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L A Pinna

Publications and source records attributed to L A Pinna.

At least 145 records · Page 8Linked to original sources
PubMed1990

An investigation of the substrate specificity of protein phosphatase 2C using synthetic peptide substrates; comparison with protein phosphatase 2A.

The synthetic phosphopeptide RRATpVA was found to be the most effective substrate for protein phosphatase 2C (PP2C) so far identified. Replacement of phosphothreonine by phosphoserine decreased activity over 20-fold and a striking preference for phosphothreonine was also observed with two other substrates (RRSTpTpVA and casein) that were phosphorylated on both serine and threonine. Replacement of the C-terminal valine in RRATpVA by proline abolished dephosphorylation, while exchanging the N-terminal alanine by proline had no effect. The preference for phosphothreonine and the effect of proline are similar to protein phosphatase 2A (PP2A). However, the peptide RRREEETpEEEAA, an excellent substrate for PP2A, was not dephosphorylated by PP2C, and substitution of the C-terminal valine in RRATpVA by glutamic acid reduced the rate of dephosphorylation by PP2C over 10-fold, without affecting dephosphorylation by PP2A. Addition of two extra N-terminal arginine residues to RRASpVA increased PP2A catalysed dephosphorylation 4- to 5-fold, without altering dephosphorylation by PP2C. These results represent the first study of the specificity of PP2C using synthetic peptides, and strengthen the view that this approach may lead to the development of more effective and specific substrates for the serine/threonine-specific protein phosphatases.

Alanine↗
PubMed1990

Ribofuranosyl-benzimidazole derivatives as inhibitors of casein kinase-2 and casein kinase-1.

5,6-Dichloro-1-(beta-D-ribofuranosyl)benzimidazole (DiCl-RB) is a powerful inhibitor of casein kinase-2 (CK-2) [Zandomeni, R. et al. (1986) J. Biol. Chem. 261, 3414-3420]. Here a series of 17 analogues of DiCl-RB has been employed for studying the specificity and the mode of action of this family of CK-2 inhibitors. The two halogen substituents on the benzene ring are shown to play a prominent role in inhibition, the 5,6-dibromo derivative (DiBr-RB) being fivefold more effective than DiCl-RB (Ki = 2 microM, with GTP as substrate), whereas the difluoro derivative (DiF-RB) is nearly as ineffective as unsubstituted 1-(beta-D-ribofuranosyl)benzimidazole. On the other hand, although some modifications of the ribose group significantly decrease the inhibitory efficiency, the sugar moiety is not strictly required, since dichlorobenzimidazole itself (DiCl-Bz) is an inhibitor almost as good as DiCl-RB. Inhibition of CK-2 by DiCl-RB and by its analogues, DiCl-Bz included, is of the competitive type with respect to the nucleotide substrate, the Ki values being lower with GTP than with ATP. The Ki values of the most potent inhibitor, DiBr-RB, with ATP and GTP, are 6 microM and 2 microM, respectively, denoting an affinity for the enzyme higher than that of the physiological substrates, ATP and GTP. DiBr-RB has been assayed for its inhibitory capacity toward several protein kinase other than CK-2. Protein kinase-C, cAMP-dependent protein kinase, the Ser/Thr protein kinase expressed by Pseudorabies virus, and four different tyrosine protein kinases from spleen, proved insensitive to DiBr-RB concentrations capable of almost entirely suppressing the activity of rat liver and maize seedling CK-2. Casein kinase-1 however is nearly as sensitive as CK-2 to DiBr-RB. Inhibition of CK-1 is also of the competitive type with respect to ATP (Ki = 14 microM). Although the inhibitory spectrum of CK-1 by the various analogues is reminiscent of that observed with CK-2, a remarkable difference is revealed by 5'-phosphorylation of ribose which increases the Ki with CK-2 while decreasing that with CK-1.

Animals↗
PubMed1989

Synthetic fragments of beta-casein as model substrates for liver and mammary gland casein kinases.

