Altered levels and protein kinase C-mediated phosphorylation of substrates in normal and transformed mouse lung epithelial cells

EXPERIMENTALCELL RESEARCH 2 0 0 , 149-155 (1992)

Altered Levels and Protein Kinase C-Mediated Phosphorylation of Substrates in Normal and Transformed Mouse Lung Epithelial Cells CLIVE M . G. M O R R I S 1 AND GARRY J . S M I T H

Department of Pathology, Carcinogenesis Research Unit, University of New South Wales, Kensington 2033, Australia

P r o t e i n p h o s p h o r y l a t i o n a n d p r o t e i n k i n a s e C (PKC) l e v e l s w e r e a n a l y z e d in i n t a c t c u l t u r e s o f s p o n t a n e o u s l y transformed, chemically transformed, and untransf o r m e d m o u s e p u l m o n a r y e p i t h e l i a l c e l l l i n e s . It w a s found that although the transformed cell lines cont a i n e d a b o u t 80% l e s s p r o t e i n k i n a s e C, m e a s u r e d as t o t a l e n z y m e a c t i v i t y o r b i n d i n g o f [3H]phorbol e s t e r , phosphorylation events after phorbol ester treatment c o u l d still b e e a s i l y d e t e c t e d . A c o m m o n l y d e s c r i b e d M r 8 0 - k D a p r o t e i n k i n a s e C s u b s t r a t e ( p 8 0 , 8 0 K, MARKS) was identified using 2D-PAGE, following p h o s p h o r y l a t i o n in i n t a c t c e l l s , a n d f o u n d to h a v e red u c e d a v a i l a b i l i t y for p h o s p h o r y l a t i o n in t h e t r a n s f o r m e d c e l l l i n e s C4SE9,C1SA5a n d N U L B 5 in c o m p a r i s o n to t h e u n t r a n s f o r m e d C 4 E t o a n d C~C~o. A v a i l a b l e l e v e l s o f p 8 0 w e r e f u r t h e r a n a l y z e d in h e a t - d e n a t u r e d e x t r a c t s f r o m all c e l l l i n e s u s i n g p a r t i a l l y p u r i f i e d bovine brain PKC and correlated well with changes seen in i n t a c t c e l l s . It w a s a l s o n o t e d t h a t all t r a n s f o r m e d c e l l lines contained large amounts of a family of phosphop r o t e i n s o f Mr 5 5 - 6 5 k D a , t h a t c o u l d n o t b e d e t e c t e d in the untransformed cell lines and whose phosphorylation state was increased by protein kinase C activation. T h i s p r o t e i n w a s f o u n d to b e l o c a t e d in t h e n u c l e u s . Hence, spontaneously and chemically transformed mouse pulmonary epithelial cells exhibit reduced levels o f P K C , a l o n g w i t h an a l t e r e d p a t t e r n o f P K C - m e d i a t e d phosphorylation.

9 1992 Academic Press, Inc.

INTRODUCTION Binding of factors to specific cellular receptors initiates signal transduction pathways, usually commencing with activation of protein kinases and terminating with alterations in gene expression, which in turn lead to events such as cell division or differentiation. Neoplastically transformed cells exhibit a decreased ability to respond appropriately to normal regulatory signals. Oncogenic activation has been detected at all levels of signal transduction, including growth factors (v-sis), growth factor receptors (erb family), guanidine 5'-tri1 To whom reprint requests should be addressed.

