ARCHIVES
Vol.
OF BIOCHEMISTRY
285, No. 1, February
AND
BIOPHYSICS
15, pp. 153-157,1991
Effect of Parathyroid Hormone on Rat Renal CAMPDependent Protein Kinase and Protein Kinase C Activity Measured Using Synthetic Peptide Substrates Ramakrishna
Nemani,‘**Tt
N irandon
Wongsurawat,t
and H. James
ArmbrechtfT$
Geriatric Research, Education, and Clinical Center, VA Medical Center, St. Louis, Missouri 63125; and Departments *Pharmacology, tlnternal Medicine, and $Biochemistry, St. Louis University School of Medicine, St. Louis, Missouri
Received
July
3, 1990, and in revised
form
September
27, 1990
The actions of parathyroid hormone (PTH) on the renal cortex are thought to be mediated primarily by CAMPdependent protein kinase (PKA) with some suggestion of a role for protein kinase C (PKC). However, present methods for assaying PKA and PKC in subcellular fractions are insensitive and require large amounts of protein. Recently, a sensitive method for measuring the activity of protein kinases has been reported. This method uses synthetic peptides as substrates and a tandem chromatographic procedure for isolating the phosphorylated peptides. We have adapted this method to study the effect of PTH on PKA and PKC activity using thin slices of rat renal cortex. PTH (250 nM) stimulated cytosolic PKA activity four- to fivefold within 30 s, and PKA activity was sustained for at least 5 min. PTH also rapidly stimulated PKC activity in the membrane fraction and decreased PKC activity in the cytosol. These changes were maximal at 30 s, but unlike changes in PKA, they declined rapidly thereafter. PTH significantly activated PKC only at concentrations of 10 nM or greater. This study demonstrates that PTH does activate PKC in renal tissue, although the duration of activation is much less than for PKA. It also demonstrates that a combination of synthetic peptides with tandem chromatography can be used as a sensitive assay procedure for protein kinase activity in biological Sampk?S. 0 1991 Academic PRESS, hc.
Parathyroid hormone (PTH)’ has numerous biological actions in the renal cortex. Two important physiological 1 To whom correspondence and requests for reprints should be addressed at Geriatric Center (lllG-JB), St. Louis VA Hospital, St. Louis, MO 63125. FAX: (314) 894-6614. * Abbreviations used: Mes: 2(N-morpholino)ethanesulfonic acid; PMSF, phenylmethylsulfonyl fluoride; EGTA, ethylene glycol bis(Paminoethylether)N,N’-tetracetic acid, PKA, CAMP-dependent protein kinase; PKC, protein kinase C; DTT, dithiothreitol; MIX, 1-methyl-3isobutylxanthine. 0003-9661/91$3.00
Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
of 63104
effects of PTH are to increase the production of 1,25dihydroxyvitamin D (l), the biologically active form of vitamin D, and to inhibit phosphate transport (2). PTH is known to increase CAMP production and CAMP-dependent protein kinase (PKA) activity in the renal cortex, and this pathway is thought to be responsible for most of the effects of PTH on vitamin D metabolism and phosphate transport (3, 4). Recently, there has been increasing evidence that PTH may also stimulate protein kinase C (PKC) in addition to PKA. PTH is known to stimulate Ca2+ influx and inositol phosphate turnover in the kidney (5). It has been reported that PTH causesa translocation of PKC activity in rat osteosarcoma cells (6) and cultured renal cells (7). The physiological significance of PKC activation is not clear. However, PKC, in addition to PKA, may play a role in regulation of renal phosphate transport (8,9) and 1,2Sdihydroxyvitamin D production (10). To assessthe role of PKA and PKC in mediating the actions of PTH, a simple and sensitive method for measuring protein kinase activity in renal tissue is needed. Unfortunately, the present methods for assaying protein kinase activities in tissue samples have high backgrounds and require large amounts of protein sample. Recently, a tandem chromatographic column method for measuring protein kinase activity has been described (11). This method, which uses synthetic peptides as substrates, is very sensitive, but it has only been used to measure the activity of purified enzyme. We report here the application of the tandem chromatographic column method, using synthetic peptides as substrates, to measure PKA and PKC in renal tissue. Using isolated renal slices, we demonstrate that PTH treatment results in the rapid stimulation of PKC activity in the membrane fraction and decreased PKC activity in the cytosol. We also compare the effect of PTH on renal PKA and PKC activity in terms of dose and time course. 153
154 MATERIALS
NEMANI.
AND
WONGSURAWAT.
