tissue-specific posttranslational modification of the small heat shock protein hsp27 indrosophila

EXPERIMENTAL CELL RESEARCH 223, 1–8 (1996)ARTICLE NO. 0052

Tissue-Specific Posttranslational Modification of the SmallHeat Shock Protein HSP27 in Drosophila

RAQUEL MARIN,* JACQUES LANDRY,† AND ROBERT M. TANGUAY*,1

*Centre de recherche du CHUL and Laboratoire de genetique cellulaire et developpementale, RSVS, Pavillon C. E. Marchand, UniversiteLaval, Ste-Foy, Quebec, Canada G1K 7P4, and †Centre de Recherche en Cancerologie, Universite Laval,

L’Hotel-Dieu de Quebec, 11 Cote du Palais, Quebec, Canada G1R 2J6

INTRODUCTION

Drosophila sHSPs (small heat shock proteins) are ex-pressed in the absence of stress in specific regions of In contrast to the families of high-molecular-weightthe central nervous system and in gonads of young HSPs (heat shock proteins, HSP70 and HSP90) whoseadult flies. In these two organs, the sHSPs show a cell- participation in a number of basic cellular processesspecific and developmental stage-specific pattern of has been well documented [1, 2], the function(s) of theexpression suggesting distinct regulation and func- small HSPs remains unclear. Evidence for the involve-tion(s) of each individual sHSP (R. Marin et al., Dev. ment of human and Drosophila HSP27 in protectionGenet. 14, 69–77, 1993). Since mammalian HSP27 has against thermal and oxidative stresses has been pre-been reported to be phosphorylated through a com- sented [3–7], and gene transfection studies have shownplex novel cascade implicating distinct kinases, we ex- that overexpression of HSP27 in mammalian cells is aamined whether two of the sHSPs (HSP27 and HSP23)

sufficient condition for conferring thermoresistance [7–exist in different isoforms as a result of posttransla-16]. Mammalian HSP27 behaves in vitro as an actin-tional modification in vivo. HSP27 and HSP23 were an-capping protein, suggesting that this protein may bealyzed in various tissues in unstressed and heat-involved in the regulation of the dynamics of actin mi-shocked flies. Four isoforms of HSP27 were found tocrofilaments [7–9]. Human HSP27, murine HSP25,be constitutively expressed in the nervous system andand bovine a-crystallin have also been proposed to actin testes and two in ovaries. The proportion of theseas molecular chaperones [10]. Under heat shock condi-isoforms relative to each other was specific to a giventions, these sHSPs bind unfolding proteins, preventingtissue. In the case of HSP23, two isoforms were ex-nonspecific aggregation of the substrate protein. Phos-pressed in the heads and in testes of unstressed flies.phorylation is another interesting property of sHSPsIn ovaries, a low level of a single isoform of HSP23 wasand is observed in response to a number of differentfound. Heat shock caused an increase in the amountstimuli. This phenomenon has been particularly wellof preexisting HSP27 and HSP23 and the appearance

of additional isoforms in ovaries. Susceptibility to documented for mammalian HSP27 [4, 10–14, 17],phosphatase treatment indicated that isoforms of where phosphorylation occurs rapidly following expo-HSP27 were phosphoproteins. This was further sup- sure to stress conditions, and in unstressed cells uponported by in vitro experiments in which Drosophila stimulation with mitogens and other inducers of differ-sHSPs were incubated with purified Chinese hamster entiation [for a review, see 18]. Phosphorylation ofHSP27 kinase. Only HSP27 was shown to be a substrate mammalian HSP27 occurs at the same serine residuesof this mammalian HSP27 kinase. The present data in vivo in different organisms, and these sites are lo-suggest that tissue- and HSP-specific posttranslational cated within a common sequence motif R-X-X-S, sug-modification systems may modulate the function of gesting that the same protein kinase is involved [13].these proteins in different cell types. Furthermore, the HSP27 protein kinase appears to be homologous to thesignal transduction pathways leading to phosphoryla- MAP kinase-activated protein kinase II (MAPKAP ki-tion of the sHSPs are conserved between mammals and nase II) [19, 20]. The activity of HSP27 kinase is highlyDrosophila, and the sHSP kinase cascade may be de- sensitive to treatment with protein phosphatases [20]velopmentally regulated. q 1996 Academic Press, Inc. and can be activated in vitro by mitogen-activated pro-

tein MAP kinase (MAPK), indicating that HSP27 ki-nase may be linked to signal transduction pathwaysinvolving MAPK. It has also been suggested that thestate of phosphorylation may be an important modula-1 To whom correspondence and reprint requests should be ad-

dressed. Fax: 418-656-7176. E-mail: [email protected]. tor of the protective function of the small HSPs in cellu-

1 0014-4827/96 $18.00Copyright q 1996 by Academic Press, Inc.

All rights of reproduction in any form reserved.