phosphate-binding proteins (ras family), cytoplasmic serine/threonine protein kinases (mos, raf), and nuclear transcriptional regulatory proteins (fos, myc, jun) (for review, see [1]). Protein kinase C (PKC) plays a key role in early events resulting from binding of growth factors to specific receptors, and transformed cell lines generally show a decrease in PKC activity [2, 3], although this is not always the case [4]. However, little is known about how neoplastic transformation affects the availability or activity of substrates for PKC. The type II pneumocyte acts not only as the producer of pulmonary surfactant, but has other major roles, acting as a precursor for type I epithelium in response to tissue damage; in the metabolism of xenobiotics and the transepithelial movement of water [5]. In mouse lung, the type II pneumocyte is thought to be the target cell in the development of adenomas and carcinomas in response to chemical insult (for review see [6]). Previous work from this laboratory led to the establishment of an epithelial cell line from normal mouse lung, closely related to the type II pneumocyte (NAL1A), which underwent spontaneous malignant transformation in vitro [7]. We now have for comparison three nonmalignant cell clones derived from the original NAL1A cell line (C4E,0, CIC10, and BsD3), their spontaneously malignant "siblings" (C4SEg, CISAs, and BsSF11), and cell lines derived from N U L l , established from a urethaneinduced pulmonary adenoma (NULB5 and NULB3) [8, 9]. Previous studies indicated that our transformed cell lines have an activating mutation of Ki-ras [11] and reduced amounts of fibronectin and epidermal growth factor (EGF) receptor [10]. Little is known of how spontaneous transformation affects expression of PKC and its substrates in mouse lung epithelial cell lines. Here, we describe alterations in PKC and availability of PKC substrates in intact cells of our cloned malignant and nonmalignant lung epithelial cell lines. METHODS AND MATERIALS Cell culture. All cell lines were maintained in CMRL 1066 medium with a-glutamine (ICN-Flow Laboratories, Sydney, Australia) containing 10% fetal bovine serum (Commonwealth Serum Laboratories, 149

0014-4827/92 $3.00 Copyright 9 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

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Parkville, Australia), 100 IU/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 ttg/ml amphotericin B {Sigma) and kept at 37~ in an atmosphere of 5% CO2 and passaged weekly. Stock cultures were maintained in 25-cm 2 tissue culture flasks (ICN-Flow Laboratories). Phosphorylation in intact cells. Cells were seeded into 30-mm wells in 6-well tissue culture plates {Flow Laboratories) at a density of 2 • l0 s cells per well. Cultures were refed after 2 days and used at confluence {normally 5 days). Confluent cultures were washed three times in phosphate-free DMEM (Flow Laboratories) and incubated in this medium containing 100 #Ci/ml [a2Pi] (Amersham Australia) for 3.5 h, followed by treatment with 200 nM phorbol-12,13-dibutyrate (PBt2) or carrier for 15 min. After this time, cultures were washed rapidly with cold phosphate-buffered saline (PBS) and extracted with extraction solution (2% sodium dodecyl sulfate (SDS), 5% fi-mercaptoethanol, and 10% glycerol). Extracts were heated at 100~ for 10 min and then stored at -20~ until resolution by 2-dimensional gel electrophoresis. For analysis of heat-stable phosphoproteins, cytosolic proteins were extracted with extraction buffer (20 m M Tris, pH 7.5, 2 mM EDTA, 1 mM EGTA, 10 m M NaF, 1 m M PMSF, and 0.5% Triton X-100) without scraping and heated to 100~ for 10 rain and centrifuged at 13,000 rpm for 5 min to pellet heat-denatured proteins [12]. An aliquot of supernatant was resolved by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), as described below. S D S - P A G E . Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was carried out essentially according to O'Farrell [13], using isoelectric focusing in the first dimension and S D S - P A G E (8% gel) in the second. For 1-dimensional SDS-PAGE, samples were routinely mixed with an equal volume of 2• SDS sample buffer (130 mM Tris, pH 6.8, 20% glycerol, 10% fi-mercaptoethanol, 4% SDS), heated at 100~ for 5 rain and run on an 8% S D S - P A G E gel. After electrophoresis, gels were stained, destained (20% methanol, 10% acetic acid), dried, and autoradiographed using Fuji X-ray film (Fuji Photo Film Company, Japan). Phosphorylation in cell extracts by PKC. Protein kinase C was partially purified from frozen bovine brain using ammonium sulfate precipitation and chromatography on phenyl-Sepharose CI-4B (Sigma (pfs)), as described by Walsh et al. [14]. Fractions were assayed for PKC activity using histone III-S as substrate [15] in the presence and absence of PKC activators. PKC-containing fractions were pooled and stored as aliquots at - 7 0 ~ in 20% glycerol. Confluent cultures of cells on 15-cm tissue culture dishes (Flow Laboratories) were washed and scraped into 200 ttl extraction buffer (see above), passed 10 times through a 25-G needle and centrifuged for 10 min at 13,000 rpm. Supernatants were placed at 100~ for 10 min and centrifuged for 10 min at 13,000 rpm to pellet heat-denatured proteins. Assay of PKC substrates in heat-treated cell extracts [16] was performed in a total volume of 80 #1 containing 20 mM Tris, pH 7.5, 7.5 m M magnesium chloride, 10 ttg heat-treated cell extract, 2 t*g PKC preparation, and 30 tLM [~-a2p]adenosine 5'-triphosphate (ATP) (2 ttCi); in the presence and absence of 80 ttg/ml phosphatidyl-L-serine, 200 nM PBt 2, and 0.4 m M CaCl2 (activators). The reaction was started by the addition of ATP and stopped after 5 min by the addition of 80 ttl of 2• SDS sample buffer and heating at 100~ for 5 min. Phosphorylated proteins were then resolved by SDS-PAGE. Protein kinase C assay. Protein kinase C was partially purified from extracts of 20-50 million cells (20 m M Tris-HCl, pH 7.5, 0.25 M sucrose, 2 m M EDTA, 2 m M EGTA, 1 m M PMSF, 5 m M DTT) using batch elution from a 1-ml column of DE-52 (2 ml of 20 m M Tris-HC1, pH 7.5, 2 mM EDTA, 1 m M EGTA, 1 m M PMSF, 1 m M DTT, 130 mM NaC1), essentially as described by Ase et al. [17]. Assay was performed using 40 #l PKC eluate and histone H1 as substrate [15], in a total volume of 200 #1 (20 m M Tris-HC1, pH 7.5, 5 m M MgC12, 50 ~M ATP (2 #Ci), 60 t~g histone) in the presence and absence of PKC activators (80 ~g/ml PS, PBt 2 100 ng/ml, 0.5 mM CaC12). After appro-

