, BNAME:'jOB 11 PAGE: 1 SESS:" OUTPUT: Tue Dec 7 06:56:39 1993 1!305/teaml/science/originall238alI56I8

JT I JS(v)

.. REPORTS--________________________________ __

. Association of Transcription Factor APRF < and Protein Kinase Jak 1 with the IL-6

Signal Transducer gp 130

ate-earlyaenet (12-14). The bindinc spec­ifidty of APRF is shared by the interfaut .., (IFN-'Y)-.ctivation factor (GAF) (14), which .. Identical to the ISGF-la p91 protein (8, 10). Because APRF croD-IaCII with an antiserum raised apinst the ISGF-3a p91 NH1-terminus, it is likdy ID be related to p91 (J3). Thus. the ........ pathway tX tu; may be similar ID _

Claudia LUtticken, Ursula M. Wegenka, Juping Yuan, Jan Buschmann, Chris Schindler, Andrew Ziemieci<i, Ailsa G. Harpur, Andrew F. Wilks, Kiyoshi Yasukawa. induCed by other cytOkines.

Tetsuya Taga, TadamHsuKishimoto, Giovanna Barbieri; Sandra Pellegrini, Michael Sendtner, Peter C. Heinrich,

Because activation of ISGF-la by IfN.cI involves tyrOSine phosphorylation of the pIU, p91, and pM components and GM activation by IFN-'Y requires the lyIOIine phosphorylation of p91 (9, 10), wc ima­tigated whether APRF becomes tyroIiDe .phosphorylated in response to IL-6. I.ysara from untreated or IL-6-tteated human ~ atoma (HepG2) ceDs were aubjecud 10 immunopredpitation with an IllUiser-. to the NH1-terminus of p91 and pM (md­p91,p84) and analy%ed .by an immtlDlDYot wtthmonoclonal antibodies to plw.ispboq.. rosine. The IL-6 induced the appearance.

. Friedemann Horn· .

Interleukln-8. leukemla inhibitory facIDr, oncostatin M. Interleukln-11, and cilialy neur0-trophic factor bind ~ recaptor complexes that share the signal transducer gp130. Upon binding. the ligands rapidly activate DNA binding of acute·phase response factor (APRF). a protein antigenicalyrelaled to the p91 subun/t of the interferon-stimulated gene factor~ (JSGF-3a). These c:ytokines caused 1JroSine phosphorylation of APRF and ISGF-3a p91. Protein klnases oflheJak familywereal;so rapidly tyrosine phosphorylated, and boIh APRF and Jak1 associated with gp130. These data indicate that Jak family protein k1nases may participate in Il-6 signaJing and thalAPRF may be activated in a complex with gp130.

Interleukin-6 (11.-6). leukemia inhibitory .factor (UF), oncoaatin M (OSM), and ciliary . neurotrophic factor (CNn) are members of a family of cytolcines and neu­ronal differentiation factors (I) or neu- ' rolcines (2). These f.tctors exen pleioaopic effects on multiple cell types and bind to composite receptOlS cOntaining the signal nansducer gp130 (3). The neurolcine Keep­tors belong to a mperfamily of cyIIikine receptors sharing bod\ structural and imc­tional features (4). Cytokine receptolS ini­tiate related signaling pathways chaclcter­ized by association with and activation of protein tyrosine ldnases of the JaIc family (5, 6) and the reauitment of latent cyto­plasmic transcription factors of the ISGF-3a family by tyrosine phosphorylation and subsequent ngdear ttanslocation (7-10).

