Abstract
Interleukin 1 (ILl) increased phosphorylation of the small heat-shock protein (hsp 27) in MRC5 fibroblasts. The increase was maintained for at least 30 min, but levels had returned to pre-stimulation values by 2 h. When hsp 27 was metabolically labelled with [3H]leucine, about 15 % was phosphorylated in resting confluent cells; this rose to 90 % upon stimulation by ILl. Peptide maps of the three differently charged phosphorylated forms were consistent with their arising by phosphorylation of increasing numbers of serine residues. ILl had the same effect on hsp 27 in pig articular chondrocytes, endothelial cells from human umbilical vein and an epidermoid carcinoma cell line (KB). Certain other agents were found selectively to increase phosphorylation of hsp 27 in MRC5 cells besides ILl [and tumour necrosis factor (TNF)]. Platelet-derived growth factor had a similar effect to that of ILl; bradykinin, acid fibroblast growth factor and ATP caused an intermediate effect; phorbol myristate acetate (PMA) and I-oleoyl-2-acetylglycerol had smaller effects. Dibutyryl cyclic AMP and forskolin had no effects on hsp 27 phosphorylation. When cells had been depleted of protein kinase C (PKC) by prolonged treatment with PMA, stimulation by ILl, TNF or bradykinin still increased hsp 27 phosphorylation. The stimulation by all three agents was also unaffected by the PKC inhibitor staurosporine. ILl, TNF and bradykinin each caused hsp 27 phosphorylation by a pathway independent of PKC. The results are consistent with ILl activating a serine kinase which remains to be identified.
INTRODUCTION
Interleukin 1 (ILl) is a potent inflammatory cytokine which mediates many local and systemic features of inflammation (Dinarello, 1988) . It shares a number of its biological actions with tumour necrosis factor (TNF), but acts through different receptors. Whereas there has long been general agreement over the biological effects of ILl and their importance, the biochemical basis of its action on cells has remained controversial (Mizel, 1990; O'Neill et al., 1990) . Some investigators have found that ILl stimulates cellular accumulation of cyclic AMP (Chedid et al., 1989) or diacylglycerol (Rosoffet al., 1988) , but others have found no evidence for this (Carroll, 1986; Didier et al., 1988) . It has been argued that ILl may work by activating protein kinase A (Mizel, 1990) , but it has also been suggested that it could activate protein kinase C (PKC) (Rosoff et al., 1988; Ostrowski et al., 1988) . Similar uncertainty exists regarding the intracellular signalling mechanisms involved in the action of TNF. It too may activate PKC (Meichle et al., 1990) .
INTRODUCTION
Despite the continuing uncertainty over the importance of recognized second messengers in responses of cells to ILl and TNF, there is clear evidence that these two cytokines cause rapid increases in protein phosphorylation in stimulated cells. It is therefore likely that they work by regulating protein kinases. The receptor for epidermal growth factor (EGF) was the first protein whose state of phosphorylation was shown to be increased upon stimulation of cells by ILl or TNF (Bird & Saklatvala, 1989 . Recently, we reported that the small heat-shock protein (hsp 27) was another polypeptide whose state of phosphorylation was increased in fibroblasts stimulated by either of these two cytokines (Kaur et al., 1989) . We have now characterized this response, and show that it is caused by ILl in a variety of cell types. Other agents, including certain growth factors, bradykinin and ATP, also cause changes in phosphorylation of hsp 27. These findings have implications for the signalling mechanism used by the cytokines.
MATERIALS AND METHODS

Materials
ILla and TNFa were human recombinant proteins made in Escherichia coli as described elsewhere . Human recombinant platelet-derived growth factor (PDGF) (csis) was from Amersham International, Aylesbury, Bucks., U.K. Acidic fibroblast growth factor (FGF) (bovine brain) was from Biogenesis, Bournemouth, U.K. Receptor-grade EGF, bombesin, bradykinin, ATP, calcium ionophore A23 187, phorbol myristate acetate (PMA), 1-oleoyl-2-acetylglycerol (OAG), TosPhe-CH2CI-treated trypsin, proteinase and phosphatase inhibitors, Protein A-agarose and t.l.c. plates (Sigmacell type 100) were all from Sigma, Poole, Dorset, U.K. Ampholines were from Pharmacia LKB, Milton Keynes, U.K. Chemicals for electrophoresis were all of the best available grade from BDH, Poole, Dorset, U.K., or Fisons, Loughborough, Leics., U.K. Culture media were from Flow Laboratories, Rickmansworth, Herts., U.K.; foetal calf serum (FCS) The rabbit antiserum to hsp 27 was raised to a protein antigen that had been highly purified from HeLa cells; it has been characterized previously (Arrigo & Welch, 1987) . This antiserum was generously given by Dr. W. J. Welch, San Francisco General Hospital, San Francisco, CA, U.S.A.
