Activation of phosphatidylinositol 3-kinase and phosphatidylinositol 4-kinase during rat parotid acinar cell proliferation

40

Biochimica et Biophysica Acta, 1178 (1993) 40-48 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4889/93/$06.00

BBAMCR 13424

Activation of phosphatidylinositol 3-kinase and phosphatidylinositol 4-kinase during rat parotid acinar cell proliferation Karnam R. Purushotham a Yoichi Nakagawa a Pawels Kurian b5 Rajiv Patel b Fulton T. Crews b and Michael G. Humphreys-Beher a,b Departments of a Oral Biology and b Pharmacology and Experimental Therapeutics, University of Florida, Gainesville, FL (USA)

(Received 26 February 1993)

Key words: Salivarygland; Signal transduction; Galactosyltransferase;Tyrosine phosphorylation; Epidermal growth factor We have recently shown that/3-adrenergic agonist, isoproterenol-induced parotid acinar cell proliferation is in part mediated by elevated levels of surface galactosyltransferase which undergoes interaction with the EGF-R. The receptor subsequently undergoes autophosphorylation on the tyrosine residues in a manner similar to its 'receptor-ligand' interaction (Purushotham et al. (1992) Biochem. J. 284, 767-776). In this study, we provide evidence for phosphatidylinositol 3-kinase and 4-kinase as cytoplasmic signalling proteins involved in both the isoproterenol and EGF-stimulated signal transduction upon in vitro and in-vivo stimulation of parotid acinar cells. Total cell lysate activity for the Ptdlns 4-kinase was 2- and 3-fold higher than unstimulated control cells, while the Ptdlns 3-kinase was 1.4- and 2.8-fold higher following stimulation by isoproterenol or EGF, respectively. Increases of 6- and 2-fold in phosphatidylinositol 3-kinase were observed in anti-phosphotyrosine-antibody-immunoprecipitated cell lysates upon in-vitro growth stimulation with isoproterenol or EGF, respectively. There was an increase in tyrosine phosphorylation of the holoenzyme and association of the p85 subunit of phosphatidylinositol 3-kinase with EGF-R in response to both isoproterenol and EGF treatments. This corresponded with the mobilization of p85 from the cytoplasm to the plasma membrane upon growth stimulation. These results further implicate the phosphoinositide metabolites in the second messenger signalling pathways of isoproterenol-induced rat parotid cell proliferation. The parallel utilization of EGF indicate that the post-transductional mechanisms of isoproterenol-induced acinar cell proliferation are similar to the growth-factor-mediated activation of intracellular signalling pathways for cell growth.

Introduction I n recent years, considerable progress has been made in identifying the pathways that regulate the events of signal transduction from the cell surface to the cell cytoplasm. Binding of growth factor with its specific receptor induces tyrosine autophosphorylation which in turn induces the physical association between the receptor and the cytoplasmic signalling protein components. These signal-transducing proteins include phos-

Correspondence to: K.R. Purushotham, Department of Oral Biology, Box 100424, J.H. Miller Health Center, University of Florida, Gainesville, FL 32610-0424, USA. Abbreviations: Ptdlns, phosphatidylinositol; Ptdlns 3-kinase, phosphatidylinositol 3-kinase; Ptdlns 4-kinase, phosphatidylinositol 4kinase; PLCr, phosphatidylinositol specific phospholipase C~; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; cAMP, cyclic adenosine monophosphate; EGTA, ethylenebis(oxyethylenenitrilo)tetraacetic acid; ISO, isoproterenol; Gal Tase,/3-1,4 galactosyltransferase.

pholipase C~, Ras GTPase-activating protein (GAP) and Ptdlns 3-kinase among others [1-3]. Growth-factor-initiated signals include the generation of second messengers such as Ins(1,4,5)P 3 and diacylglycerol which are involved in the mobilization of intracellular calcium and activation of calcium-phospholipid dependent protein kinase C, respectively [4-6]. E G F is known to stimulate Ptdlns turnover in various cell types through phosphorylation and activation of phospholipase C [1,7,8]. In addition, tyrosine phosphorylation has been implicated in coupling the activated growthfactor receptors to the Ptdlns [1,9]. The phosphoinositide kinases which catalyze the phosphorylation of the inositol ring from Ptdlns are designated as type I (Ptdlns 3-kinase) and type II (Ptdlns 4-kinase) depending on the phosphorylation of the inositol ring at the D3 or D4 position, respectively [10,11]. Although Ptdins 4-kinase is involved in the production of second messengers, a unique Ptdlns 3-kinase has recently been shown to become associated with certain activated