c. LUIIick8n. u. M. Wegda. J. Yucwl. J. BuscDnam. P. c. Heinrich. F. HaD. Instibje for Biochemistty, RWT1i Aachen. Pamebataasse 3), 0-52057 Aachen. Gtwmany. c. Sc:hirder, ~ ~ I'f¥icIans and SlJrgIans cl CoUnbia University, IlapaItrneIt of MedicN. New YOtk. NY 10032. A Ziemiecki, l..abotatory for Clinical and Elcpe&ineIItaI • Cancer Research, UrMrsiIy cl Berne. Te'alaus­trasse 120, CH-3OO4 e.m. Switzerland. A G. HaIpu" and A F. Wiles. ludwig InstUe for Cancer ReseM;h. RoyJJ MeRxune Hospital. ~ 3050. Australia. K. YasUcawa, e;otec::hnoklgy Reseatch l.atDaIcIy, Tosoh COIpoIaliOIi. 27&1 Hayakawa, Ayaa. !(ana. gawa 252, Japan. . T. Taga. InsIIlM for MaIecUar and CeIUar lIoIogy, Osaka \kliverIiIy, 1-3; YamadaoIca. SWa. 0SIIIIa 565, . Japan. T. Kishimolo. 0epa(iJ._ 01 Mecicine ID. 0SIIIIa ~ wrSity Medical SchOoI.2-2, Yamadaoka. Suit&. Osaka 565.Japan. G. BaItIieri and S. Pllegrinl.lk1ile' INS8IoI 276. InsIN Pastai'. 75n4 Paris Cedex 15. Ftanoe. M. SendIner, Mal( PIcn:.i; InslihAe for PsychiIIry, Am KJopIerspiIz. ().82152 IIIIrtnsried. Germany.

The IL-6 signal transduction pathway has not yet been elucidated, but the in­volvement of Jalc family members has been proposed (ll). A latent cytoplasmic tran­scription factor, APRF, is rapidly actiwted in response to IL-6, UF, OSM. nAl, and CNTF (12. 13). Alter activation, the 89-kD protein binds to IL-6 response elements identified in the promoter regions of various IL-6-induced plasma-protein and immedi-

p91 APRF

p91

B

116

97.4

two major tyrosine-phospborylated prouin bands of 91 and 89 ID (Fig. W. The 91-kD band was also observed aCta- JFN..y treatment tXHepG2 cells (Fig. lA) and .. immunopredpitated by other antisea 10 p91, demonstrating its identity with ISGF-3a p91 (lS). Tyrosine-phosphorylatecl p91 has a higher apparent molecular abe IIfiu SDS-polyacrylamide gel elec:tlupluesis (pAGE) than unphosphorylated p91 (10). In fact, when immunoblotted with 8DDse-

C Anl-PTtJa) - ~ ~ .!!. Pr --+-.-.

66~:::":::===:=I

Fig. 1. Tyrosine phosphory1ation of APRF and ISGF-3a p91 in response to IL~ (A) HepG2 eels (10') were treated for 20 min without or wiIh tunan IL~ (100 unitll/ml) or IFN-y (251l9'm1). eels were rinsed with cold phosphate-buffered sa&le and lysed in O.5-ml1ysis bulfer (24) for 20 oWl at O'C. Proteins in the Iysates were irnrTlJnopnlCipilated with anti-p91.p84 (4 pJ) (25). Irrm.ne couple .. were separated by SOs.PAGE (7% geI), transferred to poIyvinyIdifluoride membrane (Qiabrane, Diagen). and probed with monocIonaI anlibocIies to phospholyrosine (PY20. ICN). To wrify application of equal protein anoc.rts. we sIripped and rEiprobed the blot with antisenm to p91 (25). The unphosphorylated and phosphorylated forms of p91 (lower and I..W8f band. r~ are incfacated by arrowheads (26). 19G. im'an)g1obul'1fl G. Molecular size markers are indicated on fie . left in kiIodaltoos. (8) Phosphoamino acid analysis of APAF. The 32p..Jabeled HepG2 eels were stimulated with IL~ or IFN-y as atJo.te. The APRF and p91 were irnmunoprecipiated with anti-p91.p84, protein irnn'ulobIoUed. and visualized by autoradiography (Jower panel). the phosphorylated APRF band (brackeCs) was excised and subjected to phosphoamino acid analysis . (27) (upper paneI). The positions 01 utileled phosphoamino acid standards are indieaIed S, · serine; T, threonine; and Y. tyrosine. (C) Inhibition of bincflllg of APRF to DNA by arllilodies to phosphotyrosine. Nuclear extracts were prepared from HepG2 cells l1eaIed for 15 min wiII\ I.~ (100 units/m!) as described (12) and were incubated 0Iiemight at O"C with allbldes to phosphotyrosine (anti-PT) in the absence or presence of phosphotyrome (1 mM). The DNA bindng of APRF was examined in a gel reta'daIion assay (28).

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IIIDl to p91. the IL-6- a~d IFN-l-induced ~ce of • retarded p91 band was observed (Fig. lA). Activation of p91 DNA binding by fL.6 was also demonstrat­ed by eel retardation assays (I S).