TABLE 1 B i n d i n g of [3H]PBt2 to I n t a c t Cells Binding Cell line

cpm/106 cells

(SEM)

7435 2076 7344 1782 1413

(855) a (240) b (247) (400) (345)

C4ElO C4SE9

CIClo ClSA~ NULB5

a Standard error of the mean. Results are the mean of at least three independent experiments performed in duplicate. b Assuming a normal distribution, each transformed line had significantly fewer [3H]PBt2 binding sites than either C1C10 or C4E10 (P <: O.O5).

priate incubation times, total protein was precipitated in 10% TCA on ice for 5 rain. Pellets were dissolved in 0.5 ml 1 M NaOH and mixed with an equal volume of scintillant and radioactivity was quantified in a scintillation counter. Binding of [3H]PBt~ to intact cells. Confluent cultures in 30-mm wells (6-well dishes from Flow Laboratories) were washed three times in binding medium (CMRL with 1 mg/ml bovine serum albumin and 50 m M Hepes, pH 7.4) and incubated in this medium with 30 nM [aH]PBt~ ([20(n)-3H]PBt2 17.8 Ci/mmol, Amersham Australia) in the presence and absence of a 500-fold excess of cold PBta [18]. After 30 min at 37~ cultures were washed rapidly with cold PBS and solubilized in 1 ml 0.1 N NaOH, 1% Na2CO 3 and cell-associated radioactivity was determined. Specific binding of PBt2 was calculated as that remaining after subtraction of nonspecific counts (500-fold excess) and normalized for cell number. Protein estimation. Protein was estimated in duplicate samples using Bio-Rad (Bio-Rad Laboratories, Australia) protein assay solution, with absorbance being measured at 595 nm.