41 tyrosine-kinase-possessing receptors [3,12]. Recent evidence indicates that PtdIns 3-kinase activity not only phosphorylates PtdIns but also PtdIns(4)P and PtdIns(4,5)P 2 to generate two additional novel phospholipids, PtdIns(3,4)P 2 and PtdIns(3,4,5)P3, respectively [13]. These novel phosphoinositides are detected in many cell types including non-transformed proliferating cells. In addition to growth factors such as PDGF, CSF and EGF, PtdIns 3-kinase has been shown to associate with oncoproteins containing tyrosine kinase activity, namely polyomavirus middle-T antigen and the pp60 c-src complex [14-17]. Chronic administration of the/3-adrenergic agonist, isoproterenol (ISO), causes parotid gland hypertrophy and hyperplasia in rats and mice [18,19]. In attempting to elucidate the signalling pathway in the ISO-induced hyperplasia, we have reported earlier observations of increases in cAMP, phosphatidylinositol turnover and possible receptor crosstalk dependent upon acinar cell tyrosine kinase activity which results in elevated levels of Ins(1,4,5)P 3 [20,21]. An important early event in the transition to active acinar cell proliferation is the significant elevation of /3-1,4 galactosyltransferase (Gal Tase - typically identified as a marker enzyme of the Golgi complex) at the surface which interacts with a plasma-membrane glycoprotein, the EGF-R, in a manner similar to 'receptor-ligand' interactions [22]. We have observed the occurrence of an EGF-independent mechanism of receptor activation dependent upon cell-surface Gal Tase which resulted in cell proliferation, involving the generation of intracellular second messengers mediated in part by the activation of PLCr [22]. The present investigation is aimed at further understanding the role of Ptdlns kinases in the ISO-induced proliferating parotid acinar cells. Parallel experiments using EGF to stimulate cell growth were performed to provide evidence in support of the hypothesis that the Gal-Tase-mediated responses to ISO mimic the 'EGF-EGF-R' second messenger pathway. Materials and Methods

Materials. D,L-Isoproterenol, phosphatidylinositol, EGF and EGF-R monoclonal antibody (clone 29.1) were purchased from Sigma (St. Louis, MO). [y_32p]. ATP (6000 Ci/mmol) and Sepharose-conjugated monoclonal antibody against phosphotyrosine were obtained from Amersham (Arlington Heights, IL). Polyclonal antibody against the 85-kDa subunit of PtdIns 3-kinase was obtained from Upstate Biotechnology, NY. All chemicals and reagents were purchased through commercial sources and were of ultrapure quality. Animals. Female Sprague-Dawley rats (170-200 g) were purchased from University of Florida breeding colony. Animals, after acclimatization for one week to

laboratory conditions, were grouped as untreated control animals, ISO-treated or EGF-treated (experimental groups). Experimental groups of animals received intraperitoneal injections of ISO (25 mg/kg) or EGF (25 /zg/kg) for 15 min or 1 h. For 24-h and 72-h treatments, the animals were injected twice daily (i.e., every 12 h). Control animals received injections of 0.5 ml saline twice a day. Food and water were provided ad libitum. Food was withdrawn 12 h prior to killing. At the appropriate time interval, rats were killed by exsanguination after being anesthetized with pentobarbital. The parotid gland was subsequently identified by gross morphology, freed of connective tissue and lymph nodes and used for experimental analysis of Ptdlns 3-kinase and 4-kinase activity. Plasma-membrane preparation. The parotid gland thus isolated was homogenized by a Biospec tissue disperser followed by Dounce glass homogenization at 4°C in 10 mM Tris-HC1 buffer (pH 8.0) containing phosphatase and proteinase inhibitors: Na-orthovanadate (1 mM), PMSF (4 /zg/ml), and aprotinin (10 /zg/ml). Unlysed cells were removed by low-speed centrifugation at 500 × g for 5 min and the resulting slurry was centrifuged at 100 000 x g for 1 h to pellet the total membrane and supernatant (cytoplasm) fractions. The plasma membranes were isolated by the method of Arvan and Castle [23]. The purity of the membranes thus prepared is documented elsewhere [24]. The plasma-membrane fraction was shown to be enriched 10-fold over total membranes for y-glutamyltranspeptidase, an enzyme marker appropriate for rat parotid acinar cells [23].

Sucrose-gradient fractionation of parotid homogenates. Isolated rat parotid glands were weighed and homogenized in 5 vols. of 0.25 M sucrose in 3 mM imidazole buffer (pH 7.4) and centrifuged at 12 000 x g for 10 min to pellet low-speed microsomes; the supernatant was centrifuged at 100000 x g for 60 min to pellet high-speed microsomes. These low- and highspeed microsomes were resuspended and applied to a 1.06-1.25 g / c m 3 sucrose-density linear gradient (28 ml) that was made above a 4-ml cushion of 1.22 g / c m 3 sucrose. After centrifugation for 12 h at 100000 Xg, 1-ml fractions were collected from the top using a Buchler Auto Densi-Flow fractionator (Buchler Instruments, KS). The activity levels of/3-1,4 galactosyltransferase and y-glutamyltranspeptidase in the above 1.0-ml fractions were measured following the methods described elsewhere [25,26]. Approx. 25 /xl of alternate fractions containing an equal quantity of protein were run on SDS-PAGE for p85 detection. Acinar cell preparation. The parotid glands from the rats were finely minced, and subjected to enzymatic digestion by following the method described by Baum et al. [27] for in vitro stimulation studies. In brief, the minced tissue was incubated at 37°C in Hanks' bal-

