©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Protein Kinase CK2 Mutants Defective in Substrate Recognition
PURIFICATION AND KINETIC ANALYSIS (*)

(Received for publication, December 22, 1995; and in revised form, February 14, 1996)

S. Sarno (1)(§)(¶) P. Vaglio (1)(§)(**) F. Meggio (1) O.-G. Issinger (2) L. A. Pinna (1)(§§)

From the  (1)Dipartimento di Chimica Biologica, Università di Padova and Centro per lo Studio della Fisiologia Mitocondriale del Consiglio Nazionale delle Ricerche, Padova, 351321 Italy and the (2)Biokemisk Institut, Odense Universitet Campusvej 55, 5230 Odense, Denmark

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Five mutants of protein kinase CK2 alpha subunit in which altogether 14 basic residues were singly to quadruply replaced by alanines (K74A,K75A,K76A,K77A; K79A, R80A,K83A; R191A,R195A,K198A; R228A; and R278A, K279A,R280A) have been purified to near homogeneity either as such or after addition of the recombinant beta subunit. By this latter procedure five mutated tetrameric holoenzymes were obtained as judged from their subunit composition, sedimentation coefficient on sucrose gradient ultracentrifugation, and increased activity toward a specific peptide substrate as compared with the isolated alpha subunits. The kinetic constants and the phosphorylation efficiencies (V(max)/K) of all the mutants with the parent peptide RRRADDSDDDDD and a series of derivatives, in which individual aspartic acids were replaced by alanines, have been determined. Three mutants, namely K74A,K75A,K76A,K77A; K79A,R80A, K83A; and R191A,R195A,K198A display dramatically lower phosphorylation efficiency and 8-50-fold higher K values with the parent peptide, symptomatic of reduced attitude to bind the peptide substrate as compared with CK2 wild type. Such differences either disappear or are attenuated if the mutants R191A,R195A, K198A; K79A,R80A,K83A; and K74A,K75A,K76A,K77A are assayed with the peptides RRRADDSADDDD, RRRADDSDDADD, and RRRADDSDDDAA, respectively. In contrast, the phosphorylation efficiencies of the other substituted peptides decrease more markedly with these mutants than with CK2 wild type. These data show that one or more of the basic residues clustered in the 191-198, 79-83, and 74-77 sequences are implicated in the recognition of the acidic determinants at positions +1, +3, and +4/+5, respectively, and that if these residues are mutated, the relevance of the other acidic residues surrounding serine is increased. In contrast the other two mutants, namely R228A and R278A,K279A, R280A, display with all the peptides V(max) values higher than CK2 wild type, counterbalanced however by somewhat higher Kvalues. It can be concluded from these data that all the five mutations performed are compatible with the reconstitution of tetrameric holoenzyme, but all of them influence the enzymatic efficiency of CK2 to different extents. Although the basic residues mutated in the 74-77, 79-83, and 191-198 sequences are clearly implicated in substrate recognition by interacting with acidic determinants at variable positions downstream from serine, the other basic residues seem to play a more elusive and/or indirect role in catalysis.


INTRODUCTION

Protein kinase CK2, formerly termed casein kinase-2 (or -II), is a ubiquitous Ser/Thr protein kinase normally composed by the tight association of two catalytic (alpha and alpha`) and two non catalytic beta subunits that appears to play a central albeit still enigmatic role in cell regulation(1, 2) . The presence among the myriad of its substrates of many proteins implicated in gene expression and signal transduction (3) , the increase of CK2 activity in transformed and proliferating tissues(4) , and development of leukemias in transgenic mice transfected with CK2alpha subunits (5) suggest the involvement of CK2 in both normal and uncontrolled cell proliferation. Though CK2 is endowed with basal catalytic activity toward most of its substrates and by contrast to previous reports, it seems not to be subjected to any kind of direct regulation by growth factors(6) , its activity can be modulated by polycationic effectors acting through its beta subunit, which has been shown to exert a dual function of positive as well as negative regulation over the catalytic subunit(7, 8, 9) . The negative effect of the beta subunit, especially evident with some substrates exemplified by calmodulin, is mediated by an acidic cluster located in the N-terminal part of the molecule. This would imply the interaction with basic residue(s) of the catalytic subunit. Other properties of CK2 would imply the presence of crucially relevant basic residues in the catalytic subunit, namely inhibition by heparin (10) and other polyanionic compounds, like poly(Glu, Tyr)4:1 (11) and substrate specificity. This latter is invariably determined by multiple acidic residues located at positions between -2 and +5 (and probably farther) relative to the target amino acid (mostly Ser and rarely Thr)(12) . Heparin inhibition is reduced but not abolished by mutations affecting lysyl residues 74/75 (13) and 75/76(14) . On the other hand the substitution of Asp for His, homologous to PKA (^1)Glu (interacting with Arg at position -2 in PKA substrates)(15, 16, 17) , affects the phosphorylation of peptide substrates whose recognition is partially dependent on an acidic residue at position -2(18) . In contrast the most powerful determinants of CK2 specificity normally are acidic residues located downstream from serine, the ones at positions +3 and +1 playing an especially crucial role, although they have also been shown to be effective at more remote positions(12, 19, 20) .