RESULTS Protein Kinase

C Levels

P K C is t h e m a j o r b i n d i n g s i t e f o r p h o r b o l e s t e r s i n mammalian cells, mediating the actions of these tumor p r o m o t e r s [20, 21]. W e m e a s u r e d [ 3 H ] P B t 2 b i n d i n g t o cultures of untransformed and transformed cell lines as an estimate of levels of PKC in intact cells. Confluent c u l t u r e s i n 3 0 - m m w e l l s w e r e i n c u b a t e d w i t h 30 n M [ 3 H ] P B t 2 f o r 30 m i n , a f t e r w h i c h t i m e c e l l - b o u n d r a d i o a c t i v i t y w a s r a p i d l y d e t e r m i n e d . O v e r 30 m i n , 30 n M PBt2 causes the translocation of PKC to the cell memb r a n e w h e r e t h e b i n d i n g a f f i n i t y f o r P B t 2 is h i g h [22]; and therefore any initial differences in the cellular distribution of PKC between the cell lines would have no bearing on the final amount of [3H]PBt2 bound. As s h o w n i n T a b l e 1, t h e r e w e r e a b o u t 8 0 % f e w e r [ 3 H ] P B t 2 binding sites in both the spontaneously transformed cell l i n e s C4SE9 a n d ClSA~, a n d i n N U L B 5 , i n c o m p a r i s o n t o C 4 E l o or C1Clo ( P < 0.05). PKC activity was also assayed

directly in extracts

CHANGES IN PKC SUBSTRATES AFTER TRANSFORMATION from untransformed CIC10and transformed ClSA5cells, following batch elution from DE-52 cellulose and using histone as a phosphate acceptor. It was calculated that the activity in CaSA5 cells was 0.083 pmol phosphate/ min/106 cells, while that in CaCao was 0.53 pmol phosphate/min/106 cells (total of cytosolic and particulate PKC; mean of two experiments). These results compare well with those obtained using phorbol ester binding, with CaSA5having more than 80% less activity than CaCao.

PKC-Dependent Phosphorylation in Intact Cells

151

regions of autoradiographs from the untransformed cell lines, nothing could be detected. Phosphorylation of this protein family was increased by activation of PKC, since treatment with PBt2 resulted in both an increase in density of labeling and a marked acidic shift in pL When crude cellular fractionation was performed prior to 2D-PAGE, it was revealed that these proteins are predominantly found in the nucleus, as shown in Fig. 2. A third phosphoprotein of molecular weight 95 kDa (p95, Fig. 1) was seen to vary between cell lines and with phorbol ester treatment. Because no consistent, or phenotype-specific, effect of PKC activation on p95 was seen (treatment of CaC~0 and CaSA5 cells with PBt2 resulted in loss of p95 phosphorylation, while in C4Ea0 cells the opposite occurred), it is unlikely to be a direct physiological substrate of PKC.

