THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 264, No. 15, Issue of May 25, pp. 8951-8955, 1989 Printed in U.S.A.

Hyaluronic Acid Stimulates Protein Kinase Activity in Intact Cells and in an Isolated Protein Complex*

(Received for publication, May 13, 1988)

Eva A. TurleyS From the Oncology Research Group, Department of Pharmacology, Faculty of Medicine, Uniuersity of Calgary, Calgary, Alberta, Canada T2N 1N4

The addition of hyaluronate to intact chick embry- onic heart fibroblasts enriched with a hyaluronate- binding protein (HABP) stimulated phosphorylation of tyrosine and serinelthreonine residues in cellular pro- teins. A protein complex containing a hyaluronate- binding protein (cell-HABP) was isolated from the cul- tured heart fibroblasts. The isolated complex (Mr - l X 10‘) contained phosphoproteins that exhibited pro- tein kinase activity specifically stimulated by hyalu- ronate. Both tyrosine and serine residues in the protein complex were phosphorylated in response to this gly- cosaminoglycan. The hyaluronate-stimulated protein kinase activity was tightly associated with cell-HABP in vitro; enzyme activity co-immunoprecipitated with cell-HABP using a monospecific anti-HABP antibody and co-eluted with cell-HABP when chromatographed on a column of Sephacryl S-1000 in 2.0 M guanidine hydrochloride. The uniqueness of the cell-HABP-as- sociated protein kinase activity was suggested by both its specific response to hyaluronate, relative to related glycosaminoglycans such as heparin and chondroitin sulfate or to growth factors such as epidermal growth factor or insulin, and its antigenic distinction from other protein kinases such as growth factor receptors. These results point to a new mechanism by which glycosaminoglycans, such as hyaluronate, may modify cell behavior.

Molecules of the extracellular matrix have been implicated in the regulation of cell growth, recognition, cytodifferentia- tion, and motility during normal and disease processes (1-4). The manner in which these molecules do so has been ascribed primarily to their differential adhesive properties and to their influence, via interactions with specific cell surface receptors, on the architecture of the cytoskeleton (1-4). For instance, hyaluronate is believed to influence cell migration, differen- tiation, growth, and immune regulation by reducing cell ad- hesiveness (1, 5), altering the structure of the extracellular matrix (5), and/or masking other glycoconjugates (3, 5-12). Several hyaluronate-binding proteins have been isolated that may act as receptors for this glycosaminoglycan (13-17). One such protein, originally isolated from spent culture medium ( E ) , is concentrated in the blebs and ruffles of actively locomoting cells where it codistributes with newly bound

* This work was supported by grants from the Medical Research Council of Canada and National Cancer Institute of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Recipient of a Senior Scientist Career Award from the National Institute of Canada.

exogenous rhodamine-labeled hyaluronate (17). Addition of this soluble protein(s) to embryonic fibroblasts, together with hyaluronate, stimulates random cell movement (18). This report describes both the ability of hyaluronate to stimulate the phosphorylation of cellular proteins in this model system (18) and the preliminary isolation of a complex containing both a protein that is antigenetically related to HABP’ (17) and hyaluronate-stimulated protein kinase(s) activity.

EXPERIMENTAL PROCEDURES

Isolation of Cell-HABP

Cell-HABP was extracted from subconfluent chick heart fibroblast monolayers grown in roller bottles (16) that had been extensively washed with defined DMEM to remove serum from culture media. Such cultures contained endogenous cell-HABP and were therefore not supplemented with this protein. (see “Protein Kinase Activity”). For extraction, cell layers were incubated for 15 min in 20 ml of DMEM containing 0.1% Triton X-100 and the proteolytic inhibitors aprotinin, leupeptin, and anti-trypsin (Sigma, 20 mg/ml). The ex- tracted material was diluted to 200 ml with DMEM and incubated, on a rotary shaker (30 rpm), with anti-HABP rabbit IgG coupled to Sepharose (15). Protein was eluted from the gel with 1.0 M acetic acid that was immediately neutralized. The eluted protein solution was dialyzed, lyophilized, and stored a t -70 “C. Yields of extractedprotein were approximately 10-20 pg/mg cell protein. To assess its hyaluro- nate binding capabilities, cell-HABP was iodinated as described (16) and chromatographed on hyaluronate-Sepharose (19). Approximately 90% of isolated lZ5I-protein bound to hyaluronate-Sepharose (data not shown). This isolated material was routinely used for assays of enzyme activity.