Cell culture
Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10 % (v/v) heat-inactivated FCS, and kept at 37°C in a humidified atmosphere of C02/air (1:19).
Cell culture
Metabolic labelling with 132PIP; Confluent cells were washed for 20 min in phosphate-free DMEM containing 10 % FCS (dialysed) and 20 mM-Hepes and adjusted to pH 7.4. Cells were next incubated for 8 h in the same fresh medium (0.5 ml/30 mm dish) containing 100 ,uCi of [32P1P /ml. They were then stimulated by adding the chosen agent for the required time. Stimulation was terminated by rapidly washing the cells twice in ice-cold phosphate-buffered saline (PBS) consisting of 0.15 M-NaCl, 3 mM-NaH2PO4 and 18 mM-Na2HPO4 at pH 7.4. Unless the sample was to be immunoprecipitated, the proteins were next rapidly precipitated by adding 0.5 ml of 3 % trichloroacetic acid to the cell layer. Precipitated material was scraped into 1.5 ml vials and centrifuged in a Microfuge (2 min, 10000 g). The pellets were taken up in 50,1u of 8 M-urea containing 1 % (v/v) Nonidet P40 (NP40), then diluted 1: 1 in isoelectric focusing sample buffer [10% (v/v) 2-mercaptoethanol, 20% (v/v) glycerol, 8 M-urea, 0.8 % (v/v) Ampholines pH 5-7 and 0.2% (v/v) Ampholines pH 3-10].
Cell culture
Heat shock and labelling with 13l-Ileucine Newly confluent cells were washed for 20 min in leucine-free DMEM containing 10 % FCS (dialysed). Next they were incubated for 30 h in the same fresh buffer containing 100 1Ci of [3H]leucine/ml. Cells were then heat-shocked at 45°C for 20 min by immersing the flask in a water bath, and subsequently maintained for a further 8 h at 37°C under 5 % CO2. Some were then stimulated with ILl for 10 min, rapidly washed with icecold PBS and then processed for immunoprecipitation.
Immunoprecipitation
After stimulation by cytokine, the cells were rapidly washed in ice-cold PBS, scraped into 1.5 ml vials and centrifuged. Pellets were solubilized in ice-cold 10 mM-triethanolamine, pH 8.0, containing 0.5 M-NaCl, 1 mM-EGTA, 50 mM-NaF, 100 4uM-sodium orthovanadate, 0.1 % (w/v) SDS, 0.2 % (w/v) sodium deoxycholate, 1 % (v/v) Triton X-100, 10 jug of leupeptin/ml, 1O ug of pepstatin A/ml, 20,ug of aprotinin/ml and 1 mmphenylmethanesulphonyl fluoride. Lysates were clarified by centrifuging and then mixed (10 min, 4 IC) with an equal volume of solubilizing buffer containing Protein A-agarose beads [50 % (v/v) slurry]. After centrifuging, the supernatant was incubated with antiserum to hsp 27 (3 ,tl/500 ,ul of lysate) for 3 h at 4 'C.
Immunoprecipitation
Finally, Protein A-agarose was added and mixed for 1 h at 4 'C. The immunosorbent was centrifuged, washed three times in 10 mM-triethanolamine, pH 8.0, containing 0.5 M-NaCl, 1 % (v/v) NP40 and 0.25 % (w/v) sodium deoxycholate, and then washed twice in 50 mM-Tris/HCl, pH 6.8. Absorbed proteins were solubilized by adding the sample buffer for two-dimensional electrophoresis to the agarose.
Electrophoresis
Isoelectric focusing was carried out in gel rods (10 cm x 2.5 mm) containing 8 M-urea, 3.5 % acrylamide (acrylamide/NN-methylene bisacrylamide, 19:1), 4% (v/v) Ampholines pH 5-7 and 1 % (v/v) Ampholines pH 3-10. Samples were focused overnight (250 V for 15 min, 400 V for 16 h and 1000 V for 1 h). Reservoirs contained 25 M-NaOH (upper) and 20 mM-H3PO4 (lower).