The results presented above suggest t h a t less PKC is present in our transformed cell lines. However, previous results from this laboratory indicated that phorbol ester treatment of intact cells induced a similar degree of transmodulation (decreased affinity for ligand resulting Analysis of Heat-Stable Phosphoproteins from phosphorylation by PKC [23]) of the EGF recepAfter labeling with [32Pi] and treatment with PBt2, tot in both transformed and untransformed cell lines cultures were extracted with buffer containing 0.5% [10]. In order to investigate the effects of PKC activaTriton X-100. Extracts were placed at 100~ for 10 min, tion further, we decided to look at the effect of PBt2 denatured proteins removed by centrifugation, and samtreatment on protein phosphorylation in intact cells laples of supernatant subjected to SDS-PAGE. As shown beled with carrier-free [32Pi]. Extracted proteins were in Fig. 3, a heat-stable protein of 80 kDa was specifically resolved by 2D-PAGE, as shown in Fig. 1. labeled in extracts of C1C,0 in response to treatment A phosphoprotein of molecular weight 80 kDa and pI with PBt2 or vasopressin, while no p80 band could be of approximately 4.5 was present in large amounts in seen in extracts of CSA5 or NULB5. This heat-stability the two untransformed cell lines C4Elo and CICa0. Alin Triton X-100 solubilized extracts is a characteristic though this protein was quite noticeable in autoradioproperty of p80 proteins [24, 16]. graphs from untreated cultures, its degree of phosphorylation increased dramatically upon treatment with Phosphorylation by Exogenous PKC in Cell Extracts PBt2. In the spontaneously transformed cell lines C,SA~ In order to determine whether the decrease (C4SE9, and C4SE9, however, this phosphoprotein was barely visible before treatment and the degree of phosphorylation ClSAs) and disappearance (NULB5) of p80 phosphoryachieved after PBt2 treatment was small in comparison lation (Fig. 1) was due to deficiencies in PKC or to a to that seen even in untreated untransformed cell lines. change in the availability of this PKC substrate, we deIn the tumor cell line NULB5, this p80 phosphoprotein cided to investigate substrates of PKC in heat-denawas not apparent either before or after treatment with tured cell extracts (i.e., in the absence of endogenous PBt2. On the basis of molecular weight, p/, and phorbol kinases and phosphatases). PKC, partially purified ester-dependent phosphorylation it appears that this from bovine brain, was added to solutions containing protein is related to the protein(s) frequently described [~-32P]ATP and 10 tLg heat-treated extracts from conas the major PKC substrate (80 K, 87 K, MARKS) in fluent cultures of untransformed and transformed cells, many cells and tissues and often used as a marker of both with and without the addition of PKC activators. Figure 4 shows proteins specifically phosphorylated by PKC activation [24-27]. The density of the p80 spot on autoradiographs was PKC, since lanes containing no PKC activators show no quantified using a scanning densitometer and expressed phosphoprotein bands. Each of the three untransas a fraction of a spot that was unaffected by PBt2 treat- formed cell lines tested contained a large amount of ment. While the spontaneously transformed cell lines available p80 PKC substrate, while their transformed contained much less phosphorylatable p80, the relative siblings contained a dramatically reduced amount, and magnitude of the increase in labeling after PBt 2 treat- NULB5 contained almost no detectable phosphorylatment was similar to that found in the untransformed able p80. Coomassie blue staining of the same gel cell lines (between three- and five-fold). showed no differences in protein loading. This reduction in phosphorylatable p80 in our transIn both the spontaneously transformed and the urethane-induced adenoma cell lines, a group of proteins of formed cells was not due to an increased level of endogemolecular weight 55-65 kDa (p55-65) and pI 5.3-5.6 nous phosphorylation, since this would have been obwas prominently phosphorylated. In the corresponding served in the equilibrium conditions used for obtaining

152

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CHANGES IN PKC SUBSTRATES AFTER TRANSFORMATION 9 --116 --Ii0

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153 4 I 3

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% r-4

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FIG. 2.

Sucellular fractionation following phosphorylation. Confluent cultures of C,SA5 in 30-mm wells were washed and incubated for 3.5 h in phosphate-free DMEM containing 100 ttCi/ml [32Pi], as described above. After this time, cultures were extracted without further treatment. For the pattern shown on the right hand side of the figure, cultures were extracted directly with extraction solution prior to isoelectric focusing. For the left-hand side, cultures were scraped and pelleted in PBS, and the pellet was resuspended in low salt lysis buffer (20 mM Hepes, 5 mM KC1, 5 mM MgCle, 0.5% NP-40, 0.1% Na deoxycholate, 0.1 mM PMSF, pH 6.8). The suspension was passed 5• through a 25-G syringe and nuclei were pelleted by centrifuging at 1000g for 5 min. Nuclei were washed in lysis buffer and extracted with extraction buffer prior to IEF. Both IEF gels were run on the same 8% SDS-PAGE gel, with isoelectric focusing from left (pH6) to right (pH 4.3). Arrowheads indicate the position of p55-65. The photograph represents results from two independent experiments each giving similar results,