Chromatography and SDS-PAGE

The cell-HABP complex was further characterized by SDS-PAGE, immunoblotting, and gel chromatography. Twenty pg of protein was electrophoresed, under reducing conditions, on either a 12% or a 3- 12% gradient SDS-PAGE gel and either stained with silver or elec- trically transferred to a nitrocellulose membrane (16). Nitrocellulose membranes were incubated with polyclonal antibodies to HABP (1.0 pg/ml (16)), and protein was visualized using the peroxidase method as described (16). Forty pg of protein was also chromatographed on Sephacryl S-1000 (1 X 60 cm; flow rate, 6 ml/h) in 2.0 M GdnHCl before or after reduction and alkylation (20). Vo was determined from sized fragments of DNA kindly denoted by Dr. G. Dixon, University of Calgary. The included volume was determined with Orange G. Fractions were dialyzed, and the protein concentration in each frac- tion was quantitated either a t 280 nm or with a y counter when radiolabeled protein was used.

The abbreviations used are: HABP, hyaluronate-binding protein; cell-HABP, hyaluronate-binding protein complex extracted from monolayers with Triton X-100; GdnHCl, guanidine hydrochloride; DMEM, Dulbecco’s modified Eagle’s medium; soluble HABP, hya- luronate-binding protein isolated from the supernatant medium cul- tures; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electro- phoresis; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.

8951

8952 Hyaluronate Stimulates Protein Kinase Activity

Protein Kinase Activity

Phosphorylation of Proteins in Intact Cells-Confluent contact- inhibited cultures of chick heart fibroblasts (60-mm dishes containing approximately 1 mg of cell protein) were labeled with 32Pi (inorganic phosphate, 1 mCi/ml, Amersham Corp.) for 24 h in Ringer's solution containing glucose, but no serum supplements, at 37 "C and supple- mented with HABP as described (18). Confluent cultures of fibro- blasts contain little cell-HABP (17, 18), and supplementation with HABP was necessary to increase the binding of hyaluronate to monolayers and to elicit a biological response (18) to this glycosami- noglycan. Cells were exposed to hyaluronate (10 pg/ml DMEM) or to buffer alone for 10 min, then extracted with radioimmune precipita- tion buffer ((21) 0.15 M sodium chloride, 0.01 M sodium phosphate a t pH 7.2, 1% deoxycholate, 1% Triton X-100, 0.1% sodium dodecyl sulfate) or 0.1 N NaOH containing aprotinin (50 pg/ml), iodoaceta- mide (4 mM, Sigma), sodium vanadate (100 p ~ , Sigma), and ZnC12 (10 p ~ , Sigma). Extracts were either electrophoresed on a 3-12% gradient SDS-PAGE gel or extracted with phenol (21), hydrolyzed with 5.7 N HCl, and processed for phosphoamino acid analysis (22). Total 32Pi phosphate incorporation into cell monolayers varied for each experiment but was unrelated to treatment protocols and was in the range of 1-2 X lo6 cpm/mg cell protein. Protein extracted with phenol from hyaluronate-treated cell extracts was redissolved, and samples were digested with one of the following enzymes: Strepto- myces hyaluronidase to remove material that might be nonspecifically trapped by this polymer (23), deoxyribonuclease I (Worthington Enzymes), ribonuclease A and B (Worthington Enzymes) (24), or Pronase (23) to remove "P-labeled contaminants or protein, respec- tively. Samples were precipitated with 10% trichloroacetic acid and compared to samples treated with buffers only. The first three treat- ment protocols included bovine serum albumin and the above protease inhibitors to control for proteases; treatment with these enzymes had no effect on the trichloroacetic acid precipitation of 32P-labeled ma- terial relative to buffer controls, but Pronase removed 92-100% of trichloroacetic acid-precipitable counts relative to buffer controls suggesting that the majority of the sample represented phosphola- beled protein.