The octapeptide Glu-Ser-Leu-Ser-Ser-Ser-Glu-Glu, corresponding to the 14-21 sequence of bovine beta-casein A2 and 11 shorter and/or modified derivatives were synthesized and used as model substrates for three casein kinases: rat liver casein kinases 2 and 1 and a casein kinase isolated from the golgi-enriched fraction of lactating mammary gland (GEF-casein kinase). Casein kinase-2 readily phosphorylates the octapeptide at its Ser-4 residue with a Vmax value comparable to those obtained with protein substrates and Km values of 85 microM and 11 microM in the absence and presence of polylysine, respectively. These are the most favourable kinetic parameters reported so far with peptide substrates of casein kinase-2. Stepwise shortening of the octapeptide from its N terminus promotes both a gradual decrease of Vmax and an increase of Km, this being especially dramatic in passing from the hexapeptide Leu-Ser-Ser-Ser-Glu-Glu (Km 210 microM) to the pentapeptide Ser-Ser-Ser-Glu-Glu (Km 2630 microM). The tetrapeptide Ser-Ser-Glu-Glu is the shortest derivative still phosphorylated by casein kinase-2, albeit very slowly, and the tripeptides Ser-Glu-Glu and Glu-Leu-Ser were not substrates at all. Furthermore, the pentapeptide Ser-Ser-Ser-Glu-Glu was found to be a better substrate than Ser-Ser-Ala-Glu-Glu, Ser-Ala-Ser-Glu-Glu and Ser-Ala-Ala-Glu-Glu by virtue of its lower Km value. These data, while confirming that the motif Ser-Xaa-Xaa-Glu is specifically recognized by casein kinase-2, strongly suggest that additional local structural features can improve the phosphorylation efficiency of serine-containing peptides which are devoid of the large acidic clusters recurrent in many phosphorylation sites of casein kinase 2. In particular, predictive structural analysis as well as NMR and C18 reverse-phase HPLC elution profile data support the hypothesis that a beta-turn conformation is responsible for the remarkable suitability of the octapeptide Glu-Ser-Leu-Ser-Ser-Ser-Glu-Glu and some of its shorter derivatives to phosphorylation mediated by casein kinase-2. While neither the peptide Glu-Ser-Leu-Ser-Ser-Ser-Glu-Glu nor any of its derivatives were affected by casein kinase-1, a rapid phosphorylation of the octapeptide by GEF-casein kinase at Ser-5 (not Ser-4) was obtained.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1989

A synthetic peptide substrate specific for casein kinase I.

The synthetic peptide, Asp-Asp-Asp-Glu-Glu-Ser-Ile-Thr-Arg-Arg, derived from the phosphorylation site of casein kinase-1 (CK-1) in beta-casein A(2), is readily phosphorylated by CK-1, but not by casein kinase-2(CK-2), cyclic AMP-dependent protein kinase, protein kinase C, phosphorylase kinase and protein kinase FA. Phosphorylation by CK-1 occurs only at Ser-6, Thr-8 being unaffected. The Km for the peptide is higher (1 mM) than for beta-casein A(2) (40 microM), while the Vmax is quite comparable. This is the first synthetic peptide substrate for CK-1 described so far, and can be used for the rapid and specific estimation of CK-1 activity in crude extracts.

Amino Acid Sequence↗
PubMed1989

Synthetic peptides reproducing the EGF-receptor segment homologous to the pp60v-src phosphoacceptor site. Phosphorylation by tyrosine protein kinases.