METHODS
Materials. ATP, CAMP, CM-Sephadex cation exchange resin (bead size 40-120 pm (CZS-120)), Dowex AGl-X8 anion exchange resin (loo200 mesh), DTT, phosphatidylserine, and 1,2-diolein were obtained from Sigma Chemical Co. (St. Louis, MO). [y-32P]ATP (25 Ci/mmol) was purchased from International Chemical and Nuclear Corp. (Irvine, CA). The PTH used in these experiments was synthetic rat PTH (l-34) (5100 U/mg) purchased from Bachem Inc. (Torrance, CA). Kemptide, a synthetic heptapeptide substrate for A-kinase, H,N-LeuArg-Arg-Ala-Ser-Leu-Gly-COOH (12), was obtained from Sigma Chemical Co. (St. Louis, MO). GS-peptide, a synthetic dodecapeptide of glycogen synthase which is a substrate for the protein kinase C, HzNPro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala-Lys-Lys-COOH (13), was purchased from the Department of Medicine, Repatriation General Hospital, West Heidelberg, Vie 3081, Australia, as previously described (11). Animals. Male Fisher 344 rats aged 6-8 weeks were purchased from Harlan Industries (Indianapolis, IN). Rats were fed Purina rodent laboratory chow. The effect of PTH on protein kinase activity Tissue preparation. was measured using renal slices (14). Animals were killed by decapitation. The excised kidneys were decapsulated and washed in cold saline, and renal cortical slices were prepared as described elsewhere (15). Thin slices of renal cortex were preincubated for 15 min at 37°C in 1 ml of Krebs-Ringer bicarbonate buffer and then 20 min in buffer containing 1 mM 1-methyl-3-isobutylxanthine (MIX), a cyclic nucleotide phosphodiesterase inhibitor. At the end of the second incubation, PTH was added to the slice incubation medium for the indicated time, Slices were then quickly homogenized in the appropriate ice-cold buffer, and the homogenate was centrifuged as indicated to obtain a supernate and/or membrane fraction as needed. For the Measurement of CAMP-dependent protein kinase activity. measurement of PKA activity, slices were homogenized in 1 ml of icecold 5 mM potassium phosphate buffer (pH 6.8) containing 2 mM EDTA, 0.5 mM MIX, 0.5 mM PMSF, and 150 mM sodium chloride. Tissue was homogenized for six strokes using a glass-teflon Potter-Elvehjem homogenizer at 600 rpm (14). The homogenate was centrifuged at 20,OOOg for 20 min, and the capacity of the supernate to phosphorylate Kemptide was determined in the presence and absence of CAMP according to the method of Ghazarian and Yanda (16) at 30°C. Kemptide is a specific substrate for PKA under these conditions (12). The reaction mixture consisted of Kemptide (30 pg), 0.05 mM CAMP, 0.100 mM ATP, 0.5 PCi [y-32P]ATP, 0.3 mM EDTA, 1 mM magnesium acetate, 4.8 mM theophylline, 20 mM Mes, and supernate sample all in a final volume of 160 pl at pH 6.2. Assays were performed in 13 X loo-mm glass test tubes. The assay was run for various times as indicated, and it was terminated by adding 4 ml of a 2.5 mM ATP solution, pH 6.8. The reaction mixture was processed as described below. Measurement of protein kinase C actiuity. For measurement of PKC activity, slices were homogenized in 1 ml of 20 mM Tris-HCl, 0.25 M sucrose, 1 mM EDTA, 0.5 mM PMSF, 2 mM EGTA, 0.1 mM DTT, 20 gg/ml leupeptin, pH 7.5, as described by Tamura et al. (7). The homogenate was centrifuged at 35,000g for 30 min, and the supernatant was used as the cytosolic fraction. The pellet was resuspended in the original volume of (1 ml) homogenizing buffer, and Triton X-100 was added to a final concentration of 0.025%. After sonication (4 X 1 min on ice with Tekamar Sonic Disruptor), the suspension was left on ice for 1 h and then centrifuged at 35,000g for 30 min. The resulting supernatant was used as the membrane fraction. The cytosolic and membrane fractions were diluted appropriately with the homogenizing buffer just before assay. Protein kinase C was assayed according to the method of Nishizuka (17) using the GS-peptide as a substrate. The GS-peptide is a specific substrate for PKC under these conditions (13). The reaction mixture (160 ~1) contained 20 mM Tris-HCl at pH 7.5,5 mM magnesium acetate, 0.100 mM ATP with radiolabeled ATP, 10 fig GS-peptide, 0.1 mM CaCl,, 2 fig phosphatidylserine, 0.2 pg of diolein, and enzyme fraction. Phos-
AND
ARMBRECHT
phatidylserine and diolein were mixed in small volumes of chloroform and dried under a stream of nitrogen. The residue was then sonicated in 20 mM Tris, pH 7.5, and added to the reaction mixture as lipid micelles. Basal activity was measured in the presence of 0.5 mM EGTA, and CaCl,, phosphatidylserine, and diolein were omitted from the reaction mixture. Basal activity was subtracted from the Ca’+/phospholipidstimulated activity, and the difference was taken as protein kinase C activity. The assay was run for various times as indicated, and it was terminated by adding 4 ml of a 2.5 mM ATP solution, pH 6.8. The reaction mixture was then processed as described below. Processing of reaction mixture. The phosphorylated peptides were separated from the reaction mixture according to the method of Egan et al. (11). The radiolabeled Kemptide or GS-peptide was recovered from the reaction mixture by sequential processing of the reaction mixture through tandem chromatographic columns of cation and anion exchange resins. Briefly, the reaction mixture was poured onto a CMSephadex column (l-ml bed volume), and the test tubes were allowed to drain for a few minutes. The columns were then washed with a solution of 2.5 mM ATP, pH 6.8. After the liquid had drained from the CMSephadex resin, the columns were mounted over the Dowex AGl-X8 columns (l-ml bed volume). Eight milliliter, of 30% acetic acid was applied to the CM-Sephadex columns, and the total eluates from the AGl-X8 columns were collected in scintillation vials. The amount of radioactivity present was determined by liquid scintillation counting. Protein was determined by the method of Protein determinations. Bradford (18). Bovine serum albumin was used as a standard. Statistics. The data from these experiments are reported as the means of f standard error (SE) of the indicated number of measurements. Statistical analyses were performed using Student’s two-tailed t test (19), and a confidence level of 95% or greater was considered significant.
