Biochimica et Biophysica Acta, 1094(1991) 243-245

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© 1991 Elsevier '~;ciencePublishers B.V. All rights reserved 0167-4b¢~9/91/$03.5~, ADONIS 016748899100239Y BBAMCR 10281

Rapid Report

Phorbol ester induces phosphorylation and down-regulation of connexin 43 in WB cells Saw Yin Oh, Chris G. Grupen

and Andrew W. Murray

School of Biological Sciences, Flinders Unicersity, Adelaide (Australia)

(Received 4 Jul,, 1991) Key words: Phorbol ester; Connexin43; Phosphorylation Western blot analysis indicated that WB cells contained multiple proteins of molecular mass 42-47 K which reacted with anti-connexin 43 antibody. Incubation of cells with 12-O-tetradeeanoylphorbol-13.acetate (TPA) for 5 rain caused the inhibition of dye transfer and the appearance of reactive material of 50 K which was abolished by acid phosphatase. Longer term incubation with TPA caused loss of conner,ln 43 protein.

Junctional channels (connexons) which permit the exchange of small molecules between cells, are composed of a family of related proteins called connexins. The best characterised of these are connexin 32 from rat liver and connexin 43 from rat heart [1]. The amino acid sequences of both of these proteins is known and models have been proposed for their structural organ±sat±on in membranes [2-6]. Little is known about the regulation of connexon permeability, but several studies have suggested either a dtrect or indirect role of PKC. Thus, compounds which activate PKC such as phorbol esters or diacylglyceroi cause a rapid inhibition of junctional transfer in many cultured cells [7-11]. In the present p a p e r we show that in WB cells TPA-induced inhibition of junctional transfer is accompanied by phosphorylation of connexin 43, followed by down regulation of the protein. The phorbol ester T P A caused a rapid decrease in the transfer of microinjected lucifer yellow to contacting WB cells (Table I). Inhibition was observed aRer 5 min and was maintained for at least 1 il. After prolonged exposure to T P A the cells became refractory to the inhibitory effects as has been frequently observed in other cells [10]. This may be due to down regulation of PKC, a major cellular target for TPA. Earlier studies have shown that exposure of W B cells to T P A for 6

Abbreviations: DMSO, dimethyl sulphoxide; PKC, protein kinase C; TPA, 12-Ootetradecanoylphorbol-13-acetate. Correspondence: A.W. Murray, School of Biological Sciences, Flinders University, GPO Box 2100, Adelaide, S.A. 5001, Australia.

TABLE 1 Effect of TPA on lucifer yellow transfer between WB cells

WBF344 cells (passages 13-26) obtained from Dr. J.W. Grisham were maintained in D medium (Gibco) as described 119]. The cell line was isolated from rat liver and has a phenotypie resemblance to 'oval' cells [19]. For microinjectionexperiments confluent cultures in 35 mm dishes were injected with lucifer yellowCH (10% in 0.33 M LiCl2) as described [20] using a Leitz micromanipulatorcoupled to a Nikon epifluorescencemicroscope,Recipient cells were scored 2 min after injection. The cell cultures were incubated with TPA (100 nM) or solvent (DMSO; final concentration, 0.1%) for appropriate periods hetore microinjection. Results are the means (±S.E.) of 12-25 individual injections. Time (min)

Recipient cells/injection

5 20 00 120 240 12h

21.4±1.9 16.4±1.9 17.1±1.9 21.4~z6 16.4±2.2 18.7±L7

0.4±0.2 0.4±0.1 0 1.7±0~ ~5±0.9 15.5±1~

h causes a marked loss of beth particulate and soluble PKC activity [11]. Western blot analysis indicated that WB cells contain proteins recognised by an antibody to a pert±de corresponding to amino acids 314-322 of rat heart connexin 43 (Fig. 1). No immunologically reactive bands were seen using pre-immune serum (data not shown). In DMSO-tleated cells multiple bands of molecular mass 42-47 K were observed, together with more slowly migrating bands of > 100 K which are likely to be aggregated forms of connexin 43 [12]. As reported

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a direct effect of P K C on connexon permeability or assembly. L o n g e r t e r m exposure to T P A ( 1 - 2 h) c a u s e d a m a j o r loss of immunologieally reactive eonnexin 43 protein (Fig. 2). This loss is unlikely to be d u e to phosphorylation of a m i n o acid residues in the connexin 43 epitope recognised by the antibody, as acid phosp h a t a s e - t r e a t m e n t of the alkali-enriched samples f r o m 2 h T P A - t r e a t e d cells did not g e n e r a t e reactive m a t e rial ( d a t a not shown). S o m e r e a p p e a r a n c e of i m m u n o logically reactive protein o c c u r r e d a f t e r 4 h a n d a f t e r 12 h the a m o u n t o f protein was similar to control ceils. T h e s e d a t a correlate with the recovery of dye transfer c o m p e t e n c e at these times (Table I). Incubation o f W B

