorylation site (78-93) (Km 280 microM vs 33 microM). This peptide is readily phosphorylated by CK2 as well, although it lacks the canonical consensus sequence for CK2 and its Ser174 is almost unaffected by CK2 in intact I-2. These data provide the clear-cut demonstration that the consensus sequence with N-terminal prephosphorylated residue(s), SerP/ThrP-Xaa-Xaa-Ser/Thr, [Flotow, H., Graves, P. R., Wang, A., Fiol, C. J., Roeske, R. W. & Roach, P. J. (1990) J. Biol. Chem. 265, 14264-14269; Meggio, F., Perich, J. W., Reynolds, E. C. & Pinna, L. A. (1991) FEBS Lett. 283, 303-306] is not always required to achieve efficient and high-affinity phosphorylation by CK1. They also show that the specificity determinants for I-2 phosphorylation by either CK2 or CK1, but not by GSK3, are entirely grounded on local structural features of the phosphoacceptor site, being only marginally affected by the overall structure of I-2.

Amino Acid Sequence↗
PubMed1994

Design and synthesis of two new peptide substrates for the specific and sensitive monitoring of casein kinases-1 and -2.

The available information about the specificity determinants of casein kinases-1 and -2 (CK1 and CK2) has been utilized to obtain two new peptide substrates optimally suited for the specific monitoring of these two pleiotropic enzymes. The best substrate developed for CK1 is the inhibitor-2 derived peptide RRKDLHDDEEDEAMSITA which is superior in every respect to all the non phosphorylated CK1 peptide substrates used so far. Its Km is 172 microM and the Vmax is 6-fold higher than that of casein. The dodecapeptide RRRADDSDDDDD, on the other hand, is totally refractory to CK1 while it is an excellent substrate for CK2, exhibiting, under basal conditions, a Km value of 19 microM and a Vmax higher than those obtained with all the routinely used substrates of CK2. Both the novel CK1 and CK2 peptide substrates are suited for the phosphocellulose paper assay.

Amino Acid Sequence↗
PubMed1994

Substrate specificity of protein kinase CK2.

Unlike most Ser/Thr protein kinases which recognize phosphoacceptor sites specified by basic residues, protein kinase CK2 is extraordinarily acidophilic in nature. By combining the analysis of more than 100 CK2 natural phosphorylation sites with the kinetic behaviour of a large number of model peptide substrates, it can be concluded that although the most crucial specificity determinant is an acidic residue (Glu, Asp, TyrP, or SerP) at position +3, additional acidic residues at positions spanning from -2 to +7 (and probably farther) also act as positive specificity determinants for CK2, whereas basic residues at these positions, prolyl residue at position +1, and a bulky hydrophobic doublet at position +1 and +2, are powerful negative determinants. It also appears that the nature of the acidic determinants may variably influence their effect depending on the position occupied: Thus, multiple aspartic acids are, in general, determinants as good as, or even better, than an equivalent number of glutamic acids; an individual Asp at position +3 flanked by Glu residues is ineffective; and phosphorylated residues appear to be much more effective if adjacent to the target residue (positions -2 to +2). In some instances, the local determinants alone are insufficient to account for the phosphorylation efficiency of the substrate which is greatly improved by the overall protein conformation, as illustrated by the examples of CK2 beta-subunit and protein p53, the latter exhibiting no consensus sequence around its phosphorylation site.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