N-terminal sequence analysis of bovine dentin phosphophoryn after conversion of phosphoseryl to S-propylcysteinyl residues.

Bovine dentin phosphophoryn (BDP), a protein rich in aspartyl (Asp) and O-phosphoseryl (Ser[P]) residues, is synthesized by odontoblasts and is believed to be involved in matrix-mediated biomineralization of dentin. We have purified BDP, using selective precipitation and ion exchange chromatography, from an EDTA soluble dentin extract and converted the Ser(P) residues to S-propylcysteinyl residues that are stable to Edman degradation, facilitating the determination of the amino acid sequence of the N-terminal 38 residues. After the initial Asp-Ser(P)-Pro-Asn-Ser(P)-Ser(P)-Asp-Glu-Ser(P)-Asn-Gly-, the sequence contained the repeated motifs Asp-Ser(P) and Asp-Ser(P)-Ser(P). Purified BDP migrated as a single band on gradient SDS-PAGE with an apparent molecular weight of 156 kDa. This value was consistent with the molecular weight of the dephosphorylated protein of 105 kDa determined by means of MALDI mass spectrometry.

Epitope analysis of the multiphosphorylated peptide alpha s1-casein (59-79).

The multiphosphorylated tryptic peptide alpha(s1)-casein(59-79) has been shown to be antigenic with anti-casein antibodies. In an approach to determine the amino acyl residues critical for antibody binding we undertook an epitope analysis of the peptide using overlapping synthetic peptides. With alpha(s1)-casein(59-79) as the adsorbed antigen in a competitive ELISA only two of five overlapping synthetic peptides at 1 mM significantly inhibited binding of the anti-casein antibodies. Peptides Glu-Ser(P)-Ile-Ser(P)-Ser(P)-Ser(P)-Glu-Glu and Ile-Val-Pro-Asn-Ser(P)-Val-Glu-Glu inhibited antibody binding by 20.0+/-3.6% and 60.3+/-7.9%, respectively. The epitope of Glu63-Ser(P)-Ile-Ser(P)-Ser(P)-Ser(P)-Glu-Glu70 was further localised to the phosphoseryl cluster as the peptide Ser(P)-Ser(P)-Ser(P) significantly inhibited binding of the anti-casein antibodies to alpha(s1)-casein(59-79) by 29.5+/-7.4%. Substitution of Ser(P)75 with Ser75 in the second inhibitory peptide Ile-Val-Pro-Asn-Ser(P)75-Val-Glu-Glu also abolished inhibition of antibody binding to x(s1)-casein (59-79) demonstrating that Ser(P)75 is also a critical residue for recognition by the antibodies. These data show that the phosphorylated residues in the cluster sequence -Ser(P)66-Ser(P)-Ser(P)68 and in the sequence -Pro73-Asn-Ser(P)-Val-Glu77- are critical for antibody binding to x(s1)-casein(59-79) and further demonstrate that a highly phosphorylated segment of a protein can be antigenic.

Common and differential recognition of structural features in synthetic peptides by the catalytic domain and the Src-homology 2 (SH2) domain of pp60c-src.

The relative efficiencies of the catalytic domain of the src-family kinase pp60c-src in phosphorylating four peptide substrates including (i) src-optimal peptide (AEEEIYGEFEAKKKK), (ii) "-YEEI-peptide" (KKTHQEEEEPQYEEIPIYL), (iii) cdc2(6-20) (KVEKIGEGTYGVVYK), (iv) src-autophosphorylation site peptide (ADFGLARLIEDNEYTARG) and the relative efficiencies of its SH2 domain in binding the phosphorylated forms of these peptide substrates were compared. The results show that the src-optimal peptide, "-YEEI-peptide," cdc2(6-20) peptide were phosphorylated by the catalytic domain with high efficiency and that the phosphorylated form of all three peptides could bind the SH2 domain of the kinase, confirming the hypothesis proposed by Songyang and co-workers that the catalytic domain of pp60c-src phosphorylates sites which are recognized by its own SH2 domain (Songyang et al. (1995) Nature 373, 536-539). The four peptides were phosphorylated by the kinase with relative efficiencies in the order of Src-optimal peptide > "-YEEI-peptide" > cdc2(6-20) >> src-autophosphorylation site peptide. However, the Tyr(P)-Src-optimal peptide and [pY]15cdc2(6-20) bound to the SH2 domain of the kinase with an affinity at least an order of magnitude lower than that of the tight-binding peptide, "-pYEEI-peptide." Thus, our study suggests that the catalytic and SH2 domains of pp60c-src recognize overlapping but not identical determinants in the local structure around the tyrosine phosphorylation site of the substrate peptides.