t h e r e s u l t s s h o w n in Fig. 1. W h e n s a m p l e s w e r e t r e a t e d with alkaline phosphatase (calf intestine, BoehringerMannheim) prior to treatment with PKC, there was no change in the relative levels of p80 phosphorylation s e e n i n Fig. 4. T h e s e r e s u l t s a n d t h o s e p r e s e n t e d a b o v e i n d i c a t e t h a t t r a n s f o r m a t i o n h a s r e s u l t e d in a l o s s o f available p80 PKC substrate. I t is i n t e r e s t i n g t o n o t e t h a t w h e n P K C w a s a d d e d t o untreated total cellular extracts (not heat-denatured), the only protein specifically phosphorylated after addit i o n o f P K C a c t i v a t o r s w a s p 8 0 (i.e., n o P K C - s p e c i f i c phosphorylation of p55-65 could be detected). DISCUSSION N i c k s et al. [28] d e t e r m i n e d t h a t a c h e m i c a l l y t r a n s formed lung adenoma cell line contained less PKC activi t y t h a n o n e o f o u r u n t r a n s f o r m e d cell l i n e s . R e s u l t s presented here extend this observation to include c l o s e l y r e l a t e d , s p o n t a n e o u s l y t r a n s f o r m e d cell l i n e s . F o r t h e C1C1 o a n d C~SA5 cell l i n e s u s e d in t h e s e e x p e r i ments, total PKC activity correlated well with binding

FIG. 3. Heat-stable phosphoproteins in intact cells. Confluent cultures of C1C10 (Lanes 1-3), C,SA5 (Lanes 4-6), and NULB5 (Lanes 7-9) in 30-mm wells were washed and incubated for 3.5 h in phosphate-free DMEM containing 100 ttCi/ml [3~Pi], followed by treatment with PBt 2 (200 nM) (Lanes 2, 5, and 8), vasopressin (50 nM) (Lanes 3, 6, and 9), or carrier alone (Lanes 1, 4, and 7) for 15 rain. After this time, cultures were extracted with 0.5% Triton extraction buffer and heated at 100~ for 10 rain and supernatants subjected to SDS-PAGE. The dark arrows indicate the distance migrated by the molecular weight markers glutamate dehydrogenase (55.4 kDa) and phosphorylase b (97.4 kDa) (combitek, Boehringer-Mannheim, Australia). The figure is representative of three experiments, all giving similar results.

o f [3H]PBt2 t o i n t a c t cells. P r e v i o u s r e s u l t s f r o m t h i s laboratory demonstrated that PBt2 treatment of both untransformed and transformed cells caused a similar degree of transmodulation of the EGF receptor, indicating that a PKC-mediated response could be elicited in t h e t r a n s f o r m e d p h e n o t y p e [10]. T h e r e s u l t s p r e s e n t e d here indicate that exposure of cultures of intact untransformed and transformed cells to phorbol ester res u l t e d in o b v i o u s P K C - s p e c i f i c p h o s p h o r y l a t i o n e v e n t s . Although available levels of the p80 protein were much r e d u c e d i n t h e s p o n t a n e o u s l y t r a n s f o r m e d cell l i n e s (Fig. 1), t h e f o l d i n c r e a s e in l a b e l i n g a f t e r P B t 2 t r e a t m e n t w a s v e r y s i m i l a r t o t h a t s e e n in t h e u n t r a n s f o r m e d cell l i n e s . W e a r e p r e s e n t l y i n v e s t i g a t i n g w h e t h e r t h e s e easily observable PKC-mediated events, coinciding with reduced PKC activity, are due to altered expression of PKC isozymes; or whether this reduced amount o f P K C is s u f f i c i e n t t o e l i c i t t h e r e s p o n s e s s e e n . I n b o t h s p o n t a n e o u s l y t r a n s f o r m e d (C4SE9 a n d ClSAs) a n d c h e m i c a l l y t r a n s f o r m e d ( N U L B 5 ) cell l i n e s , w e o b served large amounts of a group of phosphoproteins of

