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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.

20 synthetic peptides, each of which includes a tyrosyl residue flanked by either neutral or acidic amino acids in different proportions and at variable positions, have been employed as model substrates for investigation of the site specificity of three tyrosine protein kinases previously isolated from spleen [Brunati, A. M. & Pinna, L. A. (1988) Eur. J. Biochem. 172, 451-457] and conventionally termed TPK-I, TPK-IIB and TPK-III. Comparison of the phosphorylation efficiencies shows that each tyrosine protein kinase is considerably different from the others in both the stringency and the nature of its specificity determinants. By considering, in particular, the kinetic constants obtained with the pentapeptides AAYAA, EEYAA, AEYAA, EAYAA, with the tetrapeptides AYAA and EYAA and with the tripeptides AYA and EYA, it turns out that N-terminal acidic residue(s) are only essential with TPK-IIB for efficient phosphorylation with multiple residues displaying a synergistic effect. The very similar Km (130 microM) but 14-fold-different Vmax values with YEEEEE vs. EEEEEY indicate that an N-terminal rather than C-terminal location of acidic residues is required for a high phosphorylation rate with, though not for binding to TPK-IIB. Acidic residues decrease the phosphorylation rate with TPK-I, a kinase related to the src family which is immunologically indistinguishable from the lyn TPK; they are nearly ineffective, however, with TPK-III, the least specific of the tyrosine protein kinases, which exhibits appreciable activity toward tripeptides and dipeptides like GAY and AY which are not significantly affected by TPK-I and TPK-IIB. While the peptide substrate specificity of TPK-I is similar to that of TPK-IIA, a spleen tyrosine protein kinase previously considered [Brunati, A. M., Marchiori, F., Ruzza, P., Calderan, A., Borin, G. & Pinna, L. A. (1989) FEBS Lett. 254, 145-149], the remarkable requirement of TPK-IIB alone for acidic peptides may suggest the involvement of this enzyme, which is also unique in its failure to autophosphorylate, in the phosphorylation of the highly conserved and quite acidic phosphoacceptor sites of the src family protein kinases.

The substrate specificity of the different forms of the polycation-stimulated (PCS, type 2A) protein phosphatases and of the active catalytic subunit of the ATP, Mg-dependent (type 1) phosphatase (AMDC) was investigated, using synthetic peptides phosphorylated by either cyclic-AMP-dependent protein kinase or by casein kinase-2. The PCS phosphatases are very efficient toward the Thr(P) peptides RRAT(P)VA and RRREEET(P)EEE when compared with the Ser(P) analogues RRAS(P)VA and RRREEES(P)EEEAA. Despite their distinct sequence, both Thr(P) peptides are excellent substrates for the PCSM and PCSH1 phosphatases, being dephosphorylated faster than phosphorylase a. The slow dephosphorylation of RRAS(P)VA by the PCS phosphatases could be increased substantially by the insertion of N-terminal (Arg) basic residues. In contrast with the latter, the AMDC phosphatase shows very poor activity toward all the phosphopeptides tested, without preference for either Ser(P) or Thr(P) peptides. However, N-terminal basic residues also favor the dephosphorylation of otherwise almost inert substrates by the AMDC phosphatase. Hence, while the dephosphorylation of Thr(P) substrates by the PCS phosphatases is highly favored by the nature of the phosphorylated amino acid, phosphatase activity toward Ser(P)-containing peptides may require specific determinants in the primary structure of the phosphorylation site.

The hexapeptide (Arg)2-Pro-Thr-Pro-Ala (T1) and octapeptide (Arg)2-Pro-Thr-Pro-Ala (T5), reproducing the phosphorylatable site of protein phosphatase inhibitor-1, a physiological target of cAMP-dependent protein kinase, and five related peptides were synthesized by the method in solution. The phosphorylation rates of such peptides by cAMP-dependent protein kinase and their kinetic parameters have been determined and compared with those of the hexapeptide (Arg)2-Ala-Ser-Val-Ala, reproducing the phosphorylatable site of rat liver pyruvate kinase. The results obtained show that both the presence of threonine instead of serine and the adjacent C-terminal proline represent highly unfavourable factors seriously impairing the protein kinase reaction by both increasing Km and depressing V. On the other hand the N-terminal proline is compatible with high phosphorylation rates and the row of four rather than two consecutive arginines improves the phosphorylation efficiency by lowering tenfold the Km, without affecting the V. The extension of the hexapeptide T1 on its C-terminal side to give the derivative (Arg)2-Pro-Thr-Pro-Ala-Thr-Val-Ala has no significant effect on the kinetic parameters. Moreover no relationship between the phosphorylation efficiency and the predicted secondary structures around the target residue could be evidenced. Therefore the local structural features of the phosphorylatable site of inhibitor-1 cannot fully account for the fast phosphorylation of this regulatory protein. Other factors must optimize the protein kinase reaction.