Phosphorylation of the Cell-HABP Complex in Vitro-Protein ki- nase activity was determined as described (25) by measuring the amount of [3ZP]ATP incorporated into the isolated cell-HABP com- plex in the presence or absence of increasing concentrations of purified high molecular weight hyaluronate (0.01-100 pg/ml Healon, Pharmacia LKB Biotechnology Inc.). Some samples were also incu- bated with hyaluronate that had been exposed to either protease-free leech hyaluronidase (courtesy of Dr. J. R. Couchman, University of Alabama, 50 units/ml, pH 4.7) or to Pronase (10 mg/ml, Sigma, type VII) at 37 "C for 24 h. The standard kinase assay contained 5-10 pg of cell-HABP in 40 p1 of the reaction buffer (20 mM Hepes, pH 7.4, 3 mM MnC12, 10 pM ZnCl,, 0.4 p M dithiothreitol, 0.2% Nonidet P-40, and 50 pg/ml aprotinin (19)). Cell-HABP was preincubated for 10 min at 4 'C in either buffer alone or with 0.01 pg of untreated or enzyme-treated hyaluronate, other glycosaminoglycans, or growth factors. The kinase reaction was started by adding 0.1 p M [32P]ATP (150 mCi/mM, Amersham, Oakville, Ontario) for 10 min at 4 "C and was stopped by either boiling in SDS-PAGE loading buffer (16) or by immunoprecipitation. Heat-denatured cell-HABP or soluble HABP (15) served as controls. Samples were electrophoresed on either a 3-12% gradient or 12% SDS-PAGE; the radiolabeled HABP was cut out and counted in a y counter.

Phosphoamino Acid Analysis

SDS-PAGE gels were placed in plastic Ziploc bags and processed for autoradiography. Cell-HABP was electrically eluted from SDS- PAGE gels, dissolved in 5.7 N HCl, and hydrolyzed for 1 h at 110 "C in the presence of standard phosphoamino acids. Phosphoamino acids were separated as described (22). Recovery was estimated relative to that of authentic phosphoamino acids treated in an identical manner. 3ZPiinc~rp~ration into amino acids was identified by autoradiography or by y scintillography.

Immunoprecipitation

After either labeling with [32P]ATP or, as in the case of intact cells, "Pi, cell-HABP was chromatographed on a Sephadex G-25 column (Bio-Rad, 1 X 8 cm, flow rate 0.1 ml/min) to remove unin- corporated 32P and then immunoprecipitated with the following an- tibodies: polyclonal antibody to HABP (1.0 pg/ml, affinity purified

with protein A- and HABP-Sepharose); monoclonal antibody to the same (0.1 pg/ml); polyclonal antisera to the exterior domain of the epidermal growth factor receptor (4.0 pglml); and rabbit polyclonal antibody prepared against phosphotyrosine (1.0 pg/ml, a kind gift of Dr. J. Goren, University of Calgary). Background precipitation was quantitated using rabbit preimmune or nonimmune sera. The immune complexes were precipitated with goat or mouse anti-rabbit antibody (1:lOOO dilution, Miles Laboratories Inc.). Precipitates were washed in radioimmune precipitation buffer (21) containing 50 pg/ml apro- tinin and then counted in a y counter.

RESULTS

Protein Phosphorylation in Intact Cells-Hyaluronate stim- ulated a 14-fold increase in 32P, incorporation into protein extracted from cells enriched with HABP (Table I, A). Cells that were treated in an identical manner but were not supple- mented with HABP showed marginal changes in cell behavior reported previously (18) and in protein phosphorylation (ie. 1.2-1.5-fold stimulation). These results were commensurate with the lesser ability of untreated monolayers to bind hya- luronate (18).