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.

Solid phase synthesis of pp60src-related phosphopeptides via 'global' phosphorylation and their use as substrates for enzymatic phosphorylation by casein kinase-2.

The seven phosphopeptide derivatives based on the native -NEYTA- sequence of the pp60src protein kinase family, Asn-Glu-Tyr(P)-Ser-Ala, Ala-Glu-Tyr(P)-Ser-Ala, Ala-Ser-Tyr(P)-Ser-Ala, Ala-Ser(P)-Tyr-Ser-Ala, Ala-Thr-Tyr(P)-Ser-Ala, Ala-Thr(P)-Tyr-Ser-Ala and Ala-Ser(P)-Tyr(P)-Ser-Ala, were prepared in good yield using the "global' "phosphite-triester' phosphorylation method. The peptide resins were assembled using the Fmoc mode of solid phase peptide synthesis (PyBOP coupling method) with specific Ser-, Thr-, or Tyr-residues incorporated as their side chain free Fmoc-derivatives. The final "global' phosphorylation of the peptide resins was accomplished using di-tert-butyl N, N-diethylphosphoramidite followed by m-chloroperoxybenzoic acid oxidation of the resultant di-t-butyl phosphite triester intermediate. Subsequent resin cleavage and deprotection of the phosphorylated peptide resins was effected by treatment with 5% anisole: TFA and gave the seven phosphopeptides in high yield and purity. The use of the seven synthetic phosphopeptides in enzymatic (casein kinase-2) phosphorylation studies showed that, (A) the change of the target Thr site to Ser resulted in markedly improved phosphorylation of the peptide substrates, (B) that the Tyr(P) residue in the - 1 position was significantly more important than the Ser(P)/Thr(P) residue in the - 2 position for efficient seryl phosphorylation, and (C) that an acidic residue in the - 2 position relative to the target site facilitated phosphorylation of the downstream seryl residue irrespective of the nature of the acidic residue in the -Xxx-Tyr(P)-Ser- and -Xxx-Tyr-Ser- sequences {Xxx = Ser(P), Thr(P), Glu}. In addition to the Tyr(P) residue directing phosphorylation to the +1 position, the good phosphorylation of both ASY(P)SA and ATY(P)SA by casein kinase-2 indicated that the Tyr(P) residue was also able to direct phosphorylation to a Ser/Thr in the - 1 position.

Anticariogenicity of calcium phosphate complexes of tryptic casein phosphopeptides in the rat.

Casein phosphopeptides (CPP) stabilize calcium phosphate through the formation of casein-phosphopeptide amorphous calcium-phosphate complexes (CPP-CP). The ability of CPP-CP to reduce caries activity was investigated by use of specific-pathogen-free rats inoculated with Streptococcus sobrinus. The animals consumed a defined cariogenic diet free of dairy products. Solutions (100 microL) of the CPP-CP (0.1, 0.2, 0.5, 1.0% w/v) were applied to the animals' molar teeth twice daily. Other groups of animals received solutions containing 500 ppm F, the non-phosphorylated peptides of a casein tryptic digest (0.5% w/v), or the calcium-phosphate complex of a synthetic octapeptide, Ac-Glu-Ser(P)-Ile-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-NHMe, corresponding to the common sequence in the CPP. The CPP-CP significantly reduced caries activity in a dose-response fashion, with 1.0% CPP-CP producing 55% and 46% reductions in smooth surface and fissure caries activity, respectively, being similar to that of 500 ppm F. The anticariogenic effects of CPP-CP and fluoride were additive, since animals receiving 0.5% CPP-CP plus 500 ppm F had significantly lower caries activity than those animals receiving either CPP-CP or fluoride alone. The tryptic digest of casein with the phosphopeptides selectively removed showed no anticariogenic activity. The synthetic octapeptide-calcium phosphate complex significantly reduced caries activity, confirming that this calcium-phosphate-stabilizing portion of the casein phospho-peptides is associated with anticariogenicity. The CPP-CP did not significantly affect the level of S. sobrinus in fissure plaque.(ABSTRACT TRUNCATED AT 250 WORDS)

