A cyclic AMP dependent protein kinase in

A cyclic AMP dependent protein kinase in
of 11
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  Vol. 108, No. 3, 1982 8lOCHEMlCAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS October 15, 1982 Pages 1210-1220 A CYCLIC AMP DEPENDENT PROTEIN KINASE IN DICTYOSTELIUM DISCOIDEUM Charles L. Rutherford, Robert D. Taylor, Lynn T. Frame, and Roxanne L. Auck Department of Biology, School of Arts and Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061 Received September 2, 1982 A cyclic AMP-dependent protein kinase was found to appear during the time course of development of Dictyostelium discoideum. No cyclic AMP dependency was observed at any stage of development in crude 110,000 X G soluble extracts. After partial purification, however, extracts from post-aggregation stages contained enzyme that was activated up to 6-fold by cyclic AMP, whereas protein kinase from earlier stages was not affected by cyclic AMP. Likewise, cyclic AMP binding activity increased from the aggregation to the slug stage of development. Approximately one-half of the total cyclic AMP binding activity co-purified with the cyclic AMP dependent protein kinase. The enzyme from Dictyostelium showed similarities to mammalian protein kinases with respect to its kinetic properties but differed in its behavior on ion-exchange chromatography. INTRODUCTION Adenosine 3':5'-monophosphate (CAMP) is known to act a chemoattractant during the aggregation stage of development of Dictyostelium (1). In addition CAMP may be involved in the subsequent differentiation of the two cell types, spore and stalk cells, although the mechanism of action of CAMP at this stage of development is not understood. Cyclic AMP levels are not detectable in non- developing vegetative amoebae, show a sharp increase at the aggregation stage, then continue to increase during the subsequent stages of cell differentiation (2-5). The enzymes which regulate CAMP levels, adenylate cyclase (AC) and phosphodiesterase (PDE) have been studied by several laboratories and have been shown to exhibit stage-dependent changes in their activities. Adenylate cyclase activity is not detectable in non-differentiating cells then sharply increases in activity at aggregation, coincident with the increased level of CAMP (5-8). The regulation of this membrane-bound enzyme is not well understood although its 0006-291X/82/191210-11$01.00/0 Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. 1210  Vol. 108, No. 3, 1982 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS activity is thought to be affected by CAMP itself (9) as well as a stage dependent inhibitor (IO). The activity of PDE also increases upon aggregation of the amoebae (11-13) and is apparently regulated by interaction of a catalytic subunit (50,000 MW) and a regulatory subunit (47,000 Md) (14, 15). During development of the two cell types PDE is active in stalk cells but masked by the regulatory subunit in prespore cells (16). In relation to the amount of information available about the regulation of CAMP levels by adenylate cyclase and PDE, little is known about the biochemical effect of CAMP itself on the developing cell. In mammalian systems the major and perhaps the only receptors for CAMP are CAMP-dependent protein kinases (17-19). However, whether such an enzyme exists in Dictyostelium has been a controversial subject. In an early report Sampson (20) described CAMP-dependent phosphorylation of histone VII-S by two peaks of activity which appeared in the salt elution of DEAE-cellulose. However, subsequent investigations did not support the existence of such a CAMP-dependent enzyme (21-24). In the latter reports no CAMP-dependent phosphorylation was found in material which eluted from ion-exchange columns nor was any kinase activity associated with CAMP binding activity which eluted from these columns. However in a recent paper (25) a CAMP binding protein (Mr = 41,000) from Dictyostelium was described which was capable of inhibiting a catalytic subunit of beef heart CAMP-dependent protein kinase. This result suggests that the CAMP binding protein from Dictyostelium is a dissociated regulatory subunit and therefore provides indirect evidence for the existence of a CAMP-dependent protein kinase. In this report we show that CAMP-dependent protein kinase does indeed exist in Dictyostelium, it can be fractionated to co-purify with CAMP binding activity, is developmentally regulated, and can be obtained with a high yield of CAMP dependency. METHODS Preparation of cell-free extracts - Growth and differentiation of Dictyostelium discoideum NC4, was carried out as previously described (26). At the desired stage of development the cells were removed from an agar surface with cold distilled water were washed by 1211  Vol. 108, No. 3, 1982 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS centrifugation at 500 X g for one minute and were resuspended (IO volumes buffer/ml packed cells) in 50 mM tris-HCl buffer (pH 7.5) containing 2 mM mercaptoethanol, and 0.02% sodium azide (TAM). Most of the preparations contained 6-8 gm wet weight of cells (approximately 200 mg protein). The cells were then evenly distributed by two strokes of a Potter-Elvehjem tissue grinder, then were disrupted by three 45 second exposures to a one centimeter probe of a sonic cell disrupter (Model 150, Virtis, Gardiner, N.Y.) at a setting of 80. The homogenate was then centrifuged at 110,000 x g for 60 min and the supernatant removed for further purification. Column Chromatography The 110,000 x g supernatant, usually 60-80 ml (200 mg protein) was applied to a DE-52 cellulose column (1.6 X 13 cm) at a flow rate of 80 ml/h. The material which did not bind to the resin (flow-through) was allowed to completely elute from the column as determined by the return to the baseline on a column monitor. Proteins which were bound to the resin were then eluted for 4 h with a O-O.3 M NaCl gradient (in TAM, flow rate of 80 ml/h) as