Electrophoresis
After focusing, gels were extruded and equilibrated in SDS sample buffer for 20 min. For measurement of the pH gradient, some gels were sliced after extrusion and the slices were eluted in degassed water. For the second dimension on SDS/PAGE, the buffer system described by Laemmli (1970) was used. The separating gel was 12.5 % acrylamide, and the stacking gel was 5%.
Electrophoresis
After electrophoresis gels were stained in Coomassie Brilliant Blue solution, destained, processed for fluorography if necessary and dried.
Fluorography and autoradiography
For detection of 3H, stained gels were soaked for 1 h in two changes of dimethyl sulphoxide, then for 30 min in 22 % (w/v) 2,5-diphenyloxazole in dimethyl sulphoxide, then washed in water and dried. Gels were exposed for the required time at -80°C to RX medical X-ray film (Fuji). For detection of 32p, dried gels were exposed with an image intensifying screen.
Fluorography and autoradiography
Autoradiographs were scanned in an LKB Gelscan system. Incorporation of 32P into specific proteins was quantified by means of the LKB 2400 Gelscan XL software package.
Two-dimensional phosphopeptide mapping
Two-dimensional gels were autoradiographed without drying in order to localize proteins. The piece of gel containing the required protein spot was excised, crushed and eluted with 50 mM-NH4HCO3 containing 0.1 % SDS and 5 % 2-mercaptoethanol. Protein was precipitated from the eluate by trichloroacetic acid in the presence of carrier y-globulin (40,ug/ml), washed, oxidized with performic acid and digested with Tos-Phe-CH2Cl-treated trypsin, all as described by Beemon & Hunter (1978) . Lyophilized phosphopeptides were dissolved in 1% NH4HCO3, pH 8.9, applied to 20,um cellulose t.l.c. plates and electrophoresed in this buffer (1 kV for 25 min). Ascending chromatography was carried out in n-butanol/acetic acid/pyridine/water (15:3:12:10, by vol.) for 4 h. Plates were autoradiographed as described for two-dimensional electrophoresis gels.
Two-dimensional phosphopeptide mapping
EGF radiolabelling and binding EGF was radioiodinated with 125I to a specific radioactivity of about 2200 Ci/mmol by a method employing chloramine T (Carpenter & Cohen, 1976 Phosphorylation of heat-shock protein 27 EGF/ml, with or without 50 ng of unlabelled EGF/ml. After 3 h at 4°C, the buffer was removed and cells were washed four times with fresh chilled binding buffer (2 ml/well), lysed with 1 % (v/v) Triton X-100/0.1 M-NaOH (1 ml/well) and bound radioactivity counted.
RESULTS

ILl induces phosphorylation of most newly synthesized hsp 27
Previous experiments (Kaur et al., 1989) had shown that stimulation of fibroblasts by ILl causes an early increase in their content of three phosphorylated forms of hsp 27. In order to assess whether this increase involved modification of a significant amount of hsp 27, cells were metabolically labelled with either [3H]leucine (hsp 27 has only a single internal methionine) or
ILl induces phosphorylation of most newly synthesized hsp 27
[32P]P . Confluent cells were incubated with [3H]leucine for 30 h to allow equilibration with the intracellular pool of leucine. They were then subjected to heat shock (45°C, 20 min), and incubated for a further 8 h. This procedure was necessary to label hsp 27 because it is synthesized at only a very low rate in unstressed cells. For labelling with phosphate, cells were incubated with the radioisotope for 8 h. The cells labelled in these two ways were stimulated for 10 min with ILl, by which time phosphorylation was optimal. The distribution of the labels in hsp 27 is shown in leucine-labelled hsp 27 was unphosphorylated, and was focused at a position corresponding to pH 6.5 (Fig. la, spot a) . By contrast, the hsp 27 from the cytokine-stimulated cells consisted largely of the two more basic putative phosphorylated forms ( I1-induced phosphorylation is sustained for at least 30 min It was shown previously (Kaur & Saklatvala, 1988 ) that the cytokine-induced phosphorylation of hsp 27 is an early event. It became detectable after 3-5 min of stimulation, and was very marked by 10 min. Fig. 2 shows that the phosphorylation induced by ILl was maintained for at least 30 min, but after 2 h of stimulation the amount of phosphorylated hsp 27 had apparently returned to the level present before stimulation.