42 anced salt solution with Hepes (pH 7.5), containing 0.01% BSA, 100 U / m l collagenase, 0.2 mg/ml hyaluronidase. The tissue was dispersed by pipeting and gassed with 95% 0 2 and 5% CO 2 for 10 s. The cells were washed three times with the above Hanks' buffer and the resulting suspension was used for further experiments.

Determination of Ptdlns 3- and Ptdlns 4-kinase activities The Ptdlns 3-kinase and Ptdlns 4-kinase activities were measured in control and ISO-or EGF-treated rat parotid gland lysates by following the method described by Shibasaki et al. [28]. Briefly, the reaction mixture (50 /xl of total volume) for Ptdlns 3-kinase activity consisted of 50 mM Tris-HCl (pH 7.5), 5 mM MgCI 2, 0.5 mM EGTA, 50 mM NaC1, 1 mM Ptdlns, 400/zM Phosphatidylserine, 100/xM [T-32p] ATP and the cell lysates (protein 50 Izg) as the source of enzyme. After incubation for 10 min at 30°C, the reaction was stopped by the addition of 2 ml chloroform/ methanol/12 M HC1 (200:100: 1, v/v). Subsequently 0.5 ml of 1 M HCI was added, vortexed and centrifuged at 2000 × g for 15 min. The upper phase was discarded and the lower phase was washed once with water, vortexed and centrifuged again. The resulting lower phase was transferred to a scintillation vial and the radioactivity associated with it measured by liquid scintillation counting. The Ptdlns 4-kinase was similarly assayed in a reaction mixture containing 50 mM TrisHCI (pH 7.5), 20 mM MgC12, 1 mM EGTA, 0.4% Triton X-100, 1 mM Ptdlns and 100/xM [3,-32p]ATP. The reaction products of the two kinases obtained by these conditions were confirmed by HPLC using authentic standards [28]. In separate reactions to differentiate Ptdlns 3-kinase from Ptdlns 4-kinase, varying concentrations (from 0.1% to 5%) of NP-40 were included [28]. Immunoprecipitation of Ptdlns 3-kinase Following isolation of intact cells by hyaluronidase and collagenase digestion, acinar cells were preincubated in RPMI 1640 containing 5% fetal bovine serum for 30 min at 37°C and stimulated by the addition of 1 /xM ISO or 100 nM EGF for 15 min. The media was removed and the cells were immediately frozen in dry ice to terminate cell stimulation. The ceils were then solubilized in 0.5 ml of lysis buffer (20 mM Tris-HC1 (pH 8.1), 137 mM NaC1, 1 mM MgC12, 1 mM CaCI 2, 10% glycerol (v/v), 1% nonidet P-40 (v/v) 1 mM Na-orthovanadate, 1 mM PMSF and 10 /zg/ml of aprotinin) for 40 min at 4°C. The cell lysates were clarified by centrifugation at 13000 × g for 15 min. Aliquots of supernatants containing 150 /xg of total cellular protein were immunopreciptated by adding 10 ixl of Sepharose conjugated phosphotyrosine monoclonal antibody (Amersham). After incubation on a

