Involvement of calmodulin in hormone-stimulated cyclic AMP production and degradation in mouse B 16 melanoma. SIMON W. WALKER,* SHEILA MAcNEIL,*.
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Involvement of calmodulin in hormone-stimulatedcyclic A M P production and degradation in mouse B 16 melanoma SIMON W. WALKER,* SHEILA MAcNEIL,*
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R. MICHAEL SHARRARD,? LISA CAWOODJ BARRY L. BROWN,? STEPHEN TOMLINSON* and STANLEY S . BLEEHENS *Department of Medicine, ?Department of Human Metabolism and Clinical Biochemistry and $Department of Dermatology, University of Shefield Medical School, Shefield SIO 2RX,
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'Z We have previously studied the hormonal regulation of a 2 highly pigmented B16 melanoma cell line by a-melanotropin 0.08and related peptides (MacNeil et al., 1981). The increase 0 in intracellular cyclic AMP produced by a-melanotropin -0 is controlled by adenylate cyclase and phosphodiesterase ?5 activities, both of which, in some tissues, are regulated by calmodulin (Wang 8~ Waisman, 1979). Accordingly, we investigated the role of calmodulin in both the production and .- 0.06the degradation of cyclic AMP in B 16 melanoma. 6 Icl In cultured B16 cells 34% of the total cell calmodulin was 0, located in the particulate fraction. Calmodulin was measured E" with a highly calmodulin-responsive pig brain phosphodifc 0.04esterase by using the assay method described by Thompson et .-> al. (1979). The concentration of calmodulin in particulate .-c preparations (0.72 pg/mg of particulate protein) was not d 0 significantly lowered by sequential washing with EGTA. 0 Adenylate cyclase activity measured in such preparations was 6 0.02not affected by the addition of exogenous calmodulin, even up to P lOpg/mi. However, calmodulin antagonists markedly dex creased the enzyme activity. Brief preincubation of particulate preparations with calmodulin antagonists increased the degree of inhibition observed with these drugs: Fig. 1 shows almost . 0total inhibition of the. adenylate cyclase response to a-melanoT I I I 0 0.1 1 .o 10 100 tropin in a particulate preparation that had been preincubated Concn. of a-melanotropin ( p ~ ) with the calmodulin antagonist N-(6-aminohexyl)-5-chloronaphthalene- 1-sulphonamide (compound W7) (Hidaka et al., 1978) for 3min. In contrast, compound W7 had com- Fig. 1. Efect of the calmodulin antagonist compound W7 on paratively little effect on basal or fluoride-stimulated adenylate hormone-responsive adenylate cyclase activity of cultured B16 melanoma cells cyclase activity. The inhibition produced by compound W7 could be reversed by the addition of calmodulin. Thus, in one Particulate preparations of B16 melanoma cells were prewith either buffer (.) or 125p~-compoundW7 (0) experiment, addition of compound W7 (final concn. 5 0 ~ ~incubated ) decreased enzyme activity from 1.61 f0.16 to 0.76 +0.09nmol for 3 min at 4°C immediately before assay of adenylate cyclase of cyclic AMP/lOmin per mg of particulate protein, but this activity. returned to 1.60f0.12nmol/lOmin per mg on the addition of 10pg of calmodulin/ml (mean s.D.,n = 3). In cultured B16 cells at least 95% of the total homogenate cyclic AMP phosphodiesterase activity was located in the cytosol. Over a 5min incubation period hydrolysis of cyclic increasing to 5.65 f 0.14nmol/3 min per mg on the addition of AMP by three preparations of B 16-cell cytosol was decreased calmodulin (1 pg/ml). An attempt was made to measure the by 45.5+3.2% (mean+ s.D., n = 3) by the addition of a proportion of total phosphodiesterase activity that is cal. maximally inhibiting concentration of compound W7 ( 5 0 ~ ~ )modulin-dependent: on the basis of the difference between Addition of calmodulin (1 pg/ml) to these preparations