RESULTS
Measurement of CAMP-Dependent Protein Kinase in Renal Cytosol. The linear conditions for assay of PKA in renal cytosol were established in the presence of CAMP. With regard to protein concentration, phosphorylation of Kemptide was linear from O-10 pg of protein using a 15min timepoint. With regard to time, phosphorylation of Kemptide was linear from O-30 min at a protein concentration of 2.5 pg. Therefore, all subsequent assays were performed using 2.5 fig of protein for 15 min at 30°C. No measurable endogenous protein phosphorylation was detected in the absence of Kemptide. Effect of PTH on PKA The effect of PTH on renal PKA was studied using renal cortical slices. The protein kinase activity in control (no PTH) was 148 f 4 and 651 k 22 pmol/min/mg protein without and with CAMP, respectively. The protein kinase activity in slices treated with PTH (250 nM for 30 s) was 511+ 14 and 517 ? 48 pmol/min/mg protein without and with CAMP, respectively. When PKA inhibitor (synthetic peptide of rabbit skeletal muscle) was added to the incubation, the CAMP-dependent protein kinase activity was reduced to the basal level. The time course of PKA activation induced by PTH is shown in Fig. 1. PKA activity was expressed as the ratio
EFFECT
0.0 I 0
I 15
$0
OF
PARATHYROID
I 60
HORMONE
IlO
300
Time (SC)
FIG. 1. Effect of PTH on the time course of PKA activation. Thin slices of rat renal cortex prepared as described under Materials and Methods were preincubated at 37’C for 15 min in the Krebs-Ringer bicarbonate buffer and then 20 min in the buffer containing 1 mM MIX. At the end of second incubation, PTH (2.5 X 10m7 M) was added to the slice incubation and the slices were exposed for the indicated time. After, the slices were quickly homogenized (in ice-cold 5 mM potassium phosphate buffer (pH 6.8) containing 2 mM EDTA, 0.5 mM MIX, 0.5 mM PMSF, and 150 mM sodium chloride) and the homogenate was centrifuged for 20 min at 20,OOOg at 4°C. The supernatant was used to determine the PKA activity measured using 2.5 pg protein incubated for 15 min, as described under Materials and Methods. Each datum represents mean SE + for three separate slice incubations.
ON
RAT
RENAL
PROTEIN
155
KINASES
f 1 pmol/min/mg protein in the cytosol and membrane fractions respectively. With PTH (250 nm for 30 sec)treated rat renal slices, the protein kinase basal activity in the cytosol and membrane fractions was 69 f 3 and 103 + 3 pmol/min/mg protein. The activity with Ca2+/ phospholipids was 132 + 4 and 262 f 3 pmol/min/mg protein for the cytosolic and membrane fractions, respectively. Thus, PTH decreased cytosolic PKC from 175 f 9 pmol/min/mg to 63 + 7 pmol/min/mg and increased membrane PKC from 68 + 2 pmol/min/mg to 159 -+ 6 pmol/min/mg. The time course of PKC activity induced by PTH is shown in Fig. 3. PKC was maximally activated in the membrane fraction and depressed in the cytosol fraction by PTH at 30 s. Activity returned toward basal levels in both the cytosolic and the membrane fractions by 4 min. Maximal activity ratio (PKC specific activity of membrane/specific activity of cytosol) of PKC occurred at 30 s with a return to basal levels by 4 min (Fig. 4). The doseresponse relationship for activation of PKC is shown in Fig. 5. The PKC activity ratio increased in a dose-dependent manner, with a significant increase at lOpa M. DISCUSSION
(-cAMP/+cAMP) of activity in the absence (-CAMP) and presence (+cAMP) of CAMP (14). PKA was maximally activated by 30 s and remained activated for at least 5 min. The dose-response curve for PKA activation is shown in Fig. 2. Maximal activation of PKA occurred between lo-’ and 10-s M and did not increase at higher concentrations of PTH. However, PTH produced a slight but significant increase even at a concentration of lo-l1
M.