rocker platform at 4°C for 2 h, the immune complexes were washed as follows: twice with PBS containing 1% nonidet-P-40, twice with 0.5 M LiCI in 100 mM TrisHC1 (pH 7.6), and once with 10 mM Tris-HCl (pH 7.4) containing 100 mM NaCI and 1 mM EDTA. Finally, the phosphotyrosyl-containing proteins were eluted in a buffer containing 15 mM phenylphosphate by vortexing every 5 rain for 20 min at 4°C. The sample thus obtained was used separately for (a) the detection of EGF-R using monoclonal antibody to EGF-R (29.1) by Western blot, (b) detection of PtdIns 3-kinase using antibody to p85 subunit (UBI) by Western blot and (c) PtdIns 3-kinase assay by TLC (see below). Separate immunoprecipitations using anti-EGF-R (10 /xg/ml) were also carried out for the measurement of PtdIns 3-kinase assay by TLC. Detection of EGF-R. The immunoprecipitable protein complexes obtained above by using anti-phosphotyrosine antibody were run on a 9% SDS-PAGE [29]. The proteins were electrophoretically transferred to nitrocellulose at 17 V for 12 h. Following transfer, the separated proteins were blocked by 5% BSA and 5% nonfat milk in 10 mM Tris (pH 7.4) with 100 mM NaC1 for 2 h. The nitrocellulose was incubated overnight with mouse anti-EGF-R clone 29.1 [22] diluted 1 : 1000 in the blocking solution. After washing the blot three times for 30 rain each with 10 mM Tris (pH 7.4) containing 200 mM NaCI and 3% Tween-20, the nitrocellulose was incubated in 125I-labeled antimouse secondary antibody for 2 h. The nitrocellulose was washed as above, dried and exposed to X-ray film and developed after 1 day. Detection of p85. The anti-P-Tyr antibody treated immune complexes isolated following stimulation by ISO or EGF both by in vitro culturing or anti-EGF-R antibody treated immunocomplexes following in-vivo drug treatment were run on SDS-PAGE and transferred to nitrocellulose as described above. The blot was incubated overnight with anti-rat PtdIns 3-kinase (which recognizes p85 subunit of the 190-kDa holoenzyme) rabbit polyclonal antibody (1:500). Following washes, the nitrocellulose was incubated in the blocking solution with either anti-rabbit IgG alkaline phosphatase conjugate (1:1000) or ~25I-labeled protein A for 1 h. The nitrocellulose was washed and the immunoreactive proteins were detected in 10 mM Tris (pH 9.6) containing 0.1 M NaC1, 2 mg NBT and 1 mg BCIP when using the alkaline phosphatase-antibody conjugate. Similarly, the p85 subunit of PtdIns 3-kinase was detected in lysates from various time points after ISO or EGF administration. The cytoplasm and plasma membrane from control and 72 h ISO or EGF treated samples also were separately used for p85 detection. In-vitro lipid kinase assay. Lipid kinase assays were performed directly on the anti-P-Tyr immune complexes bound to sepharose beads (Amersham). The

43 immunoprecipitates obtained above were incubated at 4°C for 20 rain with PtdIns (1 mg/ml) as substrate (previously dried with phosphatidylserine 0.25 mg/ml, under nitrogen, sonicated and resuspended in 20 mM Hepes buffer containing 1 mM EDTA). PtdIns 3-kinase activity was measured by the method of Whitman [11] with the following modifications. Kinase reactions were initiated by adding [y-32P]ATP (15 /zCi per reaction) to a reaction mixture 120 ~ M ATP, 30 mM MgC12 and 60 mM Hepes (pH 7.4) in a final volume of 120 /zl. Tubes were incubated at room temperature for 30 rain and the reactions were stopped by adding 350/zl of 1 M HCI. Subsequently, 400 p~l of chloroform/methanol (1 : 1, v/v) was added. Samples were mixed by vortex and centrifuged briefly to separate the phases. The lower organic phase was dried under nitrogen and resuspended in 50 /zl chloroform. Samples were spotted onto oxalate-pretreated silica-coated thin-layer chromatography plates (EM Science, N J) and separated by ascending chromatography in a solvent system consisting of chloroform/methanol/acetone/glacial acetic acid/water (60:20 : 23 : 18: 11). The solvent was run within 3 cm of the top of the 20-cm plate and then air dried. Radioactive spots were visualized by autoradiography, identified by comparison with the unlabeled lipid standards (Sigma), stained with iodine vapor and quantitated by liquid scintillation counting by scraping the spot. Using this assay, PtdIns(3)P in the neonatal rat neurons stimulated by insulin has been confirmed by HPLC [30]. Phosphatidylinositol(4)P was used to identify the position of PtdIns(3)P, a product of PtdIns 3-kinase. The two lipids have been reported to migrate with similar Rf values; PtdIns(3)P migrated slightly lower than PtdIns(4)P [11].

Other methods. Protein assays where appropriate were made at one time by a modification of the Lowry method with bovine serum albumin as the standard [31]. Statistical analysis. The paired Student's t-test was used to determine the significant differences between the means. Results

Type I (Ptdlns 3) and type II (Ptdlns 4) lipid kinases were assayed in fl-adrenergic agonist (isoproterenol)or growth factor (EGF)-stimulated rat parotid glands. Chronic in-vivo administration of ISO or EGF caused an increase in the level of the Ptdlns 4-kinase (92% and 171% over controls, respectively) and Ptdlns 3kinase (44% and 188% over controls, respectively; Table I) activities. The enzyme activities showed linearity over 30 rain under the assay conditions for both control and experimental samples (data not shown). The differentiation of Ptdlns 3-kinase activity from Ptdlns 4-kinase activity was further confirmed by inclusion of various concentrations (0.01% to 5%) of NP40 in the reaction mixture. Ptdlns 3-kinase showed a dose dependent inhibition in that a 50% inhibition of the 3-kinase was noted at 0.05% NP40 and 90% inhibition when 2% detergent was present in the reaction mixture. Similar sensitivity to the detergent was not observed for Ptdlns 4-kinase activity. The Ptdlns 3-kinase inhibition observed in the present study is similar to that reported by Shibasaki [28] in bovine thymus. To test for activation of the enzymes by tyrosine phosphorylation, following ISO or EGF in-vitro stimulation of cells, isolated primary cultures were incubated for 15