Further studies into the Boc/solid-phase synthesis of Ser(P)- and Thr(P)-containing peptides.

The Ser(P)-containing peptide corresponding to phospholamban 11-19, Ac-Ala-Ile-Arg-Arg-Ala-Ser(P)-Thr-Ile-Glu-NH2, was prepared by the use of Boc-Ser(PO3Ph2)-OH in Boc/solid-phase peptide synthesis followed by HF cleavage of the peptide from the polystyrene resin and subsequent platinum-mediated hydrogenolytic cleavage of the phenyl phosphate groups. A study of the HF deprotection step showed that extensive dephosphorylation of the Ser(PO3Ph2)-residue occurred using three commonly used HF conditions and gave rise to large quantities of the Ser-containing peptide. The subsequent study of model peptide systems under standard HF conditions established firstly that the extent of dephosphorylation was dependent on the HF-contact time, and secondly that the Ser(PO3Ph2) residue underwent dephosphorylation at a slightly higher rate than the Thr(PO3Ph2) residue.

Efficient Fmoc/solid-phase synthesis of Abu(P)-containing peptides using Fmoc-Abu(PO3Me2)-OH.

The synthesis of the two 4-phosphono-2-aminobutanoyl-containing peptides, Leu-Arg-Arg-Val-Abu(P)-Leu-Gly-OH.CF3CO2H and Ile-Val-Pro-Asn-Abu(P)-Val-Glu-Glu-OH.CF3CO2H was accomplished by the use of Fmoc-Abu(PO3Me2)-OH in Fmoc/solid-phase peptide synthesis. The protected phosphoamino acid, Fmoc-Abu(PO3Me2)-OH, was prepared from Boc-Asp-OtBu in seven steps, the formation of the C-P linkage being effected by the treatment of Boc-Asa-OtBu with dimethyl trimethylsilyl phosphite. Peptide synthesis was performed using Wang Resin as the polymer support with both peptides assembled by the use of PyBOP for the coupling of Fmoc amino acids and 20% piperidine for cleavage of the Fmoc group from the Fmoc-peptide after each coupling cycle. Cleavage of the peptide from the resin and peptide deprotection was accomplished by the treatment of the peptide-resin with 5% thioanisole/TFA followed by cleavage of the methyl phosphonate group by 1 M bromotrimethylsilane/1 M thioanisole in TFA.

Hydroxyapatite mineralization and demineralization in the presence of synthetic phosphorylated pentapeptides.

A constant composition (CC) method was used to compare the influence of statherin-like N-terminal 5-residue fragments having different amino acids in the terminal position on hydroxyapatite (HAP) growth and dissolution. The CC experiments were done in solutions containing 4.00 x 10(-4) mol/l calcium and 2.40 x 10(-4) mol/l phosphate. The solutions used in the crystallization studies were supersaturated only with respect to HAP (pH = 7.40, sigma HAP = 3.60). The CC dissolution studies were done in solutions undersaturated with respect to HAP (pH = 6.00; sigma HAP = -0.39). The HAP mineralization and demineralization processes were markedly inhibited by the negatively charged pentapeptides. Those containing a phosphorylated terminal residue inhibited dissolution to a greater extent than the native statherin fragment having aspartate as the N-terminal residue. Strong dependencies of the degree of inhibition of growth and dissolution reaction rates on the extents of reaction were noted. As the reactions proceeded, the rate inhibition decreased in the case of crystal growth and increased for dissolution.

Efficient Fmoc/solid-phase peptide synthesis of O-phosphotyrosyl-containing peptides and their use as phosphatase substrates.