/ m4861$3057 01-24-96 00:14:42 eca AP: Exp Cell

2 MARIN, LANDRY, AND TANGUAY

organs from 50 heat-shocked or non-heat-shocked flies were placedlar physiology although it may not be essential for thein tubes containing 150 ml of pH9/NP40 buffer (25 mM Tris–Hcl, pHchaperone activity of HSP25 [15].9.0, 1 mM EDTA, 1 mM PMSF, and 1% NP40), and homogenized

HSP27 and HSP23 are two members of the family of with a micro-tissue grinder (Fisher Scientific). After centrifugationsmall heat shock proteins (sHSPs) of Drosophila mela- at 13,000 rpm for 30 min at 47C, the soluble phase was recovered

and lyophilyzed in a SpeedVac concentrator. Dried pellets were re-nogaster clustered with other sHSP genes within a 12-suspended in 2-D lysis buffer (9.5 M urea, 1% NP-40, 2% ampholineskb stretch of DNA at locus 67B [21, 22]. In response to(75% Bio-lyte 5–7, 25% Bio-lyte 3–10), 5% b-mercaptoethanol), andheat shock, the sHSPs show a coordinate pattern ofhomogenized until complete solubilization was obtained. In the case

induction [23]. Some of the sHSPs have also been re- of 1-D isoelectric focusing (IEF) gels, the dissected organs (heads,ported to be transcribed in the absence of stress [24, testes, ovarioles from early and late stages, embryos, and thoracic

muscles) were homogenized and resuspended in the IEF lysis bufferreviewed in 25]. During development, some sHSPs are(1% Chaps, 2% ampholines, 5% b-mercaptoethanol, 1 mM PMSF, 1transcribed in late larval and late pupal stages [24].mM EDTA, saturated with urea).They are also inducible in tissue culture cells by the

Gel electrophoresis and immunodetection. One-dimensional IEF-steroid molting hormone ecdysterone [26–28]. Thus inPAGE (3.3% acrylamide:piperazine diacrylamide (28.3:1.6), 9.0 Mcontrast to their coordinated synthesis following heat urea, 1% Chaps, 3.6% Bio-lyte 5–7, and 0.4% Bio-lyte 3–10) was

shock, the small HSPs of Drosophila show a tissue- carried out on horizontal slab gels in a Multiphor II System (Phar-specific and developmental-stage-specific pattern of ex- macia LKB), as previously described [20]. The electrophoretic migra-

tion was done at 300 V for 4 h. Two-dimensional gels were run ac-pression in unstressed organisms [24, 29–33]. Thecording to the method of O’Farrell [35]. Equal amounts of proteinspecificity in the constitutive patterns of expression ofwere loaded for all samples. For immunoblot assays, proteins weresome of these proteins suggest that, in addition to their electrophoretically transferred onto nitrocellulose membranes (Gel-

role during stress, sHSPs may play additional func- man). 32P-labeled isoforms of in vitro phosphorylated Drosophilations in the normal unstressed cell. HSP27 were separated on 1-D IEF gels, transferred onto Immobilon-

P (Millipore) membranes, and autoradiographed prior to immu-In cultured cells of D. melanogaster, HSP27 andnoblotting. Membranes were incubated with 2C8 or 7B12 in BLOTTOHSP26 are phosphorylated after treatment with ec-[36] for 2 h at room temperature, and washed in PBT (PBS / 0.2%dysterone [34]. However, little is known about the post- Tween 80) as described previously [33]. In some cases, membranes

translational modifications of the small HSPs under were first treated with the anti-HSP27 antibody (2C8), exposed, andnormal physiological conditions. Here we have exam- then reblotted with the anti-HSP23 monoclonal antibody (7B12).

Membranes from 1-D IEF gels were then incubated with an anti-ined the distinct isoforms of HSP27 and HSP23 presentmouse IgG horseradish peroxidase-conjugated secondary antibodyin the brain and in the gonads of both unstressed and(ECL, Amersham) diluted 1:2500 in BLOTTO for 1 h at room temper-heat-shocked flies. We report the presence of distinct ature and processed for detection according to the manufacturer’s

isoforms of these two sHSPs in different tissues. In the protocol. Membranes from 2-D gels were treated with a 125I-labeledcase of HSP27 these isoforms correspond to phosphory- goat anti-mouse IgG [28] (2 1 105 cpm/ml in BLOTTO).lated forms of the protein. This suggests that each indi- Alkaline phosphatase treatment. Gonads from 20 males were dis-

sected in Ringer’s solution and homogenized with a micro-tissuevidual sHSP may have distinct mechanisms of post-grinder in 25 ml of phosphatase buffer (100 mM Tris, pH 8.3, 10 mMtranslational modifications, and that this modulationMgCl2, 10 mM ZnCl2). Dephosphorylation was initiated by addingmay be important for cell-specific functions.12 U of the enzyme dissolved in stock solution (phosphatase buffer/ 50% glycerol). The reaction mixture was incubated at 377C for 1or 2 h, and terminated by the addition of 25 ml of 21 IEF lysis buffer.MATERIALS AND METHODSControl reactions were performed with 1 ml of heat-denatured (957C,5 min) enzyme in the same buffer.