ILl induces phosphorylation of most newly synthesized hsp 27
Multiple forms of hsp 27 probably arose due to differences in phosphorylation It was previously shown (Kaur et al., 1989 ) that the three phosphorylated forms of hsp 27 contained phosphoserine, but not phosphothreonine or phosphotyrosine. The differences in charge between the four forms of hsp 27 are large: on isoelectric focusing, one pH unit separates the unphosphorylated protein (spot a in Fig. 1 ) from the most acidic form (spot d in Fig. 1 ). Experiments were carried out to see if these differences could be attributed solely to phosphorylation, or whether some other modification of the protein was occurring.
ILl induces phosphorylation of most newly synthesized hsp 27
Attempts to dephosphorylate the [3H]leucine-labelled protein by treating immunoprecipitated protein with alkaline phosphatase were unsuccessful because the protein was lost, probably as a result of degradation. As a second approach, tryptic phosphopeptide maps were made from each of the three phosphorylated forms eluted from two-dimensional gels (Fig. 3) . The least acidic phosphoprotein contained five major phosphopeptides (Fig. 3c) . The intermediate-phosphorylated form (Fig. 3b) showed marked intensification in the region of peptide 3; this could have been due to a new peptide adjacent to peptide 3. The most acidic form (Fig. 3a) showed pronounced intensification of peptide 5. The maps suggest the initial phosphorylation of serine residues to generate the protein of spot b (Fig. 1) , with further phosphorylations causing the appearance of a new phosphopeptide in each of the two more acidic forms (spots c and d). The number of serine residues phosphorylated in spot b is unknown. Digestion with chymotrypsin, thermolysin or a mixture of thermolysin and trypsin all generated at least three major phosphopeptides (results not shown). This evidence, together with the change in pl of 0.5 unit, suggests that phosphorylation of more than one serine residue is involved in the generation of the first phosphorylated form, spot b. There are 21 serine residues in the sequence of hsp 27 (Hickey et al., 1986; Carper et al., 1990 ).
Effect of ILl on hsp 27 in other cell types
Cells considered to represent important physiological targets for ILl were tested. The cytokine increased phosphorylation of hsp 27 in human vascular endothelial cells (Figs. 4a and 4b ) and in chondrocytes from rabbit articular cartilage (Figs. 4c and 4d ). The identity of the chondrocyte proteins could not be confirmed by immunoprecipitation because an antiserum to rabbit hsp 27 was lacking. Nevertheless, the highly characteristic appearance of the three phosphoprotein spots makes it probable that they are hsp 27. ILl also increased hsp 27 phosphorylation in the KB epidermoid cell line (Figs. 4e and 4J ).
Effects of other agents upon phosphorylation of hsp 27
The ability of various agents to stimulate phosphorylation of hsp 27 was tested with a view to obtaining some indication of whether or not ILl might be acting through a known signalling pathway. ILl shares many biological actions with the tumour promoter PMA, a substance which works by activating PKC. PMA (or OAG) increased hsp 27 phosphorylation, but only weakly in comparison with the effect of ILl (Fig. 5) . In particular, the very large increase in spot d was not seen after stimulating MRC5 fibroblasts with PMA or OAG. Fig. 6 shows the effects of some other agents that were found to markedly increase phosphorylation of hsp 27. Besides TNF (Kaur & Saklatvala, 1988) , only PDGF produced a response comparable with that of ILl (Fig. 6) . FGF, ATP and bradykinin all produced smaller (b-d in Fig. 1) were excised. Elution, digestion by trypsin, electrophoresis and chromatography were done as described in the Materials and methods section. Autoradiographs show (a) the most acidic form (spot d in Fig. 1 range of concentrations and stimulation times, had no discernible effect on the phosphorylation of hsp 27 (results not shown). These were: dibutyryl cyclic AMP (up to 1 ,UM), dibutyryl cyclic GMP (1 4uM), forskolin (to 1/ M), arachidonic acid (10 ZM), bombesin (6 nM), EGF (50 ng/ml) and insulin (50 ng/ml). The relative amounts of the three phosphorylated forms of hsp 27 from cells treated with various agents were measured by scanning densitometry of the autoradiographs and were expressed as a percentage of the response to ILl (Table 1) . This shows a ranking order with ILl and PDGF having the largest effect, FGF, bradykinin and ATP having an intermediate action, and OAG and PMA eliciting the smallest response. Because of the biological similarity of ILI and PMA and the fact that activation of PKC in cells by PMA or OAG can be associated with increased hsp 27 phosphorylation, it seemed possible that ILl might be acting in part through PKC. Thus MRC5 fibroblasts were incubated with PMA for 24 h to deplete them of regulatable PKC. During the last 8 h, the cells were labelled with [32P]P1 and their responses to ILl, TNF and bradykinin were examined by two-dimensional electrophoresis and compared with the responses of cells not pretreated with PMA (Fig. 7) . The phosphorylation of hsp 27 and the increases caused by all three agents were unaffected by the prior treatment of the cells with PMA (Fig. 7) , suggesting that neither the cytokines nor bradykinin was causing the phosphorylation-via a route involving PKC.