A general synthetic method for the efficient preparation of Tyr(P)-containing peptides is described by the use of Fmoc-Tyr(PO3tBu2)-OH in Fmoc/solid-phase synthesis followed by simultaneous cleavage of the peptide from the resin and peptide deprotection by acidolytic treatment. The applicability of this approach is demonstrated by the synthesis of H-Ser-Ser-Ser-Tyr(P)-Tyr(P)-OH.TFA and the synthesis of the phosphorylated forms of the two physiological peptides, angiotensin II and neurotensin 8-13. In addition, the three phosphorylated peptides were used as substrates in the study of the local specificity determinants of T-cell protein tyrosine phosphatase. In a competition assay using 32P-radiolabeled [Tyr(P)]4-angiotensin II, both unlabeled synthetic [Tyr(P)]4-angiotensin II and Ser-Ser-Ser-Tyr(P)-Tyr(P) reduced the release of 32P and indicated that they efficiently competed as substrates for the phosphatase. Conversely, [Tyr(P)]4-neurotensin 8-13 was ineffective as a competitive substrate and indicated that this particular Tyr(P)-containing peptide sequence was not recognized by the enzyme. The marked difference in the recognition of Asp-Arg-Val-Tyr(P)-Ile-His-Pro-Phe and Arg-Arg-Pro-Tyr(P)-Ile-Leu is consistent with the presence of an acidic residue in the -3 position relative to the Tyr(P) residue.

The synthesis and use of pp60src-related peptides and phosphopeptides as substrates for enzymatic phosphorylation studies.

A series of peptides and phosphopeptides corresponding to the auto-phosphorylation site of pp60src, -Asn-Glu-Tyr416-Thr-Ala-, were prepared by either Boc/solution or Fmoc/solid phase peptide synthesis and used as substrates to study their enzymatic phosphorylation by various casein kinases. The Tyr(P)-containing peptide, Asn-Glu-Tyr(P)-Thr-Ala, was prepared by the use of Fmoc-Tyr(PO3Bzl2)-OH in Fmoc/solid phase peptide synthesis followed by acidolytic treatment of the peptide-resin with 5% anisole/CF3CO2H. Both Asn-Glu-Tyr-Thr-Ala and Asn-Glu-Ser(P)-Thr-Ala were prepared by the Boc/solution phase peptide synthesis and employed hydrogenolytic deprotection of the protected peptides. Enzymatic phosphorylation studies established that (A) the Tyr residue acted as an unusual positive determinant for directing phosphorylation to the Thr-residue, (B) the rate of Thr-phosphorylation was markedly facilitated by a change from the Tyr-residue to the Tyr(P)-residue, and (C) a Ser(P)-residue was as effective as the Tyr(P)-residue in facilitating Thr-phosphorylation. A subsequent structure-function study using Asn-Glu-Phe-Thr-Ala, Asn-Glu-Tyr(Me)-Thr-Ala (prepared by Fmoc/solid phase peptide synthesis) and Asn-Glu-Cha-Thr-Ala (prepared by hydrogenation of Asn-Glu-Tyr-Thr-Ala) established that the rate of Thr-phosphorylation was influenced by the extent of hydrophobic-hydrophobic interactions by the aralkyl side-chain group (either aromatic or aliphatic) of the 416-residue with casein kinase-2; the rate of Thr-phosphorylation being decreased by the introduction of methyl or hydroxyl groups at the 4-position of the aromatic group (i.e. Tyr(Me) and Tyr respectively) but enhanced by the introduction of the hydrophilic phosphate group (i.e. as Tyr(P)).

Strategies for nonradioactive methods in the localization of phosphorylated amino acids in proteins.