Measurement
of Protein
Kinase
1.4 ,
I
C in Renal Tissue
The linear conditions for assay of PKC in renal tissue were established in the presence of Ca2+ and phospholipid. The phosphorylation of GS-peptide by the renal cytosol and solubilized membrane fractions was linear from O-5 pg of protein and O-20 min of time. Therefore, all subsequent PKC assays were performed at 2 pug of protein for 10 min at 3O“C. Enzyme activity was not stimulated by Ca2+ alone in the absence of phosphatidyl serine and diacylglycerol, which are specifically required for PKC activity. No measurable endogenous protein phosphorylation was detected in the absence of GS-peptide. Effect of PTH
These studies demonstrate that the tandem chromatographic column method can be usedto study the hormonal regulation of protein kinases in tissue samples. The method offers several advantages over the existing methods. First, since the method has a low radioactive background (15-20 cpm), it is very sensitive in detecting pro-
on PKC
The effect of PTH on renal PKC activity was studied using renal cortical slices. The protein kinase basal activity in control (no PTH) cytosol and membrane fractions was 80 + 3 and 83 f 1 pmol/min/mg protein and the activity with Ca2+/phospholipids was 255 f 6 and 151
0.0
I 0
Ib-11
lo-'0 ’
10-a PTH (M)
I
10-e
I
10-7
I
2.5x10-’
FIG. 2. The dose-response curve of PKA activation by PTH. Thin slices of rat renal cortex prepared as described in the legend to Fig. 1 were incubated at 37’C in Krebs-Ringer bicarbonate buffer. PKA activation was measured 5 min after exposure to PTH at different concentrations in the presence of 1 mM MIX as described under Materials and Methods. At the end of 5 min, the slices were quickly homogenized in the ice-cold homogenization buffer and centrifuged. The capacity of the supernate to phosphorylate Kemptide was determined as described under Materials and Methods. Each datum represents the mean of three separate slice incubations using 2.5 pg protein incubated for 15 min. There was significant stimulation by PTH at all concentrations. Standard error was smaller than symbols.
NEMANI,
WONGSURAWAT,
AND
ARMBRECHT
2.0
I
0
15
30
60
120
240
0
I 0
I 15
I 30
50
FIG. 3. Effect of PTH on the time course of renal PKC activity in cytosol and membranes. Thin slices of rat renal cortex prepared as described under Materials and Methods were preincubated at 37°C for 15 min in 1 ml of Krebs-Ringer bicarbonate buffer as described in the legend to Fig. 1. At the end of the second incubation, PTH (2.5 X 10-r M) was added to the slice incubation medium and the slices were exposed for the indicated time. At the end of the indicated time, the slices were quickly homogenized (in 20 mM Tris-HCl buffer (pH 7.5) containing 0.25 M sucrose, 1 mM EDTA, 0.5 mM PMSF, 2 mM EGTA, 0.1 mM DTT, 20 pg/ml leupeptin) and the homogenate was centrifuged for 30 min at 35,000g at 4°C. The supernatant was carefully removed. The pellet (particulate fraction) was homogenized in the original volume (1 ml) of homogenizing buffer containing 0.025% Triton X-100. After sonication, the suspension was left on ice for 1 h and then centrifuged as described above. The resulting supernatant was used as the membrane fraction. Just before assay, the cytosolic and membrane fractions were diluted with the homogenizing buffer. The enzyme activities were determined as described under Materials and Methods, using 2 pg of protein incubated for 10 min.
tein kinase activity. Using previous methods, it has been reported that PKC activities were undetectable in kidney (20), but with the present method we could detect significant activity in renal tissue. A second advantage is that only a small amount of protein (2-5 pg) is needed for the assay of PKA and PKC. The current methods require a high amount of protein (40-100 pg) (16, 21) to overcome the high background. This small protein sample requirement is especially useful when measuring the protein kinase activity of subcellular fractions or cultured cells. We have used this technique to measure protein kinase activity in rat adrenal glands, renal mitochondria, and cultured renal cortical cells (data not shown). A third advantage of this method is the selectivity gained by using synthetic peptides as specific substrates. In a recent study, Giembycz and Diamond (22) have shown that Kemptide is a suitable phosphate acceptor for the measurement of soluble PKA activity in bovine tracheal smooth muscle and in guinea pig lung-parenchyma. With Kemptide as the substrate for PKA, it was found that non-PKA activities accounted for ~5% of the total Kemptide phosphorylating activity (11, 22). In terms of hormonal stimulation, the tandem chromatographic column method with synthetic peptide substrate allowed a more sensitive characterization of the
1 240
120
Time (Set)
Time
(Sac)
Effect of PTH on PKC activity ratio. FIG. 4. of membrane to cytosol was plotted as function Fig. 3.
Ratio of specific activity of time using data from
effect of PTH on renal PKA compared to previous studies (14). In the present study, the unstimulated PKA ratio was 0.18 (Fig. 2). This compares with an unstimulated protein kinase ratio of 0.48 obtained with histone as a substrate and precipitation as the method of collection (14). Since PTH stimulated the PKA ratio to a maximum of about 1.0 regardless of method, use of the synthetic substrate resulted in a four- to fivefold increase in response to PTH compared to the onefold increase seen using histone. In terms of the dose response to PTH, both methods reported a large increase in PKA activity between 1 and 10 nM, although the fold increase was much larger when the synthetic substrate was used (Fig. 2). In addition, the use of the synthetic substrate combined with the columns detected a significant increase in PKA activity even at 0.01 nM PTH (Fig. 2).