A 2-, 3-, and 5-fold increase in the radiolabeling of serine, threonine, and tyrosine residues, respectively, was observed after phosphoamino acid analysis of phenol-extracted cell proteins (Table I, A, Fig. 1). Its resistance to digestion with DNase or RNase and susceptibility to Pronase indicated that phenol-extracted material was largely protein. SDS-PAGE of cell protein extracts revealed a general increase in radiolabel- ing of protein bands but also the specific appearance of a radiolabeled 21-kDa protein band in response to hyaluronate (Fig. 1) and a protein that was immunoprecipitated by an anti-HABP polyclonal antibody (Table I, A) suggesting it to be cell-HABP. The efficiency of this antibody, using purified cell-HABP, was approximately 30-40%. Phosphoamino acid analysis of the 21-kDa band, from treated cultures, indicated a high level of phosphotyrosine (Table I). This was in contrast to a 15-kDa protein that labeled primarily on serine/threo- nine. The total recovery of radiolabel after phosphoamino acid analysis was similar for both protein bands (21 kDa, 50%; 15 kDa, 58%) and for authentic phosphoamino acids treated in the same manner.

Isolation of a Cell-HABP Complex-A protein that was cross-reactive with a characterized soluble HABP (15-17) was originally detected on cell monolayers using a polyclonal antibody (17). It was removed with 0.1% Triton X-100 from the lamellae and blebs of motile chick heart fibroblasts ((17) data not shown). Immunoaffinity chromatography of the Tri- ton extract, using the above antibody (15), isolated a protein that bound to hyaluronate-Sepharose and chromatographed on Sephacryl S-1000 in 2.0 M GdnHCl as a peak at the position expected for a M, = 100,000 protein (Fig. 2). This protein cross-reacted with 15 monoclonal and 30 polyclonal antibod- ies to soluble HABP in dot blot and enzyme-linked immuno- sorbent assays (examples of these are shown in Table I1 and Fig. 2). Preimmune and nonimmune IgG precipitated approx- imately 10% that precipitated by immune IgG (Table 11). The immunoprecipitated protein electrophoresed as a diffuse band at the top of the separating gel in either a 12% or a 3-12% gradient SDS-PAGE gel (Fig. 2), and an immunoblot, using anti-HABP antibody as a probe, revealed only one cross- reactive protein band (Fig. 2B). Monoclonal antisera precip- itated approximately 65% of the purified complex (Fig. 2) while polyclonal antisera immunoprecipitated approximately 40-50% of the complex.

Phosphorylation of Cell-HABP Complex-In the absence of hyaluronate, the cell-HABP complex incorporated [32P]ATP in a protein kinase assay (Table I, B). Radiolabel comigrated with the protein band in SDS-PAGE gels. Phosphoamino acid

Hyaluronate Stimulates Protein Kinase Activity

TABLE I Characterization of protein phosphorylation in response to hyaluronate and immunoreactivity of cell-HABP

8953

For in uiuo assays, confluent cultures of chick heart fibroblasts were prelabeled with 1 mCi of ”P,/ml of Ringer’s without sera, supplemented with supernatant HABP to improve hyaluronate binding since cultures produce little endogenous HABP at culture confluence, and exposed to either buffer alone or to 100 pg of hyaluronate/106 cells for 10-15 min. The cell layer was dissolved with radioimmune precipitation buffer and either extracted for protein, processed for immunoprecipitation, or electrophoresed on 12% SDS-PAGE. Phosphoamino acids extracted from either the cell protein or radiolabeled bands excised from SDS-PAGE were analyzed as described under “Experi- mental Procedures.” Values represent mean f S.E. ( n = 3). For in uitro assays, 5-10 pg of cell-HABP, extracted from sparse cultures that were washed free of serum and which were not supplemented with HABP, were purified by immunoaffinity chromatography and incubated with 0.1 p~ [32P]ATP in the presence of intact glycosaminogly- cans, glycosaminoglycans treated with enzymes, growth factors, or buffer alone. All reactions were carried out at 4°C for 10 min. The protein was immunoprecipitated with polyclonal antibodies to soluble HABP and then electrophoresed on SDS-PAGE gels cut out, and counted in a y counter. For phosphoamino acid analysis, the cell- HABP complex was electroeluted from the gel and processed as described under “Experimental Procedures.” Radioactivity co-migrating with phosphoamino acid standards, which were detected with ninhydrin, was cut out and counted in a y counter. The peak fraction of protein eluted from Sephacryl S-1000 in 2.0 M GdnHCl was dialyzed and assayed for enzyme activity. As a control, an identical amount of protein was exposed to GdnHCI, dialyzed, and also assayed for enzyme activity. Values were calculated as cpm/wg protein originally added to the assay and represent the mean k S.E. ( n = 6) .

