BIOCHEMICAL

Vol. 182, No. 3, 1992 February 14, 1992

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1460-1465

THE COMPARATIVE EFFICIENCIES OF THE Ser(P)-, Thr(P)- and Tyr(P)-RESIDUES AS SPECIFICITY DETERMINANTS FOR CASEIN KINASE- 1 Flavio Meggio*, John W. Perich+, Oriano Marin* and Lorenzo A. Pinna* 1 *Dipartimento di Chimica Biologica and Centro per lo Studio della Fisiologia Mitocondriale de1 Consiglio Nazionale delle Ricerche, Universita di Padova, Italy +Centre CNRS-INSERM Received

December

23,

de Pharmacologic-Endocrinologie, Montpellier, France

1991

The P-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 B-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)>>Tyr(P) and this order is markedly different from Tyr(P)>Ser(P)>>Thr(P) which was previously established for casein kinase-2 [Meggio et al. (1991) FEBS Lett. 279, 307-3091. 0 1992 Academic Press, Inc.

While

most Ser/Thr protein kinases recognize basic residues as specificity

determinants, a small and heterogeneous group of such enzymes, which include casein kinase-2 (CK2) [l, 21, glycogen synthase kinase-3 (GSK3) [3], a casein kinase from the golgi enriched fraction of lactating mammary gland (GEF-CK) [4], casein kinase-1 (CKl) [5] and the P-adrenergic receptor protein kinase (P-ARK) [6], appear to be acidophylic in nature since their targets are specified by acidic residues at definite positions relative to the phosphorylatable residue(s). In the case of CK2, though, the acidic determinants are normally carboxylic 1

To whom correspondence via Trieste, 75, 35131

should Padova,

be addressed Italy. Fax:

at Dipartimento 049-8073310.

aminoacids (either

di

Chimica

Biologica,

Abbreviations: CKl, casein kinase-1; CK2, casein kinase-2; Ser(P), phosphoserine; Thr(P), phosphothreonine, Tyr(P), phosphotyrosine. 0006-291X/92 Copyright All rights

$1.50

0 1992 by Academic Press, Inc. of reproduction in any form reserved.

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Glu or Asp) and can be effectively replaced by phosphorylated residues, both in synthetic peptide substrates [7, 81 and physiological targets [9]. Conversely, GSK3 and CKl

behave as typically

“phosphate

directed”

side chains cannot replace or can only specificity determinants [ 10, 111. Since both volved would tute

CKl

and CK2

very

are ubiquitous

protein

weakly enzymes

in the regulation of a variety of cell functions be interesting to assess whether phosphotyrosine for

phosphoserine

the hypothesis protein kinases A positive which

was

in determining

their

that their targeting could be triggered acting on nearby tyrosyl residues. answer

found

to

to this question

was recently

actually

phosphotyrosine

prefer

glutamic acid as a specificity determinant, poorly effective [14] in comparison. Here we provide the first While the Thr(P)-residue was determinant,

specificity,

the Tyr(P )-residue

kinases

since carboxylic

surrogate

phosphoserine

as

which

are apparently

in-

(reviewed in [12, could effectively thus

rendering

or potentiated

provided over

13]), it substiplausible

by tyrosine

in the case of CK2, phosphoserine

the phosphothreonyl

residue

and being

evidence that the converse situation exist for CKl. as effective as the Ser(P)-residue as a specificity was unable to replace the Ser(P)-residue.

Experimental The synthesis of Glu-Glu-Ser(P)-Glu-Glu-Ser-Ile-Thr-NHMe and Glu-Glu-Thr(P)Glu-Glu-Ser-Ile-Thr-NHMe was accomplished by a synthetic approach [15-171 which used either Boc-Ser(POgPh2)-OH in the Boc mode of solution phase peptide synthesis followed by hydrogenation (platinum) of the protected peptides in 50% TFA/AcOH. The synthesis of Glu-Glu-Tyr(P)-Glu-Glu-Ser-Ile-Thr-OH was accomplished by a synthetic approach which used Fmoc-Tyr(P03tBu2)-OH [18] in Fmoc/solid phase peptide synthesis followed by 5% anisole/TFA deprotection of the peptide-resin [19]. The synthesis of peptides GVEAASG, GVDAASG and GVYAASG was performed by solid-phase technique from 9-fluorenylmethoxycarbonyl-protected amino acids using a manual synthesizer (model Biolinx 4175, LKB) by a procedure to be detailed elsewhere. CKl was prepared as previously described [20]. Its specific activity was 19 U/mg, one unit being defined as the amount of enzyme transferring 1 nmol P to casein per min under standard conditions. Peptide phosphorylation was performed and quantitated essentially as in [2] (procedure a) by incubating the peptides (1 mM, unless differently indicated) at 37 ‘C in a medium containing 50 mM Tris-HCl pH 7.5, 12 mM MgC12, 100 mM and 0.01-0.02 units of NaCl, 20 uM [Y~~P]ATP (sp. act. 1000-1500 cpm/pmol) enzyme. The reaction was stopped by adding HCl (6 N final concentration). After partial acid hydrolysis (4 hrs at 105 ‘C), the radiolabeled phosphoaminoacids were isolated by high voltage paper electrophoresis and quantitated as in 121. Phosphoserine invariably was the only radiolabeled aminoacid detected on the electropherograms. Results In past work we showed that the phosphopeptide EESpEESIT, which is derived from the P-casein phosphoacceptor site and includes Ser-22, was an excel1461