LL ’
0
16”
16’O
10-a
10
PTH
-9
IO
-7
2.5X10-’
(M)
FIG. 5. The dose-response curve of PKC activity ratio. Thin rat renal cortex slices were prepared as described under Materials and Methods, incubated in Krebs-Ringer bicarbonate buffer at 37°C. PKC activities were measured 30 s after exposure to PTH at different concentrations. At the end of 30 s incubation, the slices were quickly homogenized in ice-cold buffer. PKC activities were determined in the supernatant from the tissue homogenate and soluble membrane fraction, as described under Materials and Methods. Each datum represents the mean + SE for three separate slice incubations. Significant stimulation occurred at PTH concentrations of 10-s M and greater.
EFFECT
OF
PARATHYROID
HORMONE
In terms of PKC activity, these techniques demonstrated that PTH clearly increases the PKC activity from cytosol to membrane in renal tissue (Fig. 4). Previously, this effect of PTH had only been demonstrated in cultured cell lines (6, 7). PTH treatment resulted in a threefold increase in the activity ratio (Fig. 4) and was effective at a concentration of 10 nM (Fig. 5). Similar results have been reported in renal cell lines (7). In contrast to PKA, which remained activated for at least 5 min (Fig. l), PKC peaked at 30 s and then declined rapidly (Fig. 4). These studies demonstrate that PTH activates PKC in renal tissue in addition to its well-known effect on PKA. The development of a sensitive assay for renal PKA and PKC activity will help to elucidate their specific roles in mediating the actions of PTH in the kidney. This will be of considerable interest in cases where the action of PTH in the kidney appears to be blunted, such as aging (23), diabetes (24), and hypophosphatemia (25). In these pathophysiological conditions, the effect of PTH on PKA and PKC in terms of dose and time response may be especially significant. ACKNOWLEDGMENTS This investigation was supported by USPHS Grant AM32158 (H.J.A.) and by the Medical Research Service of the Department of Veterans Affairs (N.W. and H.J.A.), and the American Federation for Aging Research, Inc., (R.N.). We thank Dr. R. Gopalakrishna, University of Southern California, and Drs. T. V. Zenser and M. L. Chen, St. Louis VA Medical Center, for helpful discussions. We also acknowledge Monica Boltz for skillful technical assistance, and Carol McCleary for typing the manuscript.
REFERENCES 1. De Luca,
H. F. (1988)
FASEB.
51 2, 224-236.
2. Lau, K., Goldfarb, S., and Goldberg, M. (1980) in Renal Handling of Phosphate (Massey, S. G., and Fleisch, H., Eds.), pp. 115-135, Plenum Medical Book Company, New York. 3. Armbrecht, B. B. (1984)
H. J., Wongsurawat, Amer.
J. Physiol.
246,
N., Zenser, E102-E107.
T.
V., and
Davis,
ON
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RENAL
4. Hammerman,
PROTEIN
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KINASES
M. R., and Hruska,
K. A. (1982)
J. Biol.
Chem.
257,
992-999.
5. Hruska, K. A., Moskowitz, S., and Huskey, M. (1987)
D., Esbrit, P., Civitelli, R., Westbrook, J. Clin. Inuest. 79, 230-239.
6. Abou-Samra, A-B., Jueppner, H., Westerberg, D., Potts, J. T., and Segre, G. V. (1989) Endocrinology 124, 1107m1113. T., Sakamoto, H., and Filburn, C. R. (1989) Biochem. Bio7. Tamura, phys. Res. Commun. 159, 1352-1358. 8. Cole, J. A., Eber, S. L., Poelling, R. E., Thorne, P. K., and Forte, L. R. (7987) Amer. J. Physiol. 258, E221-E227. S., and Tenenhouse, H. S. (1989) Biochim. Bio9. Boneh, A., Mandla, phys. Acta 1012,308-316. 10. Henry, H. L. (1986) Biochem. Biophys. 500. 11. Egan, J. J., Chang, M. K., and Londos, 175, 552-561. 12. Kemp, Acad.
13. House, Chem.
Res.
Commun.
C. (1988)
Anal.
139,
495-
Biochem.
B. E., Benjamini, E., and Krebs, E. G. (1976) Proc. Nutl. USA 73, 1038-1042. C., Wettenhall, R. E. H., and Kemp, B. E. (1987) J. Biol.
Sci.
262,
772-777.
14. Armbrecht, H. J., Boltz, M. A., and Forte, L. R. (1986) Exp. Gerontol. 2 1, 515-522. 15. Siegel, N., Wongsurawat, N., and Armbrecht, H. J. (1986) J. Biol. Chem. 261, 16,998-17,003. 16. Ghazarian, J. G., and Yanda, D. M. (1985) Biochem. Biophys. Res. Commun. 132, 10951102. 17. Kitano, T., Go, M., Kikkawa, U., and Nishizuka, Y. (1986) in Methods in Enzymology (P. M. Conn, Ed.), Vol. 124, pp. 349-352, Academic Press, San Diego. 18. Bradford, M. (1976) Anal. Biochem. 72, 248-252. 19. Dixon, W. J., and Massey, F. J. (1969) Introduction to Statistical Analysis, McGraw-Hill, New York. 20. Hashimoto, Y., and Soderling, T. (1987) Arch. Biochem. Biophys. 252, 418-425. 21. Witt, J. d., and Roskoski, R. (1975) Anal. Biochem. 66, 253-258. 22. Giembycz, M. A., and Diamond, J. (1990) Biochem. Pharmacol. 39, 271-283. 23. Armbrecht, H. J., Wongsurawat, N., Zenser, T. V., and Davis, B. B. (1982) Endocrinology 111, 1339-1334. 24. Wongsurawat, N., and Armbrecht, H. J. (1985) Acta Endocrinologica 109, 243-248. 25. Nesbitt, ‘I’., Drezner, M. K., and Lobaugh, B. (1986) J. Clin. Inuest. 77, 181-187.