Cell-HABP isolated hy Phenol- [3ZP]ATP incorporation immunoprecipitation or

SDS-PAGE extracted Treatment

or antibody protein Serine Threonine Tyrosine

A. Phosphorylation in intact cells assays 1. Buffer

3. Proteins isolated by SDS-PAGE 2. Hyaluronic acid

( a ) 21 kDa ( b ) 15 kDa

B. Phosphorylation in uitro 1. Cell-HABP immunoprecipitated by

HABP antibodies Buffer Hyaluronic acid (0.1 pg/ml) Hyaluronidase-treated hyaluronic acid Pronase-treated hyaluronic acid Heparin (1.0 pg/ml) Chondroitin sulfate (1.0 pglml) Epidermal growth factor (10 ng/ml) Insulin (10 ng/ml) peak fraction Cell-HABP isolated by gel filtration in

Control 2. Supernatant HABP

Buffer Hyaluronic acid

2.0 M GdnHCl

3. Heat-denatured cell-HABP

cpmlassay or P R cell protein cpmfrgprntein cpmlrg protein

130 k 67 1,460 f 31 147 f 26 1,286 f 162 184 * 23 500 k 230 21,240 f 156 287 f 89 3,427 f 223 874 & 107

1,060 276 1203 621 521 167

25,767 f 3,568 53,494 f 5,697 19,082 f 3,641 56,705 k 4,774 21,117 f 4,831 23,477 f 2,165 20,211 k 3,123 22,388 k 3,652

4,647 k 312

3,972 k 276

300 f 45 278 f 65 187 f 24

243 f 67 2 9 1 f 67 1 1 5 + 26 369 k 108 826 f 213 1032 f 285

analysis of this band revealed 32P incorporation primarily into threonine and tyrosine residues (Table I, B). The presence of endogenous phosphotyrosine in the isolated complex was confirmed by cross-reactivity with antibodies to phosphoty- rosine (Table 11, B). [”PIATP incorporation into the complex was increased 2-3-fold in the presence of high molecular weight hyaluronate (Table I, B). Increased incorporation into threonine (3-fold), tyrosine (9-fold), and, to a slight extent, serine (1.5-fold) was observed. Treatment of hyaluronate with leech hyaluronidase, which degraded hyaluronate to tetra- and hexasaccharides, prevented this stimulation while Pro- nase treatment of hyaluronate had no effect (Table I, B). Other glycosaminoglycans and the mitogens, epidermal growth factor or insulin, whose receptors contain protein kinase activity, did not stimulate the cell-HABP-associated protein kinase activity (Table I, B). Further, the cell-HABP complex differed antigenically from protein kinases associated with growth factor receptors (Table 11). Heat-denatured cell- HABP and soluble HABP incorporated very little, if any, radioactivity (Table I, B).

That the cell-HABP complex and the hyaluronate-stimu- lated protein kinase were tightly associated was suggested by

several properties of the peak fraction of cell-HABP complex isolated by gel filtration in 2.0 M GdnHC1; this fraction cross- reacted with either polyclonal or monoclonal antibodies to soluble HABP (Fig. 2) and contained protein kinase activity (Table I, B). The kinase activity in this fraction was approx- imately 30% of the kinase activity observed for material that had not been denatured with GdnHC1. However, this level of incorporation was equal to that of protein that had been exposed to GdnHCl but not chromatographed.

