Interaction of calmodulin and glycogen phosphorylase

40

Biochimica etBiophysicaActa, 757 (1983) 40-46 Elsevier Biomedical Press

BBA 21438

INTERACTION OF CALMODULIN AND GLYCOGEN PHOSPHORYLASE CARLOS VILLAR-PALASI a, DIANNE L. OSHIRO b and ROBERT H. KRETSINGER b

Departments of a Pharmacology and b Biology, University of Virginia, Charlottesville, VA 22908 (U.S.A.) (Received October 7th, 1982) (Revised manuscript received February 2nd, 1983)

Key words: Calmodulin; Phosphorylase," Enzyme-calmodulin interaction

We have demonstrated the interaction of 1251-1abeled calmodulin with glycogen phosphorylase by four techniques: polyacrylamide gel overlay, sucrose density centrifugation, gel filtration chromatography, and affinity chromatography. Phosphorylase b has more affinity for calmodulin than does phosphorylase a. Under all conditions tested, the presence of calmodulin affects neither the enzymatic activity nor any kinetic characteristics of phosphorylase a or b. We present these results as evidence that while binding between calmodulin and phosphorylase clearly exists, it may not have a physiological role.

Introduction Calmodulin-binding proteins can be identified on SDS-polyacrylarnide gels following electrophoresis, fixation, incubation with 125I-labeled calmodulin and autoradiography [ 1,2]. This method has been used to identify putative calmodulinbinding proteins in postsynaptic density membranes [3,4], in isolated microfilament cores of intestinal microvilli [1] and in chick lens gap junction [5]. Burgess [6] noted in gel overlay procedures that 125I-labeled calmodulin labels phosphorylase b from molecular weight calibration kits in a calcium-dependent manner. Calmodulin mediates the effect of calcium on two enzymes in the control of glycogen metabolism, as one of the subunits of phosphorylase b kinase [7], and as an activator

Abbreviations: Pipes, piperazine-N,N'-bis(2-ethanesulfonic acid); EGTA, ethyleneglycol bis(fl-aminoethyl ether)-N,N'-tetraacetic acid. 0304-4165/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers

of one of the glycogen synthase kinases [8]. As calmoduhn had not previously been implicated in any direct involvement with the phosphorylase, we pursued this investigation. Glenney and Weber [1] noted that the proteins in the polyacrylamide gels are at least partially denatured. We considered the possibility that the interaction of calmodulin and phosphorylase requires denaturation of phosphorylase by SDS present during electrophoresis. We found that calmodulin also interacts with phosphorylase b in a calcium-dependent manner in the nondenaturing conditions of gel filtration chromatography, sucrose density centrifugation, and affinity chromatography. Calmodulin does not affect the enzymatic activity of either phosphorylase a or phosphorylase b under a wide range of experimental conditions. Phosphorylase does not prevent the calmodulin activation of cyclic nucleotide phosphodiesterase. We conclude that the binding of calmodulin to phosphorylase is relatively weak, and either that it has no physiological function, or that it has a function which we have been unable to detect.

