Effect of parathyroid hormone and cyclic AMP on protein phosphorylation in rabbit kidney cortex

372

Biochimica et Biophysica Acta, 451 (1976) 372--381 Q Elsevier/North-Holland Biomedical Press

BBA 28105 E F F E C T OF P A R A T H Y R O I D HORMONE AND CYCLIC AMP ON PROTEIN P H O S P H O R Y L A T I O N IN RABBIT KIDNEY CORTEX

D. AUSIELLO, J. HANDLER and J. ORLOFF Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, Bethesda, Md. 20014 (U.S.A.)

(Received May 24th, 1976)

Summary Suspensions of renal cortical tubules were incubated with 33Pi and exposed to parathyroid hormone (40 pg/ml) or 1 mM dibutyryl cyclic AMP. In other experiments homogenates of renal cortex were assayed for protein kinase and phosphoprotein phosphatase activity using [~/-32p]ATP with or without 5 mM cyclic AMP. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and phosphorylation of proteins measured by liquid scintillation counting of gel slices. The pattern of protein phosphorylation was similar in control tissue from both tubule suspensions and homogenates. In intact tubules, parathyroid hormone stimulated the phosphorylation of four proteins with molecular weights of approx. 150 000, 125 000, 100 000 and 50 000 by 28%, 24%, 13%, and 20%, respectively. Results with dibutyryl cyclic AMP were comparable but more variable. Stimulation of phosphorylation by cyclic AMP in homogenates was more generalized with the major effect on a 50 000 dalton protein (50% stimulation). No effect of cyclic AMP on dephosphorylation of proteins was observed. The results are interpreted as indicating that increased phosphorylation of cell proteins is part of the cyclic AMP-mediated response of the renal cortex to parathyroid hormone.

Introduction It has been established that the action of parathyroid hormone on the kidney is mediated by cyclic AMP [1--4]. Studies on the role of cyclic AMP as a "second messenger" for hormone action in many tissues have led to the theory that the final effects of the nucleotide may be a consequence of activation of protein kinases [5--7]. Cyclic AMP-dependent protein kinase activity [7--12] and endogenous protein substrates for the enzyme [13--21] have been found in broken cell preparations of a wide variety of tissues. The effect of hormones on

373 the phosphorylation of proteins in intact cells, however, has been studied in only a few tissues; these include liver (glucagon) [22], nucleated erythrocytes (~-adrenergic agents) [23], and toad urinary bladder (vasopressin) [21,24--26]. Specific phosphorylation of protein substrate accompanied by change in enzymatic activity of the substrate has been demonstrated for skeletal muscle phosphorylase kinase [27], glycogen synthetase [28] and adipose tissue triglyceride lipase [29]. In the present investigation, suspensions of intact tubules or homogenates of renal cortex were used to study the effect of parathyroid hormone and of cyclic AMP on the phosphorylation of endogenous proteins. Materials and Methods

Preparation of reagents Parathyroid hormone. Purified bovine parathyroid hormone (2000--3000 U/mg) was generously supplied by Dr. Bryan Brewer of NHLI, NIH. Synthetic bovine parathyroid hormone 1-34 (3000 U/mg) was obtained from Beckman Instruments, Inc. Both hormones were stored frozen in 0.1 M acetic acid. 33p04 Ringer solution. Carrier-free inorganic 33po4 in 0.2 M HC1 was obtained from New England Nuclear Corp. and added to a phosphate-free Krebs-Ringer bicarbonate solution containing, in mmol per l: NaC1, 115; KC1, 5; NaHCO3, 25; sodium acetate, 10; MgSO4, 1.2; CaCl2, 1.0; glucose, 5.5. The solution was bubbled with 95% O2/5% CO2 gas; pH was 7.4. Radioactivity of the final solution varied from 150 to 25 pCi/ml.

Rabbit kidney cortex preparations (a) Cortical slices. New Zealand white male rabbits weighing approximately 2 kg were killed by decapitation. Slices of approximately 0.5 mm in thickness were made from outer cortex of the kidney using a Stadie-Riggs apparatus as previously described [30]. Each slice contained approximately 4 mg of protein. (b) Separated cortical tubules. A suspension of separated renal cortical tubules was prepared by a modification of the method of Burg and Orloff [31]. The suspension was poured through double-layered nylon mesh and centrifuged at 4 ° C for 1 min at 50 × g. The pellet was resuspended in phosphate-free KrebsRinger bicarbonate solution and recentrifuged. This washing procedure was repeated three times. In studies of phosphoprotein metabolism, the final pellet was resuspended in 33pO4-containing Ringer solution to yield a concentration of about 4 mg of tubule protein per ml. In other experiments, cell cyclic AMP content in response to parathyroid hormone was measured. The tubules were incubated in phosphate-free KrebsRinger solution for 50 min. Theophylline was added to a final concentration of 1 mM and the incubation continued for an additional 10 min. Control aliquots (2 ml) were taken followed by the addition of purified bovine parathyroid hormone (40 pg/ml final concentration). Additional 2 ml samples were obtained at 2, 5, and 15 min. Each 2 ml aliquot was added to 0.2 ml of 50% trichloroacetic acid in ice and homogenized with a Brinkman polytron for 45 s. Samples were centrifuged and the supematant solutions collected. Cyclic AMP was separated [32] and then was measured by the method of Gilman [33].