The 89-kD phosphoprotein band in­duced by 1L-6 axresponds to APRF (16).

205

116

.7.4

66

e L-6 ,. C • • is. 8 2 5 ' 15 30 60 mIn

Jak1

IgG

. J -~~ ..... ~ .... -I... Jak1 116 _ f!II!!"""" <.: . .... - , , .f"

AnIhJak1 imn'ulobiot

B '. e L-6

~ i ~ 5 15 30 00' rrin

116~. t4 .lii.r!yk2 AnIi-phosphltJrosIe inmunobIoI

I _ _ .;.. __ .. ~Tyk2 116-1 !

'--""''''''''~-~Tyt2~immunobIot-----'

c HepG2 FAO h u.. _

I.L I- _

::; 5 ~

I .. - ", I· .W~ ··--r

Jak1

.~~~:~F fig. 2. Tyrosre phosphorylation of Jak family protein kinases in response to IL-6 and other cytokines, CA) Tme course of Jakl phosphoryl- . ation in response to IL-6, Lysates from HepG2 <:ells treated wih L-6 (200 units/(T11) for various periods (IabeIed in minutes) were imn'ulopre­cipiIaled with a1tiserum to Jakl (6) or preim­fTUl8 senm (pee) and irnnulobIotted with an-. tibodies to phosp/"lotyI:osine (upper paneI). The position of Jakl (130 kO) is indicated. We verified equal protein load by reprobing with antiserum 10 Jakl (lower panel). (8) TIfTl8 eotne of Ty1<2 phosphorylation in response to !L-6. anaIyzed as described above for Jakl (29). The posiioo of Tyk2 (134 kO) is il'l!icated. (C) Induction d Jakl . APRF. and ISGF-3a p91 tyrosine phosphoIytalion by different cytokines. The HepG2 eels were slirrulated for 10 min wiIhout or will tunan IL-6 (200 units/ml). OSM (20 ngImI). or FN-y (25 1'9'mI). and FNJ cells (30) were treated for 10 min with tunan UF (25 1l!)tnI). rat CNTF (20 nglmI). or tunan IL-ll (SO 1'9'mI). CeI tjsaIes were immunoprecipitated with antiserun 10 Jakl or anli-p91.p84 and irmulc>bIoned with antibodies to phospholy­rosine (31).

After immunopm:ipitadon from 1L-6-treated. lZp_labeIed Hep02 celIs, phospho­serine and phospbotyrosine but no phoa­phothreonine weft detected in an APRF prote!A hydrolysat£ (Fig. 18). The presence 01 phosphcxyrosine in the DNA-binding form of APRF .. con6nned by the obser­vation that incuI:.don with antiphosphoty­rosine antibodies spedfically interfered with the formation oI1he APRF-DNA complex (Fig. IC). These findings show that both 15GF-3a p91 and APRF are tyrosine phoa­phoaylated in raponse to IL-6. The time course cl this df'ect (IS) Corresponded closely CO that deunnined (or the induction 01 APRF DNA-llinding activity (12), indi­cating that latmt APRF is activated by tyrosine phosphorylation.

Tyrosine pro«dn kinase activity copre­cipitates with gp130 from IL-6-treated celIs (In. Antisera co the Jak family members T y1c2 and Jalcl ftre used to test whether

: _' <§ fegp130 " : . ' ,i! .

: . "feJak1 116 • It .

IrmUlO- ~ AnlhJak1 blot: phosjA.."osme

fig. 3. AssociaIian of Jakl with the IL-6 signal transducer gpl30. Untreated HepG2 eels (right panel) or eels treated for 5 min with L-6 (200 UliIsImI) (leA panel) were washed wiIh icEH:oId phosphaIe buffered saline and cdIect­eel. CeIs were aoss-linked (32) and lysed. and proteins were imuloprecipitated with preirn­mune senm (pe) 01 anbbod"l8S to gp130 or Jak1 as in<icated. The croSs-&nker was then cleaved upon boiling in sat11'Ie buffer. and proteins were anaIyzed by imrTulobIotting wiIh antiboOes to pho6photyrosine or Jak1, as n. caled.