In order to identify the basic residues responsible for substrate recognition, inhibition, and intrinsic down-regulation, we have applied the ``charged-to-alanine'' scanning mutagenesis strategy (21) to a number of basic residues of the human alpha subunit that are conserved across various species but divergent from the homologous residues of other protein kinases. Six such mutants in which collectively 16 residues have been singly to quadruply mutated to alanines have been obtained, and three of them have been shown to be seriously defective in catalytic activity(18) . Here we describe the purification of five of these mutants, either as such or combined with the beta subunit to give heterotetrameric holoenzymes, and we analyze their kinetic properties with a set of peptide substrates varying for the replacement of individual aspartyl residues between positions -2 and +5 within the structure of the reference peptide RRRADDSDDDDD.


EXPERIMENTAL PROCEDURES

Materials

Synthetic peptide substrates were prepared as described or referenced in (22) . [-P]ATP (2 mCi/ml) was from Amersham Corp. P11 phosphocellulose was from Whatman. MonoQ HR 5/5 column was from Pharmacia Biotech Inc. Antiserum against CK2 alpha subunit was raised using the peptide CVVKILKPVKKKKIKREIKILE, reproducing the sequence 66-86. The peptide was coupled to keyhole limpet hemocyanin using m-maleimidobenzoyl-N-hydroxysuccinimide ester (Pierce). Nitrocellulose membrane (0.2 µm) was from Bio-Rad. All the other reagents were of the highest purity available. Recombinant human CK2 alpha and beta were prepared as described in (22, 23, 24) .

Expression and Purification of CK2 alpha Mutants

Expression of the mutants in Escherichia coli was performed as previously described(22, 23, 24) . For subsequent purification, 2 g of bacteria pellets were resuspended in 20 ml of buffer A (25 mM Tris-HCl, pH 8.5, and 7 mM 2-mercaptoethanol) and sonicated (6 times 20 s) in ice. After sonication the bacteria extract was centrifuged for 15 min at 80,000 times g. The supernatant was adjusted at the salt concentration of 0.2 M NaCl and loaded on a P11 phosphocellulose column (20 ml) previously equilibrated with buffer A + 0.2 M NaCl. The column was eluted with a linear gradient of 2 times 100 ml 0.2-1.5 M NaCl. 2-ml fractions were collected. 15-µl aliquots were analyzed by 12% SDS-PAGE to identify the presence of the protein. The fractions with alpha subunits were collected, dialyzed against 25 mM Tris containing 0.1 M NaCl, and stored in small aliquots at -20 °C.

In order to obtain CK2 holoenzymes with mutated alpha subunits, 1.5 g of bacteria pellets expressing mutated alpha subunits were resuspended and sonicated together with 1.5 g of bacteria expressing the wild type beta subunit in 30 ml of buffer A. The same purification procedure was applied as for alpha subunit alone, but after the phosphocellulose column a further purification step was performed by pooling all the fractions containing both the alpha and beta the subunits (as judged by SDS-PAGE) and subjecting them to MonoQ fast protein liquid chromatography. The column was eluted with a linear gradient from 0.1 to 1 M NaCl, and the eluent was analyzed by OD monitor. The fractions in the OD peaks were assayed for CK2 activity, and the presence of the holoenzyme was assessed by 12% SDS-PAGE showing both the alpha (mutated) and the beta subunits. The reconstituted enzyme was generally eluted from the column at a salt concentration of 0.5-0.6 M (corresponding to the prominent peak of both OD and catalytic activity). The fractions containing the purified holoenzyme were pooled and dialyzed for 4-5 h against 5 mM Tris-HCl, pH 7.5, 50 µM phenylmethylsulfonyl fluoride, and 50% glycerol and stored at -20 °C. The specific activities of CK2 holoenzymes were: CK2 wild type, 300 units/mg; K74A,K75A,K76A,K77A, 27 units/mg; K79A,R80A,K83A, 93 units/mg; R191A,R195A,K198A, 40 units/mg; R228A, 450 units/mg; and R278A,K279A,R280A, 370 units/mg. One unit is defined as the amount of enzyme transferring 1 nmol of phosphate to the peptide substrate RRRADDSDDDDD per min under the experimental condition detailed below.