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Fig. 1. Phosphatidylinositol 3-kinase activity in anti-P-Tyr and E G F - R immunoprecipitates of rat parotid acinar cells. Cells were stimulated with 1 /zM ISO or 100 n M E G F for 15 min at 37°C. Solubilized supernatant protein (150/Lg) from the cell lysates were immunoprecipitated with either anti-P-Tyr antibody (A) or anti-EGF-R antibody (B). The Ptdlns 3-kinase activity in the immunoprecipitable complexes wase m e a s u r e d as described in Materials and Methods. Each value is a m e a n + S.D of three observations. * P < 0.05; * * P < 0.001, compared to control.

44 min in the presence of the appropriate stimuli. They were then immunoprecipitated with monoclonal antiphosphotyrosine antibody and the resulting pelleted proteins analysed for Ptdlns kinase activity. As shown in Fig. 1A, a small amount of kinase activity was associated with unstimulated control cells following immunoprecipitation by the anti-P-Tyr antibody. Quantitation of the radioactive spots adjacent to PtdIns(4)P standard revealed a 6.2- and 2.2-fold enhancement in the 3-kinase product in the anti-P-Tyr fraction of cells upon ISO or EGF treatment, respectively. A similar increase in the Ptdlns 3-kinase activity (4.8 and 2.7-fold in ISO or EGF treatment, respectively) was observed when antibody to EGF-R (29.1 clone) was used instead of P-Tyr antibody in the immuneprecipitation (Fig. 1B). Thus stimulation by ISO or EGF caused a significant induction of Ptdlns 3-kinase activity compared with unstimulated cells in both the antiP-Tyr and anti-EGF-R immunoprecipitated samples. The immunocomplexes obtained using anti-P-Tyr also showed NP40 detergent sensititivy similar to the lysates (data not shown). In addition to the increase in Ptdlns 3-kinase activity, activation of intrinsic tyrosine kinase activity of the EGF-R could be detected by measurement of tyrosine autophosphorylation in an immunoblot using antibody to EGF-R. In in-vitro-untreated dispersed cell cultures and those treated with ISO or EGF, an immunoreactive band corresponding to approx. 170 kDa was observed when the anti-P-Tyr immunoprecipitates were run on SDS-PAGE and detected by antibody to EGF-R in Western blotting (Fig. 2A). Using non-immune serum in the immunoprecipitation, no increase in the Ptdlns 3-kinase activity was observed suggesting that the EGFor ISO-induced Ptdlns 3-kinase was observed in the

TABLE I

Effect of ISO and EGF on the parotid acinar cell phosphatidylinositol kinase activity Ptdlns 3-kinase and Ptdlns 4-kinase in the control and 72-h ISO- or EGF-treated rats were measured in a reaction mixture as described in the Materials and Methods section, utilizing total cell lysate protein (50 /zg) as the source of enzyme. After incubation, lipids were extracted with c h l o r o f o r m / m e t h a n o l / H a (200:100:1) and the phases separated. The lower phase was washed and taken for radioactivity and measurement directly. Values are mean + S.D. of four observations. nmol/mg protein per h:

Control ISO % change over control EGF % change over control

Ptdlns 4-kinase

Ptdlns 3-kinase

0.476 + 0.06 0.912+0.09 ** + 92 1.290+0.10 ** + 171

0.500 + 0.08 0.729+0.06 * + 44 1.466+0.11 ** + 188

* P < 0.05; ** P < 0.001, compared to control.

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49 32 Fig. 2. (A) Detection of EGF-R in the anti-P-Tyr immunoprecipitates of parotid acinar cells following stimulation in in-vitro-dispersed cell culture. Equal amounts (150 ~g protein) of solubilized supernatant from the untreated (a), ISO (b) or EGF (c)-treated acinar cell lysates were used to obtain the immunoprecipitable (anti-P-Tyr) complexes. Subsequently the proteins were separated by SDS-PAGE, transferred to nitrocellulose and blotted with anti-EGFR antibody. Following washes, the protein was detected by incubation with 125I-labeled Protein A and by autoradiography. Blots were exposed for 1 day at -80°C. (B) Detection of p85 in the above anti-P-Tyr immuno-precipitates. The proteins of the immunoprecipitable complexes were resolved by electrophoresis on 9% SDS-PAGE and detected by the Western blot technique [45] by incubation with anti-p85 antibody. After washes, the immunoblots were incubated with [125I]Protein A followed by autoradiography. Blots were exposed for 1 day at -80°C. Prestained molecular-weight standards are: myosin, 205 kDa; /3-galactosidase, 116 kDa; bovine serum albumin, 80 kDa; ovalbumin, 49 kDa; carbonic anhydrase, 32 kDa.