374

Cortex homogenates. Kidneys were excised and perfused as for preparation of separated tubules, and immersed in 0.25 M sucrose at 4 ° C. All further homogenization procedures were carried out at this temperature. Approximately 350 mg of outer cortex were minced and suspended in 10 ml of 0.25 M sucrose. After 10 strokes in a 15 ml Dounce homogenizer (B pestle), the cells were disrupted further with 20 strokes of a m o t o r driven teflon pestle (1800 rev./ min). The homogenate was filtered through double-layered nylon mesh. Phase microscopic examination revealed that more than 95% of the cells were broken. The final concentration of homogenate was approximately 4 mg protein per ml. Protein phosphorylation Cortex slices. Single slices of cortex were placed in 1 ml of 33PO~-containing Ringer solution in Erlenmeyer flasks, equilibrated with 95% 02/5% CO2 gas, and shaken continuously for 60 min. At the end of the incubation, slices were withdrawn and immediately placed in 1 ml of boiling 5% sodium dodedyl sulfate and 10 mM sodium phosphate buffer pH 7.2. After 5 min at 100 °C, the visible tissue remaining in the sodium dodecyl sulfate solution was dissolved by homogenizing briefly with a m o t o r driven teflon pestle. Aliquots were taken for protein determination [34] using bovine serum albumin as standard. Samples were prepared for gel electrophoresis by addition of the following (final concentration): 10% sucrose, 8% 2-mercaptoethanol and 0.002% Pyronin Y, and were then kept at 100°C for 2 min. Separated cortical tubules. The separated cortical tubules were suspended in 33pO4-containing Ringer solution at 23°C in special silonized incubation flasks [31]. Suspensions were stirred by continuous bubbling with 95% 02/5% CO2 gas. At various times 1 ml aliquots of the suspension were added to 200 pl of 20% sodium dodecyl sulfate in a boiling water bath and kept at 100°C for 5 min. The visible tissue remaining was dissolved in the sodium dodecyl sulfate by homogenizing with a m o t o r driven teflon pestle. The sample was dialyzed overnight against 3.3% sodium dodecyl sulfate and 10 mM sodium phosphate buffer pH 7.2 in order to remove most of the non-protein radioactivity and to reduce the ionic strength of the solution. Samples were then prepared for electrophoresis as indicated for cortex slices. Cortex homogenates. 50 pl of homogenate (4°C) were mixed with 100 pl of buffer composed of 100 mM sodium acetate and 20 mM magnesium acetate, pH 6.4, for 2 min at 23°C to allow for temperature equilibration. The phosphorylation reaction was initiated by the addition of [~/-32p]ATP (ICN Corp.), with or without cyclic AMP. The final composition was: 50 mM sodium acetate and 10 mM magnesium acetate, pH 6.4; 5 pM [~-32p]ATP (20 Ci/mmol), 5 pM cyclic AMP {where added) and approximately 200 pg of homogenate protein in a total volume of 200 pl. Incubations were ended by adding 100 pl of a solution composed of 3% sodium dodecyl sulfate, 30 mM Tris • HC1 (pH 8.0), 3 mM EDTA, 30% sucrose, 120 mM dithiothreitol, and 0.006% Pyronin Y. Samples were immediately placed in a boiling water bath for 5 min.

Sodium dodecyl sulfate.polyacrylamide gel electrophoresis Polyacrylamide gel electrophoresis, utilizing a high pH sodium dodecyl sul-