Materials. Specific monoclonal antibodies to HSP27 and HSP23Preparation of purified sHSPs for in vitro treatment with HSP27were prepared by immunization of BALB/c mice with sHSP fusion

kinase. Drosophila sHSPs fusion proteins were produced in theproteins produced in an expression vector, as described previouslypET-3c expression vector [37] expressed in E. coli. For protein extrac-[33]. The 2C8 (anti-HSP27) and 7B12 (anti-HSP23) monoclonal anti-tion, cells were lysed by sonication, resuspended in SDS samplebodies were used at dilutions of 1:100 and 1:10, respectively, in im-buffer [0.075 Tris–HCl (pH 6.8), 2.3% (w/w) SDS, 5% (v/v) b-mercap-munoblotting assays. Alkaline phosphatase (Escherichia coli) wastoethanol, 10% (w/v) glycerol and 0.005% (v/v) bromophenol blue],purchased from Pharmacia. Ampholines (Bio-lyte) were obtainedand heated at 957C for 5 min. Proteins were separated on one-dimen-from Bio-Rad.sional SDS–PAGE [38], and each of the four Drosophila sHSP bandsTissue preparation. Heads, gonads (ovaries or testes), and tho-electroeluted against the elution buffer (30 mM Hepes (pH 7.0), 25raxes (mostly thoracic muscles) from cold-anesthetized flies of themM NaCl, 1 mM MgCl2, 0.1 mM EGTA) in an electroelutor (ISCO,Oregon-R stock of D. melanogaster raised at 237C were dissected inmodel 1750).Ringer’s solution. For heat shock treatments, flies were left for 1 h

at 357C in an Eppendorf tube. To prepare 2-D gel samples,2 dissected In vitro HSP27 phosphorylation. Recombinant DrosophilaHSP27 (4 mg) and recombinant Chinese hamster HSP27 (0.02, 0.05and 0.1 mg) were phosphorylated with 7.5 munits of highly purifiedmammalian HSP27 kinase [39] in 10 ml of 10 mM Mops (pH 7.0), 152 Abbreviations used: 1-D, one-dimensional; 2-D, two-dimensional;mM MgCl2, 40 mM para-nitrophenylphosphate, 1 mM DTT, 0.1 mMIEF, isoelectrofocusing; PMSF, phenylmethylsulfonyl fluoride; NP40,PMFS, and 100 mM [g-32P]ATP (22,000 cpm/mmol). After incubationNonidet P-40; PBS, phosphate-buffered saline; PAGE, polyacryl-for 1 h at 307C, a 2-ml aliquot was analyzed immediately using SDS–amide gel electrophoresis; cpm, counts per minute; Hepes, N-2-hy-PAGE. The rest of the reaction mixture was rapidly frozen on drydroxyethylpiperazine-N*-2-ethanesulfonic acid; Mops, 3-(N-morpho-

lino) propanesulfonic acid. ice and kept at 0807C for IEF electrophoresis.

/ m4861$3057 01-24-96 00:14:42 eca AP: Exp Cell

3SMALL HSP ISOFORMS IN Drosophila ORGANS

FIG. 1. Distinct isoforms of HSP27 and HSP23 found in the brain and in gonads of Drosophila adults. Protein extracts from heads andgonads (testes and ovaries) of non-heat-shocked (237C) flies were separated on 2-D PAGE, and transferred to nitrocellulose membranes.HSP27 a, b, c, and d, and HSP23 a and b isoforms were revealed by blotting with 2C8 (a-HSP27) and reblotting the same membrane with7B12 (a-HSP23) monoclonal antisera. The most acidic forms (c and d for HSP27 and b for HSP23) are shown on the right.

RESULTS are enriched in HSP23 while HSP27 is mainly ex-pressed at later stages (OII). The isoforms of HSP27 (a

Drosophila HSP27 and HSP23 Show Distinct and c) detected during early oogenesis are the same asIsoforms in the Brain and in Gonads of Nonheat- those found during later stages. Only one isoform ofshocked Adult Flies HSP23 is observed at early or late stages of oogenesis.

Early (0 to 5 h) embryos (Fig. 2, E1) have an isoformThe sHSPs of Drosophila are expressed in the ab-pattern of HSP27 identical to that of late oocytes, con-sence of stress in the brain and in gonads [29, 31–33].sistent with the maternal origin of this HSP [42]. InTo find out whether different isoforms of HSP27 andthese embryos HSP23 increases, and this may be re-HSP23 were expressed during development, proteinslated to the expression of this HSP in midline precursorfrom dissected brains and gonads of young (0–6 days)cells observed during early embryogenesis [25, 32]. Theadult flies were separated on 2-D gels and the distinctpresence of both HSP27 and HSP23 decreased in lateisoforms revealed by immunoblotting. In unstressed(5 to 12 h embryos (Fig. 2, E2).flies, four distinct HSP27 species (a–d) and two HSP23

isoforms (a and b) were present in heads and testes(Fig. 1; Fig. 2, H and T). The b and d isoforms of HSP27and the b isoform of HSP23 were absent in ovaries fromnon-heat-shocked flies (see also O in Figs. 2 and 3).These results suggest that a complex posttranslationalsystem may be acting to modulate the modifications ofHSP27 and HSP23 in a tissue-specific manner.