immunoprecipitates only when the EGF-R was present. An analysis of P-Tyr immunoprecipitates using monospecific antibody to Ptdlns 3-kinase detected an immunoreactive band with a molecular mass of 85 kDa in the untreated control, which showed an increase upon ISO or EGF treatment (Fig. 2B). To gain additional evidence on the association between the Ptdlns 3-kinase and EGF-R, and also to compare the above in-vitro results with in-vivo conditions, we assayed p85 levels in cell lysates from untreated and 72 h ISO or EGF treated rats. A timecourse response in activation of p85 after chronic ISO or EGF administration was also determined. Equal amounts of parotid cell lysate protein from untreated, ISO- or EGF-treated rats for 15 min, 1 h, 24 h and 72 h, were separated by SDS-PAGE, and probed for p85 by Western blotting. A significant amount of Ptdlns 3-kinase was detected in the non-stimulated control lysates. Upon either ISO or EGF treatment, the amount of p85 associated protein increased at 24 and 72 h treatment (Fig. 3). Scanning of these bands corresponding to p85 by densitometer showed areas of peaks, 25 and 88% higher, at 24 h and 72 h, respectively, for ISO compared to control. Similarly, the areas of the bands at 24 h (110%) and 72 h (105%) post-EGF treatments show increase over the control. When the lysates from control and 72 h ISO or EGF treatment were immunoprecipitated first using anti-p85 antibody and subsequently detected by the anti-EGF-R antibody

45

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EGF

B

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Fig. 3. Time-course in the detection of ~85. Total parotid acinar cell lysates from untreated, 15 min, l-h, 24-h and 72-h ISO-or EGFtreated rats were lysed as described in Materials and Methods. Equal amounts of protein (35 pg) from solubilized supernatants were run on a 9% SDS-PAGE and transferred to nitrocellulose. After incubation with polyclonal anti-p85 and anti-rabbit IgG alkaline phosphatase conjugate as described in Materials and Methods, the immunoreactive proteins in the blot were detected by alkaline phosphatase. Prestained molecular-mass standards are: myosin, 205 kDa; p-galactosidase, 116 kDa; bovine serum albumin, 80 kDa; ovalbumin, 49.5 kDa; carbonic anhydrase, 32.5 kDa.

using Western blot, a protein band with an apparent M, 170 kDa corresponding to EGF-R was observed which was found to have undergone increased phosphorylation upon IS0 or EGF treatment (Fig. 4A). Although the anti-p85 antibody immunoprecipitated tyrosine phosphorylation associated with the EGF-R, it failed to detect tyrosine-phosphorylated protein associated with ~85. Consistent with this observation the antibody to EGF-R coimmunoprecipitated the 85-kDa protein which can be recognized by using antibody to ~85 in an immunoblot (Fig. 4B). Densitometric analysis of the band areas corresponding to M, 85 kDa showed that the coimmunoprecipitated p85 increased after growth stimulation when equal amounts of protein from the lysates were used in the initial immunoprecipitation (data not shown). Since the PtdIns 3-kinase substrates are membrane constituents, it was of interest to determine the distribution of the enzyme upon stimulation by IS0 or EGF. This was done by isolating the plasma membrane and cytoplasm from control and 72 h IS0 or EGF treated rat parotid gland and detecting PtdIns 3-kinase using the antibody to ~85. Fig. 5 is an autoradiogram from the above experiment. In control cells, there was a very small amount of PtdIns 3-kinase protein associated with the plasma membrane whereas there were signifi-

Fig. 4. (A) Association of p85 with the EGF-R following in vivo stimulation. Parotid acinar cell lysates from untreated (a), 72 h IS0 (b)- or EGF (c)-treated rats were lysed as described in Materials and Methods. After normalizing for protein content (3.5 pg), samples were immunoprecipitated with anti-p85 antibody. The immune complexes were run on 9% SDS-PAGE, transferred to nitrocellulose and the blot was probed with anti-EGF-R (29.1) followed by detection of ‘2SI-labeled Protein A. (B) The samples lysed as described above were immunoprecipitated with anti-EGF-R (29.1) and the immunoreactive complexes separated by 9% SDS-PAGE, transferred to nitrocellulose and the blot incubated with anti-p85 antibody followed by detection with alkaline phosphatase. Prestained molecular-weight standards are same as shown for Fig. 2.

cantly higher levels of enzyme localized in the cytoplasm. This low level of p85 in control plasma membrane is consistent with the low PtdIns 3-kinase activity in the total cell lysates immunoprecipitated with anti-

pl.mem abc

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06

B

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32 Fig. 5. Targetting of p85 from cytoplasm to plasma membrane upon growth stimulation. The plasma membrane (35 Fg) and the cytoplasmic proteins (70 pg) from untreated (a,d), 72-h IS0 (b,e)- or EGF (&-treated rat parotid gland lysates were separated by 9% SDSPAGE, transferred to nitrocellulose and incubated with antibody to p85 and detected by ‘2SI-labeled Protein A. Prestained molecularweight standards are same as given in legend for Fig. 2.