Identification of O-phosphorylated amino acids within the primary structure of regulatory proteins is important in understanding the mechanisms by which their functions are regulated. In many cases radioactive labeling with [32P]phosphate is tedious or sometimes impossible. Therefore, we have established a series of new non-radioactive methods that permit the localization of phosphoserine, phosphothreonine, and phosphotyrosine. After partial hydrolysis of a phosphopeptide or phosphoprotein, phosphoserine, phosphothreonine, or phosphotyrosine are determined by capillary electrophoresis as their dabsyl-derivatives. Chemical modification transforms phosphoserine or phosphothreonine to S-ethyl-cysteine or beta-methyl-S-ethyl-cysteine, respectively, allowing their localization during sequence analysis. We apply solid-phase sequencing to overcome the limitations of the gas-phase sequenator in the case of phosphotyrosine-containing peptides. Liquid chromatography on-line connected to an electrospray mass spectrometer is a powerful new method of increasing importance in the protein chemistry field. It is especially well suited for identification of phosphoserine- or phosphothreonine-containing peptides in a proteolytic digest of a phosphoprotein. In this article we will describe how to work with these new methods practically.

Efficient solution phase synthesis and use of multiple O-phosphothreonyl-containing peptides for calcium phosphate binding studies.

The protected phosphothreonine derivative Boc-Thr(PO3Ph2)-OH was prepared in high yield from Boc-Thr-OH by a simple three-step procedure which involved 4-nitrobenzylcarboxyl protection, either phosphorotriester (diphenyl phosphorochloridate) or "phosphite-triester" (diphenyl N,N-diethylphosphoramidite) phosphorylation of the threonine hydroxyl group of Boc-Thr-ONb followed by hydrogenolytic carboxyl deprotection. The three Thr(P)-containing peptides, H-Thr(P)-Glu-Glu-NHMe.TFA, H-Thr(P)-Thr(P)-Glu-Glu-NHMe.TFA and H-Thr(P)-Thr(P)-Thr(P)-Glu-Glu-NHMe.TFA, were prepared in high yield by the use of Boc-Thr(PO3Ph2)-OH in the Boc mode of peptide synthesis (mixed anhydride method) followed by platinum-mediated hydrogenolytic deprotection of the Thr(PO3Ph2)-containing peptides. The use of the phosphopeptides in calcium phosphate binding studies showed that the triple Thr(P)-cluster was a basic structural requirement, since only the pentapeptide was able to bind calcium phosphate efficiently.

Specificity of T-cell protein tyrosine phosphatase toward phosphorylated synthetic peptides.

The local specificity determinants for a T-cell protein tyrosine phosphatase (TC-PTP) have been inspected with the aid of a series of synthetic peptides, either enzymically or chemically phosphorylated, derived from the phosphoacceptor sites of phosphotyrosyl proteins. The truncated form of T-cell PTP, deprived of its C-terminal down-regulatory domain, readily dephosphorylates submicromolar concentrations of eptapeptides to eicosapeptides, reproducing the C-terminal down-regulatory site of pp60c-src (Tyr527), the phosphorylated loop IV of calmodulin and the main autophosphorylation site of two protein tyrosine kinases of the src family (Tyr416 of pp60c-src and Tyr412 of p51fgr). However, phosphopeptides of similar size, derived from phosphoacceptor tyrosyl sites of the abl and epidermal-growth-factor receptor protein tyrosine kinases, the phosphorylated loop III of calmodulin, and phosphoangiotensin II undergo either very slow or undetectable dephosphorylation, even if tested up to 1 microM concentration. The replacement of either Ser-P or O-methylated phosphotyrosine for phosphotyrosine within suitable peptide substrates gives rise to totally inert derivatives. Moreover, amino acid substitutions around phosphotyrosine in the peptides src(412-418), src(414-418) and abl-(390-397) deeply influence the dephosphorylation efficiency. From these data and from a comparative analysis of efficient versus poor phosphopeptide substrates, it can be concluded that acidic residues located on the N-terminal side of phosphotyrosine, with special reference to position -3, play a crucial role in substrate recognition, while basic residues in the same positions act as negative determinants. In any event, the presence of at least two aminoacyl residues upstream of phosphotyrosine represents a necessary, albeit not sufficient, condition for detectable dephosphorylation to occur. By replacing the truncated form of TC-PTP with the full length TC-PTP, the dephosphorylation efficiencies of all peptides tested are dramatically impaired. Such an effect is invariably accounted for by a substantial increase in Km values, accompanied by a more or less pronounced decrease in Vmax values. These data support the concept that the C-terminal regulatory domain of TC-PTP exerts its function primarily by altering the affinity of the enzyme toward its phosphotyrosyl targets.