41 Materials and Methods

Reagents. Commercial sources of reagents and materials were as follows: lactoperoxidase, Calbiochem; Na125I, Amersham; phosphorylase a, batch P-1261, bovine serum albumin (Fraction V), ATP, Trizma base, Pipes, 5'-AMP, cyclic AMP, bovine heart cyclic AMP phosphodiesterase, Crotalus adamanteus 5'-nucleotidase, EGTA, glucose 2-phosphate, rabbit liver glycogen, Sigma; [U-14C]glucose-l-phosphate, New England Nuclear; molecular weight calibration kit, CNactivated Sepharose 4B, Pharmacia; sodium dodecyl sulfate (SDS) and glycine, Matheson, Curtis, and Bell; acrylamide, bisacrylamide, Bio-Gel P-150, Bio-Rad; XAR-5 X-ray film, Kodak; Cronex lightening plus enhancing screens, Dupont. Other chemicals were reagent grade. Phosphorylase b, lyophilized, was kindly given to us by Dr. E. Krebs. Phosphorylase b kinase was a gift of Dr. A. dePaoli-Roach. Proteins. Calmodulin was prepared from bovine brains by differential ammonium sulfate precipitation at pH 4.1 and DEAE-cellulose chromatography, in the absence and presence of calcium, as described by Burgess et al. [9]. The purified calmodulin was iodinated with Na~25I and lactoperoxidase by the method of Richman and Klee [10]. The reaction was stopped, and the iodinated calmodulin (1.0 ml) desalted by passage through a 25 ml PD-10 sieve chromatography column. The specific activity of the calmodulin was l0 s cpm//~g; it was calculated to contain about 2.5.10 -3 mol 125I/mol calmodulin. Gel overlay calmodulin-binding. Protein samples in 2% SDS were electrophoresed in linear 5-15% polyacrylamide slab gels (0.75 × 110 × 160 mm) containing 0.2% SDS. After electrophoresis, the proteins in the gels were fixed with 25% isopropanol/10% acetic acid. The gels were then washed in 50 mM Tris-HC1 (pH 7.6)/0.2 M NaCI containing either 1.0 mM CaC12 or 2.0 mM EGTA for 6 h with four changes. The gels were further washed for 2 h in the same buffer containing 1.0 mg/ml bovine serum albumin. For the binding of calmodulin, the gels were incubated in buffer containing 10/~g 12SI-labeled calmodulin/ml, without bovine serum albumin, for 6 h. The gels were washed next with buffer (no protein) for 6 h with

four changes. The gels were then stained with Coomassie blue, destained, dried, and the proteins containing ~25I-labeled calmodulin revealed by exposure to X-ray film for 48 h at - 8 0 ° C [2]. Phosphorylase activity assays. The test of phosphorylase activity was based on the incorporation of [14C]glucose from [U-14C]glucose 1-phosphate into glycogen primer, and counting the radioactivity incorporated into the glycogen. The usual test mixture was: 50 mM Pipes, (pH 6.5)/150 mM [14C]glucose 1-phosphate/2.0% rabbit liver glycogen (deionized by passage through a MB-3 Amberlite column). 50/~1 of this mixture were mixed with 20/~1 50 mM Pipes (pH 6.5) or 20 ~tl 50 mM AMP/50 mM Pipes (pH 6.5), and the reaction was started by addition of 30 /~1 phosphorylase, appropriately diluted in 50 mM Pipes (pH 6.5)/2% mercaptoethanol/1.0 mg bovine serum albumin/ml. The reaction mixtures were incubated at 30°C for 10 min. Concentration of the components of the mixture varied when the kinetic parameters were tested in the l~resence or absence of added calmodulin. At the end of the incubations, 75/tl aliquots of the reaction mixtures were deposited into 2 × 2 cm squares of Whatman ET31 paper, and the glycogen precipitated by immersion into 66% ethanol. After three changes of ethanol (10-rain wash each), and one of acetone, the papers were dried and the radioactivity measured by scintillation counting in vials containing 0.5% 2,5-diphenyloxazole in toluene. Sucrose density gradient centrifugation. Sucrose solutions were prepared in 50 mM Pipes, pH 6.5, containing either 0.2 mM CaC1z or 2.4 mM EGTA. Continuous gradients of sucrose, 5-25% were made in a total volume of 5.0 ml. Fresh solutions of phosphorylase a and b (8-10 mg/ml) were prepared in 25 mM Pipes (pH 6.5)/50 mM mercaptoethanol. For the centrifugation, mixtures containing 0.4-0.5 mg phosphorylase/0.05 mg 125I-labeled calmodulin/25 mM Pipes (pH 6.5) and either 0.2 mM CaCI 2 or 2.4 mM EGTA, in a total volume of 125/~1, were incubated for 30 min at room temperature; the sucrose gradients were overlaid with 100 ttl of mixture, and the samples centrifuged at 40000 × g for 20 h at 5°C. 5-drop fractions (about 73 /~1) were collected from the bottom of the tubes at the end of centrifugation. 125I-labeled calmodulin was identified by gamma