Vol.
OF BIOCHEMISTRY
285, No. 1, February
AND
BIOPHYSICS
15, pp. 153-157,1991
Effect of Parathyroid Hormone on Rat Renal CAMPDependent Protein Kinase and Protein Kinase C Activity Measured Using Synthetic Peptide Substrates Ramakrishna
Nemani,‘**Tt
N irandon
Wongsurawat,t
and H. James
ArmbrechtfT$
Geriatric Research, Education, and Clinical Center, VA Medical Center, St. Louis, Missouri 63125; and Departments *Pharmacology, tlnternal Medicine, and $Biochemistry, St. Louis University School of Medicine, St. Louis, Missouri
Received
July
3, 1990, and in revised
form
September
27, 1990
The actions of parathyroid hormone (PTH) on the renal cortex are thought to be mediated primarily by CAMPdependent protein kinase (PKA) with some suggestion of a role for protein kinase C (PKC). However, present methods for assaying PKA and PKC in subcellular fractions are insensitive and require large amounts of protein. Recently, a sensitive method for measuring the activity of protein kinases has been reported. This method uses synthetic peptides as substrates and a tandem chromatographic procedure for isolating the phosphorylated peptides. We have adapted this method to study the effect of PTH on PKA and PKC activity using thin slices of rat renal cortex. PTH (250 nM) stimulated cytosolic PKA activity four- to fivefold within 30 s, and PKA activity was sustained for at least 5 min. PTH also rapidly stimulated PKC activity in the membrane fraction and decreased PKC activity in the cytosol. These changes were maximal at 30 s, but unlike changes in PKA, they declined rapidly thereafter. PTH significantly activated PKC only at concentrations of 10 nM or greater. This study demonstrates that PTH does activate PKC in renal tissue, although the duration of activation is much less than for PKA. It also demonstrates that a combination of synthetic peptides with tandem chromatography can be used as a sensitive assay procedure for protein kinase activity in biological Sampk?S. 0 1991 Academic PRESS, hc.
Parathyroid hormone (PTH)’ has numerous biological actions in the renal cortex. Two important physiological 1 To whom correspondence and requests for reprints should be addressed at Geriatric Center (lllG-JB), St. Louis VA Hospital, St. Louis, MO 63125. FAX: (314) 894-6614. * Abbreviations used: Mes: 2(N-morpholino)ethanesulfonic acid; PMSF, phenylmethylsulfonyl fluoride; EGTA, ethylene glycol bis(Paminoethylether)N,N’-tetracetic acid, PKA, CAMP-dependent protein kinase; PKC, protein kinase C; DTT, dithiothreitol; MIX, 1-methyl-3isobutylxanthine. 0003-9661/91$3.00
Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
of 63104
effects of PTH are to increase the production of 1,25dihydroxyvitamin D (l), the biologically active form of vitamin D, and to inhibit phosphate transport (2). PTH is known to increase CAMP production and CAMP-dependent protein kinase (PKA) activity in the renal cortex, and this pathway is thought to be responsible for most of the effects of PTH on vitamin D metabolism and phosphate transport (3, 4). Recently, there has been increasing evidence that PTH may also stimulate protein kinase C (PKC) in addition to PKA. PTH is known to stimulate Ca2+ influx and inositol phosphate turnover in the kidney (5). It has been reported that PTH causesa translocation of PKC activity in rat osteosarcoma cells (6) and cultured renal cells (7). The physiological significance of PKC activation is not clear. However, PKC, in addition to PKA, may play a role in regulation of renal phosphate transport (8,9) and 1,2Sdihydroxyvitamin D production (10). To assessthe role of PKA and PKC in mediating the actions of PTH, a simple and sensitive method for measuring protein kinase activity in renal tissue is needed. Unfortunately, the present methods for assaying protein kinase activities in tissue samples have high backgrounds and require large amounts of protein sample. Recently, a tandem chromatographic column method for measuring protein kinase activity has been described (11). This method, which uses synthetic peptides as substrates, is very sensitive, but it has only been used to measure the activity of purified enzyme. We report here the application of the tandem chromatographic column method, using synthetic peptides as substrates, to measure PKA and PKC in renal tissue. Using isolated renal slices, we demonstrate that PTH treatment results in the rapid stimulation of PKC activity in the membrane fraction and decreased PKC activity in the cytosol. We also compare the effect of PTH on renal PKA and PKC activity in terms of dose and time course. 153
154 MATERIALS
NEMANI.
AND
WONGSURAWAT.