Efficient solution-phase synthesis of multiple O-phosphoseryl-containing peptides related to casein and statherin.

The multiple Ser(P)-containing peptides, H-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-NHMe.TFA, H-Asp-Ser(P)-Ser(P)-Glu-Glu-NHMe.TFA and H-Glu-Ser(P)-Ser(P)-Glu-Glu-NHMe.TFA were prepared by the use of Boc-Ser(PO3Ph2)-OH in the Boc mode of solution phase peptide synthesis followed by platinum-mediated hydrogenolytic de-protection of the Ser(PO3Ph2)-containing peptides. The protected peptides were assembled using the mixed anhydride coupling methods with 40% TFA/CH2Cl2 used for removal of the Boc group from intermediate Boc-protected peptides.

Efficient solid phase synthesis of mixed Thr(P)-, Ser(P)- and Tyr(P)-containing phosphopeptides by "global" "phosphite-triester" phosphorylation.

The synthesis of the mixed Thr(P)/Tyr(P)-containing peptide, Ala-Thr(P)-Tyr(P)-Ser-Ala, was accomplished by "phosphite-triester" phosphorylation of the resin-bound Thr/Tyr-containing peptide using di-t-butyl N,N-diethylphosphoramidite as the phosphitylation reagent. The pentapeptide-resin was assembled by Fmoc/solid-phase peptide synthesis with the use of PyBOP as coupling reagent and the hydroxy-amino acids incorporated as side-chain free Fmoc-Tyr-OH and Fmoc-Thr-OH. "Global" bis-phosphorylation of the peptide-resin was accomplished by treatment with di-t-butyl N,N-diethylphosphoramidite/1H-tetrazole followed by m-chloroperoxybenzoic acid oxidation of the intermediate di-t-butylphosphite triester. Simultaneous peptide-resin cleavage and peptide deprotection was effected by treatment of the peptide-resin with 5% anisole/TFA and gave the Thr(P)/Tyr(P)-containing phosphopeptide in high yield and purity. In addition, the tyrosyl residue was found to be phosphitylated in preference to the threonyl residue since the phosphitylation of the pentapeptide-resin using only 1.1 equiv. of di-t-butyl N,N-diethylphosphoramidite gave Ala-Thr-Tyr(P)-Ser-Ala as the major product and both Ala-Thr(P)-Tyr(P)-Ser-Ala and Ala-Thr-Tyr-Ser-Ala as minor products.

Role of phosphorylated aminoacyl residues in generating atypical consensus sequences which are recognized by casein kinase-2 but not by casein kinase-1.

Casein kinase-2 (CK-2) is a ubiquitous Ser/Thr specific protein kinase that recognizes phosphorylatable residues located upstream of acidic determinants, its consensus sequence being Ser(Thr)-Xaa-Xaa-Acidic. Here we show that the phosphotetrapeptide AcSer(P)-Ser(P)-Ser-Ser(P), which is devoid of the canonical consensus sequence, is nevertheless phosphorylated by CK-2 with rates comparable to that of typical peptide substrates Ser-Glu-Glu-Glu-Glu-Glu and Arg-Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu routinely employed for assaying CK-2 activity. The phosphopeptide AcSer(P)-Ser-Ser(P) [but not Ac-Ser-Ser(P)-Ser(P) or AcSer(P)-Ser(P)-Ser] is also phosphorylated albeit less efficiently than AcSer(P)-Ser(P)-Ser-Ser(P). Further N-terminal elongation with additional phosphoseryl residues to give the peptides AcSer(P)-Ser(P)-Ser(P)-Ser-Ser(P) and AcSer(P)-Ser(P)-Ser(P)-Ser(P)-Ser-Ser(P) does not improve but rather slightly decreases the phosphorylation efficiency by CK-2. These two peptides are conversely excellent substrates for CK-1, which does not appreciably phosphorylate either AcSer(P)-Ser-Ser(P) or AcSer-(P)-Ser(P)-Ser-Ser(P). Either individual or multiple replacement of the phosphorylated residues with glutamic acid in the peptide AcSer(P)-Ser(P)-Ser-Ser(P) drastically reduces the phosphorylation efficiency by CK-2, the phosphoseryl residue at position -2 playing an especially crucial role which cannot be surrogated by glutamyl residues.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of polylysine on casein-kinase-2 activity is influenced by both the structure of the protein/peptide substrates and the subunit composition of the enzyme.

