JOURNAL OF CELLULAR PHYSIOLOGY 152240-244 (1992)

Identification of Multiple PKC lsoforms in Swiss 3T3 Cells: Differential Down-Regulation by Phorbol Ester ANDREE R. OLlVlER

AND

PETER J. PARKER*

Protein Phosphorylation Laboratory, Imperial Cancer Research Fund, London WC2A 3PX, United Kingdom The cxpression of members of the Ca’+ and phospholipid-dependent protein kinase (PKC) family were studied in murine Swiss 3T3 cells. In addition to PKC-a, the presence of immunoreactive PKC-8, -E, and 5 was detected. Treatment with 500 n M 12-0-tetradecanoylphorbol-13-acetate (TPA) led to the down-regulation of a,6, and E isoforms, but not thal of 5 . tiigher concentrations of TPA similarly had no effect on the level of PKC-5. In contrast to PKC-a, the membrane localization of PKC-f, -E, and -5 was not enhanced by extraction in Ca’+-containing buffers, whereas acute TPA treatment increased membrane association of PKC-a, -6, and -E but not that of PKC-j. o 1992 WiIev-Liss, Inc. Swiss 3T3 cells, a “normal” murine fibroblast cell line, has been extensively used as a model system in studying cell growth (Rozengurt, 1986). When the fibroblasts are deprived of serum, they cease to grow and accumulate in the G,-G, stage of the cell cycle. Readdition of serum or defined growth factors triggers a number of early responses a t the membrane as well a s in the cytosol culminating in the initiation of DNA synthesis and cell division. One of the early agonist-induced responses frequently observed is phosphoinositide hydrolysis by phospholipase C that leads t o the generation of two second messengers diacylglycerol (DAG) and inositol 1,4,5 trisphosphate (Ins1,4,5P3), recently reviewed in (Meldrum et al., 1991). Ins1,4,5,P3 causes the release of Ca2+ from internal stores (Streb e t al., 1983) and both Ca2+and DAG are thought to play a n important role in the activation of protein kinase C (PKC), the phospholipid, DAG, and Ca2+ dependent serinei threonine kinase first described by Nishizuka and colleagues (reviewed in Nishizuka, 1986). PKC is now known to be comprised of a family of enzymes, and these are thought to play a critical role in the control of many cellular processes (reviewed in Nishizuka, 1988; Parker et al., 1989). The current family numbers seven genes (a-q) encoding a t least 10 polypeptides (some apparently derived as alternative splice products) (Coussens et al., 1987; Kubo et al., 198713; Ono et al., 1987). The enz mes can be broadly divided into two classes; the Cap’ dependent forms ( a , p, y) and those that probably do not require Ca2+for activation (6, E, 1;, q). The presence of PKC-a in Swiss 3T3 cells has been well documented and has been thought to be the only isoform expressed in these cells (RoseJohn et al. 1988). The subsequent identification of additional members of the PKC family (Bacher et al., 1991; Ohno et al., 198813; Ono et al., 1988; Osada et al., 1990; Schaap e t al., 1989), prompted us t o investigate the expression of these enzymes in this model system. 0 1992 WILEY-LISS, INC.

MATERIALS AND METHODS All reagents were from BDH unless stated otherwise. Antipeptide antisera to the predicted COOH-sequences of a,p, y,E, 6 and 4 PKCs were prepared essentially as previously described (Marais et al., 1991; Olivier et al., 1991; Schaap et al., 1989; Ways et al., 1992). The antisera to PKC-q was raised against a C-terminal peptideKLH conjugate as previously described (Dekker, Parker and McIntyre, in preparation). All these PKC antisera recognise the proteins expressed from their cognate cDNAs following transient expression in COS-1 cells (Olivier et al., 1991; Pears et al., 1990; Schaap et al., 1989; Ways et al., 1992; Dekker, Parker and McIntyre, in preparation). Peroxidase-linked donkey-antirabbit IgG antibodies and the ECL Western blotting detection system were from Amersham Int., U.K. 12-0-tetradecanoylphorbol-13-acetate(TPA) was from Sigma. Cell culture and extraction Swiss 3T3 cells (kindly provided by M.M. Burger, Basel) were seeded a t 3.3 x lo4 cells per 6 cm dish in Dulbeccos modified Eagles medium (DMEM) containing 10% fetal calf serum (FCS) (Gibco). After 72 hours the cells were refed with DMEM containing 31% Weymouths medium and 6% FCS. Eight days after seeding the cells were judged quiescent and were either lysed or treated with 0.05% DMSO, or with TPA dissolved in DMSO. For extraction the medium was removed and the cell layer washed two times with 5 ml of ice-cold phosphate-buffered saline (PBS). The cells were then lysed in 100 pl of 2 x concentrated SDS-sample buffer

