Volume lY91

0

ADONIS

291, number 2, 359-362 Federatlon

of European

FEES 10287

Blochemlcal

Sosletlts

00145793/91/53

October

1991

50

0014579391009952

imcreases turnover of 90

Heat shock

k

a heat

protein

shock

phosphate

groups in He%a cells Vincent

Eegagneux

Laboratowe de Blologre MoleWuwe

*, Michel Morange

amcl Olivier Bensaude

du Stress, Beprtemerzt de Broiogle. Ecole Normale Superreure, i’5230 ParIs Cedex 0.5, France Received

3 1 July

46 Ylled’Uitn.

1991

The 90 kDd heat shock protem (hsp90) IS a major phosphoprotcrn which associates various other ccllulae polypeptldes such ds actm, cdtmoduhn. steroid hormone receptors and certam protem-kmases LIttIe IS known about the function of hsp90 m recovery from stress In this report, we describe d dramatIc Increase m the rate of both phosphate uptake and dephosphorylatlon of hsp90 m HeLa cells submltted to acute stresses This Increased turnover of hsp90 phosphate groups might reflect a greater protem bmdmg activity of hsp90 m stressed cells Heat

shock,

Heat

shock

protem,

1. I[NTRODUCTION Heat shock as well as chemical stresses are known to have toxic effects on cellular functions These effects are mostly due to enzymatic inactlvatlon, a consequence of protein denaturatlon caused by cellular stresses [l-3] When cells are allowed to recover by replacing them in normal culture conditions, cellular functions are progressively rescued Recovery 1s probably due, at least partially, to the accumulation, in response to stress, of a specific set of proteins, the heat shock proteins (hsps) [4,5] Most heat shock proteins are already present m the absence of stress and ensure physiologlcal functions. For Instance, hsp60 and proteins of the 11~~70family are known to associate with polypeptldes and to catalyze folding and unfolding reactions in an ATP-dependent manner [6,7] Hsp90 associates with various protems such as, for Instance, actm [8], calmodulm [9], src-type protein kindscs [lo] and stelold hormone receptors [I 1,121 These complexes correspond to an actlvatlon step of these proteins, which involves conformation and/or location changes. Recent evtdences suggest that m stressed cells, heat shock proteins belonging to the hsp70 family are drrectly involved m renaturation processes, by forming

Protem

phosphorylatlon,

DCpartement de Blologle et GCnCtque du DCvclopUeduheu, Univcrslt& de Rcnncs I, 35042 Rcnncs

Carrcsporrdctr~c~ rrrlrlran 0 Moltcul,urc Sup&curc, 4329 8 172

du Stress, 4G tued’ulnr,

Uensdudc, Ldboratolre dc B~ologle Dcpdrtcnrcnt dc Blologq Ecolc Norm& 75230 Parts Ccdcx OS, rldnCC Fdx (33) (I)

cell

complexes with denatured polypeptldcs m an ATP-dependent manner [ 13,141. However, httle is known about the possible mvolvement of hsp90 m such reactions In this report, the dynamics of hsp90 phosphate exchange was found to be altered during and after an acute heat shock m HeLa cells. 2 MATERIALS

AND METHODS

2 I Cell culture cmd hur shk condmorrs HeLa cells (MRL2 stram) wet-c grown at 37°C and 12% CO2 in Dulbecco’s modified Edgle’s medmm (DMEM) supplemented with 10% fetal calf serum Hedt shocks were performed by lmmersmg the culture flasks m wdter bath 2 2 Cell lubell~ng and protm uttulym 32P-tdbelhng wds performed by mcubatmg thecells with 0 25 mWm1 [‘2P]orthophosphdtc rn culture medium After Idbcttmg, cells were washed twice m phosphate buffel sdlme dnd lysed m LSB (2 3% sodium dodecyl sulfate, 10% glyLcrol, 1% mercdptoethdnol, 125 mM Tns-HCI, pH 6 8) Chdse experrmcnts were performed by removmg the ‘2P-contdinmg medium and keeping the cells in stdnddrd DMEM with serum for variable times before lysls Sdmples were andlysed by 10% potydcrytdmlde get electrophoresis [15] or 2D get electrophoresls [16] as previously dcscrlbed Phosphoryldtcd proteins weredetected by dutoradiography or fluorogldphy with mtcnslfymg screens Protein synthesis was dndlysed by Idbcltlng the cells with 1OOflCllml [‘5S]methlonme (L200 Cilmmol) In stdnddrd DMEM

3 RESULTS *Prc~rr u&res\ pcment, Cdmpus Ccdcx, France

HeLa

AND DISCUSSION

In vivo phosphate uptake into protetns was analyzed in HeLa cells by metabolic 32P-labelling and 2D gel electrophoresis (Fig. 1) The ‘*P-labcllcd hsp90 position was detcnnmd by cornrgratm with silver-stamed hsp90 spot (Fly. ID) After 16 h of continuous labelling at 37”C, hsp90 appeared as 3 major phosphorylated