The mechanism by which polybasic peptides stimulate the activity of casein kinase 2 (CK2) has been studied by comparing the effect of polylysine on the phosphorylation of a variety of protein and peptide substrates by the native CK2 holoenzyme and by its recombinant catalytic alpha subunit, either alone or in combination with the recombinant non-catalytic beta subunit. Calmodulin is not phosphorylated by the CK2 holoenzyme, in either the native or the reconstituted form, unless polylysine is added. In the presence of polylysine, it becomes a good substrate for CK2 (Km 14.2 microM, Kcat 4.6 mol.min-1.mol CK2-1). The recombinant alpha subunit, however, spontaneously phosphorylates calmodulin, this phosphorylation being actually inhibited rather than stimulated by polylysine. The calmodulin tridecapeptide, RKMKDTDSEEEIR, reproducing the phosphorylation site for CK2, is spontaneously phosphorylated by either CK2 holoenzyme or the recombinant alpha subunit with 5.8-fold and 2.8-fold stimulation by polylysine, respectively. The recombinant beta subunit of CK2 is itself a good exogenous substrate for the enzyme, its phosphorylation, however, is inhibited rather than enhanced by polylysine. On the contrary, the phosphorylation of the nonapeptide, MSSSEEVSW, reproducing the beta-subunit phosphoacceptor site, is dramatically stimulated by polylysine. Using a variety of small peptide substrates, it was shown that phosphorylation rate is diversely stimulated by polylysine. The observed stimulation, moreover, is variably accounted for by changes in Vmax and/or Km, depending on the structure of the peptide substrate. Maximum stimulation with all protein/peptide substrates tested requires the presence of the beta subunit, since the recombinant alpha subunit is much less responsive than CK2 holoenzyme, either native or reconstituted. While the phosphorylation of the peptide RRRDDDSDDD by CK2 is stimulated 2.8-fold, with 15 nM polylysine being required for half-maximal stimulation, a stimulation of only 1.9-fold, with 80 nM polylysine required for half-maximal stimulation, is attained with recombinant alpha subunit. The concentration of polylysine required for half-maximal stimulation is comparable to CK2 concentration and increases by increasing CK2 concentration, suggesting that polylysine primarily interacts with the enzyme, rather than with the peptide substrate.

The comparative efficiencies of the Ser(P)-, Thr(P)- and Tyr(P)-residues as specificity determinants for casein kinase-1.

The beta-casein derived phosphopeptide, Glu-Glu-Ser(P)-Glu-Glu-Ser-Ile-Thr-NHMe and two derivatives in which the Ser(P)-residue is replaced by the Thr(P)- and Tyr(P)-residue have been compared for their susceptibility to phosphorylation by casein kinase-1. While both the Ser(P)- and Thr(P)-peptides are good substrates with similar kinetic constants, the Tyr(P)-peptide is a substrate as poor as the unphosphorylated derivative EEEEESIT, exhibiting a 21-fold higher Km and 6-fold lower Vmax values. While prior dephosphorylation of the Ser(P)-peptide caused a marked loss in its phosphoacceptor capacity, prior dephosphorylation of the Tyr(P)-peptide caused no significant change in its poor phosphoacceptor capacity. Thus the order of efficiency of phosphoaminoacids as specificity determinants for casein kinase-1 was found to be Ser(P)=Thr(P) much greater than Tyr(P) and this order is markedly different from Tyr(P) greater than Ser(P) much greater than Thr(P) which was previously established for casein kinase-2 [Meggio et al. (1991) FEBS Lett. 279, 307-309].