Received September 5,1991; accepted March 9,1992.

*To whom reprint requests/correspondence should be addressed.

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RESULTS A Western analysis of the isoforms of PKC expressed in Swiss 3T3 cells is shown in Figure 1. It is evident 200from the immunoreactivity with the specific sera that PKC-a, -6, +, and -5 are present. PKC-5 appears to be very susceptible to proteolysis, which results in the ad11 6ditional detection of two proteolytic fragments that are competed by antigen (see also Ways et al., 1992). PKC9 7PI,-p2, y, and q could not be detected when using either monoclonal antisera to y or polyclonal antisera to PI, pz,and -q (data not shown). 66The expression and down-regulation of PKC-a in Swiss 3T3 cells has been widely monitored in defining the role of PKC in agonist-induced responses (e.g., Rodriguez-Pena et al., 1986). I n order to determine the validity of such approaches, the TPA-induced downregulation of these PKC isoforms has been studied. A 43time-dependent loss of PKC-a, -6, and -E is observed on exposure to TPA (Fig. 2). Interestingly, the rate of loss of the individual isoforms is not uniform. PKC-a and PKC-8 follow similar rates of down-regulation, whereas the loss of PKC-e is %fold slower than PKC-a. In contrast to this characteristic down-regulation, no loss of PKC-5 is observed. This differential sensitivity is not a reflection of the phorbol ester dose employed since no PKC-( down-regulation is seen over a range of TPA concentrations (Fig. 3). As noted for the rate of loss of PKC-a, -6, and -el there is also a similar difference in the down-regulation sensitivity to TPA of these isoforms. Thus, PKC-a and -6 show a similar sensitivity, Fig. 1. PKC isoforms present in Swiss 3’r3 cells. An aliquot of total whereas PKC-Eis less sensitive. protein from quiescent Swiss 3T3 cells was subjected to SDS-PAGE Since PKC-c was insensitive to TPA-induced downand Western blotting as described in Materials and Methods. Blots were probed with antisera specific to PKC-a (A), PKC-8 (B), PKC-E regulation, it was appropriate to determine whether (C), and PKC-< (D) in the presence ( + ) or absence ( - ) of competmg TPA could induce stabilisation of the membrane-associpeptide antigen, The competed, immunoreactive bands in A-C repreated enzyme. Cytosol and particulate fractions from sent the intact polypeptides. In D in addition to the full length PKC-I control or TPA-treated 3T3 cells were thus analysed for (-75 kDa) are two proteolytic fragments of -60 kDa and -35 kDa, which are competed by antigen. This is one of over a dozen experi- isoform content (Fig. 4A, B). In untreated cells all 4 ments showing the same pattern of expression. isoforms are predominately cytosolic, with PKC-E and -5 showing some membrane association. As demonstrated previously, PKC-a and -E are both significantly retained in the particulate fraction following a 10minute TPA-treatment. PKC-6 is only partially retained and surprisingly PKC-< shows no evidence of (Laemmli, 1970) and the lysate was heated to 95°C for TPA-induced membrane association. The poor recovery 10 minutes. An aliquot was taken for protein determi- of PKC-a and PKC-6 following a 10-min TPA treatment nation (Peterson, 1977) and the rest stored in liquid of the cells is due to the proteolysis of these enzymes nitrogen. For fractionation into cytosolic and particu- during fractionation; on lysis of cells directly into SDS, late fractions, extracts were prepared as previously de- no loss of PKC-a or PKC-6 is observed at short times of scribed (Olivier et al., 1991). TPA treatment (see above). This translocation of PKC-a and PKC-6 in response to TPA has been observed on multiple occasions in Swiss 3T3 cells and Immunoblot analysis PKC-6 transfected COS-1 cells (unpublished data). For whole cell extracts, a n aliquot equivalent t o 35 In parallel extractions, the Ca2+sensitive membrane pg of total protein was subjected to 10% SDS-PAGE and association of the different isoforms was also tested transferred to Immobilon (Millipore) as previously de- (Fig. 4A, C). Only PKC-a was associated with the memscribed (Olivier et al., 1991). For cytosolic and particu- brane fraction when extracted in the presence of Ca2+, late fractions, equal cell equivalents (1-5 x lo5 cells) whereas 6, E, and 5 remained in the cytosol. This is were loaded to allow direct comparison of PKC content. consistent with the behaviour of PKC-Eand PKC-6 preAntipeptide antiserum was diluted 15,000 and incu- viously described (Kiley et al., 1990; Olivier et al., bated with the membrane for 1.5hours at RT. The blots 1991). were washed and then incubated with horse radish perDISCUSSION oxidase conjugated antisera and the immunoreactive bands visualised using the ECL detection system acThe expression of PKC-a in Swiss 3T3 cells has been cording to the manufacturers instructions. well documented and indeed the murine homologue of