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EESpEESlT EETpEESIT

0

20

IO

30

40

50

time (min)

Figure 1. Time course of synthetic peptides phosphorylation by CKI. Sp, Tp, Yp denote Ser(P), Thr(P) and Tyr(P), respectively. Peptides EEYEESIT (A) and EESEESIT (0) were obtained by enzymatic dephosphorylationof EEYpEESIT (A) and EESpEESIT (o), respectively, with potato acid phosphatase,as in [7]. Previous enzymatic dephosphorylationof EETpEESIT (0 ), to give EETEESIT, also prevented subsequentphosphorylation by CKl (not shown). With all peptides the only radiolabeted phosphoaminoacid was Ser(P). No radioactive phosphothreonine and phosphotyrosine could be detected.

lent substrate for CKI in virtue of the Set(P)-residue at position -3 relative to the target seryl residue [lo]. The derivatives of this peptide in which Ser(P)-residue has been replaced by either Thr(P)- or Tyr(P)-residue

have been now synthesized

and compared to the parent peptide for their susceptibility CU.

to phosphorylation by

The time course experiment of Fig.1 clearly shows that while the Thr(P) substitution of Ser(P) is well tolerated, Tyr(P)

substitution is very detrimental, giving

rise to a peptide substrate almost as poor as the Glu peptide (EEEEESIT-NHMe). The marked superiority of the phosphoryl side chain of phosphoserine over that of phosphotyrosine

is further

illustrated

by the finding

that (a) prior

dephos-

phorylation of the phosphoseryl peptide is dramatically harmful and generates a peptide which is no longer a substrate for CKl. The same detrimental effect of dephosphorylation was observed with the phosphothreonyl peptide (not shown); (b) prior dephosphorylation of the Tyr(P)-peptide causes no reduction in its modest phosphoacceptor capacity (Fig. 1). These data suggest there may be a weak yet significant role of tyrosine residues in determining CR2 targeting. Such a point of view would be a!so consistent with the finding that the heptapeptide GVYAASG, reproducing the putative autophosphorylation site of c-fes TPK, is significantly affected by CKI at its Ser-5 residue. In contrast, the phosphorylation of its derivatives in which the Tyr-3 residue had been replaced by either Glu or Asp is hardly detectable (see Table 1). 1462

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BIOCHEMICAL

Tyrosine

is more

AND BIOPHYSICAL

effective than ficity determinant

Peptide

RESEARCH COMMUNICATIONS

aspartic and glutamic for CKl

Phosphorylation

rate

G&4AsG

81

GQ4A!SG

80

GmAASG

acid

as speci-

(cpmemin-l)

239

The radioactivity incorporated was entirely accounted for tide GVYAASG. The results presented represent individual of three separate determinations.

by Ser(P) also in pepvalues that are typical

In order to assess whether, and to what extent, alterations of either Km or Vmax

could account for the different

phosphorylation rates outlined by the ex-

periment of Fig.1,

the kinetic constants of the peptide substrates were determined. As shown in Table 2, the Thr(P)-peptide is a substrate as good as the Ser(P)-peptide in virtue of almost identical values of both Vmax and Km. On the other hand the low phosphorylation efficiency of the Tyr(P)-peptide is accounted for by both a 21-fold increased Km and a 7-fold decreased Vmax. AS a result of these two concomitant detrimental effects the phosphotyrosyl peptide displays a very low phosphorylation efficiency, comparable to that of the non phosphorylated substrate with a glutamic acid replaced for phosphoserine. Discussion The experiments described here support the concept that CKl is a Ser(P)- and Thr(P )-directed

Table 2. Kinetic Peptide

protein kinase and m

a Tyr(P)-directed

constants of CKl for a series of peptides the specificity determinant Vmax (nmol/min/mg)

protein

varying

kinase. These

for the nature of

Km (mM)

Efficiency (Vmax/Km)

EESpEESlT

24.6

0.083

296.3

J=TPWW

25.8

0.075

344.0

EEYpEQlT

5.7

1.800

3.1

FFFFESE

5.7

1.173

4.8

Sp, Tp and Yp denote phosphoserine, phosphothreonine spectively. The phosphorylatable serine is underlined. threonine was detected in any peptide.