42 counting of the fractions. 10/~1 aliquots were used to measure phosphorylase activity. Gel filtration chromatography. A 0.9 × 59 cm column of Bio-Gel P-150, 100-200 mesh, was equilibrated with 0.1 M KCI/0.2 mM CAC12/25 mM Pipes (pH 6.5). Mixtures of 60 /~1 of phosphorylase b (2 mg dissolved in 120/~1 of 25 mM Pipes (pH 6.5)/15% sucrose/50 mM mercaptoethanol/0.2 mM CaC12) and 60/~1 of 125I-labelled calmodulin, 1.75 mg/ml, were applied to the colu m n . The molar ratio [ p h o s p h o r y l a s e ] / [calmodulin] was approx. 2. Total 12sI cpm in the samples applied was about 1.4. 1 0 6 . The column eluted with the equilibration buffer at a flow rate of 2.6 ml/h. A void volume of 215 drops was collected followed by 5-drop fractions (250 /~1). Radioactivity was determined in the tubes by gamma counting and phosphorylase activity using 10/~1 aliquots of the fractions. Affinity chromatography. Purified bovine brain calmodulin was coupled to cyanogen bromide activated Sepharose as directed by Pharmacia. A column containing 9.5 mg bound calmodulin (3.5 ml bed volume) was equilibrated with 25 mM Pipes (pH 6.5)/1.0 m M CAC12/15 m M mercaptoethanol (buffer A). Solutions of phosphorylase a or b, 10 m g / m l were prepared in this buffer, and 200/~1 aliquots applied to the column. Five-drop fractions were collected. After washing with buffer A (10 ml), the columns were eluted with solutions of buffer A containing increasing concentrations of NaC1 or with 30 mM EGTA instead of calcium. The column was washed between runs with at least 20 vol. of 0.1 M Tris-HC1 (pH 8.5)/0.5 M NaC1, followed by 20 vol. of 0.1 M sodium acetate (pH 4.5)/0.5 M NaC1, before re-equilibrating with buffer A.

Test of phosphodiesterase activation by calmodufin. Solutions of activator-deficient bovine heart cyclic AMP phosphodiesterase (0.2 units/ml), 5'nucleotidase (4.0 u n i t s / m l ) , and purified calmodulin (1.0 mg/ml) were prepared in 20 mM Tris-HC1 (pH 7.5)/1.0 mM MgC12/1.0 mM imidazole (buffer B). The test mixtures contained: 500 /tl of 2.5 mM cyclic A M P / 5 0 mM Tris/50 mM imidazole (pH 7.5)/6 mM MgC12+ 50 /~1 5'-nucleotidase solution + 30/xl of 1 mM CaC12 or 8.0 mM EGTA + 50/~1 of calmodulin solution or buffer B, and buffer B to 850 /~1. The reactions

were started by the addition of 50 /zl of phosphodiesterase solution. After incubation at 30°C for 20 min, the reactions were ended by addition of 100 #1 of 50% trichloroacetic acid, centrifuged, and 800 #1 aliquots of the supernatants were removed for determination of inorganic phosphate by the Fiske-SubbaRow [11] method in a final volume of 2.0 ml. Phosphorylase, when added, was used as a solution of 2.0 m g / m l in buffer B, and 100/~1 were used in the test. Results