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Time (hours) Fig. 2. PKC down-regulation in response to TPA. Quiescent cells were stimulated with 500 nM TPA and harvested at the time points shown (see Materials and Methods). A portion of each lysate was subjected to immunoblotting as described in Materials and Methods. The intensities of the autoradiograph corresponding to PKC-(Y( o ) ,

PKC-8 (a), PKC-c (A),and PKC-5 iA ) were scanned using an LKB Ultrascan XL. 100%is equivalent to the amount of each PKC present in quiescent cells. The data shown are from one of two complete downregulation studies.

this gene has been isolated from this source (Rose-John et al., 1988). It is demonstrated here that in addition to PKC-cx other isoforms are also expressed in this cell line. It is likely that these other isoforms were previously missed since (1) PKC-6 and PKC-• are not detected by the typical PKC histone kinase assay (Olivier et al., 1991; Schaap et al., 1990) and neither is PKC-I; (Ways et al., 1992), ( 2 ) cDNA cloning of PKC from a Swiss 3T3 library was carried out at a stringency that would have precluded detection of -6, -c, and -5 cDNAs (Rose-John et al., 19881, and (3) the low levels of PKC-E present was previously missed due to the relative insensitivity of the 1251-protein A detection technique (unpublished results). The presence of this variety of PKC isoforms in 3T3 cells is of interest in view of the use of these cells as a model system for defining the action of growth factors and in particular the role of PKC in such responses. Assessment of PKC action in this instance and many others has frequently involved the use of TPA-induced down-regulation as a means of “removing” the PKC element of any response (e.g., Chen e t al., 1991). Here i t is shown that PKC-6 and -E like PKC-a are indeed down-regulated in response to TPA albeit with distinct kinetics and sensitivities. Therefore studies where a response was lost after chronic TPA treatment and attributed to PKC could be mediated by either PKC-a, PKC-6, or PKC-E alone or in combination. By contrast,