Assay of CK2 Activity

Phosphorylation experiments were performed by incubating CK2 enzymes for 10 min at 30 °C in a final volume of 30 µl containing 50 mM Tris-HCl, pH 7.5, 12 mM MgCl(2), 100 mM NaCl, and 25 µM [-P]ATP (specific activity, 500 cpm/pmol) and either casein (1 mg/ml) or peptides (100 µM, unless differently indicated) as substrate. P incorporation into casein was evaluated by trichloroacetic acid precipitation spotting 25 µl on Whatman 3MM chromatography paper and washed as described(25) . P incorporated into peptide substrates was evaluated by the phosphocellulose paper procedure(26) . Kinetic constants were determined by double reciprocal plots constructed from initial rate measurements fitted to the Michaelis-Menten equation.

Gel Electrophoresis and Immunodetection of CK2 alpha Mutants

Aliquots (1 and 2 µg, respectively) of either mutated alpha subunits or CK2 holoenzymes reconstituted with mutated alpha subunits purified as described above were subjected to 12% SDS-PAGE according to Laemmli(27) . The gels were either stained with Coomassie Blue or transblotted to nitrocellulose in a Hoefer apparatus at 250 mA for 2.30 h. The filters were blocked for 1 h at room temperature with 3% bovine serum albumin in 10 mM Tris-HCl, pH 7.5, containing 0.15 M NaCl and treated with anti CK2 alpha antisera (diluted 1:2000). Immunoreactive proteins were incubated with donkey anti-rabbit Ig, biotinylated, and detected by incubation with streptavidin alkaline phosphatase conjugate.

Other Methods

Protein concentration was determined by the method of Bradford (28) using bovine serum albumin as standard. Sucrose density gradient ultracentrifugation was performed as described previously(29) .


RESULTS

Five mutants of CK2 alpha subunit (22) have been purified by submitting to phosphocellulose chromatography the extracts of bacteria expressing the mutated alpha subunits (see ``Experimental Procedures''). The Coomassie-stained SDS-PAGE gels of the final preparations are shown in Fig. 1A. All mutants display a prominent 44-kDa band with the same mobility as alpha wild type. In several cases a doublet rather than a single band is visible, probably indicative of limited proteolysis occurring during the isolation and purification procedure. This conclusion is corroborated by the finding that anti-alpha antibodies (see ``Experimental Procedures'') recognize not only the 44-kDa band but also the ones with lower molecular masses (not shown).


Figure 1: SDS-PAGE analysis of CK2 mutants. The Coomassie-stained gels are shown. A, mutants of CK2 alpha subunit after phosphocellulose purification. The arrow denotes the position of wild type alpha subunit. The identification of mutated alpha subunits was also corroborated by immunoreaction with anti-alpha subunit antiserum (not shown). Also the minor bands with slightly higher mobility immunoreacted, consistent with their identification as proteolytic derivatives of the alpha subunit (see text). Lane 1, wild type-alpha subunit; lane 2, R228A; lane 3, R191A, R195A,K198A; lane 4, R278A,K279A,R280A; lane 5, K74A,K75A, K76A,K77A; lane 6, K79A,R80A,K83A. B, CK2 holoenzymes reconstituted with mutants of the alpha subunit and wild type beta subunit and purified by phosphocellulose chromatography and MonoQ fast protein liquid chromatography (see ``Experimental Procedures''). The arrows denote the positions of wild type alpha and beta subunits. Lane 1, wild type CK2; lane 2, R228A; lane 3, R191195K198A; lane 4, K74A,K75A, K76A,K77A; lane 5, R278A,K279A,R280A; lane 6, K79A,R80A,K83A.