FIG. 1. PKC-stimulated phosphorylation of proteins in intact cells. Confluent cultures in 30-ram wells were washed and incubated for 3.5 h in phosphate-free DMEM containing 100/~Ci/ml [~2Fi],treated with 200 nM PBt2 or carrier, and subjected to 2D-PAGE, as described under Methods and Materials. Isoelectric focusing in the first dimension was from left (pH 6) to right (pH 4.3), with an 8% SDS-PAGE gel in the second dimension. The upper part of each panel represents phosphorylation in the absence (control), and the lower to phosphorylation in the presence of PBt~2. Arrows indicate the position of p80 ( / ) , p95 (~--), p55-65 (-), and a spot unaffected by PBt2 treatment (-~). P80 was quantified using a scanning densitometer (Hoeffer Scientific Instruments) and expressed as a fraction of the unaffected spot. Photographs represent results from three independent experiments each giving similar results.

154

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-

+

I-

+

I-

+ ~ - - + "

-

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+l-

B5D 3 B5SF11 NULB5 -

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I -

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p80--~

p80-~

FIG. 4. Substrates of PKC in heat-stable cell extracts. Heatstable 0.5% Triton extracts of cultures were prepared and 10-#g aliquots incubated with partially purified bovine PKC and [-y-32P]ATP (2 pCi) in the absence (-) and presence (+) of PKC activators for 5 min, before resolution of samples by SDS-PAGE, as described under Methods and Materials. The top panel represents an 18-h exposure and the bottom panel a 4-day exposure of the same gel. The cell line used in each pair of lanes is indicated at the top of the figure. The top arrow indicates the position of p80, and the bottom arrow to another family of heat-stable substrates (M, 41-48 kDa). The figure represents the results of one experiment from at least five, all giving identical results.

Mr 55-65 k D a , t h a t could n o t be d e t e c t e d in t h e unt r a n s f o r m e d cell lines. A l t h o u g h t h e s e p r o t e i n s were c o n s t i t u t i v e l y p h o s p h o r y l a t e d , t h e y also u n d e r w e n t PKC-dependent phosphorylation after PBt 2 treatment, indicated b y b o t h an increase in d e n s i t y of labeling a n d a shift in pI. B e c a u s e p 5 5 - 6 5 p h o s p h o r y l a t i o n was n o t d e t e c t e d in t o t a l cellular e x t r a c t s t r e a t e d w i t h P K C (and activators) a n d t h e s e p r o t e i n s are p r e d o m i n a n t l y nuclear, p 5 5 - 6 5 m a y n o t be a direct physiological subs t r a t e of P K C . T h e r e is s t r o n g evidence t h a t t h e acidic Mr 80,000 p r o t e i n (p80) identified here as a P K C s u b s t r a t e , is closely r e l a t e d to t h o s e p r e v i o u s l y r e p o r t e d in m a n y cells a n d tissues: i.e., its e l e c t r o p h o r e t i c mobility, isoelectric point, a n d p h o s p h o r y l a t i o n a f t e r PBt2 t r e a t m e n t of i n t a c t cells [24-26], t h e r m a l stability in cell ext r a c t s [24, 16], a n d p h o s p h o r y l a t i o n b y e x o g e n o u s l y a d d e d P K C in h e a t - d e n a t u r e d cell e x t r a c t s [25, 26, 16]. A l t h o u g h t h e purification [16, 29], m o l e c u l a r cloning [30-32], a n d p h o s p h o r y l a t i o n - r e g u l a t e d b i n d i n g to calm o d u l i n [27] of p r o t e i n s of this family h a v e b e e n reported, no specific function h a s as yet b e e n described. I t h a s b e e n r e p o r t e d t h a t as m o u s e J B 6 epithelial cells