Coomassie blue-stained SDS-polyacrylamide gel electrophoretic patterns and the corresponding autoradiograms are shown in Figs. 1 and 2. These results show that, by the gel overlay technique, z25I-labeled calmodulin binds to phosphorylase a, phosphorylase b, troponin I, phosphodiesterase, and the a and ), subunits of the phosphorylase b kinase. No binding was detected to the/3 subunit of phosphorylase b kinase, or to any other of the proteins used as molecular weight standards (bovine serum albumin, ovalbumin, bovine erythrocyte anhydrase, soybean trypsin inhibitor, and bovine milk lactalbumin). The intensity of bands on the autoradiograms showing binding of 125I-labeled calmodulin to phosphorylase was decreased if the concentration of NaC1 in the incubation and wash buffers of the overlay was increased from 0 to 0.2 M (Fig. 2). Binding of calmodulin was detected at NaC1 concentrations up to 0.2 M when 1.0 mM CaCI: is present, but only up to 0.1 M NaCI when 2.0 mM EGTA was present. Phosphorylase appears as a single peak near the bottom of the tube after 5-25% sucrose density gradient centrifugation. In a separate experiment under identical conditions, calmodulin appears as one peak near the top. When phosphorylase b was mixed with l:5I-labeled calmodulin at a molar ratio 2.0 in the presence of 0.2 mM CaC12, calmodulin appears in two peaks, one in the normal position at the top, the other corresponding to the phosphorylase peak. In the presence of 2.4 mM EGTA or if excess non-radioactive calmodulin was added, no 125I-labeled calmodulin bound to phosphorylase b was detected. Iodinated calmodulin does not bind to phosphorylase a either in the presence of CaC12 or EGTA in these sucrose

43 10

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density gradient centrifugations (Fig. 3). When phosphorylase b and calmodulin were applied in separate experiments to the same Bio Gel P-150 gel filtration column, the profiles of elution of the two proteins did not overlap. However, if the two proteins were applied together in the presence of 0.2 mM C a C 1 2 , part of the 1251l a b e l e d calmodulin appeared at the front peak together with the phosphorylase activity (Fig. 4). Phosphorylase b is retained in calmodulin-Sepharose columns in low salt buffer containing 1.0

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mM CaC12; both E G T A and relatively high NaC1 concentrations (0.2 M) are required to elute the bound activity. Nearly 80% of the applied phosphorylase b is eluted in these conditions. Phopshorylase a on the other hand, does not bind to any significant extent to these affinity columns (Fig. 5). Effect of calmodulin on the kinetic parameters of phosphorylase a and b. A complete survey of possible effects of calmodulin on the Vmax, K m for glycogen and glucose 1-phosphate, and Ka for AMP, both in the presence of Ca 2+ or EGTA, was done with phosphorylase a and b. The test described in Methods was used, varying as required the concentrations of the components of the mixtures. Calmodulin, when added, was at a concentration of 20/~g in 100 ttl test; phosphorylase a was 5.0 ttg and phosphorylase b was 4.0/~g in the 100 /~1 test mixture, or molar ratios of calmodulin/phosphorylase subunits equal to 21 and 27 for phosphorylase a and b, respectively. Calcium concentration was 0.5 mM and EGTA was 1.0 mM. Addition of Ca 2÷ or EGTA at these concentrations did not change phosphorylase activity.

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No effect of calmodulin was found on any of the kinetic parameters of phosphorylase a and b activity, either in the presence of Ca 2+ or EGTA.

Effects of phosphorylase on calmodulin-dependent brain phosphodiesterase. Although calmodulin binding to phosphorylase does not affect the activity of the enzyme, we considered that phosphorylase b could act by trapping calmodulin and thereby decrease the activity of systems requiring

the presence of this protein. Activator-deficient brain cyclic A M P phosphodiesterase was assayed as a test of this model. The effect of phosphorylase a and b (2.0 m g / m l ) on the activation of phosphodiesterase was tested with concentrations of calmodulin between 1.0 ktl per ml of test mixture, or molar ratios of phosphorylase to calmodulin from 20 to 200. Phosphorylase does not affect the activation of phosphodiesterase by calmodulin in these conditions.

Discussion

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We c o n f i r m e d the interaction between calmodulin and phosphorylase in gel overlay experiments. The extent of protein renaturation in the gels after fixation and washing is not known, so we do not know the molecular conformations of proteins to which calmodulin binds. We evaluated the binding of calmodulin to phosphorylase by techniques designed to maintain the native state of proteins. Gel filtration and affinity chromatography experiments indicate a preferential binding of calmodulin to phosphorylase b rather than phosphorylase a in the presence of calcium. Calmodulin does not effect phosphorylase activity in the presence of absence of calcium. The amount of calmodulin is skeletal muscle has been estimated to be 50 mg [12] or 30 mg [13] per 1000 g of muscle. It has been shown that about half of this amount is bound in phosphory-