The phosphotransferase activity of the purified mutants, either as such or after addition of equimolar amounts of pure beta subunit, was determined using the peptide substrate RRRAADSDDDDD(29) . As shown in Table 1two mutants, R228A and R278A,K279A,R280A, display an activity significantly higher to that of alpha w.t., whereas the phosphorylation rate by the other mutants is much lower, being too low for a reliable measurement in the case of mutant K79A,R80A,K83A. Upon addition of equimolar amounts of beta subunit, the catalytic activity of alpha w.t. increases, as already observed(30) . A similar or even higher increment of activity is observable adding beta subunit to all the mutants. This also causes the appearance of significant activity with the mutant whose activity is undetectable in the absence of the beta subunit.



These data would indicate that all the mutants are still capable of associating with the beta subunit to give the heterotetrameric holoenzyme. This conclusion was corroborated by sucrose gradient ultracentrifugation experiments showing that the addition of the beta subunit causes a change in the sedimentation coefficient similar to that induced by alpha w.t., consistent with the reconstitution of alpha(2)beta(2) tetramers (Fig. 2).


Figure 2: Sucrose density gradient ultracentrifugation of CK2 holoenzymes reconstituted with variably mutated alpha subunits. Equimolar amounts of beta subunit were combined with 18 µg of alpha subunits prior to sucrose gradient ultracentrifugation. Analysis of CK2 activity was done as described in (20) using casein (1 mg/ml) as substrate. CK2 alpha w.t. (box), K74A,K75A,K76A,K77A (up triangle), K79A,R80A, K83A (), R191A,R195A,K198A (times), R228A (), and R278A,K279A, R280A (circle). The arrows indicate the positions of wild type alpha subunit alone and reconstituted CK2 holoenzyme (alpha2beta2).



Once established that all the mutants are still capable of associating with the beta subunit, a strategy was developed for preparing mutated holoenzymes for sake of comparison with CK2 w.t., either recombinant or native. The most successful approach was to mix together the bacteria expressing the mutated alpha subunit and those expressing the wild type beta subunit and to apply the normal purification procedure of CK2 (see ``Experimental Procedures''). In such a way all the five mutants could be purified to near homogeneity as heterotetrameric holoenzymes, as judged from both their SDS-PAGE Coomassie patterns, showing the alpha and the beta subunits in approximately the same ratio as CK2 w.t. (Fig. 1B) and sucrose gradient ultracentrifugation revealing peaks of activity with the same sedimentation coefficient as CK2 w.t. (see Fig. 2). Heat denaturation curves, another criterion for judging the reconstitution of normal CK2 holoenzyme that is much more heat stable than the isolated alpha subunit(30) , are shown in Fig. 3. Four mutants exhibited heat stability comparable with that of CK2 w.t. holoenzyme; the mutant K79A,R80A,K83A, however, exhibited a reduced heat stability, suggesting that susceptibility to protection by the beta subunit is partially compromised in it. All mutants displayed K(m) values for ATP comparable with that of CK2 w.t. (17 µM) ranging between 10 and 25 µM.


Figure 3: Thermal stability of CK2 holoenzymes. The catalytic activities of CK2 w.t. (box), K74A,K75A,K76A,K77A (up triangle), K79A,R80A,K83A (), R191A,R195A,K198A (times), R228A (), and R278A,K279A,R280A (circle) were determined after preincubation of 0.1 µg of each enzyme at 40 °C for the time indicated. The samples were immediately ice-cooled, and the residual activity was determined as described under under ``Experimental Procedures.''



The kinetic constants of all the CK2 mutants with the optimal peptide substrate RRRADDSDDDDD and with a series of six peptide derivatives in which individual aspartic acids have been replaced by alanines were determined and compared with CK2 w.t. The V(max) and K(m) values as well as the overall phosphorylation efficiencies expressed by the V(max)/K(m) ratios are summarized in Table 2.



All the Asp Ala substitutions, with only the exception of the one at position +2, are more or less detrimental to the phosphorylation efficiency of the peptide substrates by CK2 w.t.; two substitutions, however, are especially deleterious, namely the ones at positions +3 and +1, both causing a 10-fold drop in phosphorylation efficiency, accounted for by both a raise of K(m) and a decrease of V(max).