The octapeptide E-E-K-E-Y-H-A-E, corresponding to the amino acid sequence 841-845 of EGF receptor, whose tyrosine-845 is homologous to the main phosphorylation site of pp60v-src, has been synthesized together with seven shorter peptides encompassing variable segments around the tyrosine residue. The peptides have been employed as model substrates for inspecting the local structural determinants of three tyrosine protein kinases (TPKs), namely; TPK-IIB and TPK-III, isolated from lymphoid cells (Eur. J. Biochem. 172, 451-457 (1988] and the TPK encoded by the oncogene of Abelson murine leukemia virus. The phosphorylation order with the different peptide substrates is variable depending on the TPK used: in particular, the lysine residue at position -2 relative to tyrosine proved especially harmful with TPK-IIB, the peptides K-E-Y-H and K-E-Y-H-A-E being very poor substrates compared with their shorter derivatives devoid of the N-terminal lysine (E-Y-H and E-Y-H-A-E, respectively). Conversely, such a basic residue is well tolerated by the other two TPKs. The negative effect of the N-terminal lysine on TPK-IIB-catalyzed phosphorylation is accounted for by an increase of Km and can be overcome by the presence of additional glutamic acid(s) on that side. On the other hand, the C-terminal acidic doublet Ala-Glu specifically impairs the phosphorylation efficiency of abl-TPK, by lowering the Vmax value, the heptapeptide E-K-E-Y-H-A-E being much less readily phosphorylated than E-K-E-Y-H. Collectively, these results would indicate that the site specificity of tyrosine protein kinases results from the balance of positive and negative determinants whose influence on the catalytic activity of the individual enzymes can differ greatly.

Amino Acid Sequence↗
PubMed1989

Inhibition of tyrosine protein kinases by the antineoplastic agent adriamycin.

Adriamycin, a lipid-interacting anti-cancer agent, was found to inhibit the phosphorylation of polyGlu/Tyr (4:1) by tyrosine protein kinases either from spleen or expressed by the oncogene of Abelson murine leukemia virus. The dose dependent inhibition by adriamycin is accounted for by competition for the ATP binding site, but it is also deeply influenced by the nature and concentration of the phosphorylatable substrate, suggesting multiple interactions with the enzyme. The phosphorylation at tyrosine residues of cytosolic proteins from cells transformed by Abelson leukemia virus and the autophosphorylation of tyrosine protein kinases are also inhibited by adriamycin. Unlike tyrosine protein kinases most serine/threonine specific protein kinases, with the notable exception of protein kinase-C, appear to be relatively insensitive to adriamycin.

Abelson murine leukemia virus↗
PubMed1989

Specificity determinants of maize casein kinase-IIB are related to but distinct from those of rat liver casein kinase-2.

The site specificity of maize seedling casein kinase-IIB, a type-2 casein kinase exhibiting an unusually low Mr, has been studied with the aid of model acidic peptide substrates for rat liver casein kinase-2. Like the animal enzyme, casein kinase-IIB also readily phosphorylates peptides SEEEEE, SEAEEE and SEEEAE, but not SEEAEE. Maize seedling casein kinase-IIB, however, is almost inactive toward peptides SAEEEE, SAEEEEE and SAAEEEEE which are good substrates for liver casein kinase-2. This indicates that casein kinase-IIB requires acidic residues not only at position +3, similar to rat liver casein kinase-2, but also at position +1, where the animal enzyme tolerates a neutral residue. This and other differences outlined in this report support the view that protein kinases of the same type from different sources may have significant differences in their substrate specificity.

Amino Acid Sequence↗
PubMed1989

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.

Casein Kinases↗
PubMed1989

Metabolic regulation through second-site phosphorylation.