did not enzyme activity in the presence of maximally stimulating significantly increase cyclic AMP hydrolysis. This is consistent calmodulin (1pg/ml) and activity in the presence of maximally with the high concentrations of calmodulin (0.69pg/mg of inhibitory compound W7 (SOW), 54.7+ 10% (mean? s.D., protein) measured in B 16-cell cytosol. Calmodulim-depleted n = 8 , range 32-63%) of the total cyclic AMP phosphocytosol was prepared by using a fluphenazine-Sepharose 6B diesterase activity was found to be calmodulin-dependent affinity column (Charbonneau & Cormier, 1979). Although this (substrate concentration varied in the range 10-1OOpM-cyClic column removed 92% of ['Hlcalmodulin from buffer, removal AMP, incubation time 3-6 min). of [3Hlcalmodulinfrom B16cell cytosol ranged from only 48 to 71%. As boiling of cytosol increased the amount of ['HIWe conclude that calmodulin is involved in both hormonecalmodulin removed, it is possible that calmodulin-binding stimulated cyclic AMP production and degradation in the B 16 protein present in cytosol diminished the efficiencyof the column. Despite this problem, calmodulin-depleted cytosols now showed melanoma. a response to added calmodulin. For example, in one experiment cyclic AMP hydrolysis was 3.94k0.05nmol of cyclic Charbonneau, H. & Cormier, M. J. (1979) Biochem. Biophys. Res. AMP hydrolysed/3 min per mg of protein (mean f s.D., n = 3), Cornmutt. 90, 1039-1047
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BIOCHEMICAL SOCIETY TRANSACTIONS
Hidaka, H., Asano, M., Iwadare, S., Matsumoto, I., Toksuka, T. & Aoki, N. (1978)J. Phamacol. Exp. Ther. 207,8-15 MacNeil, S., Johnson, S. K., Bleehen, S. S., Brown, B. L. & Tomlinson, S. (1981) Regul. Pept. 2, 193-200
Thompson, W. J., Terasaki, W. L., Epstein, P. M. & Strada, S. J. (1979)Adu. Cyclic Nucleotide Res. lO,69-92 Wang, J. K. & Waisman, D. M. (1979) Curr. Top. Cell. Regul. 15, 17-107
Gel electrophoresis of calmodulin purified from B 16 melanoma R. MICHAEL SHARRARD,* SIMON W. WALKER,t SHEILA MAcNEIL,? BARRY L. BROWN,* STEPHEN TOMLINSON? and STANLEY S. BLEEHENS *Department of Human Metabolism and Clinical Biochemistry, ?Department of Medicine and $Department of Dermatology, University of Shefield Medical School, Shefield SlO 2RX,
UX. The introduction of phenothiazine-Sepharose affinity chromatography has simplified the extraction and purification of calmodulin. By using a method involving fluphenazineSepharose 6B affinity chromatography (Kakiuchi et al., 1981), calmodulin was extracted from mouse B16 melanoma tumour explants. The isolated calmodulin was biologically active in an assay based on that described by Thompson et al. (1979), with the use of a calmodulin-deficient cyclic AMP phosphodiesterase. When the preparation was subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (Laemmli & Favre, 1973), preparations gave a single band with EGTA present. However, one or more bands of greater mobility were observed with Ca2+ present, a phenomenon also observed by others (Molla et al., 1981). We have therefore subjected these preparations to more detailed analysis by both single-dimension and two-dimensional gel electrophoresis. Purified calmodulin was dialysed at 4°C (1) against 200~01. of 20m~-Tris/HCIbuffer, pH 7.0, containing 1OmM-EGTA (EGTA buffer) for 48h, or (2) against 200~01.of 2 0 m ~ Tris/HCI buffer, pH 7.0, containing lOm~-calcium lactate (Ca2+buffer) for 48 h, or (3) against 200vol. of Ca2+buffer for 24 h and then against 200~01.of EGTA buffer for 24 h. Samples of these preparations, and also of the undialysed calmodulin preparation, were analysed by electrophoresis on sodium dodecyl sulphate/polyacrylamide gels containing 15% (w/v) acrylamide in the resolving gel, essentially as described by Laemmli & Favre (1973). The preparation gave a single band after