An overall examination of the data of Table 2allows a rough subdivision of the mutants into two categories: (i) mutants whose affinity for the parent peptide (expressed by K(m)) is only slightly decreased (whereas the V(max) is actually increased) and whose phosphorylation efficiency is altered by the structure of the peptide substrate in a manner similar to that of CK2 w.t. (R228A and R278A,K279A,R280A) and (ii) mutants whose affinity for the parent peptide is substantially decreased and whose phosphorylation efficiency is altered by modifications of the peptide substrate in a sharply different manner as compared with CK2 w.t. (K74A,K75A,K76A,K77A; K79A, R80A,K83A; and R191A,R195A,K198A).

In order to facilitate a comparative analysis, the relative efficiencies of CK2 w.t. and the mutants are represented in Fig. 4A as histograms normalized to the phosphorylation efficiencies of the parent peptide conventionally set equal to 1 for each mutant. It can be seen that although the profiles of mutants R228A and R278A,K279A,R280A are roughly superimposable to that of CK2 w.t., the histograms of mutants K74A, K75A,K76A,K77A; K79A,R80A,K83A; and R191A,R195A, K198A are dramatically altered. With the last mutant, e.g. the phosphorylation efficiency of the peptide lacking the acidic residue at position +1 (which is normally negligible as compared with the parent peptide) is actually the highest, surpassing by 3-fold that of the parent peptide. In contrast the relative phosphorylation efficiencies of the peptides with acidic gaps at all positions other than +1 are drastically reduced as compared with CK2 w.t. In the case of mutant K74A,K75A,K76A,K77A, the relative phosphorylation efficiencies of three peptides, with Ala for Asp substitutions at positions +3, +4/+5, and +2 are increased, whereas those of the other three peptide derivatives are decreased. The mutations occurring in K79A,R80A,K83A more specifically affect the phosphorylation of the peptide lacking the crucial acidic residue at position +3, whose phosphorylation efficiency is now comparable with that of the parent peptide, without dramatic alterations in the phosphorylation efficiency of peptides RRRADDSDADDD and RRRADDSDDDAA. In contrast the phosphorylation efficiency of peptides with acidic gaps at positions -1, +1, and -2 is drastically reduced.


Figure 4: Relative phosphorylation efficiencies of synthetic peptides by the wild type and mutated forms of CK2. The histograms have been constructed with data drawn from Table 2. The reference peptide, indicated by c (control), is RRRADDSDDDDD, and the other peptides are indicated by numbers (from -2 to +4/+5) denoting the position(s) relative to serine where aspartic acid has been replaced by alanine (see also Table 2). In A the phosphorylation efficiencies are normalized to that of the reference peptide (c) set equal to 1 for each form of CK2. Upper panel, CK2 wild type. Middle panels, mutants whose selectivity profiles are not markedly different from that of CK2 w.t. Lower panels, mutants whose selectivity profiles are deeply altered (see text). In B the mutants of the lower panels of A are further analyzed by expressing the phosphorylation efficiency of each peptide as a percentage of that of the same peptide phosphorylated by CK2 wild type.



The more specific effect of the mutation occurring in the 79-83 stretch as compared with that in the 74-77 stretch is highlighted if their phosphorylation efficiencies are expressed as percentages of the corresponding phosphorylation efficiency by CK2 w.t. (Fig. 4B). In these histograms the only outstanding efficiency bars with mutants K79A,R80A,K83A and R191A, R195A,K198A are those referring to the peptides RRRADDSDDADD and RRRADDSADDDD, respectively, whereas the outcome with mutant K74A,K75A,K76A,K77A is more promiscuous, with three peptides (having acidic gaps between +2 and +5) being represented by bars that surpass that of the parent peptide.

A summary of the mutations examined in this paper and of their effects on holoenzyme reconstitution, catalytic activity, substrate recognition, and susceptibility to polyanionic inhibitors (31) is reported in Table 3.