46 P-Tyr in rat adipocytes [32]. In response to EGF, there was an increase in the plasma-membrane level of the enzyme with concomitant decrease in the cytoplasm. The same trend of movement of the enzyme was observed in response to chronic ISO stimulation of cell proliferation. In order to delineate further the subcellular localization of the enzyme, first the low-speed microsomes (enriched with plasma-membrane sheets, lysosomes and mitochondria) and later high-speed microsomes (containing Golgi complex, endoplasmic reticulum and endosomes) were prepared as described in Materials and Methods. These two fractions were subjected to a continuous sucrose density-gradient centrifugation and subsequently fractions from the gradient were collected. An aliquot of alternate fractions representing equal amounts of protein were run on SDS-PAGE and tested for p85 subunit of Ptdlns 3kinase by using antibody to p85 in Western blot. No bands corresponding to 85 kDa in the fractions of

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Fraction number Fig. 6. (A) Marker enzymes in the sucrose density-gradient fractions. The rat parotid gland lysate was subjected to sucrose density-gradient centrifugation folowed by fractionation as described in Materials and Methods. Aliquots of alternate fractions were assayed for/3-1,4 galactosyltransferase ( l , a marker enzyme for Golgi complex) or ~/-glutamyltranspeptidase (13, a marker enzyme for plasma membrane) [25,26]. (B) Autoradiogram showing the p85 in the sucrosedensity gradient fractions. Equal amounts of protein (5 ~g) from alternate fractions (2-28) of sucrose-gradient (low-speed microsomal) fractions of control and 72 h ISO- or EGF-treated rat parotid glands were separated by 9% SDS-PAGE, transferred to nitrocellulose and the p85 associated with the samples was detected as described in Materials and Methods.

high-speed microsomes were detected (data not shown). However, the low-speed microsomal fractions showed an increase in the 85-kDa band in the plasma membrane of ISO or EGF treated samples compared to control. The marker enzymes for the identification of Golgi complex and plasma membranes in these fractions were /3-1,4 galactosyltransferase and ~/-glutamyltranspeptidase, respectively, as shown in Fig. 6A. Typical of the profile observed is that shown in Fig. 6B where upon stimulation of proliferation, p85 increased in the plasma-membrane fraction. Discussion

In the present study, we have shown that both the type I and type II (PtdIns 3- and Ptdlns 4-, respectively) Ptdlns kinases are activated in response to growth stimuli in parotid acinar cells (Table I). The Ptdlns 4-kinase activation seen here is consistent with our earlier observation on phosphatidylinositol turnover and Ins(1,4,5)P 3 formation after ISO stimulation [20]. The activation of both kinases involved in phosphatidylinositol metabolism following /3-agonist- or growth-factor-stimulated signal transduction suggests a possible overlap between the two pathways, one leading to the generation of Ins(1,4,5)P 3 and diacylglycerol (PtdIns 4-kinase) and the other forming Ptdlns(3)P (Ptdlns 3-kinase). In A431 cells, rat brain and human red blood cell [33-35] it has been reported that Ptdlns 4-kinase and PtdIns(4)P 5-kinase are associated with a specific region of the EGF-R and are tyrosinephosphorylated. However, Pignataro and Ascoli [36] reported that EGF-stimulated D-3 inositol phosphorylation is restricted to only a few cell types. Metabolites of type I Ptdlns kinase have been found in cells transformed by oncogenes or growth factors [37]. Further studies on the role of other phospholipids such as phosphatidylserine and phosphatidylcholine in regulating phosphoinositide metabolism, as well as investigations into the other kinases a n d / o r phosphatases that may be activated by Ptdlns hydrolysis are necessary to clarify the simultaneous operation of two pathways in Ptdlns second messenger generation. The association of Ptdlns 3-kinase (p85 subunit) with the EGF receptor upon its activation and the increased amount of p85 associated with the phosphotyrosine-containing protein fraction of the total cell lysates suggests that the P-Tyr containing proteins play a crucial role in signal transduction. These observations are in agreement with others [37,38], who reported in various cell lines the association of Ptdlns 3-kinase activity with growth-factor receptors and oncoproteins and the significance of phosphotyrosine in mediating the signal transduction. Other observations however indicate that the binding of the p85 SH2 domains to the EGF-R in vitro did not correlate well