Effects of other agents upon phosphorylation of hsp 27
In order to check that PKC had been depleted by the prolonged treatment with PMA, fresh PMA was added, and its ability to decrease the binding of EGF to its receptor was measured. Table  2 shows that, when PMA was added to untreated cells, it caused a 740% decrease in EGF binding; this was due to the expected PKC-induced decrease in affinity of the receptor, or 'transmodulation' (Iwashita & Fox, 1984; Davis & Czech, 1984 We also looked at the response of cells pretreated for 30 min with an inhibitor of PKC, staurosporine (100 nM). This treatment was-sufficient to inhibit PMA-induced phosphorylation of the EGF receptor ), but it had no effect on Vol. 277 
DISCUSSION

Characteristics of hsp 27 phosphorylation
The experiments carried out on [3H]leucine-labelled cells showed that a large proportion of hsp 27 was phosphorylated in response to stimulation by ILl. Metabolic labelling of the protein with an amino acid was hampered by its lack of methionine, and by the fact that the cells required to be heat-shocked to induce detectable synthesis of the protein. Therefore the experiment only gave information regarding the phosphorylation state of the hsp 27 synthesized during 8 h following heat-shock, and not of the total amount of hsp 27 in the cell. If this recently synthesized protein was representative of the whole, then IL1 increased the amount phosphorylated from 15 % to 900%.
Characteristics of hsp 27 phosphorylation
Phosphorylation reached a maximum after about 10 min of stimulation, and this was maintained for at least 30 min. In some experiments the increase lasted for at least 60 min; in others it was declining by this time. In general, the amount of phosphorylated hsp 27 appeared to have returned to the prestimulation level after 2 h. The kinetics of the response were similar to those of the phosphorylation of the EGF receptor in fibroblasts stimulated by ILl or TNF (Bird & Saklatvala, 1989 . Whether the decrease in phosphorylation was due to dephosphorylation, degradation or both is not known. In the case of the EGF receptor, there was dephosphorylation rather than degradation .
Characteristics of hsp 27 phosphorylation
The three phosphorylated forms of hsp 27 appeared to arise from increasing phosphorylation, although other covalent modifications could not be entirely excluded. To see such large differences in isoelectric point with no appearance of intermediates is unusual. The protein contains 13 threonine residues in addition to its 21 serines; however, we have detected only phosphoserine on phosphoamino acid analysis, even in protein from cytokine-stimulated cells (Kaur et al., 1989) . Three of the serine residues (at positions 15, 78 and 82) are potential phosphorylation sites for PKC (Arg-Xaa-Xaa-Ser). The motifs Ser-Arg and Ser-Xaa-Asp (Glu) both occur five times, but they do not conform to any consensus sequence for known kinases.
Characteristics of hsp 27 phosphorylation
The diverse agents that increase phosphorylation all cause the same forms to appear, suggesting that there may be one or two rather specific hsp 27 kinases upon which more than one signalling pathway may converge. Such serine kinase activity might be generated in several ways; for instance, there could be ILl causes hsp 27 phosphorylation in a variety of cell types
Characteristics of hsp 27 phosphorylation
The effect was not cell-specific. Fibroblasts, chondrocytes, vascular endothelial cells and an epidermoid cell line all displayed the response. Previously we reported it occurring in hepatoma (Hep G2) and leukaemic (U937) cell lines (the latter in response to TNF; Kaur et al., 1989) . There have also been independent reports of TNF stimulating phosphorylation of hsp 27 in Hela cells (Arrigo, 1990) and of a small stress protein in endothelial cells (Hepburn et al., 1988; Robaye et al., 1989 ). We have not so far observed it in any lympocytic lines. However, the only ILlresponsive T cell line which we have studied (EL4) is murine, and we have been unable to identify any protein corresponding to hsp 27 undergoing phosphorylation in these cells before stimulation. We have also been unable to detect any phosphorylated hsp 27 in murine fibroblasts (3T3). The antiserum to the human protein does not react with hsp 27 of other species, so cannot be used to aid detection in mouse cells. The response was prominent in other species, however. It was seen in pig and rabbit chondrocytes, and has also been observed in rat fibroblasts in response to serum or PMA (Welch, 1985) .