these kinases respaad to JL.6 stimulation cl · HepG2· celIs. Boda Jald. and, to a lesser -extent. Tykl 1fta tranJiendy . tyrOSine phosphorylated Ia.IapONe to IL-6 (Fie. 2. A and 8). Also. UF. OSM, CNTF. and 1I,11atfmulated _ tyrotine phosphoryla-tion cl Jul. as .. as cl APRF and p91 (Fig. 2C). Thus. ~tion of these proteins appean ID be eenerally induced upon activation of 11'130. The maeiUtude of the response to clif'erent C}'to1cinea var- , led. but the reIadw extents of APRF and­Jul phosphorylman changed coordinate­ly. Furthermore. die time course of L6-induced Jul and T jk2 tyrosine phospho­rylation matched wdl with the one • served for APRF (IS). Therefore, JaIc &In­ily members mar be involved in the tyrosine phospbcqbdon and activation cl APRF. . .

The tyrosine pfophorylation eX Jalcl was also induced IIy IFN..., (Fig. 2C). In contrast to '1L:-6. which only transiendy activates APRF 8Dd p91. IFN-l induces p91 DNA bindincactivity for several hours in HepG2 cells ('4). Similarly, IFN-l In­duced the tyro5iDe phosphorylation of both p91 and JAKl '- at least 1 hour (15). confirming a dole correlation between ty­rosine phosphoryLdon of members of the .Jak and ISGFJa 6milies.

To examine 1IIaethcr JaIc family mem­~ may be 8aldared with gpl30, we studied the possI6, of coprecipitations of Jul and 11>130- When lysates from IL-6-treated HepG2 cds were immunoprecipi­bated with antiII:nm co Jakl. coprecipita­don of ~ted gp130 was observed (Fig. 3) (l8). ~y, JaJct was copredpltaraf upon bnmunoprecipita­don of gpl30. Sciawlation by IL-6 was not required for dUI dect (Fig. 3). indicating that Ju1 ~ interacts either di­RCtly or indiru:dr with JP 130 and hence 11

B 1 2 3 •

FIg • . 4. ~ of gpl30 with APRF from L-6-stimJlaled hepatoma eels. (A) HepG2 or FAO cells were Rated for 10 min with tunan IL-6 (200 units/ml). OSM (20 ngImI). or UF (25 ngImI). CeD Iysates prepared in the pres­ence or abSence of dithiottveitol . (On) were irmuloprecipitated

with ~ antibodies to le ~"~"-f'=:::::;:~:::! gpl30 01 with ri-p91.p84. as ~QD __ I",..

indicaled, and were irnmJnobIot-ted with antibodies 10 phosphoty-rosit*t. The open arrowhead indicates the position of the c:opre4!1nd 19O-1d) protein after LF treatment. Lane M shows molecular size markers (IabeIed on 111ft in kiIodaItons) •. (8) The coprecipitated 1~ phosphoprotein readS with antixIdies to s;p130. HepG2 cells treated for 10 tnin wiIh IL-6 (200 ooils/ml) were lysed and Iubjected 10 imuqJIec~ with antisen.m to gpl30 (lane 1) or with anli-p91.p84 in the absence of OTT (lane 2). lbe ri-p91.p84 imrTulopre­cipitates from 2 x 107 cells were washed wiIh lysis bulfer: wi1hcU OTT (lane 3) 01 with 1 mM OTT (lane 4). The supemalants of both wash stepa were .lbjecIed to a seccncI ~ecipilation with antbodies to gp130 (33). All in'mJnoprecipites were then analysed by irTlrrunobIotting wiIh antbodies 10 phosphotyrosine. .

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li1cely to represent at least part cl me tyrOSine kinase activity copreclpitaUd 1ridl gpl30.

HomodimeriJadon « gp130 and .aiva­don of its assoc:iated potein kinase .aivity have only been obIcIwd in the .t.Dce of reducing agentI (In. Thus, disul6de bidea appear to be impottamt in the ",,,.WM' of an active receptOr complex. When ..... ~