Protein phosphorylation is the most prevalent covalent reversible modification used for the regulation of many biological functions. The dynamic phosphorylation state of cellular proteins results from the balance between the activities of protein kinases and phosphatases. Even autophosphorylation, shared by practically all protein kinases, is also considered as a mode of enzyme regulation. Phosphodephosphorylation reactions seem also to be regulated at the substrate level, and the structure of a target protein can be strongly altered through phosphorylation in a way which may either promote or depress the further action of a protein kinase or phosphatase. Second-site phosphorylation may have various consequences on the kinetic properties of the enzyme-substrate. Glycogen synthase is possibly one of the most complex examples of enzyme regulation through interactions between phosphorylation sites. An interesting example of regulation at the substrate level in a network protein phosphorylations, is provided by the effects of casein kinase-1 (CK-1), casein kinase-2 (CK-2) and protein kinase FA (PKFA) on the modulator protein of the ATP, Mg-dependent phosphatase. The crucial step in the activation of the enzyme is the transient phosphorylation of the modulator at the Thr-72 residue catalyzed by PKFA. The CK-2 mediated phosphorylation occurs at three Ser residues: Ser-86, Ser-120 and Ser-121. Although by itself CK-2 does not elicit any phosphatase activity, it potentiates the subsequent activation brought about by PKFA. The phosphorylation mediated by CK-1, probably directed toward Ser-86, blocks the activation of the ATP,Mg-dependent phosphatase mediated by PKFA.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1988

Substrate-specificity determinants for a membrane-bound casein kinase of lactating mammary gland. A study with synthetic peptides.

A tissue-specific casein kinase, purified from the Golgi-enriched-membrane fraction of guinea-pig lactating mammary gland (GEF-CK), readily phosphorylates the synthetic peptide Ser-Glu5, a good substrate of casein kinase-2, and several derivatives varying for the number and position of acidic residues on the C-terminal side of serine, except those lacking an acidic side chain at position +2. The least acidic peptide, still significantly affected by GEF-CK, is Ser-Ala-Glu-Ala3 which is not a substrate for CK-2. Conversely, the peptides Ser-Ala2-Glu-Ala2, Ser-Ala2-Glu3, Ser-Ala2-Glu5 and Ser-Glu-Ala-Glu3, all of which are more or less readily phosphorylated by CK-2, are not appreciably affected by GEF-CK. On the other hand the presence of additional glutamyl residues, besides the one in the second position, improves the affinity of the peptide substrate for GEF-CK, as indicated by the Km values of Ser-Glu5, Ser-Glu2-Ala3 and Ser-Ala-Glu-Ala3 which are 80, 950 and 3950 microM respectively. It is concluded that although both CK-2 and GEF-CK require, for optimal activity, rather extended acidic clusters on the C-terminal side of the target serine, the most critical residue in the case of GEF-CK is not the one at position +3, which is required for CK-2 catalyzed phosphorylation [Marin, O. et al. (1986) Eur. J. Biochem. 160, 239-244], but the one lying at position +2. Additional differences, concerning the site specificities of these enzymes, have been outlined using the threonyl derivative of Ser-Glu5 and the peptide Arg-Ser-Glu3-Val-Glu. The former is still phosphorylated by CK-2 but not to any appreciable extent by GEF-CK, which apparently is strictly specific for seryl residues. On the contrary, the presence of an N-terminal basic residue, which greatly reduces phosphorylation by CK-2, is tolerated rather well by GEF-CK. On the other hand a C-terminal basic residue, interrupting the acidic cluster, compromises phosphorylation by GEF-CK, as indicated by the extremely high Km value of Ser-Glu3-Lys-Glu vs Ser-Glu3-Val-Glu (13,000 and 170 microM, respectively).

Amino Acid Sequence↗
PubMed1988

Synthetic peptide substrates for casein kinase 2. Assessment of minimum structural requirements for phosphorylation.

