8ea?ivcd 2) ~&V&I 1391; rcvi%d veraian rrcciv~d 8 April WJl The phusphapcptidc SW (~)~~rr(~~~Se~(P)~BItr~OIu~9~r~a~~Ic-’fhr, rtprodueingrhc I7.4+ repmcrll~ or@smln A* ineludingthe~crylrosi& (Ser.27) which ir trrgelctdby cnrieinkinas&! ~8%ryntheaizedand t&d nr mu&l xubttr#te Tat this cnaymc.ltn phssphtxylrtian e&ienry iicar~unllyhigher thttn that @I intact &rain (rimilut YRuM und t4 ,&I vs 50 ,oM 83. Canwrrclr thP tUly drphanphorylatedpcptidcSSSE%StT ia neataff~ctcctby CK-1 10 nny detcctrblPextQntund its g&~tnmyldrrivlrtive LIW3%SfT dirphtfl # m~rd than 5O~fddhi&herRmandu 5.told lower Vm, D$eclmpared to the p;rrcnt pharphopqxide.The relevanceal the indiriduul pharphoscrylrniduewhas ken us$errCdby eomprrinp rhc phaapherylrtianctlieienfirtr tar the pbaxpkpeptidrr l%3pEESlT, ESpEEi%IT ttnd SpEEEESLT: while the Rrvt ia n substrlrto t~lmsdttts gotaduo the trie Scr (&peptido C&=62 &4), and the third ORCix rltnorxt 81 pxw BYEEEEESIT (K,lp I.55 mM), ESp%&WC displayr a intermediatecffieisncy(K,,,e,e277pM). Thcs data in eonjunctionwith the finding that the phol;phapntapq&ic Sc~(~)“Ser(B).fc~(B].ferder(r). but ncithcr Pier(~~.tcr(P)~Ser-Sir nor Scr.Ser(P).Scr(P).er~u~~~urnd hr-Al~ghl.r-%crCP).lfcr(P), iwrcrdily phawpharyllrtcd by CiGl, support ttrc.eanecptthat CLI iz II phosphate dirretcd pratcin kinw rceogniainathekr(F)-X-X&x.X tmd, lessctffcicntly.the &r(B)-X-X-X&x-X metirn. Phespltopqxidc; Protein

kinirrc$qtceificity; Q&n kinaac-I: &C:tlrein

dephosphorylation 1a

tNTRODUCTION

Casein kinase-1 and -2 (CK-1 and CK-2) are spontancously active, Ca and cyclic nucleotide independent, ubiquitous Ser/Thr specific protein kinases termed after their preference for casein and phosvicin as in vitro substrates (reviewed in [1,2]). They can be differentiated from each other by a number of features which include substrate specificity. While the specificity detcrrninants of CK-2 have been elucidated with the aid of model peptide substrates reproducing the phosphoacceptor sites for this enzyme in several protein targets 111, the study of the site specificity of CKeI has been hampered by the paucity of information available about its physiological targets. In early studies which took advantage of artificial protein substrates we were able to identify the residues phosphorylated by CM-1 in different casein fractions [3,4], these residues being found to be located downstream from acidic clusters which invariably contained both glutanzyl and Ophosphoseryl residues. Moreover the finding that prior Correspondmce

address: LA. Pinna, Dipartimento di Chimica Biologica, via Trieste, 75, 35121 PADOVA, Italy. Fax: (39) (49) 807 3310 Abbreviutions: CK-1, casein kinase-1; CK-2, casein kinase-2; Ser(P),

phosphoserine; where one-letter symbols for amino acids are used; Sp denotes phosphoserine

Published b_vElseviw Science Publishers 8. V.

of such phosphosecyl

residues,

or

their conversion into dehydroalanyl residues, prevented the subsequent phosphorylation by (X-1 [3] and that seryl residues located at the C-terminal end of sequences composed solely of glutamyl and aspartyl residues were unaffected by CK-1 [S], groinpted us to propose that CK-1 might represent a ‘secondary’ protein kinasc, which depends upon a ‘primary’ kinase creating its recognition site. However, a synthetic peptide corresponding to the phosphorylation site for CK-1 in @-casein in which the three phosphoseryl residues were replaced with aspartic acid was recently found to be a fairly good and highly specific substrate for (X-1 [6], Here we show that the phosphorylation efficiency of similar peptides is dramatically increased if the carboxylic acid residues are replaced by phosphoseryl residues as they occur in @casein sites. In particular it was found that an individual phosphoserine at position - 3 and, to a lesser extent, at position -4 produced a striking reduction of K,,, and rise of V,,,,,. 2. MATERIALS AND METHODS The Ser(P)-containing peptides were prepared by the We Qf BOCSer(POaPhz)-OH in the Boc/Bzl mode of solution-phase peptide synthesis followed by hydrogenation (platinum oxide) of the protected peptides in SO%TFAIAcOH [7-91. The peptide, BEEE~~IT+JHMe, was prepared by the Boc/Bzl mode of solution-phase peptide synthesis followed by hydrogenation (palladium) of the protected peptide in 50070 TFA/AcOH. The peptide, SSSEESIT, was prepared by the