.,

Fig. l. Effect of TPA on phosphorylation of connexin 43 protein. lmmunoblot analysis of confluent WB cells incubated for 5 min with 0.1% DMSO (lane l) or 100 nM TPA (lane 4). Aliquots of extracts from DMSO and TPA-treated cells were also incubated with acid phosphatase (lanes 2 and 5, respectively) or with acid phosphatase plus phosphatase inhibitors (lanes 3 and 6, respectively). Arrows indicate the TPA-indaced phosphorylated band (50 K) and the fastest migrating dephosphovylated band (41 K). Relative molecular mass is indicated on the left of the figure. Conflaent WB cells in 100 mm dishes were alkali extracted [21] and the insoluble material resuspended in 1 mM NaCO3 containing 1 ~itM PHSF and solubilixed in SDS sample buffer. In some experiments the suspensions were incubated with potato acid phosphatase (Boehringer; l U/ml) for 30 min with or without an inhibitor mix containing 50 mM NaF, 100 mM Na3VO4 and 10 mM KHzPO4 before solabilization in SDS. Samples (5 p.g protein) were electrophores~d in 10% gels, proteins transferred to nitrocellulose and immunoreactions carried out with affinity purified antibody to amino acids 314-322 of rat heart connexin 43 [12]. Detection was with the Amersham enhanced chemiluminescence detection system.

previously [13-16], it is likely t h a t at least some of the 4 2 - 4 7 K protein was phosphorylated, as incubation with acid p h o s p h a t a s e resulted in the a p p e a r a n c e o f a m o r e rapidly migrating b a n d o f 41 K (Fig. 1, lanes 2 a n d 5), which could be blocked by the addition o f p h o s p h a t a s e inhibitors. Exposure of cultures to T P A for 5 rain resulted in the a p p e a r a n c e of m o r e slowly migrating material of molecular mass a b o u t 50 K (Fig. 1, lane 4). This b a n d was also abolished by t r e a t m e n t with acid p h o s p h a t a s e a n d preserved if p h o s p h a t a s e inhibitors w e r e included in the incubation (Fig. 1, lanes 3 a n d 6). l ~ e likely explanation o f these d a t a is t h a t the 50 K b a n d results from P K C - m e d i a t e d p h o s p h o rylation of connexin 43 protein. It should be n o t e d t h a t these c h a n g e s o c c u r at a time w h e n dye transfer between cells is essentially completely blocked in the presence of T P A (Table I) a n d are not associated with any m a j o r alteration in the total a m o u n t of connexin 43 f~rotein present. T h e result is therefore consistent with

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Fig. 2. Effect of TPA and cycloheximide on connexin 43 in WB cells. (a) lmmunoblot analysis of connexin 43 from confluent WB cells incubated for I h with 0.1% DMSO (lane 1), 5 p,g/ml eycloheximide (lane 2) or 100 nM TPA (lane 3). Lane 4; cells incubated for 12 h with 100 nM TPA. Samples (5 ~g protein) were electrophoresod in 12.5% gels and processed as in Fig. 1. (b) lmmanoblot analysis ofI connexin 43 from confluent WB cells incubated for 2 h with 0.1%1 DMSO (lane 1), 100 nM TPA (lane 2) or 5 ~g/ml cycloheximide' (lane 3) or for 4 h with 0.1% DMSO (lane 4), 100 nM TPA (lane 5) or 5 u,g/ml cyclohexlmide (lane 6). Lane 7; alkali-enriched sample from rat heart. Samples (5 ttg protein) were electsophoresed and processed as in Fig. 1.

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wBqllD 18SFig. 3. Northern blot analysis of total RNA from rat liver(lane l), rat heart (lane 2) or confluent WB cells incubated for l h with 0.1% DMSO (lane 3) or 100 nM TPA (lane 4). The mRNA species detected is about 3.4 kb. RNA was isolated [22j and 30/tg loaded on a 1% a~arn~e-formaldehydegel. After blotting onto nitrocellulose the filter was hybridized [23] with the EcoRl fragment of rat heart connexin 43 eDNA. The filters were washed at high stringency (0.2×SSC; 0.1% SDS; 65° C) and autoradiographed. A parallel filter showed no hybridization with a rat liver connexin 32 eDNA probe. cells with cycloheximide had little effect on connexin 43 protein up to 2 h, but caused a m a r k e d depletion after 4 h (Fig. 2). Using pulse-chase experiments a half-life of about 2 h has been reported for connexin 43

in myoeytes [16]. Northern analysis indicated that WB cells contained a single R N A species of about 3.4 kb which hybridised