47 with the in-vivo p85 interactions. Hu et al. [17] also showed a lower affinity of the p85 for the EGF-R than for the PDGF-R. Although no attempt was made in the present study to detail the affinities between p85 and EGF-R, it can be said from the results that in parotid acinar cells the Ptdlns 3-kinase plays a significant role in EGF-mediated signal transduction and that the extent of p85 subunit interaction with the EGF-R is similar in both in-vivo and in-vitro conditions. This was evident for both ISO and EGF elicited growth stimulation (Table I and Figs. 1, 2 and 4). Although the quantity of novel phosphoinositide (Ptdlns(3)P) is small compared with conventional phospholipids, their detectable levels in cells indicate that they are highly labeled in vivo. Further metabolites of Ptdlns(3)P or other polyphosphoinositides are not known. It is believed that they themselves act as signals to mediate growth. Thus, the receptor-associated PtdIns 3-kinase may not have a significant role in Ptdlns hydrolysis per se, but may be important in producing the second messengers mediating the mitogenic action of ISO or EGF in parotid acinar cells. The mechanism for this involvement is not known at the present time. Although p85 seems to be associated with the activated EGF-R in vivo, tyrosine phosphorylation of p85 in response to the /3-adrenergic agonist or epidermal growth factor could not be detected. Ptdlns 3-kinase has an apparent size of 190 kDa as determined by gel filtration and sucrose density-gradient centrifugation. It exists as a dimer consisting of 85-kDa (p85) and 110-kDa (pll0) subunits [39]. The amino-acid sequence of p85 shows homology to the s r c homology region 2 (SH2) [40]. Recently, Hu et al. [17] reported that the p85 may function as a regulatory subunit of p l l 0 or it may act as an adapter molecule that targets Ptdlns 3-kinase to growth-factor receptor tyrosine kinases. We observed increased phosphorylation of a protein of 110 kDa upon ISO or EGF stimulation (Fig 2A). It would be interesting to determine whether this l l0-kDa protein is a subunit of Ptdlns 3-kinase, although a number of other proteins such as GAP have similar molecular masses. The tyrosine phosphorylation of p l l 0 may therefore have greater significance in the signal transduction. Using antibody to p85 in the present study tyrosine-phosphorylated p85 could not be detected (possibly due to low levels of phosphorylation). However it was possible to immunoprecipitate tyrosinephosphorylated p85 which showed kinase activity, a finding similar to the transformed cells overexpressing EGF-R [16,17]. This suggests that the acinar cells may behave in a manner similar to those cells which possess the high affinity EGF-R. The subcellular distribution of p85 in parotid gland indicate that the majority of the subunit of enzyme is present in the cytoplasm in the unstimulated cells and the remainder is localized to either low-density or

high-density membranes. Upon growth stimulation by ISO or EGF, the activity is predominantly associated with plasma membranes with no detectable levels in the high-speed microsomes. A large amount of Ptdlns 3-kinase existing in the cytosol has been reported for adipocytes, liver and fibroblasts [32,39,41]. The increase in plasma membrane p85 in response to stimuli would not appear to be due to the increased levels of the enzyme protein but may largely be due to the recruitment of the enzyme from the cytoplasmic pool. It is possible that the cytosolic Ptdlns 3-kinase may be phosphorylated by EGF-R tyrosine kinase and then mobilized to the plasma membrane. Further studies detailing the role of phosphorylation on the subcellular distribution of Ptdlns 3-kinase are necessary to understand the role protein tyrosine phosphorylation plays in targeting of these kinases to their plasma-membrane lipid substrates. The results of the present study suggest that in both EGF- and ISO-stimulated acinar cell proliferation, Ptdlns 3-kinase acts as an intracellular transducer molecule in perpetuating the signal from cell surface to the nucleus. Under similar experimental conditions, another molecule to which the activated EGF receptor physically associates, namely GTPase activating protein (GAP), is also activated in parotid acinar cells [42]. Both these molecules contain SH2 domains that are homologous to s r c tyrosine kinase [43]. It is possible that the Ptdlns 3-kinase and GAP could bind to the same site on the EGF-R or to separate sites located within the noncatalytic region. Recently it has been reported that in 3T3 cells, Ptdlns 3-kinase and GAP bind to different phosphotyrosine residues on t h e PDGF receptor [43]. The relative specificities of the interactions of these two SH2-containing proteins with the phosphotyrosine residues of the activated receptor are being studied to further elucidate the mechanism of ISO- or EGF-induced cell proliferation.

Acknowledgements The authors thank Mr. James Fischer for technical assistance and Ms. Marilyn Lietz for preparation of the manuscript. This work was supported by NIDR grants DE 08778 and DE 00291 to MH-B, AG10485 to FTC and DE10234-01 to KRP.

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