305

3. RESULTS A series of peptides derived from the phosphoacceptor site including &cnsein Ser-22 have been assayed as substrates for CK-1. The kinetic constants are reported in Table I. For all pcptidcs the only residue subject to phosphorylarion was the sesyl residue, the threonyl residue being completely unaffected (nor shown). The lowest K, (14 ,&I) and the highest V,,,, values are attained with the tris Ser(P)qeptide, SpSpSpEESIT, exactly reproducing the phosphoaccepror site, which is actually an even better substrate than its parent protein 8. casein AZ. Substitution of the two phosphoserines at

favaurable effect of phosphoseryl residues is due to their phosphate moiety is indicated by the deleterious effect of prior enzymatic dcphosphorylation of EESpEESIT (not shown) and by the failure of SSSEESIT (i.e. the dephospharylated derivative of the b&t subd strare) to undergo any detectable phosphoryl’ation (Table I). On the other hand the striking superiority of a phosphate group over 3. carboxylic group as a phosphorylation determinant does not appear to be merely due to its higher negative charge at pH 7,5 since it is even more evident at pH 6 where the overall charge of EESpEESIT and EEEEESIT is expected to be almost the same (see Table I, values between brackets).

Tnblc I Kinetic constants of CK.1 for a series of &cascin derived pcptidcs SubStrACe

“,dX l”rnDllbi”lmpl

p-case

A2

APP

s Km

h%P,

,nul

39.1

0.053

51.2

0.014

23,O

1.550

14.8

Glu- Ser(e)- Glu- Glu-Glu-a-Ile-Thr

35.5

0.271

128*1

Glu- Glu- Sex(g)- Glu-Glu-w-Ile-Thr

30.3 (33,80)

0.062

(0.13)

9.7 (9.72)

1.173

(4.34)

in

Se~(8)-SeE(E)-Ser(e\Ser (El -

Glu-Glu-W-Xle-Thr

Glu- Glu- Glu-Glu-Ser-Ile-Thr

Glu- Glu- Glu- Glu-Glu-&-Ile-Thr

Asp- Asp-

Asp- Glu-Glu-a-Ile-Thr-Arg-Ary

Glu- Glu- Glu- Glu-Glu-Sgg Ser-

Ser-

Ser-

Glu-Glu-Ser-Ile-Thr

En, and V,TIoxvalues were determined by double-reciprocal

25.0

1.000

N.D.

N.D.

N.D,

N-D,

743.7

3657 .I

617,7

(260.0)

8.2 (2.2) 2500

plots, constructed from initial rate measurements fitted to Michaelis-Menten equation. Average values from three or more experiments are shown. The standard error was 5 20070,Reaction conditions for peptide phosphorylation were as described in the experimental section except for the values between brackets which were determined at pH 6.0 (Tris-acetate buffer) instead of 7.5. The residue undergoing phosphorylation by CR-1 is underlined. Kinetic constants for the peptide Asp-Asp-Asp-Glu-Glu-Ser-Ile-Thr-Arg-Arg are drawn from [6]. N.D.: not determined due to the undetectable phosphorylation of the peptide up to 1 mM concentration.

304

The crucial requirement of CK-1 for a phosphoseryl residue at position - 3 is corroborated by the hehaviour of another series of short peptides composed by stretekes of variably phosphorylated serines. As shown in Table II the phosphorylation rate of SpSpSpSSp is lo-fold higher than that of SpSpSSp, while SSSSpSp, SSpSpEE, SpSSp, SSpSp and SSSp are not affected at all, 4. DISCUSSION The data presented here clearly demonstrate that the efficient phosphorylation of casein fractions by CK-I is critically dependent on the constitutively phosphorylateci serines located on the N-terminal side of those residues which are targeted by this enzyme. Not all the phosphorylated side chains however are equally important for site recognition by UC-1 as shown by variably replacing them with glutamic acid, While EESpEESIT is still almost as good as the parent tris Ser(P)-peptide, ESpEEESIT is less efficiently phosphorylated and SpEEEESIT is almost as poor a substrate as EEEEESIT. These data in conjunction with the finding that SpSpSpSSp (but neither SpSpSSp nor a series of peptides with phosphoseryl residues on the C-terminal side of serine) is a good substrate for CK-1 lead io the deduction that position - 3 and, to a lesser extenr, position -4 are the crucial residues sites, while phosphoseryl residues at position - 19 - 2 and - 5 and on the cacboxyl terminal side of serine are nearly ineffective. Seemingly however one or more residues on the Cterminal side of serine are also required considering the failure of EEEEES, but not of EEEEESIT, to undergo phosphorylation by CK-1 at Ser-5. The above conelusions are in agreement with the recent finding that