METHODS
Materials. ATP, CAMP, CM-Sephadex cation exchange resin (bead size 40-120 pm (CZS-120)), Dowex AGl-X8 anion exchange resin (loo200 mesh), DTT, phosphatidylserine, and 1,2-diolein were obtained from Sigma Chemical Co. (St. Louis, MO). [y-32P]ATP (25 Ci/mmol) was purchased from International Chemical and Nuclear Corp. (Irvine, CA). The PTH used in these experiments was synthetic rat PTH (l-34) (5100 U/mg) purchased from Bachem Inc. (Torrance, CA). Kemptide, a synthetic heptapeptide substrate for A-kinase, H,N-LeuArg-Arg-Ala-Ser-Leu-Gly-COOH (12), was obtained from Sigma Chemical Co. (St. Louis, MO). GS-peptide, a synthetic dodecapeptide of glycogen synthase which is a substrate for the protein kinase C, HzNPro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala-Lys-Lys-COOH (13), was purchased from the Department of Medicine, Repatriation General Hospital, West Heidelberg, Vie 3081, Australia, as previously described (11). Animals. Male Fisher 344 rats aged 6-8 weeks were purchased from Harlan Industries (Indianapolis, IN). Rats were fed Purina rodent laboratory chow. The effect of PTH on protein kinase activity Tissue preparation. was measured using renal slices (14). Animals were killed by decapitation. The excised kidneys were decapsulated and washed in cold saline, and renal cortical slices were prepared as described elsewhere (15). Thin slices of renal cortex were preincubated for 15 min at 37°C in 1 ml of Krebs-Ringer bicarbonate buffer and then 20 min in buffer containing 1 mM 1-methyl-3-isobutylxanthine (MIX), a cyclic nucleotide phosphodiesterase inhibitor. At the end of the second incubation, PTH was added to the slice incubation medium for the indicated time, Slices were then quickly homogenized in the appropriate ice-cold buffer, and the homogenate was centrifuged as indicated to obtain a supernate and/or membrane fraction as needed. For the Measurement of CAMP-dependent protein kinase activity. measurement of PKA activity, slices were homogenized in 1 ml of icecold 5 mM potassium phosphate buffer (pH 6.8) containing 2 mM EDTA, 0.5 mM MIX, 0.5 mM PMSF, and 150 mM sodium chloride. Tissue was homogenized for six strokes using a glass-teflon Potter-Elvehjem homogenizer at 600 rpm (14). The homogenate was centrifuged at 20,OOOg for 20 min, and the capacity of the supernate to phosphorylate Kemptide was determined in the presence and absence of CAMP according to the method of Ghazarian and Yanda (16) at 30°C. Kemptide is a specific substrate for PKA under these conditions (12). The reaction mixture consisted of Kemptide (30 pg), 0.05 mM CAMP, 0.100 mM ATP, 0.5 PCi [y-32P]ATP, 0.3 mM EDTA, 1 mM magnesium acetate, 4.8 mM theophylline, 20 mM Mes, and supernate sample all in a final volume of 160 pl at pH 6.2. Assays were performed in 13 X loo-mm glass test tubes. The assay was run for various times as indicated, and it was terminated by adding 4 ml of a 2.5 mM ATP solution, pH 6.8. The reaction mixture was processed as described below. Measurement of protein kinase C actiuity. For measurement of PKC activity, slices were homogenized in 1 ml of 20 mM Tris-HCl, 0.25 M sucrose, 1 mM EDTA, 0.5 mM PMSF, 2 mM EGTA, 0.1 mM DTT, 20 gg/ml leupeptin, pH 7.5, as described by Tamura et al. (7). The homogenate was centrifuged at 35,000g for 30 min, and the supernatant was used as the cytosolic fraction. The pellet was resuspended in the original volume of (1 ml) homogenizing buffer, and Triton X-100 was added to a final concentration of 0.025%. After sonication (4 X 1 min on ice with Tekamar Sonic Disruptor), the suspension was left on ice for 1 h and then centrifuged at 35,000g for 30 min. The resulting supernatant was used as the membrane fraction. The cytosolic and membrane fractions were diluted appropriately with the homogenizing buffer just before assay. Protein kinase C was assayed according to the method of Nishizuka (17) using the GS-peptide as a substrate. The GS-peptide is a specific substrate for PKC under these conditions (13). The reaction mixture (160 ~1) contained 20 mM Tris-HCl at pH 7.5,5 mM magnesium acetate, 0.100 mM ATP with radiolabeled ATP, 10 fig GS-peptide, 0.1 mM CaCl,, 2 fig phosphatidylserine, 0.2 pg of diolein, and enzyme fraction. Phos-
AND
ARMBRECHT
phatidylserine and diolein were mixed in small volumes of chloroform and dried under a stream of nitrogen. The residue was then sonicated in 20 mM Tris, pH 7.5, and added to the reaction mixture as lipid micelles. Basal activity was measured in the presence of 0.5 mM EGTA, and CaCl,, phosphatidylserine, and diolein were omitted from the reaction mixture. Basal activity was subtracted from the Ca’+/phospholipidstimulated activity, and the difference was taken as protein kinase C activity. The assay was run for various times as indicated, and it was terminated by adding 4 ml of a 2.5 mM ATP solution, pH 6.8. The reaction mixture was then processed as described below. Processing of reaction mixture. The phosphorylated peptides were separated from the reaction mixture according to the method of Egan et al. (11). The radiolabeled Kemptide or GS-peptide was recovered from the reaction mixture by sequential processing of the reaction mixture through tandem chromatographic columns of cation and anion exchange resins. Briefly, the reaction mixture was poured onto a CMSephadex column (l-ml bed volume), and the test tubes were allowed to drain for a few minutes. The columns were then washed with a solution of 2.5 mM ATP, pH 6.8. After the liquid had drained from the CMSephadex resin, the columns were mounted over the Dowex AGl-X8 columns (l-ml bed volume). Eight milliliter, of 30% acetic acid was applied to the CM-Sephadex columns, and the total eluates from the AGl-X8 columns were collected in scintillation vials. The amount of radioactivity present was determined by liquid scintillation counting. Protein was determined by the method of Protein determinations. Bradford (18). Bovine serum albumin was used as a standard. Statistics. The data from these experiments are reported as the means of f standard error (SE) of the indicated number of measurements. Statistical analyses were performed using Student’s two-tailed t test (19), and a confidence level of 95% or greater was considered significant.