359

Volume

291, number

FEES LETTERS

2

October 199 1

k FIN I Stmiuldtlon of hsp90 phosphorylatlon durmg heal shock HeLa cells were “P-lnbcllcd for I6 h at 37°C (A). 1 h at 37°C (6) or I5 mm dt 37°C followed bj 45 mm dt 46 5°C (C) Newly phosphoryldted protcms were dndiyzed by two-dlmcnslonnl gel clcctrophoresls The powon of hsp90, ldcntllied by sliver-stammg, IS mduted (D)

protem at steady-state (Fig IA) as previously reported [I71 Nevertheless, a pulse-labelhng of 1 h at 37°C did not allow hsp90 to mcorporate detectable levels of phosphate as compared with other phosphoprotems observable on the same gel (Fig 1B) This suggests that the late of hsp90 phosphate uptake IS lather slow. as already reported [I 81 However, when cells were submltted to a 45 mm heat shock at 465°C during the pulse-labcllmg, hsp90 phosphate uptake was greatly stimulated, this plotem becoming one of the most strongly labellcd m these condltlons (Fig 1C) This mcrease of hsp90 3’P-labelhng was not due to the syntheSIS of this protem m response to stress since tt occurs well before recovery of protern synthesis and IS not affected by the presence of cyclohexunlde m the culture medmm (ddtd not shown). Such dn mcledsed phosphate uptake results m a hyperphospholylatlon of 11~~90,which was questtoned by contmuously labelhng the cells with [?$nethlonme or “P dnd analysmg labelled protems by monodlmenslonal electrophoresls (Fig. 2) The pattern of [35§]methlonine-labelled protems was ahnost the same m control and heat-shocked cells and allowed one to determme the hsp90 posItIon on the gel (Fig, 2A) The phosphorylatlon state of hsp90 WLISnot srgnlficantly nffected neither mimedlately nor several hours after heat shock, dlthough 3’P-labellmg of some other phosphoprotems (espccldlly of low molecular weights) was either mcreased or dnnmlshed III heat-shocked cells (Fig 2R) This IndlCdteS that the stimuiatlon of hsp90 phosphdte uptake by that shock does not Involve an hyperphospholyl&lon of this protem, Dephosphorylatlon of hsp90 was studled by chase experuncnts after “I’-lnbclhng dulmg I6 h at 37°C Chase was performed clthct i\t 37°C 01 durmg and ,Ifter ‘1 45 mm heat shock at 46 5°C “P-labclhng w‘ts obscrvcd by monodlmenslonol clcctIophorcsls which LIW Iltotcs qudntGcatton Howcvcr. we checked by 2D gel clcctrophorcw that, HISah cudy I cportcd [ 171, hsp90 ~~15 the mqor 90 kD,\ phosphoprotrm obscrvablc at the 360

steady-state (see, for Instance, Fig IA) hsp90 remamcd strongly labelled after d chdse of 3 25 h at 37°C In contrast to this, hsp90 labellmg ma1 kcdly dlmlrushed, as compared with that of other phosphoprotems, when

B

I ~g 2 h\pYO phclspholyl,irlon rtdtc durmg dnd .Ilicr hc‘tlt rhoch I-IcLd cell\ WCIc Libcllcd rol 16 h dl 37°C wIIh [“S]riiclluoiimc (A)or [“I’]ol Ihopho~pli.~lc (U) Ccllc wcrc lysrd :u the end of Ihc I,~bclhnp (Idncs C) (II dllcr d 45 iii111hc.ll shock dl J6 PC lollowcd by J rccovcry of 0 II. 3 II. OI I8 h ,II 37°C (corrcspondmp LIW.)~ ‘:I’- aid “5-l.~hcllmp WIG zonunucd tlu~~np ha ~Iiock .ind rccovciy. L~bcllcd p~otcms WIG

Volume

291. number

2

FEBS

A

LETTERS

October

199 I

8%

Fig 3 hsp90 dephosphorylatlon durmg dnd dfter hedt shock (A) HeLa ceils were “P-labelled durmg 16 h at 37°C and lysed at dlfferent times after d chase m non-radloactlve medmm Chase times were 0 h (open square). 3 25 h at 37°C (open cncle), 20 mm at 46 5°C (closed square), 45 mm dt 46 5°C (closed tnangle) or 45 mm at 46 5°C followed by 2 5 h at 37°C (closed clcle) Labelled protems were electrophoresed through a 10% polydcrylannde gel (B) Residual radloactlvc phosphate m hsp90 (mdlcated by dn allow) was measured by densltomctry and results (expressed ds percents of nntlal value) were normahzed to those obtamcd for other msJor phosphoprotems (mdlcated next the autoradlogram) Residual [7’P]phosphate levels m hsp90 are plotted as d functron of chdse time Time course of hedt shock and recovery are represented at the bottom of the figure