DISCUSSION

This report describes both the ability of hyaluronate to stimulate the phosphorylation of proteins in intact cells and the preliminary isolation of an HABP complex that contains hyaluronate-stimulated protein kinase(s) activity. Evidence that hyaluronate promotes phosphorylation of proteins in intact cells and in an isolated HABP complex includes dem- onstration of an increased 32P incorporation into both cellular protein and cell-HABP isolated by immunoprecipitation and SDS-PAGE; increased incorporation of :32P into phosphoa- mino acids; and increased reactivity, in the case of the com- plex, to antiphosphotyrosine antibodies. The protein ki-

8954 Hyaluronate Stimulates Protein Kinase Activity

A. SDS - PAGE + -

' 2

' 3

B. phosphoamino acids + -

3 ."I

FIG. 1. Phosphorylation of embryonic heart fibroblast pro- teins in response to hyaluronate. Intact heart fibroblasts were prelabeled with inorganic :'V, and exposed to hyaluronate as described under "Experimental Procedures." Protein was extracted with phenol and either electrophoresed on 12% SDS-PAGE ( A ) or processed for phosphoamino acid analysis ( R ) and subsequently developed for autoradiography (+, exposed to hyaluronate; -, not exposed to hya- luronate). Equal amounts of protein were applied to the SDS-PAGE gel (approximately 50 pg). The autoradiogram was overdeveloped to emphasize low molecular weight proteins. A 21-kDa protein (closed arrowhead), which showed increased phosphorylation in response to hyaluronate, and a 15-kDa protein (open arrowhead) whose phospho- rylation was not altered by this glycosaminoglycan were excised and analyzed for phosphoamino acids. These values are given in Table I. For the phosphoamino acid analysis of total protein extracted from phenol ( R ) , the control extract was overloaded to attempt to visualize any phosphotyrosine. The molecular weight standards, designated in A were: I , phosphorylase b, 97,500; 2, bovine serum albumin, 66,200; 3, ovalbumin, 45,000; 4, carbonic anhydrase, 29,500; 5, soybean tryp- sin inhibitor, 21,500; 6, lysozyme, 14,000. In R: 1, phosphoserine; 2, phosphothreonine; and 3, phosphotyrosine.

nase(s) is unique in terms of its specific responsiveness to hyaluronate and tight association with a hyaluronate-binding protein. Like other partially purified tyrosine kinases, it also contains serinelthreonine kinase activity (22, 26, 27). Whether this kinase-HABP complex mediates the effect of hyaluronic acid on protein phosphorylation in intact cells noted here or whether, alternatively, this glycosaminoglycan activates other kinases remains to be determined. Studies correlating the concentration of cell-HABP/cell with a re- sponse to hyaluronate, not conducted here, as well as antibody neutralization experiments are required to establish whether cell-HABP mediates the effects of hyaluronate in the cell experiments.

Molecules of the extracellular matrix have generally been observed to exert their influence on cell behavior via an effect on cell adhesion and cytoskeletal organization, the latter presumed to be mediated by physical linkages between the cell surface binding site and the cytoskeleton (1-4). However, platelet-collagen interactions have previously been shown to activate protein kinases, presumably as a result of phospho- lipid breakdown (28). The stimulation of protein kinase activ- ity by a glycosaminoglycan and the association of such activity with a glycosaminoglycan binding site is a novel finding and