DISCUSSION

This paper describes the reconstitution, purification, and kinetic characterization of five CK2 mutants in which the alpha subunit underwent substitution of basic residues with alanines. Charged-to-alanine mutagenesis has been successfully used to identify residues implicated in substrate recognition by other protein kinases, namely PKA(15, 17, 21) , myosin light chain kinase(32) , and phosphorylase kinase(33) . In all these cases such residues were found to be acidic in nature consistent with the knowledge that these kinases recognize basic specificity determinants. These specificity determinants moreover are located upstream from the phosphorylatable amino acid, notably at positions -2 and -3. In contrast, the main specificity determinants for CK2 are acidic residues located on the C-terminal side of the target amino acid. Our strategy therefore was to mutate basic residues that are conserved in CK2 from different species but are replaced by nonbasic residues in other protein kinases with special reference to the basophilic ones. Consequently the basic residues mutated by us (listed in Table 3) are not homologous to residues (either acidic or basic) mutated in previous studies and in particular in the pioneering study of Gibbs and Zoller (21) in which all charged residues of yeast PKA were mutated to alanine. Two of the residues mutated in (21) , Cys and Glu, were found to be implicated in ATP binding. Both residues are highly conserved throughout the protein kinase family, CK2 included, and therefore were not modified in our study. On the other hand none of our mutations significantly modifies the K(m) for ATP nor prevents the association with the beta subunit to give tetrameric holoenzyme. In one case, however, where Lys, Arg, and Lys were mutated into alanines, the resulting holoenzyme displays a reduced heat stability as compared with CK2 w.t.. This suggests that the interactions of this mutated alpha subunit with the beta subunit, which is responsible for thermostability(24) , are weakened. Also with this mutant, however, the association with the beta subunit promotes a severalfold increase of basal activity with peptide substrate, apparently even higher than that observed with CK2 w.t., although a precise evaluation is hindered by the extremely low activity in the absence of the beta subunit (see Table 1).

The kinetic constants of all the mutants with a set of seven peptides including the optimal substrate RRRADDSDDDDD (29) and its derivatives in which the aspartyl residues acting as specificity determinants have been variably replaced by alanine were calculated and analyzed. The main outcome of this study is that one or more of the basic residues replaced in three mutants, namely K74A,K75A,K76A,K77A; K79A,R80A,K83A; and R191A,R195A,K198A, are directly implicated in substrate recognition by interacting with definite acidic determinants of the peptide substrate.

In particular it is clear that one or more of the basic residues substituted by Ala in the mutant R191A,R195A,K198A are responsible for the recognition of the acidic determinant at position +1. The substitution of Asp(+1) with Ala in the peptide RRRADDSDDDDD in fact, which is one of the most detrimental substitutions with CK2 w.t., does not decrease but actually increases the phosphorylation efficiency by mutant R191A,R195A,K198A. This conclusion is also in agreement with the knowledge that the basic residues Arg, Arg, and Lys are homologous to the PKA hydrophobic residues Leu, Pro, and Leu that interact with the hydrophobic residue found in many PKA substrates at position +1(34, 35) .

By similar arguments it can be concluded that one or more of the basic residues Lys, Arg, and Lys are specifically implicated in the recognition of another crucial specificity determinant, namely the acidic residue at position +3, because the replacement of this residue is almost ineffective with mutant K79A,R80A,K83A, whereas it is dramatically detrimental with CK2 w.t.

The case of the first part of the basic cluster 74-83 is more complicated because the mutation of the four lysyl residues 74-77 gives rise to a mutant whose low phosphorylation efficiency as compared with CK2 wild type can be improved in relative terms by the substitution of all the aspartyl residues downstream from +1 (see Fig. 4B). It seems likely therefore that this basic quartet contributes to the recognition of the determinant at position +3 (together with the triplet Lys, Arg, and Lys), but it also interacts with acidic residues downstream from this that are conversely poorly interacting with Lys, Arg, and Lys. It should be remembered in this connection that although our reference peptide stops at position +5, acidic residues have been shown to act as specificity determinants for CK2 at even more remote positions(19, 20) . The different functions of the first and second part of the 74-83 basic cluster is highlighted by the finding, summarized in Table 3, that inhibition by heparin is totally abolished by the 74-77 mutation while being unaffected by the Lys, Arg, and Lys mutation(31) .

The implication of the 74-83 region in substrate recognition by CK2 discloses a situation where the smaller lobe of protein kinases, committed with ATP binding and catalysis, also contributes to the interactions with the phosphoacceptor substrate. Up to now the residues responsible for these interactions had been identified almost exclusively in the larger lobe of PKA, with special reference to subdomains VIII, VI, and IX(16, 35, 36) . In contrast, the 74-83 basic stretch of CK2, based on the common architecture of protein kinases, would be located at the lower edge of the smaller lobe in the proximity of the cleft between the two lobes at the borderline between subdomains II and III.