dialysis against EGTA buffer (with or without prior exposure to Ca2+). If the preparation was not finally dialysed against EGTA buffer, or if Ca2+ was added to a total concentration of more than 1 mM after this dialysis (corresponding to a free Ca+ concentration of about IOnM), multiple bands were observed, all with higher mobility than that of the material dialysed against EGTA buffer. Calmodulin preparations dialysed against EGTA buffer were analysed by two-dimensional gel electrophoresis by a modification of the method of Shannon et al. (198 1). Isoelectric focusing was performed in rod gels containing 9 . 5 urea, ~ 4% (w/v) Nonidet P-40 and 2.7% (w/v) mixed Ampholines of total pH range 2.5-10. After completion of focusing, the gels were equilibrated in equilibration buffer [ 10% (w/v) glycerol/l% (v/v) 2-mercaptoethanol/ 10% sodium dodecyl sulphate/ 62.5 mM-Tris/HCl buffer, pH 6.81 containing (1) 1OmM-EGTA, or (2) 10mwcalcium lactate or (3) no additions. They were then applied to sodium dodecyl sulphate/polyacrylamide gels containing 15% (w/v) acrylamide in the resolving gel. In the absence of added Ca2+,the preparation gave a single spot, which showed a shift in the molecular-weight dimension when CaZt was present in the equilibration buffer. Addition of Ca2+to the preparation before application to the isoelectric-focusing gels, however, had no detectable effect on the final position of the spot on the gel, suggesting that the focusing procedure dissociates Ca2+from calmodulin.
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Fig. 1. Sodium dodecyl sulphatelpolyacrylamide-gel electrophoresis of calmodulin ( a ) 1, Calmodulin immediately after preparation; 2, after aging at 2OoC for 48h; 3, dialysed against Ca2+ buffer and then EGTA buffer; 4, dialysed against Ca2+ buffer only; 5 , with 10mM-Ca2+ added after boiling In the presence of sodium dodecyl sulphate; 6, with Ca2+ added before sodium dodecyl sulphate. Note the effect of EGTA in (3) on samples in adjacent wells. (b) 1, Calmodulin after dialysis against EGTA buffer; 2, after dialysis against Ca2+ buffer; 3, 4 and 5 , as 1 with respectively 1 mM-, 3 mM- and 10mM-Ca2+added after dialysis.
In some preparations, both single-dimensional and twodimensional electrophoresis showed the presence of a minor band, of mobility slightly less than that of the major calmodulin species, in the samples dialysed against EGTA buffer. This band was not observed when Ca2+was added before electrophoresis, suggesting that it also undergoes a Ca2+-dependent shift and that it may be a modified form of calmodulin. The fact that all the preparations gave multiple banding in the presence of Ca2+, and that these patterns were not altered by addition of extra Ca2+, suggests that purified calmodulin may contain different species not normally resolved in the absence of CaZ+ but distinguishable by the extent of conformational change that they undergo when Ca2+ is available. It is not known, however, why only certain preparations should contain species resolvable in the absence of Ca2+,unless proteolysis is occurring to a variable extent during purification. The presence of multiple calmodulin species that are resolved in the presence of Ca2+ suggests that calmodulin heterogeneity may be related to a physiologically significant diversity of function of this protein. Kakiuchi, S., Sobue, K., Yamazaki, R., Kambayashi, J., Sakon, M. & Kosaki, G. (1981)FEBS Lett. 126,203-207 Laemmli, U.K. & Favre, M. (1973)J.Mol. Biol. 80,575-599 Molla, A., Kilhoffer, M.-C., Ferraz, C., Audemard, E., Walsh, M. P. & Demaille, J. G. (1981) J. Biol. Chem. 25615-1 8 Shannon, P. R. M., Sharrard, R. M. & Jarvis, B. C. (1981)Plant Cell Physiol. 22, 1293- 1304 Thompson, W. J., Terasaki, W. L., Epstein, P. M. & Strada, S. J. (1979)Adv. Cyclic Nucleotide Res. 10,69-92 1983