375 fate discontinuous system [35], was carried o u t on 5--15% linear polyacrylamide gradients in 6 × 75 mm cylindrical gels with a 3% stacking gel. 150--250 pg of protein were applied to each gel and electrophoresis performed at 150 V for 3 h. After electrophoresis, sample gels were fixed in a solution containing 10% acetic acid and 25% methanol which was circulated through Dowex 1 anion exchange resin. This procedure eliminated more than 99% of low molecular weight phosphate trapped in the gel. Gels were sliced into 1 mm segments with an efficient and accurate slicer designed by Robert Bowman of NHLI, NIH. Radioactivity was determined by liquid scintillation counting. The quantity of 32p or 33p in each phosphoprotein peak was determined by summing the radioactivity in the peak slice plus that in one slice on each side of the peak to allow for any small variation in slicing or gel composition. Molecular weights of phosphorylated proteins were estimated by the method of Slater [36] utilizing the mobilities of fluorescein-labeled standards. Protease and ribonuclease study Phosph.orylated samples from separated cortical tubules and cortex homogenates, dissolved in sodium dodecyl sulfate as for electrophoresis, were mixed with crude pancreatic protease Type I, 1 mg/ml or ribonuclease-A (Sigma Chemical Co.), 1 mg/ml, and allowed to stand at room temperature for 30 min. Both enzymes were found to be active at this concentration in 5% sodium dodecyl sulfate when standard proteins and RNA were tested. Gels with phosphorylated samples that had been treated with protease before electrophoresis had less than 5% of the 33p or 32p of gels with untreated samples. This corresponded the total disappearance of Comassie Blue-stained protein bands in gels with protease-treated samples. Therefore, at least 95% of radioactive phosphate counted in gel slices was phosphoprotein phosphate. Gels with samples treated with ribonuclease before electrophoresis showed no differences from control gels. Results and Discussion Protein phosphorylation in intact cells Several phosphoprotein peaks were seen in gels containing material from cortical slices. The molecular weights of the peaks ranged from 38 000 to 150 000. Although the same peaks appeared consistently, the magnitude of their phosphorylation varied considerably in slices from the same depth in the cortex, and even in portions of the same slice. Thus slices were deemed not suitable for study of effects of parathyroid hormone, and the separated tubule preparation was used for all further experiments with intact cells. Initial studies demonstrated that the separated rabbit cortical tubules were responsive to bovine parathyroid hormone. As reported previously for separated rat renal cortical tubules [37l, the cyclic AMP content of the rabbit tubules was elevated by incubation with the hormone, 40/~g/ml, in the presence of 1 mM theophylline. Controls contained 4.8 pmol cyclic AMP per mg protein. After 2 min of incubation with parathyroid hormone, cyclic AMP levels rose to 14 pmol per mg protein and remained at this level through fifteen minutes of incubation.

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Maximum levels of phosphorylation of proteins in cortical tubules were achieved after 45 min of incubation in radioactive phosphate and remained essentially unchanged for the next 60 min. The pattern of phosphoproteins observed in the separated cortical tubules was similar to that seen in slices of cortex b u t there was much less variability. Fig. 1 illustrates the pattern of phosphorylation of control tissue and the effect of incubation with purified bovine parathyroid hormone for 15 min. The phosphorylation of four proteins with approximate molecular weights 150 0 0 0 , 1 2 5 0 0 0 , 1 0 0 000 and 50 000 was significantly stimulated by parathyroid hormone (Table I). The stimulation of phosphorylation of all four peaks occurred over a similar period. In most experiments the effect of parathyroid hormone on phosphorylation was observed as early as three minutes, reached a maximum between 5 and 15 min, and diminished toward control values by 20 min. In four experiments, synthetic bovine parathyroid hormone 1-34 (40 gg/ml) yielded similar results. Heat-inactivated bovine parathyroid hormone had no effect on protein phosphorylation in two experiments. Dibutyryl cyclic AMP (10 -3 M) stimulated significantly the phosphorylation of the 50 000 molecular weight protein in separated tubules by 18 +- 3%, n = 7.

377 TABLE I EFFECTS OF PARATHYROID HORMONE AND CYCLIC AMP ON PROTEIN PHOSPHORYLATION Protein molec.

% increase in phosphorylation -+ S.E. (P < 0.05--<0.01)

wt. X 1 0 - 3

................................................ Parathyroid hormone (separated tubules) (n= 11)

Cyclic AMP (homogenates) (n= 3)

28 ± 3 2 4 +- 4 1 3 +- 4 n.s. n,s. n.s. n.s. 20 ± 4 n.s. n.s.

16 -+ 1 20 + 1 21 -+ 2 n.s. 2 8 -+ 2 17 * 3 31 -+ 4 5 2 -+ 4 27 ± 3 2 0 +- 4

150 125 100 84 74 64 55 50 42 38 n.s., not significant.

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SLICE NUMBER F i g . 2. P r o t e i n p h o s p h o r y l a t i o n in rabbit kidney cortex homogenates and ( c A M P ) . H o m e g e n a t e s w e r e i n c u b a t e d 2 r a i n w i t h [ 7 - 3 2 p ] A T P in t h e p r e s e n c e ( of 5/~M cyclic AMP. Data are from a representative experiment.

the effect of cyclic AMP ) o r a b s e n c e (. . . . . . )

378 The mean effect on the 150 000, 125 000, and 100 000 molecular weight phosphoproteins was stimulation, but because of large variability in the responses, was not statistically significant.