Unlike the peptides SAEAAA and SEEAAA which are not substrates for casein kinase 2 (CK-2) their analogs SAAEAE and SAAEAA are still significantly phosphorylated. Their Km values, however, (13.3 and 18.9 mM, respectively) are almost two orders of magnitude higher than that of SEEEEE and their Vmax values are 3- and 14-fold lower than that of SAAEEE. The peptide ESEEEEE, but not ASEEEEE, is a slightly better substrate than SEEEEE, while both RSEEEEE and SEEEKE are very poor substrates compared to ASEEEEE and SEEEAE, respectively. SAAEAE is much more responsive to polylysine stimulation and polyphosphate inhibition than is SEEEEE. Taken together these data show that a single acidic residue at the third position from the C-terminal side of the phosphorylatable amino acid represents not only a necessary, but also a sufficient condition for site recognition by CK-2. Optimal phosphorylation efficiency, however, requires an extended C-terminal cluster of several acidic residues, and can be compromised by the presence of only a basic residue either inside the acidic cluster or adjacent to the N-terminal side of the phosphoacceptor amino acid. The structure of the phosphoacceptor site can greatly influence the efficacy of substrate-directed effectors of CK-2.

Amino Acid Sequence↗
PubMed1988

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.

Amino Acids↗
PubMed1988

Partially dephosphorylated phosphopeptide AcSer(P)-Ser(P)-Ser(P) is an excellent substrate for casein kinase-2.

The synthetic phosphopeptide AcSer(P)-Ser(P)-Ser(P), reproducing a recurrent feature of casein and other phosphoproteins, once partially dephosphorylated by acid phosphatase, serves as an efficient substrate for casein kinase-2. Previous dephosphorylation beyond 30% hinders subsequent phosphorylation and the entirely dephosphorylated peptide is not a substrate at all. The kinetic constants of the partially dephosphorylated phosphopeptide are much more favourable than those of the synthetic peptides SEEEAA, SSEE and SEE, the latter one being totally inert. Optimal phosphorylation occurs at pH values that ensure complete ionization of the phosphoseryl side chains. These data provide incontrovertible demonstration that phosphoserine can replace carboxylic amino acids as specificity determinant for CK-2, being more effective than glutamic acid itself.

Amino Acid Sequence↗
PubMed1988

Characterization of four tyrosine protein kinases from the particulate fraction of rat spleen.

Four distinct tyrosine protein kinases active on poly(Glu4,Tyr1) and angiotensin II, and operationally termed TPK-I, TPK-IIA, TPK-IIB and TPK-III have been resolved and partially purified from rat spleen particulate fraction by combining DEAE-Sepharose, heparin-Sepharose, phosphocellulose and polylysine-agarose chromatographies. Once partially purified all of them are free of Ser/Thr-specific protein kinase activity as judged using casein, histones, protamine and the peptide Arg-Arg-Ala-Ser-Val-Ala as substrates. TPK-I (apparent molecular mass 64 kDa, by gel filtration) and TPK-IIA (54 kDa) share several properties, including substrate specificity and stimulation by heparin; the latter however is much more responsive to polylysine then the former (10- and 3-fold maximum stimulation, respectively). Conversely TPK-IIB (51 kDa) is markedly inhibited by heparin and it is also characterized by its unique substrate specificity: unlike the other three tyrosine protein kinases it by far prefers the tetrapeptide Glu-Tyr-Ala-Ala over the decapeptide Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Arg-Gly and readily phosphorylates band-3 protein of red cell membrane. The unusual preference for Mg2+ over Mn2+ as activator and the capability to phosphorylate calmodulin distinguish TPK-III (61 kDa) from the other isoenzymes. Moreover TPK-III is insensitive to heparin and polylysine and is inhibited by quercetin much more efficiently than the other enzymes (I50 = 10 microM). Upon incubation with [gamma-32P]ATP, TPK-I, TPK-IIA and TPK-III give rise to alkali-stable radiolabeled components of 61, 55 and 52 kDa respectively, as evaluated by PAGE/SDS. In every case such a radiolabeling takes place also in the presence of a large excess of phosphorylatable substrate (angiotensin II) while it is readily reversed by isotopic dilution with 10-fold excess unlabeled ATP, supporting the view that it represents an autophosphorylation process. No (auto)phosphorylation product(s) could be detected in TPK-IIB even if its amount, in terms of catalytic activity, was 10-fold higher than that of the others.

Amino Acids↗
PubMed1988

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.

Animals↗