The presence of distinct isoforms of HSP27 andHSP23 under normal physiological conditions was con-firmed by an alternative immunoblotting technique us-ing 1-D IEF gels (Fig. 2). Head extracts (H) containeda smaller amount of HSP27 isoforms than gonads (Tand O), confirming previous immunohistochemical ex-periments [31–33] (see also Fig. 3). The four isoformsof HSP27 present in heads were clearly distinguishedon overexposed autoradiograms (not shown). The pat-tern of isoforms in the nervous system is similar to thatobserved in testes, suggesting similar modifications ofthese sHSPs in these two tissues.

FIG. 2. Comparison of HSP27 and HSP23 forms obtained in dif-ferent organs by 1-D IEF. Proteins from heads (H), testes (T), andSimilar Isoforms Are Expressed during Oogenesisovaries (O) of non-heat-shocked (237C) flies, and embryos of 0–5 hand Embryogenesis(E1) and 5–12 h (E2) were solubilized in IEF buffer, and fractionatedby 1-D IEF on horizontal slabs. Ovarioles were separated in earlyTo investigate whether different HSP isoforms are(from germarium to S8) (OI) and late (from S8 to S14) (OII) stages.expressed at different stages of oogenesis, ovariolesAfter being transferred to nitrocellulose, being blotted with 2C8 anti-were dissected into early (OI, from germarium to S8) body (A), and being reblotted with 7B12 antibody (B), HSP27 a, b,

and late (OII , from S8 to mature oocyte) stages [40, 41]. c, and d, and HSP23 a and b forms were visualized as individualbands. As in Fig. 1, the most acidic isoforms are shown on the right.As can be seen in Fig. 2, early stage egg chambers (OI

/ m4861$3057 01-24-96 00:14:42 eca AP: Exp Cell


isoforms observed in each tissue are summarized inTable 1.

HSP27 Isoforms Are Phosphoproteins

The main posttranslational modification of HSP27reported in other organisms is phosphorylation [10, 20,43, 44]. We therefore determined whether any of thephysiologically expressed isoforms of either DrosophilaHSP27 or HSP23 were phosphoproteins. The d and cHSP27 isoforms gradually shifted to more basic posi-tions on IEF gels with increasing time of incubationwith phosphatase (Fig. 5, lanes 1 and 2), suggestingthat they correspond to multiphosphorylated forms.The b form did not show any reduction in intensity

FIG. 3. Changes in HSP isoforms in response to heat shock. Pro- upon long incubation, and could not be shifted to thetein extracts from heads (H), testes (T), ovaries (O), and thoracica form. This may result from a reduced accessibility ormuscles (M) of non-heat-shocked (237C) or heat-shocked (357C, 1 h)susceptibility of a phosphate residue. HSP23 was notflies were separated on 1-D IEF gels and blotted with anti-HSP27

(a-HSP27) or anti-HSP23 (a-HSP23) antibodies. affected by phosphatase treatment. HSP23b may be aform where the phosphate is not accessible to phospha-tase or a form containing a different type of modifica-tion. Alternatively, HSP23b may be a product of a dis-Effects of Stress on Expression of HSP27 and HSP23 tinct HSP23-related gene [45].Isoforms

Drosophila HSP27 Is Phosphorylated in Vitro by aAs posttranslational changes of the sHSPs have beenMammalian HSP27 Kinasesuggested to be important for function in stressed cells

[reviewed in 18], we next analyzed the modifications of In previous works [19, 20, 39, 46] a novel mitogen-these sHSPs in response to heat shock. Total protein induced, second messenger-independent serine kinaseextracts of four tissues, heads (H), testes (T), ovaries has been implicated in the phosphorylation of mamma-(O), and thoracic muscles (M) from unstressed (237C) lian HSP27. Therefore, we tested whether mammalianor heat-shocked flies (357C, 1 h) were separated on 1- HSP27 kinase could modify Drosophila sHSPs. Recom-D IEF gels and immunoblotted (Fig. 3). The relative binant Drosophila HSP27, HSP26, HSP23, and HSP22amount of the basic isoform a in each tissue was gener- were incubated in vitro with a highly purified HSP27ally higher in heat-shocked than in non-heat-shocked kinase in the presence of [g032P]ATP. Figure 6A showstissues. Heat shock also induced an increase in the the Coomassie blue-stained SDS–PAGE with the fourlevel of the HSP27 b isoform in testes (T), and of the c recombinant purified sHSPs (lanes 4–7). Three differ-isoform in heads (H). In contrast with the reports in ent concentrations of the Chinese hamster HSP27 weremammals demonstrating a dramatic increase of HSP27 run concomitantly as controls of phosphorylation reac-phosphorylation within minutes of exposure to heat or tions (lanes 1–3). Recombinant HSP23 (lane 5) did notchemical stresses [4, 12] or to the addition of serum inHeLa cells [14], no further modification of HSP27 wasinduced upon heat shock in Drosophila. No strikingchanges in the HSP23 isoform’s pattern were seen inthe heads and testes of stressed flies. In thoracic mus-cles a single isoform of HSP23 was observed after heatshock, confirming earlier data [31–33].