Characteristics of hsp 27 phosphorylation
Induction of hsp 27 phosphorylation by other agents PMA and OAG were weakly active in stimulating hsp 27 phosphorylation in MRC5 fibroblasts. Other types of fibroblast may show a stronger response to these agents (Welch, 1985) . More marked effects were seen in MRC5 cells stimulated by bradykinin, ATP or FGF, but the only agent that caused a response of similar magnitude to ILl was PDGF. Bradykinin and ATP have previously been reported to stimulate phosphorylation of a 26 kDa protein in human endothelial cells . The phosphorylation followed a time course similar to the one reported here , and it is highly likely that this protein was hsp 27.
Characteristics of hsp 27 phosphorylation
We were unable to induce any change in the phosphorylation of hsp 27 with agents expected to increase the activity of protein kinase A. Evidence has been presented that ILl may activate this enzyme by elevating cyclic AMP in cells (Mizel, 1990) .
Characteristics of hsp 27 phosphorylation
The weak response of MRC5 cells in terms of hsp 27 phosphorylation to activators of PKC, and the failure of PKC depletion or staurosporine to attenuate the response, all suggested that ILl did not cause this phosphorylation via PKC. Interestingly, the effect of bradykinin was also unaffected by either PKC depletion or staurosporine. The kinin is known to cause hydrolysis of phosphoinositide, and would be expected to increase diacylglycerol production and activate PKC (Derian & Moskowitz, 1986; Lambert et al., 1986 ). However, it may also cause an increase in intracellular Ca2+, (Lambert et al., 1986 ) and its effect on hsp 27 might be via activation of a Ca2+/calmodulindependent kinase. Interestingly, hsp 27 phosphorylation was enhanced by a Ca2+ ionophore (although this was part of a general increase in the phosphorylation of many proteins) and by ATP, which also elevates intracellular Ca2+ (Hallam & Pearson, 1986; Pirotton et al., 1987) . Neither ILl nor TNF has been found to cause changes in intracellular Ca2 .
Characteristics of hsp 27 phosphorylation
PDGF and FGF receptors are tyrosine kinases (Hanks et al., 1988; Ruta et al., 1989) , but not enough is known about their signalling pathways for the significance of their ability to cause hsp 27 phosphorylation to be assessed.
Characteristics of hsp 27 phosphorylation
The only biological effect that all of these agents appear to have in common is that they stimulate prostaglandin synthesis in cells (Hong & Deykin, 1982; Forsberg et at., 1987; Habenicht et at., 1985; Dinarello, 1988 ; Liu et at., 1989).
Significance of bsp 27 phosphorylation in intracellular signalling
The function of hsp 27 is largely unknown. Its synthesis is induced by stress, but it is present in significant amounts in unstressed cells. Results of experiments in which a cDNA to hsp 27 was transfected into CHO cells suggested that it may confer upon cells a resistance to thermal injury (Landry et al., 1989) . It has been studied less than other stress proteins, perhaps because deletion of a gene coding for a homologue in yeast had no discernible effect on the cells (Petko & Lindquist, 1986) . Its striking phosphorylation in cells responding to a number of agents suggests that it may play a role in intracellular signalling. As a cytoplasmic protein it is appropriately placed for such a role. It may enter the nucleus upon heat shock (Arrigo et al., 1988) . It forms aggregates (Arrigo & Welch 1987; Arrigo et al., 1988) whose nature might be affected by phosphorylation. It is not yet known whether it interacts specifically with other proteins.
Significance of bsp 27 phosphorylation in intracellular signalling
Whatever the function of hsp 27 and the significance of its phosphorylation, it should be possible to use it as a substrate with which to identify protein kinases that are activated in response to several important agents involved in inflammation and cell growth.

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