5-

6-

D

6 -

4 -

2 -

0 0-0 "0-0-0-0-0

2 4 8 8 10 12 14 16

Fraction (ml) FIG. 2. Electrophoresis and gel chromatography of cell-

HABP complex. A, SDS-PAGE; B, transblot of cell-HABP complex purified by polyclonal antibody; C, autoradiogram of SDS-PAGE of cell-HABP; D, gel filtration of polyclonal immunoaffinity-purified ["'PIATP-labeled cell-HABP. A and B, the cell-HABP complex (lane 2 ) electrophoresed on SDS-PAGE ( A ) as a diffuse band that stained with silver. Subsequent transblotting ( B ) and staining with peroxi- dase revealed immunoreactivity of this protein with polyclonal anti- body to HABP. Molecular weight standards (shown in lane 1 ) were: I , phosphorylase b, 97,500; 2, bovine serum albumin, 66,200; 3, oval- bumin, 45,000; 4, carbonic anhydrase, 29,500; 5, soybean trypsin inhibitor, 21,500; 6, lysozyme, 14,000. C , monoclonal antibody to HABP immunoprecipitated 65% of the ["PIATP cell-HABP complex that was previously isolated by immunoaffinity chromatography with polyclonal antibody to HABP. Both immunoprecipitated material (lane 1 ) and that which resisted immunoprecipitation (lane 2) exhib- ited the same motility on SDS-PAGE. D, gel filtration chromatogra- phy of [""PIATP-labeled and unlabeled cell-HABP on a Sephacryl S- 1000 column. [:'*P]cell-HABP complex (M) chromatographed as a peak eluting at M, 1 X lo6 in 2.0 M GdnHCI. Unlabeled cell- HABP (-), detected by absorption a t 280 nm, co-chromato- graphed with ['"PIATP-labeled protein. Little unlabeled protein was detected in material precipitated with preimmune sera (0-0). V, void volume; b.d., blue dextran (M, 1 X lofi); Gg, y-globulin (M, 1 X 10"); I , = included volume.

raises the possibility that hyaluronic acid, like collagen (28), may influence cell behavior by signal transduction in addition to the previously proposed effects of its physiochemical prop- erties and interactions with the cytoskeleton (14).

Although the exact nature of the association of the kinase(s) with the cell-HABP complex is as yet unknown, the co- chromatography of enzyme activity with cell-HABP in 2.0 M GdnHCl suggests a strong interaction. The hyaluronate-stim- ulated serine/threonine kinase may be separable from the HABP complex as has been achieved for other receptor- tyrosine kinases (26). However, the recent demonstration of a yeast protein containing both serinelthreonine and tyrosine kinases as integral parts of a protein raises the possibility that both enzyme activities detected in the complex described here may be integral to the complex (27). Alternatively, both kinases as well as possibly other proteins may be complexed post-translationally.

Hyaluronate Stimulates

TABLE I1 Immunoreactiuity of cell-HABP

5-10 pg of cell-HABP were either labeled with [32P]ATP as de- scribed under “Experimental Procedures” or ‘‘’1 (16) and immuno- precipitated with antibodies (preimmune rabbit serum, 1:40 dilution; nonimmune rabbit serum, 1:40 dilution; polyclonal anti-HABP, 1 pg/ ml; monoclonal anti-HABP, 0.1 pg/ml; polyclonal anti-EGF receptor, 5 pg/ml; rabbit polyclonal antiphosphotyrosine antibody, 1 pg/ml) and protein A-Sepharose. Immunoprecipitates were washed and counted in a y counter. Enzyme-linked immunosorbent assay con- ducted as described (15) confirmed immunoprecipitation data (data not shown). Values remesent the mean k S.E. ( n = 10) samdes.

Antibody Immunopre- cipitation

A. ‘251-cell-HABP 1. Polyclonal to HABP 2. Preimmune sera

1. Polyclonal to HABP 2. Monoclonal antibodies to HABP

B. [3ZP]ATP-labeled cell-HABP

3-m-5 3-m-6

3. Preimmune sera 4. Nonimmune sera 5. Polyclonal to epidermal growth factor

6. Polyclonal to phosphotyrosine receptor (outside domain)

(a ) Without hyaluronic acid treatment ( b ) With hyaluronic acid treatment

cpm fpg protein

7,910 k 812 432 f 56

5,780 f 670

21,792 f 2,167 36,176 f 4,364

712 k 268 743 f 210 726 f 125

4,713 + 219 8.840 f 712

The physiological effects of the protein kinase activity identified in this report are uncharacterized. However, the activity of other tyrosine and serine/threonine kinases has been correlated with functions similar to that of hyaluronate including cell growth, cell differentiation, and cell transfor- mation (1, 3, 5 , 6, 22, 28-34). Although it is not clear whether all of the biological effects of hyaluronate are receptor-me- diated, the interaction of hyaluronate with HABP on fibro- blast cell surfaces has been shown to directly promote both cell motility (18) and the acquisition of morphological char- acteristics that are typical of the transformed state (5, 18). The similarity of these biological effects to those of growth factors and oncogene proteins of the kinase class (33) raises the possibility that hyaluronate regulates cell behavior, in part, via protein kinase activity.

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