Heat Shock Induces Changes in HSP27 and HSP23Isoforms in Ovaries

In contrast to the situation in heads and testes, heatshock induced a strong stimulation of HSP23 synthesis, FIG. 4. Additional isoforms of both HSP27 and HSP23 are in-

duced in ovaries specifically under stress conditions. Total proteinand the appearance of isoforms HSP27b and alsoextracts from non-heat-shocked (237C) or heat-shocked (357C, 1 h)HSP23b in ovaries (Fig. 3, O). The results obtained onovaries were resolved on 2-D gel electrophoresis, and the different1-D IEF and 2-D gels (Fig. 4) show that some modifica- isoforms obtained were blotted with monoclonal anti-HSP27 (a-

tions of HSP27 and HSP23 are induced in ovaries spe- HSP27) antibody, and reblotted with monoclonal anti-HSP23 (a-HSP23) antibody. Acidic forms are on the right.cifically under stress conditions. The different sHSP

/ m4861$3057 01-24-96 00:14:42 eca AP: Exp Cell


TABLE 1

Summary of the Different Isoforms of HSP27 and HSP23

HSP27 HSP23

Tissue 237C 357C 237C 357C

Brain a, b, c, da a, b, c, d a, b a, bTestis a, b, c, d a, b, c, d a, b a, bOvary a—c— a, b, c— a— a, bEmbryos a—c— ND a— NDThoracic muscle —b —b — a—

Note. ND, not determined.a a–d describe the isoforms oriented from more basic to more acidic positions on the gels (see also Figs. 1–3).b No sHSP forms observed.

migrate at the expected 23-kDa level. This polypeptide HSP27 isoforms (b, c, and, d) incorporated 32P (lane 2).No radioactivity signal was detected in HSP27a. Fivehas been isolated from a plasmid construction (pET-

3c-HSP23) resulting in a recombinant protein of more bands of the purified Drosophila HSP27 were recog-nized by the anti-HSP27 antibody (Fig. 7B, lane 2). Thethan 23 kDa. Autoradiographic analysis of the 32P-la-

beled proteins (Fig. 6B) showed that, among the four most acidic ones correspond to the 32P-labeled b, c, andd forms, as shown by their comigration with HSP27Drosophila sHSPs, only HSP27 incorporated 32P (lane

7), and no radioactive signals were detected for HSP26, from testes (lane 1). The 32P-labeled isoform d was de-tected on overexposed autoradiograms. The native re-HSP23, or HSP22.combinant HSP27a was resolved as two different forms

The Different Isoforms of Drosophila HSP27 Are on IEF gels. The same two bands were seen when thePhosphoproteins purified recombinant HSP27 was incubated in the ab-

sence of the kinase (lane 3), indicating that this basicThe different isoforms of purified Drosophila HSP27,band is not related to kinase activity. These resultsphosphorylated in vitro by mammalian HSP27 kinase,confirm that HSP27c and HSP27d are posttransla-were resolved on 1-D IEF gels, and compared to thetionally phosphorylated forms of the same gene prod-isoforms formed in vivo. 32P-labeled HSP27 isoformsuct, HSP27a. They further suggest that HSP27b is alsowere detected by autoradiography (Fig. 7A), and unla-a phosphoprotein that, for unknown reasons, is not ac-beled isoforms by immunoblotting (Fig. 7B). Threecessible during phosphatase treatment.

DISCUSSION

Drosophila HSP27 and HSP23 are two members ofa family of proteins that includes the small HSPs of

FIG. 6. In vitro phosphorylation of Drosophila HSP27. DifferentFIG. 5. Alkaline phosphatase treatment of HSP27 and HSP23.Total soluble proteins from testes of unstressed (237C) flies were quantities of purified recombinant Chinese hamster HSP27 and Dro-

sophila sHSPs were incubated at 307C with [g032P]ATP and highlyincubated in the presence of alkaline phosphatase (12 units) at 377Cfor 0 (lane 0), 1 (lane 1), or 2 (lane 2) h. Lane 3 corresponds to testis purified Chinese hamster kinase. After 60 min. SDS sample buffer

was added, and the reaction mixtures were analyzed by 1-D SDS–proteins incubated at 377C for 1 h in the same phosphatase bufferin the presence of heat-denatured enzyme. Equal amounts of protein PAGE and autoradiography. The different sHSPs were stained with

Coomassie blue (A) and 32P-labeled proteins were visualized by auto-were loaded in the different samples. The reaction mixtures wereanalyzed by 1-D IEF and immunoblotting with antisera specific for radiography (B). Lanes 1–3: 0.02, 0.05, and 0.1 mg, respectively, of

Chinese hamster HSP27; lanes 4–7: 4 mg of HSP22 (lane 4), HSP23Drosophila HSP27 (2C8) and HSP23 (7B12). The most acidic formsare on the right. (lane 5), HSP26 (lane 6), and HSP27 (lane 7).