p r o g r e s s e d f r o m a p r e n e o p l a s t i c to a n e o p l a s t i c p h e n o type, p80 e x p r e s s i o n d e c r e a s e d [33]. A similar effect w a s n o t e d in n o r m a l a n d t r a n s f o r m e d m o u s e fibroblasts [34]. T h e results p r e s e n t e d in this p a p e r s e e m to be in a g r e e m e n t , with CIC10, C4E10, a n d BsD 3 r e p r e s e n t i n g a preneoplastic phenotype (immortalized, but not transf o r m e d ) a n d ClSAs, C4SEg, BsSFll, a n d N U L B 5 a neoplastic p h e n o t y p e ; a l t h o u g h m e a s u r e m e n t s were p e r f o r m e d only as availability for P K C - m e d i a t e d p h o s p h o r y l a t i o n . H o w e v e r , it is unlikely t h a t this r e d u c t i o n in p h o s p h o r y l a t a b l e p80 in our t r a n s f o r m e d cell lines is due to i n c r e a s e d e n d o g e n o u s levels of p h o s p h o r y l a t i o n , since this would h a v e b e e n d e t e c t e d u n d e r t h e equilibr i u m conditions u s e d for t h e i n t a c t cell e x p e r i m e n t s a n d p h o s p h a t a s e t r e a t m e n t h a d no effect on p h o s p h o r y l a tion levels. W h e t h e r this c h a n g e is due to d e c r e a s e d a m o u n t s of protein, or to a t r a n s l o c a t i o n of p80 (whereby it is no longer available as a P K C s u b s t r a t e ) , awaits f u r t h e r investigation, p e r h a p s u s i n g a specific antisera. P r e v i o u s studies f r o m this l a b o r a t o r y indicated t h a t o u r t r a n s f o r m e d cell lines h a v e a n a c t i v a t i n g m u t a t i o n of K i - r a s [11]. R a s p r o t e i n s are m e m b r a n e - b o u n d G T P b i n d i n g p r o t e i n s t h a t are t h o u g h t to h a v e a role in signal t r a n s d u c t i o n . R e c e n t evidence suggests t h a t t h e y are involved in a c t i v a t i o n of P K C [35-37], via release of diacylglycerol f r o m p h o s p h a t i d y l c h o l i n e [38], a l t h o u g h P K C - i n d e p e n d e n t p a t h w a y s are also r e p o r t e d [39]. Act i v a t i o n of P K C leads to several events, including alterations in gene t r a n s c r i p t i o n a n d its own b r e a k d o w n [20, 21]. As t h e only genetic a l t e r a t i o n so far d e t e c t e d in t h e t r a n s f o r m e d cell lines is the ras m u t a t i o n , we are p r e s e n t l y i n v e s t i g a t i n g t h e possibility t h a t this single e v e n t is r e s p o n s i b l e for the c o m p l e t e t r a n s f o r m e d p h e n o t y p e in t h e s e cells. H e n c e , the results p r e s e n t e d herein indicate t h a t s p o n t a n e o u s l y a n d c h e m i c a l l y t r a n s f o r m e d m o u s e pulm o n a r y epithelial cell lines exhibit d e c r e a s e d t o t a l P K C activity, d e c r e a s e d availability (both in i n t a c t cells a n d in cell e x t r a c t s ) of a c o m m o n l y described M r 8 0 - k D a P K C s u b s t r a t e , a n d e x p r e s s i o n of a family of p h o s p h o p r o t e i n s of M r 55-65 kDa, t h a t could n o t be d e t e c t e d in t h e u n t r a n s f o r m e d cell lines a n d whose p h o s p h o r y l a t i o n s t a t e w a s i n c r e a s e d b y P K C activation. The authors thank Mr. Lance Smith for help with preparation of photographic material and C. Morris for his expert technical assistance. We are indebted to the NSW Cancer Council for their continued support. REFERENCES

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