, pedpitadon c:J bepaaoma~ll lysMa wiIh anti·p91,p84 was per:bmed under DCIlI'e­

duc:ing c:onditions, a pbosphoprocciD IimiIar in me (145 kO) to gpllO was copre+ \icarul from ~ted Hq(:;Z cells ~ 4A). The 14S-m protein could be releaRd fiom the immune complexa by dit:hiodrre:ird and • then immunoptecipiJlel by antibxliies ID gp13O, proving its ideotity with gpllO (Fie. 4B). Several antisera ID p91 that do DOt

recognize APRF fa&d to c:opecipitate gp130 (15). Theseuperiments do DOt show whether the association of APRF ...111'130 is dirCct· or mediated by other ",cbeL However, because c:opecipitatioo of 1P13O is observed only in the absence of ~. thrcitol. an active gp130 homodUaer seemS to be required fOr its interaction wiIh APRF. Therefore, association c:J APRF 1riIh 1P13O is lilcely to be ligand-ioIuced and .., dim::t the factor into a complex with gplJO..asso. dated tyrosine kinases. Similarly. alig3nd. induced association of p91 with, the epider­mal growth factOr lUlIep(or has reccs:td, been reported (19). Aft« tRattnent olra hepa­toma (FAO) cells with UF, gpoo and a 190-kO protein wee coprecipiaraf wiIh ·anti-p91.p84 (Fig. 4A). This prcniD, pr0b­ably represents the UF receptor, wLich bet­erodimerizes with gpl 30 upon UF ('eaI mmt (20). Although OSM has been prDpO"ed ID '

bind to a gpl30-LIF receptor hamdimcr (21), only gpl30 was coprecipiallaf fiom OSM-treated HepG2 cells (Fig. 41\).

These data indicate that the RcJWing cascade induced by 11,-6 and reWal Dcmn is similar to that initiated by ~ cytO­

kincs and is characterized by actinbon and

tyrosine phosphorylation of ISGF-la-mat· cd transcription factors and of Jak family protein kinases. The observation that me ttu)SCription factor APRF associates with the iP130 signal transducer upOn IL-6 aam­ulation suggests that a aan.scriptiOll &ctor can be regulated by Its physical intencdon with a plasma membrane receptor.

REFERENCES AND NOTES

IR •••

21. D. P. Gearing et • .• IbId. -. 1434 (1~ 22. J. A. Cooper. B. Mo SeIar\ T. HInIr. ~

EtrzymoI. Ill, 387 (1083). 23. A. YOIhInua end H. F.l.odiI!h. Mol. 011. aat tl.

70S' (1992). 24. TheIysls~COIIlltlnedSO""~(pH7.5L 15D

".. NaCI, 1 mM EDTA. o.s NP-40, 1 ....... ~~ ".. NIF. 0.75 n.. lA ,' __ ,--',' .............. 15" glycerol, ..., • JICIIIIiIf -=tI d aprOCIniI, pepmIr\, end ~

25. C. SchIndIer. x.·Y. Fu. T. __ R. ,.. ., l-E. 0erneI Jr., Aoc. NtIII. Aad. Bd. c--.. l8II

, (1992).

1. J . Bazan, Neutr:1n7, 197 (1991). 26. BecauseAPRFllnetdelectedonln ........ t.y 2. P. H. PatleraonMdH. ...... Ceo772 (--.123 d-p91.p84, orIy !he 8n'IIU1I d ~ jJidIiI

(1993). CCIIAcI be enaIyzed on .. 1*Il

3. Mo Hibi et Ill., bid. 83,1149 (1990); N. Y.Ip« .. , 27. ~(1~ ~ w: .Q:.: I 1Jid.89, 1121 (1992). end aen.m-free medk.m. MM ItindIIiIIIt ..

4.. J. Bazan, Proc. NlIII. ktId. ScI. U.SA fit, eB34 L" Icto.L.. ...... ____ (1990) '" 0( .. '..-., ..... --were IyMd. nwn.~

5. L Firmbacn.Kraft. Mo &1wt. T. Shows. R. DaIIa- =. :: ::.:~~ A::: F.-a. J . J. KroIewskI, 01r::0g6ne 5, 1329 (199O)'~was excised end hydrolysed tor 1 hcuat11O'C' ~~r,;(1~~ ~~~ U 6 N HO. PhosphoIMniIO.ad 1n8IyIi5- r:.

, 2Z7 (1~); O. SWllllloille" et·III., Prot:.~ ~~two-<linell.lorlllthD-layer ... , ... Actld. Sci. U.SA 90, 8429 (1993); L S. 28. Gel ---..,;........ done

- ~ et Ill., Cel7., 237 (1993) . , ..... _, assays ... • ..... bd .. A. F. \Wks et at., Mol. Cel BioI. 11,2057 (1991). (!2). The 32p-labelec:l ~ oil; _ h ....