/ m4861$3057 01-24-96 00:14:42 eca AP: Exp Cell


nisms could be operating in ovaries under stress condi-tions.

Another important aspect of the posttranslationalmodification of Drosophila HSP27 is the possible rela-tion between the intracellular localization of the dis-tinct isoforms and their level of phosphorylation. Previ-ous data on mammalian sHSPs reported the transloca-tion of phosphorylated forms into or on the nucleusafter heat shock [44]. In serum-stimulated HeLa cells,the phosphorylated forms of HSP27 were mainly recov-ered in a soluble fraction while the unphosphorylatedform tended to associate with the particulate fraction[14]. Using whole-mount immunohistochemical experi-ments on Drosophila, HSP27 has been localized in theFIG. 7. The different posttranslational forms of Drosophilanuclei of nurse cells in the first stages of oogenesis, andHSP27 are phosphoproteins. Purified Drosophila HSP27 was phos-

phorylated in vitro by Chinese hamster HSP27 kinase in the presence it is seen to diffuse to the cytoplasm at later stages (R.of [g032P]ATP (A, lane 2), or nonradioactive ATP (B). The phosphory- Marin and R. M. Tanguay, in preparation). One couldlation reactions were stopped by adding IEF buffer, and the distinct hypothesize that the distinct isoforms of HSP27 mayisoforms were separated on 1-D IEF gels. Drosophila HSP27 was

be implicated in the division and/or differentiation ofalso incubated in the same kinase buffer but in the absence of thecells during gametogenesis and during embryogenesis.enzyme (lane 3). As a control, Chinese hamster HSP27 was also

treated with mammalian kinase (lane 5). The purified kinase was Drosophila HSP23 is constitutively present in twoalso loaded on gels to detect possible nonspecific 32P-labeled bands forms (native HSP23a and a more acidic form HSP23b)unrelated to the phosphorylation activity (lane 4). Nonstressed testis in heads and testes. A single unmodified a form ofproteins were concomitantly run on the IEF gels (lane 1). (A) The

HSP23 is present in unstressed ovaries and, as in thedifferent 32P-labeled isoforms visualized by autoradiography. (B) Thecase of HSP27b, the HSP23b isoform is induced whendifferent HSP27 a, b, c, and d isoforms revealed after blotting with

monoclonal anti-HSP27 antibody. ovaries are submitted to a heat shock. This proteincould also be subject to posttranslational regulation.Alternatively, the a and b isoforms recognized by theanti-HSP23 antibody could correspond to two genes,essentially all organisms as well as the aA- and aB-

crystallins [47]. Seven sHSPs, four of which are classi- the HSP23 gene and an additional HSP23-related gene.A Drosophila sHSP-homologous gene [1(2)efl] has beencal sHSPs, are clustered at the same locus of the

Drosophila genome [21, 22]. They have been tradition- reported recently [45].Interestingly, Drosophila HSP27 can be phosphory-ally considered to be regulated by similar mechanisms

and to have related functions in the cell. However, it lated in an in vitro assay with a highly purified HSP27kinase from Chinese hamster. Two potential phosphor-is becoming evident that each sHSP has its own pattern

of expression and posttranslational regulation [24, ylation sites similar to those found in mammalianHSP27 (R-X-X-S) [12, 13] are found at serine 58 and29–34].

Drosophila HSP27 is constitutively present in four serine 75 of Drosophila HSP27. Their location with re-spect to the a-crystallin domain could correspond tostable forms (the unmodified HSP27a and three phos-

phoproteins HSP27b, c, and, d) in cells of the central Ser 78 and Ser 82 of human HSP27. A third potentialsite (Ser 173) of Drosophila HSP27 is found at the C-nervous system and testes. Interestingly, only native