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Table 3. Distinctive features of CKl vs. CK2 specificity determinants

crucial positiona efficacy of Glu/Asp vs. Ser(P)b order of efficacy of phosphorylated residuesc a) b) cl

CKl

cK2

-3(-4)

+3

very low

high

Ser(P)=Thr(P)>>Tyr(P)

Tyr(P)>Ser(P)>>Thr(P)

Ref. [5, 10, 161 Ref. [8, 10, 11, 141 This paper and ref. 14

features differentiate which is a specificity

CKl from CK2 in two respects: firstly phosphothreonine, determinant as good as phosphoserine for CKl, is hardly

recognized

by CK2 [8, 141; secondly

is indeed sumarized

the best known determinant for CK2 [14]. These two diversities in Table 3 along with (a) the location of the crucial determinant

and +3 for CKl

phosphotyrosine

and CK2, respectively)

which

and (b) the markedly

is ineffective

with

different

relevance

carboxylic aminoacids which are effective determinants for CK2 alone. At this stage, it remains to be ascertained whether the significantly ficient

CKl

EEEEESIT

phosphorylation and GVWAASG,

as specificity in the artificial

determinants. substrates

of peptides EEYEESIT respectively, It should studied

reflects

be recalled

and GVYAASG a positive

CKl,

role of tyrosyl

so far [5, 10, 111 a phosphorylated

of

more ef-

in comparison

in this connection

are (-3

to

residues

that although side chain

appears to be needed for efficient phosphorylation, it is hard to assume that such a requirement is invariably fulfilled in all protein targets of CKl, considering, inter alia, that inhibitor-2 of protein phosphatase-1 proved to be an excellent substrate for CKl after it had been exhaustively dephosphorylated by protein phosphatases [21]. Consequently, in the future search of non phosphorylated motifs that can replace the canonical phosphorylated consensus for CKl (SerP/ThrPX-X-Ser/Thr), tyrosyl residues my deserve some attention.

Acknowledgments This work was supported by grants to L.A.P. from Italian Minister0 per l’Universit8 e la Ricerca Scientifica e Tecnologica and Consiglio Nazionale delle Ricerche (Target Project on Biotechnology and Bioinstrumentation and grant 90.02460.CT04). J.W.P. acknowledges an NHMRC/INSERM Foreign Exchange Fellowship, the financial support of INSERM and is grateful to Dr. P. Jouin (CCIPE, Montpellier, France) for the provision of laboratory facilities. 1464

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References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

14. 15. 16. 17. 18. 19. 20. 21. 22.

Pinna, L.A., Meggio, F., Marchiori, F. and Borin, G. (1984) FEBS Lett. 171, 211-214. Meggio, F., Marchiori, F., Borin, G., Chessa, G. and Pinna, L.A. (1984) J. Biol. Chem. 259, 14576-14579. Fiol, C. J., Mahrenholz, A.M., Roeske, R.W. and Roach, P.J. (1987) J. Biol. Chem. 262, 14042-14048. Meggio, F., Boulton, A.P., Marchiori, F., Borin, G., Lennon, D.P.W., Calderan, A. and Pinna, L.A. (1988) Eur. J. Biochem. 177, 281-284. Flotow, H., Graves, P.R., Wang, A., Fiol, C.J., Roeske, R.W. and Roach, P.J. (1990) J. Biol. Chem. 265, 14262-14269. Onorato, J.J., Palczewski, K., Regan, J.W., Caron, M.G., Lefkowitz, R.J. and Benovic, J.L. (1991) Biochemistry 30, 5118-5125. Meggio, F., Perich, J.W., Johns, R.B. and Pinna, L.A. (1988) FEBS Lett. 237, 225-228. Litchfield, D.W., Arendt, A., Lozeman F.J., Krebs, E.G., Hargrave, P.A. and Palczewski, K.(1990) FEBS Lett. 261, 117-120. Girault, J.A., Hemmings, H.C.,Jr., Williams, K.R., Nairn, A.C. and Greengard, P. (1989) J. Biol. Chem. 264, 21748-21759. Meggio, F., Perich, J.W., Reynolds, E.C. and Pinna, L.A. (1991) FEBS Lett. 283, 303-306. Flotow, H. and Roach, P.J. (1991) J. Biol. Chem. 266, 3724-3727. Pinna, L.A. (1990) Biochim. Biophys. Acta 1054, 267-284. Tuazon, P.T. and Traugh, J.A. (1991) in Advances in Second Messenger and Phosphoprotein Research (Greengard, P. and Robison, G.A., eds.) vol. 23, pp. 123-164, Raven Press, New York. Meggio, F., Perich, J.W., Reynolds, E.C. and Pinna, L.A. (1991) FEBS Lett. 279, 307-309. Perich, J.W., Alewood, P.F. and Johns, R.B. (1991) Aust. J. Chem. 44, 233-252. Perich, J.W., Johns, R.B. and Reynolds, E.C. (1992) Aust. J. Chem., in press. Perich, J.W., Kelly, D.P. and Reynolds, E.C. (1992) Int. J. Peptide Protein Res., in press. Perich, J.W. and Reynolds, E.C. (1991) Synlett 577-578. Perich, J.W. and Reynolds, E.C. (1991) Int. J. Peptide Protein Res. 37, 572575. Meggio, F., Donella Deana, A. and Pinna, L.A. (1981) J. Biol. Chem. 256, 11958-l 1961. Agostinis, P., Vandenheede, J.R., Goris, J., Meggio, F., Pinna, L.A. and Merlevede, W. (1987) FEBS Lett. 224, 385-390. Marin, O., Meggio, F., Marchiori, F., Borin, G. and Pinna, L.A. (1986) Eur. J. Biochem. 160, 239-244.

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