Actually the whole inclusion of the 74-83 segment into subdomain III is an arbitrary consequence of multiple amino acid sequence alignment of the catalytic domains of 75 Ser/Thr protein kinases(37) . In contrast, if CK2alpha is manually aligned to PKA alone, the 74-79 sequence would fall into subdomain II (see Table 4). An interesting outcome of such an alignment, resting on higher similarity between CK2 and PKA, is the homology between CK2 Lys, implicated in the recognition of the acidic determinant at position +3 (see above) and PKA Lys, representing a hinge between helices B and C, whose side chain actually faces that of the aspartyl residue situated at position +3 in the inhibitor peptide (protein kinase inhibitor) bound to the catalytic subunit of PKA(16, 38) . It seems likely therefore that this residue might play a general role in the recognition of determinants at position +3. In agreement with this conclusion is the observation that PKA Lys is invariably homologous to an acidic residue in the members of the protein kinase C family, which are known to recognize basic determinants at positions +2 and +3(39, 40) . The members of the Cdc2/Cdk family, moreover, which are know to select substrates with basic residues on the C-terminal side of the crucial p+1 proline(40, 41) , display a series of acidic residues clustered at the very end of subdomain II matching the basic Lys-Lys-Lys-Lys cluster of CK2 if the manual alignment of Table 4is followed.



Our kinetic data invariably show that whenever the structural elements committed with the recognition of the crucial acidic determinants at positions +1 and +3 are mutated, then the relevance of the other acidic determinants that normally play a subsidiary role is increased. This is especially true of the two acidic residues located upstream from serine at positions -1 and, less dramatically, -2; both these positions, which are relatively unimportant with CK2 w.t., become crucial (especially the former) with mutants R191A,R195A,K198A; K79A,R80A,K83A; and K74A,K75A,K76A,K77A. These observations may also provide the structural basis accounting for the efficient phosphorylation of ``atypical'' CK2 sites lacking the acidic determinant at position +3 and in which the presence of acidic residues at position -2 and even more at position -1 is essential(42) . Using these atypical peptide substrates, it was possible to show that CK2 His contributes to the recognition of the acidic determinant at position -2(18) .

It should finally be noted that even the two mutants that are almost indistinguishable from CK2 w.t. reveal significant differences by the kinetic scrutiny of this work. Their almost unchanged phosphorylation efficiency in fact results from significantly and reproducibly higher V(max) values counterbalanced by higher K(m) values. The behavior of mutant R228A is especially intriguing because this mutation also dramatically increases CK2 sensitivity to inhibition by heparin (see Table 3). A possible interpretation is that heparin, besides inhibiting CK2 by competing with some of the substrate binding elements (namely the Lys-Lys-Lys-Lys basic quartet and the p+1 loop) might also stimulate CK2 activity by interacting with Arg. This would be consistent with the finding that the mutation of the Lys-Lys-Lys-Lys cluster not only suppresses inhibition by heparin but even induces a stimulation by it(31) , as expected assuming the existence of an up-regulating heparin binding site in CK2 alpha subunit. Additional mutations are in progress in order to check these possibilities and to identify the residues responsible for the recognition of the determinants located upstream from serine and downstream from position +5.


FOOTNOTES

*
This work was supported by Grants 94.00315CT14 (to L. A. P.) and 94.02450.CT04 (to F. M.) from the Italian Ministero dell' Università e della Ricerca Scientifica e Technologica, Consiglio Nazionale delle Ricerche, Target Project Applicazioni Cliniche della Ricerca Oncologica, Ministero della Sanità (Project AIDS), and funds from Associazione Italiana per la Ricerca sul Cancro. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
These authors contributed equally to the work.

Supported by Deutsche Forschungsgemeinschaft (SFB 246) in Homburg/Saar for part of the work.

**
Recipient of European Union Human Capital and Mobility Programme Fellowship ERBCHBGCT930390.

§§
To whom correspondence should be addressed: Dipartimento di Chimica Biologica, Via Trieste, 75, 351321 Padova, Italy. Tel.: 49-8276108; Fax: 49-8073310.

(^1)
The abbreviations used are: PKA, cyclic AMP-dependent protein kinase; PAGE, polyacrylamide gel electrophoresis; w.t., wild type.


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