7. D. S. 1<essIer, S. A. VeeIs, x..Y. Fu, D. E. Levy, 5 -GAlCCIIC1GGGMT1CCTM' . ~ Genes DBv. 4, 1753 (1990); S. f'eIIeIJti.., C. ~ rep ..... 1g .. pracimII ,.,. ...... Sc:flindler, Trends Bioctwm. Sci. 18, Dl (1aiI3); .dlheralClz~ogIabuInPflll"C*ir(lr)-A. C. Lamer et at., SciI!In::e 261, 1730 (1993); S. wed as • probe. fU.Jamison, K. a.en. s. Cohen, bid.. p. 1733; 29. Antisen.m to TykZ was railed Md·

T

, pdId H. B. Sadowski, K. StLIBi, J. E. 0arneI Jr~ '" Z. against. ~ .... paIIia GiIrnan, bid., p. 1739; It Shuai, G. R. sa.. L Mo COIllailllllg. porIiOn d human Ty1c2. J<.err. J. E. DImeI Jr., I:Jid.. p. 1744. 30. We used FNJ eels to,...... ...... ar lE.

a. T. Decker, D. J. Lew, J. MirI<oIIiIch, J. E. DarneI CNTF,and Le 11 onJak1.p91,IWldAfIIF ____ Jr ~ EMBO J. 10, 927 (1991). phosp/lOIyIaIIo because HepG2 cell ......

.. C. SchindIer, K. SIuIi, v. R. Prezioso, J. E. DameII ed poorty to Ihese cytokines (15). Jr~ Science 257, 809 (1992). ,31. In severallanas d Fig. 2C, twoAPfl" ..... 1II

10. It SOOai, C. SchindIer, V. R. PrezIoso, J. E. DarneI end 87 leD) .. Clb8erYecL lheIe .. C-' bJ. Jr~ ibid. 258, 1808 (1992). ' cIIerenI serine phoephoIyIaticn ........ N'fF

11. N. StahI and G. D. Vancopouloe, CeI 74, 587 (15). (1993). 32: Qoas.Iinking was peibmed by 11 ....... 1 rI.

12. U. M. Wegenka; J. Buscmwvl, C. liiIIic:.:Mn. P. C. peIIeted cells In p/losptIIIIe b6red ... .. . Heinrich, F. Horn. Mol. Cel BioI. 13. 216 (1993). .100 ,u IIOdUn ~ and ........ . 13. U. M. Wegenka et Ill., in prIpIW'8Iion. c:ross-Ir*8r ~ 14. J . Yuan et 1II.,1n pepaalcn (DSP. 0.5 mM) for 30 "*' at 4"C ..... bid 15. C. l..iitticken, J. Y\.a1, F. Hem, ~ cfeta. (23). 16. The same 89-kD bend_ inTrulOpiec:\oilaled 33. The ... 1nOI""'1OCb ........... 1III IIIltIxdesto gp131t -S Ir

wit! anti-p91,p841rom anAPAF pepai .... , pw!- irnrTulopI~ did net nICOQiiallJ13O ill led by specific 0NA-IIiniIy ct.O!1lIILVaph)'. mYulobIoI exper'.ments. 1hefefare. cI!KI dIIB>

~ proving Its Iderdy wIIh APAF (13). lien d gp130 on !he blot was not ~ 17. Mo Mu-akami et .. , Sciance 260, 1808 (1aiI3). 34. Supported by granIs tan .. ou... Fe. 18. Ccprecipilaliond Jak1 and gp130was~ , dulgsgemeillChall, Bom. ~.sbylll

by a cleavable ~ but ccUd Il1o be Fonds der 0Iemischen h:lJsIrIe. .. l1li* A. 'CbSeMd wIthoIA crosH1kIng. Tyk2 ..., copre- NorcIleIm and J. KtIeg for help wiIh ..,.... clpitated with gp130 from HepG2 JysaIes.IIbeIt to no acid INIysIL • smaller extent ... Jak1 (15).

19. x.·Y. Fu and J . .J. 2hang. Ce117., 1136 (1993). 20. S. [)avis et 8/., Sc:isnce260, 1805 (1~

"

' 29 September 1993; 8CC8pled 16 Iba,ta 1993

~ I