the PK-A dependent phosphorylation of Scr-7 in the glycopen synthnse l-14 peptide PLSRTLSVASLPGLamide is a prcrcquisite for subsequent efficient phosphorylarion of Ser-LOby CK-1 [12]. In that study it was also shown that by varying the spacing between the phosphorylated Scr-7 and the acceptor serine the peptide with three residue spacing was virtually not phosphorylated. The data presented here, conversely, support the view that a 3eresidue spacing is still cornpatible with fairly efficient phosphorylation by GK-1. This finding incidentally accounts quite well for the phosphorylation of cv52casein Ser-135 [3,§] at a site with a &r(P)-X-X-X-Ser rather than a Ser(P)-X-X,C-Scr structure, The apparent discrepancy between our data and those reported in [12] is probably due to technical hindrances encountered in the latter study where the amount of phosphorylated peptide available was conditioned by the preliminary enzymatic reaction with PKA. As a consequence the phosphorylation rates were determined at very low substrate concentrations (10 ,&I) and the kinetic constants of peptide substrates whose K, value exceeded 25 ,&I escaped accurate evaluation [12]. This drawback highlights the practical advantage of a methodology which uses synthetic phosphopeptides in which the various phosphorylated residues are placed at any given position with 100% certainty. While the superiority of phosphoseryl residues over carboxylic amino acids as specificity determinants for CK-1 is incontrovertible, the question remains as to whether efficient phosphoacceptor sites for CK-1 could be also created by other structural features capable of surrogating the phosphorylated residue. Actually multipie carboxylic residues encompassing the crucial - 3 and -4 positions do act as positive determinants, as 305

sl the pho~~horyl~~~d chains moreover can= not be merely attributed to their doubly negative clrnrglc (at pH 7,5) since it is fully maintained at gW 6 where the ing etfwt

phosphnscryl rcziidut:bears a reduced ncgativc elmrgc similar to that of Glu and Asp, This behaviour, among aha, differentiates CK-I from CL2, which ia very rwponaive IB chwnger of pN which reduce the nega(ivc

charge of its acidic determinants f 13,141. In conclusion, W-1 is clearly a ‘pko~pha~e.direcrcd’ protein kinnsc but apparently not so ‘scidophylic ale CK-2, which conversely displays no spccinl rcquirrment for phosphoacryl residues, whose potency as specificity doterminnnts for CKd2 is comparable to that of cnrboxylic acid residues [ lS]$ Ackl~nwl~~~otlcrrf~~ This work wns wppencd by Iwlian M.U.R,S.T. nnd C,N.R, (TRI~CI Project on Biotcchnolopy and Uioinrrrumenralion). J.W.P. nnd &,C,R, [honk ~hc Aunrralinn NaIionnl Hcahh and Mcdicnl Rcscnrch Council (Grant 890943) for finaneinl support. J.W.P. also ocknowlcdgcs the financial sitpporl of an NHMRC/INSCRM Foreign Exchnnge Fellowship nnd is grnraful IO Dr. P. Jouin (CCIPE, Monqxllicr, Frnncc) for the provision of laboratory faciliriea.

REFERENCES [I] Pinnn, L-A, (1990) Biochim. 8iophy.p. Acln 1054, 267-284.

161Awwrlnlr,

P.. Pinna, LA., Me&o, F., Marin, O., Gorir, J., Varrtlcnhc&, J.R. and Mrrlcvctle, W, (i9ol9) BEUS l&w. 353. 75-78, 171Prriulr, J.W., hlcwood, P.P. untl Johns, R,l3, (I99I) hw. J. Chum. 44,233w2$2. I81 Pcrich, f.W, and Johns, R.B. (1991) hw. J. Chum. 44,

W-403. ISI Pcrich, J.W, and Johns, R,R. (1991) hw. 405-409,

J, Chum.

44,

llW Mc#gib,B., Donclln Dcnw A. and Pinna, LA. (1981) Y. Biul. mm, 236, I 19%11961, [I I] Mcygio, I:,, Marchiori, F., norin, G., Chrana, B. and Pirrna,

L.A. (1984) J. Biol. Chem. 259, 14576-14579. 1121 Flotow, H,, Graves, P.R., Wang, A., Fiol, C.J., Rocxke, R.W. and Roach, P.J. (1990) J. Viol, Chem, 265, /4262-14269. 1131 Meggio, F,, Pcrich, J.W,, lohns, R.B. tend Pinnn, L,A. (1988) FEOS Lcrt, 237, 225-228. 114) Pcrich, J.W., Mcggio, F., Khnx, %A., Vnlcrio, R,M.,’ Johns, R,B, find Pinnn, L.A. (1990) Uiorhcm. Biophyr. Rra. Common, 170, 635-642. 1151 Litchficld, D.W,, Arcndt, A., Loreman, F-J,, Krebr, E.G., Harynve, P.A. and Palcncwski, K. (1990) FEBS Lc~I, 261, 117-120,