however, PKC-5 is not down-regulated in response to TPA and furthermore does not become membrane-associated following TPA stimulation. The role of PKC-1; can therefore not be excluded from responses that are unaffected by long term TPA treatment. It is not clear whether PKC-1; will actually respond to TPA in vivo (or in vitro), or whether the lack of stable membrane association might, for example, reflect the presence of a single (perhaps relatively low affinity) binding site; it should be noted that PKC-I; has a single cysteine-rich domain in the C1 region (Ono et al., 1989aj, which would be expected to confer its effector binding activity (Cazaubon et al., 1990; Kaibuchi et al., 1989; Ono et al., 1989b). It has previously been suggested that PKC-6 does not respond to diacylglyceroliphorbol esters in vitro (On0 et al., 1989b). However, these studies were carried out with what appears to be a proteolysed form of the enzyme (Ways e t al., 1992). Recently it was reported that in platelets PKC-{ would translocate in response to TPA (Crabos e t al., 1991); what distinguishes platelets with respect to PKC-( translocation remains to be determined. Consistent with our observations in other cell types (Kiley et al., 1990; Olivier et al., 1991), the distribution of PKC-6 and -E between the particulate and soluble fractions is not sensitive to Ca2’. This clearly contrasts with the behaviour of PKC-a (see Fig. 4)and provides corroboration for the conclusion that these species are

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661 2 3 4 5 6 7 8 Fig. 3. TPA dose response. Quiescent cells were stimulated for 5h with no TPA (lane 1).1nM TPA (lane 2),10 nM TPA (lane 3), 50 nM TPA (lane 4), 100 nM TPA (lane 5), 250 nM TPA (lane 61,500 nM TPA (lane 7), and 1 p M TPA (lane 8). An aliquot of each lysate was subjected to SDS-PAGE and immunoblotting as described in Materials and Methods. A shows a blot probed with both PKC-or ( 0 ) and PKC-E(A)specific antisera; B with PKC-u ( 0 )and PKC-1; (a) specific antisera; and C with PKC-6 ( 0 )alone. The data shown are from one of two complete sets of dose response studies.

Ca2’-independent PKC isoforms (Akita et al., 1990; Olivier et al., 1991; Schaap et al., 1990). The studies presented indicate that there is significant complexity in the species of PKC expressed in Swiss 3T3 cells. Whereas some (6 and E) behave in a fashion similar to PKC-a, it is evident that PKC-( shows distinct behaviour and may contribute t o responses elicited in “PKC down-regulated cells. It may be concluded that for those responses that are inhibited following chronic TPA exposure PKC-a, -S andor -E are likely to play a rate limiting role. Future experiments should be aimed a t evaluating directly whether the distinct isoforms elicit distinct responses and how these contribute to growth control. This could be achieved by selective knockout or selective activation of individual isoforms. Studies such as this should give further insight into the role of PKCs in cell growth.

ACKNOWLEDGMENTS We thank Dr. E. Rozengurt for critical discussion and Amanda Wilkinson for the preparation of the manuscript.

LITERATURE CITED Akita, Y., Ohno, S., Konno, Y., Yano, A,, and Suzuki, K. (1990) Expression and properties of two distinct classes of the phorbol ester receptor family, four conventional protein kinase C types, and a novel protein kinase C. J . Biol. Chem., 265;354-362. Bacher, N., Zisman, Y., Berent, E., and Livneh, E. (1991)Isolation and characterization of PKC-L, a new member of the protein kinase

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Fig, 4. Cytosol and membrane distribution of PKCs. Quiescent Swiss 3T3 cells were either untreated (A and C) or treated with 500 nM TPA for 10 mins (B). The cells were homogenized in the presence of 3 mM EGTA (A and B) or in the presence of 3 mM CaC1, (C) and fractionated into cytosolic ( c ) and membrane (m) fractions as previously described (Olivier et al., 1991). Equal volumes from each fraction were subjected to SDS-PAGE and immunoblotting with isoform specific antisera as stated in Materials and Methods. ( 0 , PKC-(Y;n, PKC-6; A, PKC-t; A , PKC-(I.