The factors determining the site recognition and phosphorylation by rat liver casein kinase-2 (CK-2) have been explored with a set of 14 related hexapeptides each including a single phosphorylatable amino acid and five acidic plus neutral residues. Such peptides are different from each other in the following features: the nature of the phosphorylatable amino acid, if any; its position relative to the critically required acidic residues; the extension and the structure of the acidic cluster. All of them were tested as substrate and/or competitive inhibitors of CK-2, and their kinetic and inhibition constants were determined. The results suggest the following conclusions. Under strictly comparable conditions Ser is by far preferred over Thr. Tyr not being affected at all. In order to carry out its role of structural determinant the critical acidic cluster must be located on the C-terminal side of the target residue, though not necessarily adjacent to it. The affinity for the protein-binding site, as deduced from Km and/or Ki values, is largely dependent on the number of acidic residues but it is also significantly enhanced if a hydroxylic residue is located on their N-terminal side. An acidic residue at position +3 relative to serine plays an especially important role for triggering phosphorylation, the peptide Ser-Glu-Glu-Ala-Glu-Glu having similar Km but negligible Vmax compared to Ser-Glu-Ala-Glu-Glu-Glu and Ser-Glu-Glu-Glu-Ala-Glu. These data provide a rationale for the substrate specificity of CK-2 and will give a helpful insight into the structure of the protein-binding site of this enzyme.

Several synthetic peptides reproducing fragments of protamines have been used as model substrates for Ca2+/phospholipid-dependent protein kinase C, tested both in the absence of any effector (basal conditions) and upon activation by either Ca2+ and phosphatidylserine (or diacylglycerol) or limited proteolysis. Only the peptide Arg4-Tyr-Gly-Ser-Arg6-Tyr [Ga(52-65)] shares the unique property of protamines of being readily phosphorylated even under basal conditions. Optimal activity in the absence of effectors is observed with Tris/HCl buffer pH 7.5; Pipes and Hepes are less effective at pH 7.5, and at pH 6.5 basal phosphorylation is reduced. Under the best conditions for basal phosphorylation of Ga(52-65), its derivative with ornithine replaced for arginine and those corresponding to its C-terminal fragments Gly-Ser-Arg6-Tyr [Ga(57-65)] and Gly-Ser-Arg3 [Ga(57-61)], as well as the peptides Pro-Arg5-Ser2-Arg-Pro-Val-Arg [Th(1-12)], Arg4-Tyr-Arg2-Ser-Thr-Val-Ala [Th(13-23)] and Arg2-Leu-Ser2-Leu-Arg-Ala are not significantly affected though all of them, like histones, are more or less readily phosphorylated upon activation of protein kinase C by Ca2+/phosphatidylserine. The peptide Ser2-Arg-Pro-Val-Arg [Th(7-12)] however, corresponding to the C-terminal part of Th(1-12), is not phosphorylated even in the presence of activators. Limited proteolysis can roughly mimic the Ca2+/phosphatidylserine effect inducing however different extents of activation depending on the nature of the peptide substrates. Our results support the following two conclusions. Basal phosphorylation by protein kinase C in the absence of any effector requires peptide substrates whose target residue(s) are included between two extended arginyl blocks and is also dependent on pH and nature of the buffer. Peptides having extended clusters of either arginyl or ornithyl residues on the C-terminal side of serine are also readily phosphorylated, but they need activation of protein kinase by either Ca2+/phosphatidylserine or limited proteolysis. The same is true of peptides having basic residues only on the N-terminal side, or even on both sides but in limited number.

Some analogues have been prepared of S-peptide, the peptide obtained together with S-protein from subtilisn-modified beef pancreatic R Nase A. The syntheses are described of [Orn10, Asn14]-S-peptide and 1epsilon, 7epsilon, 10delta-triguanidino-[Orn10, Asn14]-S-peptide. The S-peptide analogues are able to activate S-protein at the level of the parent [Orn10]-S-peptide and 1epsilon, 7epsilon-diguanidino-S-peptide respectively, although at high peptide-to-protein molar ratios. After their recombination with S-protein the buried character of Tyr-25 was restored, as judged from difference absorption and circular dichroism spectra in the near-ultraviolet region. These findings indicate that the asparaginyl residue is a possible naturally occurring substituent in the R Nase A sequences whose state of amidation in position 14 has not yet been defined.

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).