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lase b kinase [13]. Therefore, all the remaining calmodulin amounts to a concentration of 1-1.5 #M. Phosphorylase, on the other hand, represents about 2% of the total soluble proteins in skeletal muscle, or, in terms of molarity, about 8-10 # M subunits, mostly in the b form in resting muscle. The results of several of the binding experiments presented here appear to indicate that calmodulin is more tightly retained by phosphorylase b than by the a form. We considered the possibility that phosphorylase b could represent a sink or reservoir of calmodulin, and that, in being phosphorylated

and converted to phosphorylase a, the calmodulin could be released free to act on other systems, such as phosphodiesterase. Our results show that excess phosphorylase a or b, does not reduce the activation of phosphodiesterase by calmodulin, so the important factor appears to be not so much the relative concentration of phosphorylase and calmodulin, but rather the affinities of phosphodiesterase and phosphorylase b for calmodulin. Phosphorylase does not interfere with the calmodulin activation of calmodulin dependent cyclic nucleotide phosphodiesterase. We have considered an alternative possibility for a functional role in facilitating the phosphorylation of phosphorylase b to phosphorylase a. Experiments to test this idea are difficult to design and interpret because (1) calmodulin is a subunit of phosphorylase kinase, (2) additional equivalents of calmodulin activate phosphorylase kinase and (3) such evaluations require preparation of calmodulin independent kinases and phosphatases. We continue to explore the possibility of competive binding of calmodulin by phosphorylase as a possible mechanism of metabolic regulation. We conclude that either the in vitro binding of calmodulin to phosphorylase does not reflect an in vivo process, or that we have not yet been able to determine the functional role. We note that a functional interaction with calmodulin has been inferred for several enzymes and structural proteins without the demonstration of a physical association. Conversely, both the affinity chromatography and gel overlay techniques have indicated calmodulin interaction with numerous proteins yet to be characterized. We caution that a protein not be considered to be regulated by calmodulin until both physical and functional interactions have been demonstrated. Acknowledgements

This work was supported by the National Institutes of Health grants HL24487-03 (to C. V.-P.) and 5 R01 AMI 16253-11 (to R.H.K.). References 1 Gleney, J.R., Jr. and Weber, K. (1980) J. Biol. Chem. 255, 10551-10554

46 2 Carlin, R.K., Grab, D.J. and Siekevitz, P. (1980) Ann. N.Y. Acad. Sci. 356, 73-74 3 Grab, D.J., Carlin, R.K. and Siekevitz, P. (1980) Ann. N.Y. Acad. Sci. 356, 55-72 4 Carlin, R.K., Grab, D.J. and Siekevitz, P. (1981) J. Cell. Biol. 89, 449-455 5 Welsh, M.J., Aster, J.C., Ireland, M., Alcala, J. and Maisel, H. (1982) Science 216, 642-643 6 Burgess, W.H. (1981) Doctoral Thesis, University of Virginia 7 Cohen, P. Burchell, A., Foulkes, J.G., Cohen, P.T.W., Vanaman, T.C. and Nairn, A.C. (1978) FEBS. Lett. 92, 287-293 8 Payne, M.E. and Soderling, T.R. (1980) J. Biol. Chem. 255, 8054-8056

9 Burgess, W.H,, Jemiolo, D.K. and Kretsinger, R.H. (1980) Biochim. Biophys. Acta 623, 257-270 10 Richman, P.G. and Klee, C.B. (1978) J. Biol. Chem. 253, 6323-6326 11 Fiske, C.H. and SubbaRow, Y. (1925) J. Biol. Chem. 66, 375-500 12 Shenolikar, S., Cohen, P.T.W., Cohen, P., Nairn, A.C. and Perry, S.V. (1979) Eur. J. Biochem. 100, 329-337 13 Yagi, K., Yazawa, M., Kakiuchi, S., Oshima, M. and Venishi, K. (1978) J. Biol. Chem. 253, 1338-1340