chase was performed durmg and after heat shock (Fig 3) Hence heat shock stimulates hsp90 dephosphorylat1on It appears from these observations that acute heat shock Induces an increased turnover of hsp90 phosphate groups A relatlonsl11p between heat shock protem phosphoiylanon and assoclat1on wit11cellular components has been observed m physiological condmons. GRP78 (a protem related to HSP70) IS phosphorylated only when m a free form (not complexed wrth Immunoglobulm heavy chams) [ 191 Concelnmg hsp90, it has been demonstrated that 111mammary cell lmes, estladiol or TPA increase sclcctlvely the phosphorylatlon of free lisp90 (not associated wnh estIad1ol rcccptor) [20] In this case, hsp90 phosphorylatlon might bc coupled wnh the dlssoclatlon of 11sp90-receptor complex which IS an early step m steroid-Induced receptor actlvatlon [21,22] Taking account of these data and of the fact thdt steroid receptor-hsp90 complexes dre dissociated by heat [23], WCpropose thdt Increased turnover of hsp90 phosphdtc groups m heat-shocked cells nnght reflect form‘ltlon and/or dlssociatlon of complexes bctwccn hsp90 and otlicr celluldl plotems. due for mstancc to the dccurnulation of dcndturcd ptotcms m these cells Such a n1odel dots not nccessallly irlvo]ve the activation of d dlstmct protein-kmasc. Indeed, hsp90 secrns to possess Cl sclfphosphorylating activtty [24]

the Umversue Pierre et Merle Curle and Grant DGA 87-124 from the Duectlon des Recherches Etudes, ct Techmques

REFERENCES [I] Nguyen. V T , Mordnge, M and Bensdude, 0 (1989) J BIOI Chem 264 IO487- IO492 [2] Bensdude. 0 Pmto, M , Dubols M F , Nguyen, VT and Mordnge, M (1990) m Stress Protems Induction and Function (Schlesmger, Sdntoro dnd Gdracl, eds ) pp 89-99, SprmgcrVcrldg, Berlin [3] Dubon, M F , HOVdneSSldn, A G dnd Bens,tude, 0 (1991) J BIOI Chem 266, 9707-9711 [4] Lmdqmst, S (1986) Annu Rev Blochcm 55, 1151-1191 [S] Lmdqmst, S nnd Crdlg, E A (1988) Annu Rev Genet 22, 63l877 [G] Rothmm, J E (1989) Cell 59, 591-601 [7] Bcckmdnn, R PI Mmzen, L A and Welch W J (1990) Scmnce 248. 850-854 [8] Koydsu, S , Nishldd. E . Kddowdkl, T. Mdlsuzdkl. F, Ilda, K , Hdradd, I- KdWgd. M , Sakdl. I-I dnd Ydhdrd, 1 (l98G) Proc Ndtl Acdd SCI USA 83. X054-8058 [9] Nlshldd, C . Koy.lsu. S , Sdkdi, H dnd Ydl1.md, I (1986) J BIOI Chcm 261. lGO33- I6036 [IO] Bruggc, J S (1986) Curr Top Mlcloblol Immunol 123, l-22 [I I] Cuclh, M G 6Bmdrt. N , Jung-Tcstdr I . Rcnon. J M , Bauhcu. E L . I-cr.mnsco, J R ,rnd Welch. W J (1985) EMU0 J 4. 313l3115 (I!] S.mch~~, r: Iz , raft, D 0, Schlcsmger, M J dnd Prdtt, W II (IYXS) J 13101Chcm 260. 12398 12401, [I31 Gdndudrrs G A , Pap.,~var\~hou. A G . Ruhock, P . Sllverstcm, S J dnd ~c)I~CCII~JI~. M C (1990) Cclt 61. lOi3-1020 (141 SbouyrJ. I), Gco~gopoulos. C and Zyl~ce, M (1990) Ccl1 G2. 339 -944 [IS] L,~enmrh. U,K (1970) N,rt+ rc 227 4x0 685,

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FEBS LETTERS

[16] Legagneux, V .Dubols, M F , Morange, M and Fknsaude, 0 (1988) FEES Lstt 231.417-420 [I71 Kelley, PM and Schlesmger, M J (1982) Mol Cell Blol 2, 267-274 [18] Lees-Mtller. S P and Anderson, C W (1989) J BIOI Chem 264, 243 l-2437 [I91 Hendershot, L M , Tmg. J and Lee, A S (1988) Mot Cell El01 8.42504256 [ZO] Lahoott. I-I , Thorsen, T and Aakvaag, A (1990) Mol Cell Eadocrmol 74, 3343

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1991

[21] Pratt, W I3 , Sanchez, E R , Bresmck, E i-l, Meshmchl, S , &hewer, L C , Dalman, F C and Welsh, M J (1989) Cancer Res 49,2222-2229 [22] Dew, M and Gustafsson. J A (1989) Cancer Res 49, 227S2281 [23] Brcsmck, E H , Dalman, F C , Sanchez, E R and Pratt, W B (1989) J BIOI Chem 264, 4992-4997 [24] Cscrmely. P and Kahn. C R (1991) J BIOI Chem 266. 49434950