HSP27a and HSP27c forms are detectable in un- terminal end. However, none of these sites shows aperfect match with the consensus of the mammalianstressed ovaries. The b form of HSP27, which is the

major acidic isoform constitutively present in testes, is HSP27 kinase, recently described by Stokoe et al. [46].Furthermore, in vitro, the Chinese hamster HSP27 ki-absent in non-heat-shocked ovaries, but can be induced

in this organ by heat shock. In addition, in Drosophila nase has a much lower (at least 50 times) affinity forDrosophila HSP27 than for its mammalian counter-brain and testes, heat shock does not lead to increased

phosphorylation of the sHSPs as reported in cultured part. Identical Drosophila HSP27 isoforms were gener-ated in vivo by transfection of the Drosophila HSP27mammalian cells [4, 13]. In fact we observed a reduc-

tion in the proportion of most acidic forms in heat- gene in Chinese hamster cells (Y. Wu and R. M. Tan-guay, unpublished results). Thus, the same isoforms ofshocked brains and testes. These data suggest that the

mechanisms regulating phosphorylation of the differ- Drosophila HSP27 which are phosphorylated in vitroby mammalian HSP27 kinase are also generated inent sites in HSP27 are complex. As for human HSP27,

which is constitutively expressed at low levels in most vivo after transfection in Chinese hamster cells.The mammalian HSP27 kinase did not phosphory-tissues and rapidly phosphorylated at two sites in

stressed cells [11], specific posttranslational mecha- late Drosophila HSP26, a protein which has been

/ m4861$3057 01-24-96 00:14:42 eca AP: Exp Cell


11. Arrigo, A. P., and Welch, W. J. (1987) J. Biol. Chem. 262,shown to be phosphorylated in vivo in Drosophila heat-15359–15369.shocked Kc cells [34], and which resolves into at least

12. Gaestel, M., Schroder, W., Benndorf, R., Lippmann, C., Buch-six species with different isoelectric points on 2-D gelsner, K., Hucho, F., Erdmann, V. A., and Bielka, H. (1991) J.[33]. Drosophila HSP23 does not have any R-X-X-S mo- Biol. Chem. 266, 14721–14724.

tif, and it has not been observed to be phosphorylated 13. Landry, J., Lambert, H., Zhou, M., Lavoie, J. N., Hickey, E.,in vivo using 32P after heat shock [34], nor after incuba- Weber, L. A., and Anderson, C. W. (1992) J. Biol. Chem. 267,tion with hamster HSP27 kinase, as described here. 794–803.

The present data suggest that the signal transduc- 14. Mehlen, P., and Arrigo, A. P. (1994) Eur. J. Biochem. 221, 327–334.tion pathways leading to phosphorylation of Drosophila

15. Knauf, U., Jakob, U., Engel, K., Buchner, J., and Gaestel, M.HSP27 may be homologous to those controlling the(1994) EMBO J. 13, 54–60.phosphorylation of mammalian HSP27. Moreover,

16. Landry, J., Chretien, P., Lambert, H., Hickey, E., and Weber,there could be other Drosophila protein-specific mecha-L. A. (1989) J. Cell Biol. 109, 7–15.nisms controlling the behavior of each individual sHSP.

17. Lavoie, J. N., Lambert, H., Hickey, E., Weber, L. A., and Landry,In Drosophila, the HSP27 kinase cascade may be devel-J. (1995) Mol. Cell Biol. 1, 505–516.opmentally regulated, and other protein kinases may

18. Arrigo, A. P., and Landry, J. (1994) in The Biology of Heatbe implicated, such as for example those stimulated byShock Proteins and Molecular Chaperones (Morimoto, R. I., Tis-

D-raf, a Drosophila homologue of Raf-1. D-raf plays key sieres, A., and Georgopoulos, C., Eds.), pp. 335–373, Coldroles in multiple signal transduction pathways, such Spring Harbor Laboratory Press, Cold Spring Harbor, NY.as that required for the determination of cell fates at 19. Stokoe, D., Engel, K., Campbell, D. G., Cohen, P., and Gaestel,

M. (1992) FEBS Lett. 313, 307–313.embryonic termini and for development of the com-pound eye [48, 49], where HSP27 is expressed. Future 20. Zhou, M., Lambert, H., and Landry, J. (1993) J. Biol. Chem.

268, 35–43.studies will help to elucidate the mechanisms regulat-21. Corces, V., Holmgren, R., Freund, R., Morimoto, R., and Mesel-ing the tissue-specific expression, and the complex pro-

son, M. (1980) Proc. Natl. Acad. Sci. USA 77, 5390–5393.tein modification–function relationships of these22. Craig, E. A., and McCarthy, B. J. (1980) Nucleic Acids Res. 8,sHSPs during development and under stress condi-

4441–4457.tions. It will also be interesting to investigate whether23. Tanguay, R. M., and Vincent, M. (1982) Can. J. Biochem. 60,the sHSP kinase cascade is also developmentally regu-

306–315.lated in higher vertebrates.24. Mason, P., Hall, L., and Gausz, J. (1984) Mol. Gen. Genet. 194,

73–78.We are grateful to Drs. Y. Wu, E. Khandjian, and Louis Nicole,

25. Arrigo, A. P., and Tanguay, R. M. (1991) in Heat Shock andand to Dominique Mayrand for assistance. This work was supportedDevelopment (Hightower, L., and Nover, L., Eds.), pp. 106–119,by the Medical Research Council of Canada (Grant MT-11086 toSpringer-Verlag, Berlin.R.M.T. and Grant MT-7088 to J.L.). Raquel Marin is a recipient of

26. Sirotkin, K., and Davidson, N. (1982) Dev. Biol. 89, 196–210.a scholarship from the FCAR of Quebec.27. Ireland R. C., Berger, E., Sirotkin, K., Yund, M. A., Osterbur,

D., and Fristrom, J. (1982) Dev. Biol. 93 498–507.REFERENCES28. Beaulieu, J. F., Arrigo, A. P., and Tanguay, R. M. (1989) J. Cell.