Protein phosphorylation in cortex homogenates The molecular weights of proteins phosphorylated in cortex homogenates (Fig. 2) were similar to those seen in cortex slices and separated tubules, but there were significant quantitative differences. The 150 000 and 125 000 molecular weight proteins were consistently among the smallest of the phosphorylated peaks while the 100 000 and 84 000 molecular weight proteins were two of the larger peaks in the homogenates. Phosphorylation of all peaks in the homogenates reached maximum levels at 4 min and was decreased slightly at 10 min. Fig. 3 illustrates the time course for the phosphorylation of the 50 000 molecular weight phosphoprotein. Cyclic AMP (5 pM) stimulated the phosphorylation of many proteins. Its effect was most marked on the 50 000 molecular weight protein (Fig. 2, Table I). The maximum effect of cyclic AMP was reached between 10 and 30 s for all peaks and was maintained through 4 min (Fig. 3). The level of phosphorylation was diminished somewhat by 10 min in the presence and absence of cyclic AMP. Protein dephosphorylation in cortex homogenates In view of recent evidence [38] of cyclic AMP-stimulated phosphoprotein phosphatase activity in homogenates of kidney, brain, and. toad urinary bladder, the effect of cyclic AMP on dephosphorylation of proteins (loss of phosphoprotein) in homogenates was studied under three different conditions. In two, proteins were phosphorylated in the presence of Mg ~÷ and dephosphorylation studied following the addition of (1) excess unlabelled ATP, or (2) excess ATP plus EDTA. In the third study, proteins were phosphorylated in the presence of Zn 2÷, which inhibits phosphoprotein phosphatase activity [38] and dephos-

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phorylation examined following the addition of excess ATP and EDTA. The experimental details are described in the legend to Fig. 4. Cyclic AMP had no effect on dephosphorylation under all three experimental conditions. The rate of dephosphorylation was similar in samples phosphorylated in Mg ~* regardless of whether ATP alone or ATP and EDTA were added (data not shown). Fig. 4 compares dephosphorylation of the 50 000 molecular weight protein in samples phosphorylated in the presence of Mg2. or Zn 2* followed by addition of excess unlabelled ATP and EDTA. The pattern observed for other proteins was similar to that of the 50 000 molecular weight phosphoprotein seen in Fig. 4. In all experiments, the rate of dephosphorylation was more rapid over the first minute than over the next several minutes. Much greater phosphorylation of all proteins occurred in the presence of Mg ~* than in the presence of Zn 2÷. In addition, there were marked differences in the pattern of protein phosphorylation. In the presence of Zn ~÷ there was a relative increase in the phosphorylation of several large molecular weight proteins (150 000 to 500 000) with a diminution in phosphorylation of lower molecular weight proteins. Malkinson et al. [38] have reported that the dephosphorylation of a 49 000 molecular weight protein in the cytosol and the particulate fractions of rat and bovine kidney cortex is stimulated by cyclic AMP. In their study [38] the stimulation of dephosphorylation occurred when phosphorylation was conducted in the presence of Zn ~÷, and was dependent on the ionic strength, pH, and temperature of the reaction mixture. Our studies were conducted under similar conditions [38] except that whole homogenates of rabbit kidney cortex were used (not particulate or cytosol fractions), and the temperature of incubation was 23°C instead of 30 ° C. In the present study, the molecular weights of the phosphoproteins and the

380 amount of the 50 000 molecular weight phosphoprotein phosphorylated in the presence of zinc were found to be significantly altered in comparison to homogenates phosphorylated in the presence of magnesium. In other studies, (Strewler, G., unpublished observation), using incubation solutions similar to those of Malkinson et al. [38] 5 mM Zn :÷ was found to precipitate toad bladder cytosolic proteins, including a 50 000 molecular weight phosphoprotein. Consequently, studies of phosphoprotein metabolism performed with zinc should be interpreted with caution. In t h e present study the quantitative differences in protein phosphorylation in intact cells vs. homogenates and the more generalized effect of cyclic AMP in broken cell preparations is not surprising. In homogenates, effects of cell compartmentalization are eliminated and enzyme and substrate reactivity may have little relationship to that in the intact cell. Conversely, the parathyroid hormone-stimulated phosphorylation of four phosphoprotein peaks in the intact tubule cell results from a kinase-substrate relationship that may be important in mediating the physiologic effects of the hormone. Further studies will be necessary to characterize the hormonal specificity of the phosphorylation of these proteins, to identify their subcellular distribution, and to define their robe in the response to parathyroid hormone.

Acknowledgement The authors are grateful to Dr. V.C. Manganiello for performing the assays of cyclic AMP.

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