to connexin 43 cDNA and expression of this mRNA was unaffected by exposure to T P A for 1 h (Fig. 3) or for 4 h (data not shown). T h e loss of connexin 43 is therefore not due to an effect of T P A at the transcriptional level. It is consequently likely that T P A causes either increased degradation or decreased synthesis of conncxin 43 protein. T h e T P A - i n d u c e d decrease clearly occurs m u c h m o r e rapidly than in cells incubated with cycloheximide. In separate experiments we have obs e w e d a m a r k e d loss of connexin 43 protein after 20 min exposure to T P A (data not shown). We therefore propose that PKC-mediated phosphorylation of connexin 43 is followed by an enhanced rate of degradation. This could result directly from increased susceptibility of connexin 43 to proteolysis or from secondary effects of PKC on o t h e r proteins such as proteinases. T h e extent of connexin 43 degradation can not be assessed from these data as the antibody used for detection is raised to a sequence close to the C-terminus of the protein. T h e s e data indicate that T P A stimulates the rapid phosphorylation of connexin 43, presumably via activation of PKC. It has not yet been established that this phosphorylation is causal in the inhibition of junctional permeability or in the subsequent loss of connexin 43 protein. F u r t h e r clarification will require identification

of the amino acids phosphorylated by PKC in connexin 43, and site-directed mutagenesis studies. It is intriguing that connexin 43 and connexin 32 may differ in their functional response to phosphorylation by PKC. Thus, although connexin 32 is a substrate for PKC both in vitro and in vivo [17,18], phorbol ester does not inhibit junctional communication in cultured hepatocytes which express this protein [17]. We thank Dr. E. Beyer for the rat heart connexin 43 c D N A , Dr. J.W. Grisham for the WBF344 cells and Dr. D. Gros for the connexin 43-specific antibody. This work was supported by a grant from the National Health and Medical Research Council. References 1 DermietzeL R.. Hwang, T.K. and Spray, D.S. (1990) Anat. Embryol. 182, 517-528. 2 Schultz. G.E. (1988) Annu. Rev, Biophys, Chem. 17, 1-21. 3 Milks, L, Kumar, N.M., Houghton, R., Unwin, N. and Gilula, N.B. (1988) EMBO J. 7, 2967-2975. 4 Yanccy. S,B., John, S.A., Lal. R., Austin, B.J. and Revel, J.-P. (1989) J. Cell Biol. 108, 2241-2254. 5 Laird, D.W. and Revel, J.-P, (1990)J. Cell Sci. 97, 109-117. 6 Beyer, E.C., Paul, D.L and Goodenough, D.A, (1987) J. Cell Biol. 105, 2621-2629. 7 Yotti, LP., Chang, C.C. and Trosko. J.E. (1979) Science 208, 1089-1091. 8 Murray, A.W. and Fitzgerald,DJ. (1979)Bio~hem. Biophys. Rcs. Commun. 91, 395-401. 9 Enomoto. T., Sasaki, Y., Shiba, Y., Kanno, Y. and Yamasaki, H. (1981) Proc. Natl. Acad. Sci.US, 78, 5628-5632. 10 Murray, A.W. and Gainer, H.St.C. (1989)in Cell Interactions and Gap Junctions (Sperelakis, N. and Cote, W.C. eds.), Vol. 1, pp. 97-106, CRC Press, Boca Raton. 11 Oh, S.Y., Madhukar, B.V, and Trosko, J.E. (1988)Carcinogenesis 9, 135-139. 12 Aoumari, A.E., Fromaget, C., Dupont, E., Reggio, H., Durbek, P. Briand, J.-P., Boiler, K., Kreitman, B. and Gros, DJ. (1990)J. Membr. Biol. 115, 229-240. 13 Mufti. LS., Cunningham, B.A., Edelman, G.M. and Goodenough, D.A. (1990)J. Cell Biol. 111, 2077-2088. 14 Musil, LS., Beyer, E.C. and Goodenough, D.A. (19901J. Membr. Biol. 116, 163-175. 15 Kadle, R., Zhang, J.T. and Nieholson, BJ. O991) J. Mol. Cell. Biol. It. 363-369. 16 Laird, D.W., Puranam. K.L. and Revel, J.-P. (1991) Biochem. J. 273, 67-72. 17 Saez, J.C., Nairn, A.C., Czernik, A.J., Spray, D.C., Hertzberg, P. and Greenberg. P. (1990) Eur. J. Biochem. 192, 263-273. 18 Takeda, A., Saheki, S., Shimazu, T. and Takeuchi, N. (1989) J. Biochem. 106, 723-727. 19 Tsao, M.S., Smith, J.D., Nelson, 1CG. and Grisham, J.W. (1984) Exp. Cell Res. 154, 38-52. 20 Enomoto, T., Martel, N., Kanno, Y. and Yamasaki, H. (1984) J, Cell Physiol. 121,323-333. 21 Hertzberg. E.L and Skibbens. R.V. (1984)Cell, 39. 61-69. 22 Chomczyaski, P. and Sacchi, H. (1987) Anal. Biochem. 162, 156-159. 23 Sambrook, J.. Maniatis, T. and Fritsch, E.F. (1989) Molecular Cloning. A laboratory manual, 2nd Edn.. Cold Spring Harbor Laboraty, Cold Spring Harbor.