C-related gene family specifically expressed in lung, skin and heart. Mol. Cell. Biol., 11:126-133. Cazaubon, S., Webster, C . , Camoin, L., Strosberg, A.D., and Parker, P.J., (1990) Effector dependent conformational changes in protein kinase Cy through epitope mapping with inhibitory monoclonal antibodies. Eur. J. Biochem., I94:799-804. Chen, R.-H., Chung, J., and Blenis, 3. (1991) Regulation of pp90rsk phosphorylation and S 6 phosphotransferase activity in Swiss 3T3 cells by growth factor-, phorbol ester-, and cyclic AMP-mediated signal transduction. Mol. Cell. Biol., 11:1861-1867. Coussens, L., Rhee, L., Parker, P.J., and Ullrich, A. (1987)Alternative splicing increases the diversity of the human protein kinase C family. DNA, 6:389-394. Crabos, M., Imber, R., Woodtli, T., Fabbro, D., and Erne, P. (1991) Different translocation of three distinct PKC isoforms with tumourpromoting phorbol ester in human platelets. Biochem. Biophys. Res. Commun., 1781878483. Kaibuchi, K., Fukumoto, Y., Oku, N., Takai, Y., Arai, K.-I., andMuramatsu, M. (1989) Molecular genetic analysis of the regulatory and catalytic domain of protein kinase C. J. Biol. Chem., 264:1348913496. Kiley, S., Schaap, D., Parker, P.J., Hsieh, L.-L., and Jaken, S. (1990) Protein kinase C heterogeneity in GH,C, rat pituitary cells. J. Biol. Chem., 265r15704-15712. Kubo, K., Ohno, S., and Suzuki, K. (198733) Nucleotide sequence of the 3’ portion of a human gene for protein kinase C p,/p,, Nucl. Acids Res., 153179-7180. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature, 227r680-685. Marais, R.M., and Parker, P.J. (1991) Purification of protein kinase C isotypes from bovine brain. In Methods in Enzymology. T. Hunter and B.M. Sefton, eds. Academic Press, Orlando, pp, 234-241.

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Meldrum, E., Parker, P.J., and Carozzi, A. (1991) The PtdIns-PLC superfamily and signal transduction. Biochem. Biophys. Acta, 1092t49-7 1. Nishizuka, Y. (1986) Studies and perspectives of protein Kinase C. Science, 233r305-312. Nishizuka, Y.(1988) The molecular heterogeneity of protein kinase-C and its implications for cellular regulation. Nature, 334r661-665. Ohno, S., Akita, Y., Komuo, Y., Imajoh, S., and Suzuki, K. (1988133 A novel phorbol ester receptorlprotein kinase, nPKC, distantly related to the protein kinase C family. Cell, 53t731-741. Olivier, A.R., and Parker, P.J. (1991)Expression and characterization of PKC-S. Eur. J. Biochem.,2OOt805-810. Ono, Y., Kikkawa, U., Ogita, K., Fujii, T., Kurokawa, T., Asaoka, Y., Sekiguchi, K., Ase, K., Igarshi, K.,and Nishizuka, Y. (19871 Expression and properties of two types of protein kinase C: Alternative splicing from a single gene. Science, 236tlll6-1120. Ono. Y.,Fujii, T., Ogita, K., Kikkawa, U., Igarishi, K., and Nishizuka, Y. (1988) The structure, expression and properties of additional members of the protein kinase C family. J. Biol. Chem., 263t69276932. Ono, Y.,Fujii, T., Ogita, K., Kikkawa, U., Igarashi, K., and Nishizuka, Y. (1989a) Protein kinase C subspecies from rat brain: Its structure, expression and properties. Proc. Natl. Acad. Sci. USA, 86t3099-3103. Ono, Y.,Fujii, T., Igarashi, K., Kuno, T.,Tanaka, C., Kikkawa, U., and Nishizuka, Y. (1989b) Phorbol ester binding to protein kinase C requires a cysteine-rich zinc-finger-like sequence. Proc. Natl. Acad. Sci. USA, 86t4868-4871. Osada, S.,Mizuno, K., Saido, T.C.: Akita, Y., Suzuki, K., Kuroki, T., and Ohno, S. (1990) A phorbol ester receptoriprotein kinase, nPKCv, a new member of the protein kinase C family predomi-