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The Control of Phosphorylase Phosphatase by Camp-Dependent Protein Kinase

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The Control of Phosphorylase Phosphatase by Camp-Dependent Protein Kinase View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Volume 82, number 2 FEBS LETTERS October 1977 THE CONTROL OF PHOSPHORYLASE PHOSPHATASE BY CAMP-DEPENDENT PROTEIN KINASE Pi1 GERGELY and Gyijrgy BOT Institute of Medical Chemistry, University of Medicine, H-4026 Debrecen, Bern t& 18/B, Hungary Received 22 July 1977 1. Introduction tissues which inhibits the phosphatase reaction is heat- stable [ 11 ,121. This heat-stable inhibitor protein is The enzymes of glycogen metabolism are regulated phosphorylated by CAMP-dependent protein kinase by interconversion between phosphorylated and dephos- [ 131. Recently, T&h et al. demonstrated the reversible phorylated forms. Contrary to our well-defined knowl- phosphorylation and dephosphorylation of this heat- edge of the phosphorylation processes of different stable inhibitor protein in vivo [ 143. Huang and Glins- enzymes and their role in glycogen breakdown and mann demonstrated the existence of two heat-stable synthesis, the phosphatases catalyzing the reverse inhibitor proteins for phosphatase [ 15,161. processes are only now being understood. The regula- Enzymic chemical modification of phosphorylase tion of dephosphorylation reactions is still unclear. phosphatase has not been established as a means of Phosphorylase phosphatase converts ‘active’ phos- regulation [ 171. phorylase a (EC 2.4.1.1) into ‘inactive’ phosphorylase In the present communication the effect of CAMP- b by cleavage of the phosphate groups from the serine dependent protein kinase on the phosphatase reaction residues. For the control of phosphorylase phospha- (i.e., on the dephosphorylation of phosphorylase a) tase the following mechanisms are possible: has been investigated. It is demonstrated that protein (i) By ligands which affect the substrate phosphoryl- kinase in the presence of CAMP inhibits the dephos- ase a (or phosphatase). phorylation of the phosphorylase a. The experiments (ii) By proteins which affect phosphorylase a (or phos- show that the inhibitory effect is due to the regul&ory phatase). subunit. Since the regulatory subunit loses its ability (iii) By enzymatic modification of phosphatase. to inhibit the phosphatase reaction on heat-treatment It was reasoned that ligands (e.g. glucose, glucose it is distinct from the heat-stable inhibitors isolated by 6_phosphate, glycogen, AMP and caffeine) influence Huangand Glinsmann [15,16] and Cohen et al. [13]. phosphorylase phosphatase reaction by changing the conformation of the substrate phosphorylase a or by shifting the tetramer _ dimer equilibrium of phos- 2. Methods phorylase a [l-8]. It is known that different proteins can mediate the Rabbit skeletal muscle phosphorylase a was prepared phosphorylase phosphatase reaction. We have shown as previously described [8]. Phosphorylase activity was that phosphorylase kinase inhibits the dephosphoryla- assayed by the procedure of Illingworth and Cori [lg]. tion of phosphorylase a and the inhibition is competi- Specific activity of phosphorylase a was 54 units.mg-’ tive in nature [9]. The phosphorylation (or thiophos; in the presence of 0.016 M glucose l-phosphate and phorylation) of phosphorylase kinase increases its inhibi- in the absence of AMP. tor constant [lo]. Phosphorylase phosphatase was prepared according Another protein isolated from different mammalian to the method of Brandt et al. [ 191. Phosphorylase North-Holland Publishing Company - Amsterdam 269 Volume 82, number 2 FEBS LETTERS October 1977 phosphatase assay was carried out as described previ- - 601 ously [8]. 2 CAMP-dependent protein kinase was prepared from 2 - 50 rabbit skeletal muscle according to Beavo et al. [20]. The separation of regulatory and catalytic subunits of protein kinase was carried out on a blue-dextran- Sepharose column [21]. Enzymic activity of protein kinase was determined by the method of Hofmann et al. [22]. The isolated protein kinase holoenzyme, the separated regulatory and catalytic subunits were homogenous by 0.1% SDS-7 5% polyacrylamide gel electrophoresis. The gel electrophoresis experiments were carried out in the manner described by Weber and Osborn [23]. The preparations (phosphorylase a, phosphorylase phosphatase, CAMP-dependent protein I kinase and its regulatory subunit) were free of phos- 2 4 6 8 phorylase kinase and did not contain heat-stable Time (min) inhibitors. The [32P]tetradecapeptide incorporating the.phos- Fig.1. Effect of CAMP-dependent protein kinase on the phorylated site was isolated after chymotryptic dephosphorylation of phosphorylase a. Experimental condi- tions are described in Methods. Activity of residual phosphoryl- digestion of [32P]phosphorylase a by the method of ase a in the absence of protein kinase Co----- o), in the Nolan et al. [24]. Dephosphorylation of the [32P]tetra- presence of 0.65 mg/ml protein kinase (A- A), 0.65 mg/ml decapeptide by phosphorylase phosphatase was mea- protein kinase + 5.10m6 M CAMP (A------- A) and 1.1 mg/ml sured at 30°C in 0.04 M Tris/O.OO:! M EDTA/O.OOOS M protein kinase + 5.10w6 M CAMP (m-9). dithiothreitol buffer, pH 7.4 [5]. Protein was determined by the modification of the biuret procedure [25]. dephosphorylation process. However, protein kinase in Inhibition of phosphorylase phosphatase reaction the presence of CAMP strongly inhibits the dephos- by CAMP-dependent protein kinase was assayed by the phorylation of phosphorylase a. The greater the following manner: 1 .O mg/ml phosphorylase a was concentration of protein kinase in the presence of incubated in 0.04 M Tris/0.002 M EDTA/0.0005 M CAMP the greater is the inhibitory effect on the phos- dithiothreitol buffer (pH 7.4) with phosphorylase phatase reaction. phosphatase in the presence of various concentrations It is known that CAMP dissociates the protein of protein kinase or regulatory/catalytic subunit. The kinase holoenzyme into regulatory and catalytic reaction mixtures were incubated at 30°C aliquots subunits. The inhibition of phosphatase reaction (50 /..d) were removed at different points of incubation observed only in the presence of CAMP indicates that and the reaction was stopped by the addition of 0.1 the inhibition could be due to the free subunits and M NaF/0.04 M glycerophosphate/0.002 M EDTA, not to the holoenzyme. (CAMP, alone does not effect pH 6.8, to attain a dilution in which the activity of the rate of phosphorylase a dephosphorylation, not residual phosphorylase a could still be measured. documented.) Therefore the inhibitory effectiveness of isolated regulatory and catalytic subunits were also investigated (fig.2). 3. Results and discussion It can be seen that the presence of catalytic subunit of CAMP-dependent protein kinase does not influence The effect of CAMP-dependent protein kinase on the dephosphorylation of phosphorylase a. Whereas the dephosphorylation of phosphorylase a by phos- the presence of regulatory subunit considerably phatase is shown in fig. 1. It is seen that protein kinase moderates the dephosphorylation reaction. Increasing in the absence of CAMP does not affect the rate of the the concentration of regulatory subunit increases the 270 Volume 82, number 2 FEBS LETTERS October 1977 + regulatory subunit + catalytic subunit 60 1 1 I I 1 I I 1 I I I 2 4 6 8 2 4 6 8 Time (min ) Fig.2. Dephosphorylation of phosphorylase a in the presence of regulatory and catalytic subunits. Experimental conditions are described in Methods. Activity of residual phosphorylase a in the absence (o -0) and in the presence of 0.23 mg/ml (A-A), 0.46 mg/ml (e -¤), 0.7 mg/ml (e---- 0) regulatory or catalytic subunits, respectively. Activity of phosphoryl- ase a in the presence of heat-treated regulatory subunit (X-X). The heat-treatment was carried out according to Brandt et al. [ 121 at 90°C for 5 min and the protein concentration after this procedure was 0.37 mg/ml. inhibition. The data presented in fig.1 and 2 demon- dephosphorylate the [32P]tetradecapeptide but this strate that only the regulatory subunit is a potent peptide does not show conformational change in inhibitor in the dephosphorylation process of phos- contrast to phosphorylase a. phorylase a, the catalytic subunit and the protein Figure 3 demonstrates the dephosphorylation of kinase holoenzyme have no inhibitory function. The [3?]tetradecapeptide by phosphatase. It is seen that regulatory subunit lost its inhibitory effectiveness after neither protein kinase in the presence of CAMP nor the heat-treatment (fig.2). This observation suggests that isolated regulatory subunit do alter the rate of the the inhibition caused by the regulatory subunit is dephosphorylation. These results show that the inhibi- distinct form the different heat-stable inhibitors. tion of phosphatase reaction by the regulatory subunit The inhibition of phosphorylase phosphatase reac- is substrate directed. tion by the regulatory subunit can be explained by Our observations point to a new feature of CAMP- two different mechanisms. On the one hand, the dependent protein kinase, i.e., not only its catalytic regulatory subunit can modify the conformation of subunit has an important control role in the glycogen the substrate (phosphorylase a). On the other hand, it metabolism but its regulatory subunit, too. The results can influence the enzyme (phosphorylase phosphatase) of this paper provide evidence for the regulation of itself. To distinguish these two supposed mechanisms phosphorylase a-b interconversion by the regulatory [32P]tetradecapeptide was used as a substrate of the subunit of the CAMP-dependent protein kinase. phosphatase reaction. Phosphorylase phosphatase can In light of experimental data presented here the 271 Volume 82, number 2 FEBS LETTERS October 1977 16, References [l] Bot, Gy. and D&a, I. (1971) Acta Biochim. Biophys. Acad. Sci. Hung. 6, 73-87. [2] De Barsy, T., Stalmans, W., Laloux, M., De Wulf, H. and Hers, H. G. (1972) Biochem. Biophys. Res. Commun. 46,183-190. [ 31 Bailey, M. J. and Whelan, W. J. (1972) Biochem. Biophys. Res. Commun. 46,191- 197. [4] Bot, Gy. and Gergely, P.
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