Sci. 92, 29–36.1. Nover, L. (1990) Heat Shock Response. CRC Press, Boca Raton,FL. 29. Glaser, R. L., Wolfner, M. F., and Lis, J. T. (1986) EMBO J. 4,

747–754.2. Morimoto, R. I., Tissieres, A., and Georgopoulos, C. (1994) TheBiology of Heat Shock Proteins and Molecular Chaperones, Cold 30. Glaser, R. L., and Lis, J. T. (1990) Mol. Cell. Biol. 10, 131–137.Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

31. Pauli, D., Tonka, Ch-H., Tissieres, A., and Arrigo, A. P. (1990)3. Berger, E. M., and Woodward, M. P. (1983) Exp. Cell. Res. 147, J. Cell. Biol. 111, 817–827.

437–442.32. Haass, C., Klein, U., and Kloetzel, P. M. (1990) J. Cell. Sci. 96,

4. Landry, J., Chretien, P., Lambert, H., Hickey E., and Weber, 413–418.L. A. (1989) J. Cell. Biol. 109, 7–15.

33. Marin, R., Valet J. P., and Tanguay, R. M. (1993) Dev. Genet5. Rollet, E., Lavoie, J. N., Landry, J., and Tanguay, R. M. (1992) 14, 69–77.

Biochem Biophys. Res. Commun. 185, 116–120.34. Rollet, E., and Best-Belpomme, M. (1986) Biochem. Biophys.6. Mehlen, P., Briolay, J., Smith, L., Diaz-Latoud, C., Fabre, N.,

Res. Commun. 141, 426–433.Pauli D., and Arrigo, A. P. (1993) Eur. J. Biochem. 215, 277–35. O’Farrel, P. H. (1975) J. Biol. Chem. 250, 4007–4021.284.36. Johnson, D. A., Gautsch, J. W., Sportsman, J. R., and Elder,7. Lavoie, J. N., Gingras-Breton, G., Tanguay, R. M., and Landry,

J. H. (1984) Gene Anal. Technol. 1, 3–8.J. (1993) J. Biol. Chem. 268, 3420–3429.37. Studier, F. W., Rosenberg, A. H., and Dunn, J. J. (1990) Methods8. Lavoie, J. N., Hickey, E., Weber, L. A., and Landry, J. (1993)

Enzymol. 185, 60–89.J. Biol. Chem. 268, 24210–24214.38. Thomas, J. O., Kornberg, R. D. (1975) Proc. Natl. Acad. Sci.9. Miron, T., Vancompernolle, K., Vanderkerckhove, J., Wilchek,

USA 72, 2626–2630.M., and Geiger, B. (1991) J. Cell Biol. 114, 255–261.10. Jakob, U., Gaestel, M., Engel, K., and Buchner, J. (1993) J. 39. Huot, J., Lambert, H., Lavoie, J. N., Guimond, A., Houle, F.,

and Landry, J. (1995) Eur. J. Biochem 227, 416–427.Biol. Chem. 268, 1517–1520.

/ m4861$3057 01-24-96 00:14:42 eca AP: Exp Cell


40. King, R. C., Rubinson, A. C., and Smith, R. F. (1956) Growth 45. Kurzik-Dumke, U., and Lohmann, E. (1995) Gene 154, 171–175.20, 121–157.

46. Stokoe, D., Caudwell, B., Cohen, P. T. W., and Cohen, P. (1993)41. Cummings, M. R., and King, R. C. (1969) J. Morphol. 128, 427–Biochem. J. 296, 843–849.442.

47. Hickey, E., Brandon, S. E., Potter, R., Stein, G., Stein, J., and42. Zimmerman, J. L., Petri, W., and Meselson, M. (1983) Cell 32,Weber, L. A. (1986) Nucleic Acids Res. 14, 4127–4145.1161–1170.

48. Perrimon, N., Engstrom, L, and Mahowald, A. P. (1985) Dev.43. Arrigo, A. P. (1990) Mol. Cell. Biol. 10, 1276–1280. Biol. 110, 480–491.

49. Nishida, Y., Hata, M., Ayaki, T., Ryo, H., Yamagata, M., Shim-44. Arrigo, A. P., Suhan, J. P., and Welch, W. J. (1988) Mol. Cell.Biol. 8, 5059–5071. izu, K., and Nishizuka, Y. (1988) EMBO J. 7, 775–781.

Received July 3, 1995Revised version received October 30, 1995

/ m4861$3057 01-24-96 00:14:42 eca AP: Exp Cell

tissue-specific posttranslational modification of the small heat shock protein hsp27 indrosophila

Documents