formed from a programmable chromatography pump (Dialagrad, Model 382, ISCO). The active fractions were pooled, concentrated to 3 ml by ultrafiltration (PM10 membrane, Amicon) then applied to a Sephacryl S-300 column (1.6 cm X 90 cm, flow rate of 30 ml/h). Protein Kinase * CAMP Binding Assay -- Protein kinase activity was assayed in a total volume of 50 ul with 25 ul of the enzyme sample and 25 ul of a reaction mixture which contained 50 mM potassium phosphate buffer (pH 6.5), 3 mM dithiot&eitol, 10 mM MgCl , 0.8 mg/ml histone VII-S (Sigma Chemical Co.), and 25 uM [a- a ATP (0.4 Ci/mmo?) either with or without 20 uM CAMP. After incubation at 25 C for 15 min the entire reaction volume was removed to 1 cm square pieces of filter paper (Whatman 3 MM) which were immediately transferred to cold 10% TCA. After 15 min the papers were removed to hot 5% TCA, 5 min, cold 5% TCA, 5 min, acetone, 5 min, then dried for determination of radioactivity. Cyclic AMP binding activity was measured in a total volume of 125 ul containing 100 ul of the protein sample and 25 ul of 4 reaction mixture containing 25 mM dithiothreitol, 5 mM MgC12, 150 nM [ H] CAMP (130 Ci/mmol) in 50 mM tris-HCl buffer (pH 7.5). After incubation for 15 min the entire reaction mixture was removed to a Millipore filter reservoir containing 5 ml of ice cold 50 mM Tris-HCl (pH 7.5). The solution was immediately filtered through a Gelman GN-6 filter (0.45 urn) by vacuum filtration. The filter was washed twice in the same buffer then removed and dried for determination of radioactivity. Gel Electrophoresis and Autoradiography - - Enzyme sample and reactionOmixture (described above) were allowed to incubate for l-10 minutes at 23 C with and without 20 uM CAMP. The reaction was stopped by adding 50 ul of a stopping solution (9% SDS, 15% glycerol, 30 mM Tris, pH 7.8, 0.05% bromophenol blue) and boiling for 5 minutes. After cooling to room temperature, 50 ul of dithiothreitol (initial concentration 75 mg/ml) was added. Samples were then subjected to SDS-plyacrylamide slab gel electrophoresis, protein staining and/or autoradiography according to the method of Rudolph and Krueger (27). Quantitative differences in protein phosphorylation due to CAMP addition were determined using a Quick-Scan, Jr. densitometer from Helena Laboratories. RESULTS Chromatography on DE-52 Cellulose & Sephacryl S-300. Ion exchange -- chromatography of a 110,000 x g supernatant fraction from the slug stage of 1212  Vol. 108, No. 3, 1982 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COAM4UNlCATlONS 0 20 40 60 80 Y 1.5 z 4 q a 0.5 0 FRACTION NUMBER Figure 1. DE52 cellulose chromatography of a 110,000 x g soluble fraction of the slug stage of development. The NaCl gradient is in moles/liter. The faint solid line indicates the absorbance profile of the column monitor. Protein kinase without added CAMP (A) and with 10 UM CAMP (O), and CAMP binding activity (a) are shown. development produced the elution pattern shown in Figure 1. Approximately SO% of the activity capable of phosphorylating histone Vll-S was found in the flow- through volume while the remainder of the activity eluted at 140 mM NaCl. In twelve separate experiments the flow-through activity was stimulated two-fold by inclusion of 10 uM CAMP in the reaction mixture while the salt elutable activity was not affected by CAMP addition. Likewise, no CAMP stimulation of activity occurred in the 110,000 x g supernatant which was applied to the column. The appearance of activity in the flow-through volume was not due to overloading the column or to the flow-rate at which the sample was applied to the resin. In addition, the activity did not bind to DE52 cellulose at pH 6.8, 7.5 or 8.6. The lack of a CAMP effect on the salt eluting kinase activity was not due to destruction of added CAMP by CAMP phosphodiesterase as PDE does not bind to DE52 cellulose under the conditions of the experiment and PDE is inhibited by the amount of dithiothreitol which is included in the reaction mixture (16). In all experiments the kinase activity and the CAMP binding activity which were bound by the resin were found in separate fractions of the salt elution. The CAMP binding protein which eluted in the salt gradient was concentrated and applied 1213  Vol. 108, No. 3, 1982 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS c9 0.6 0.4 : 2 ii 0 In 0.2 z 0 FRACTION NUMBER Figure 2. Sephacryl S-300 chromatography of the active CAMP-dependent protein kinase fractions from the DE52 column. The elution profiles which are shown are for absorbance (faint solid line), CAMP-binding activity (0) and protein kinase activity without CAMP (A) and with 10 uM CAMP in the reaction mixture (0). to a Sephadex G-100 column. The activity eluted at a volume corresponding to a molecular weight of 40,000 and showed high specificity for CAMP over 5'AMP. These properties are quite similar to those of the CAMP binding protein described earlier (28, 29) and to the CAMP binding protein reported by Leichtling et al. (25) which appeared to be a regulatory subunit of CAMP dependent protein kinase. The flow-through volume from the DE-52 cellulose column, which contained both CAMP binding activity and CAMP-stimulated protein kinase, was concentrated and then applied to a Sephacryl S-300 column. Figure 2 shows that the two activities co-eluted from this column. Furthermore, the fractions containing kinase activity were stimulated approximately six fold by CAMP whereas the material applied to the column (DE52 flow-through) showed two-fold stimulation. As mentioned before the unfractionated 110,000 x g soluble fraction was not affected by CAMP addition. We do not believe that the enhanced CAMP stimulation which is observed upon purification is due to removal of CAMP-independent protein kinase activity for we did not observe any major peaks of kinase activity that was not activated by the addition of CAMP. It is also doubtful 1214
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