RESULTS
Measurement of CAMP-Dependent Protein Kinase in Renal Cytosol. The linear conditions for assay of PKA in renal cytosol were established in the presence of CAMP. With regard to protein concentration, phosphorylation of Kemptide was linear from O-10 pg of protein using a 15min timepoint. With regard to time, phosphorylation of Kemptide was linear from O-30 min at a protein concentration of 2.5 pg. Therefore, all subsequent assays were performed using 2.5 fig of protein for 15 min at 30°C. No measurable endogenous protein phosphorylation was detected in the absence of Kemptide. Effect of PTH on PKA The effect of PTH on renal PKA was studied using renal cortical slices. The protein kinase activity in control (no PTH) was 148 f 4 and 651 k 22 pmol/min/mg protein without and with CAMP, respectively. The protein kinase activity in slices treated with PTH (250 nM for 30 s) was 511+ 14 and 517 ? 48 pmol/min/mg protein without and with CAMP, respectively. When PKA inhibitor (synthetic peptide of rabbit skeletal muscle) was added to the incubation, the CAMP-dependent protein kinase activity was reduced to the basal level. The time course of PKA activation induced by PTH is shown in Fig. 1. PKA activity was expressed as the ratio
EFFECT
0.0 I 0
I 15
$0
OF
PARATHYROID
I 60
HORMONE
IlO
300
Time (SC)
FIG. 1. Effect of PTH on the time course of PKA activation. Thin slices of rat renal cortex prepared as described under Materials and Methods were preincubated at 37’C for 15 min in the Krebs-Ringer bicarbonate buffer and then 20 min in the buffer containing 1 mM MIX. At the end of second incubation, PTH (2.5 X 10m7 M) was added to the slice incubation and the slices were exposed for the indicated time. After, the slices were quickly homogenized (in ice-cold 5 mM potassium phosphate buffer (pH 6.8) containing 2 mM EDTA, 0.5 mM MIX, 0.5 mM PMSF, and 150 mM sodium chloride) and the homogenate was centrifuged for 20 min at 20,OOOg at 4°C. The supernatant was used to determine the PKA activity measured using 2.5 pg protein incubated for 15 min, as described under Materials and Methods. Each datum represents mean SE + for three separate slice incubations.
ON
RAT
RENAL
PROTEIN
155
KINASES
f 1 pmol/min/mg protein in the cytosol and membrane fractions respectively. With PTH (250 nm for 30 sec)treated rat renal slices, the protein kinase basal activity in the cytosol and membrane fractions was 69 f 3 and 103 + 3 pmol/min/mg protein. The activity with Ca2+/ phospholipids was 132 + 4 and 262 f 3 pmol/min/mg protein for the cytosolic and membrane fractions, respectively. Thus, PTH decreased cytosolic PKC from 175 f 9 pmol/min/mg to 63 + 7 pmol/min/mg and increased membrane PKC from 68 + 2 pmol/min/mg to 159 -+ 6 pmol/min/mg. The time course of PKC activity induced by PTH is shown in Fig. 3. PKC was maximally activated in the membrane fraction and depressed in the cytosol fraction by PTH at 30 s. Activity returned toward basal levels in both the cytosolic and the membrane fractions by 4 min. Maximal activity ratio (PKC specific activity of membrane/specific activity of cytosol) of PKC occurred at 30 s with a return to basal levels by 4 min (Fig. 4). The doseresponse relationship for activation of PKC is shown in Fig. 5. The PKC activity ratio increased in a dose-dependent manner, with a significant increase at lOpa M. DISCUSSION
(-cAMP/+cAMP) of activity in the absence (-CAMP) and presence (+cAMP) of CAMP (14). PKA was maximally activated by 30 s and remained activated for at least 5 min. The dose-response curve for PKA activation is shown in Fig. 2. Maximal activation of PKA occurred between lo-’ and 10-s M and did not increase at higher concentrations of PTH. However, PTH produced a slight but significant increase even at a concentration of lo-l1
M.
Measurement
of Protein
Kinase
1.4 ,
I
C in Renal Tissue
The linear conditions for assay of PKC in renal tissue were established in the presence of Ca2+ and phospholipid. The phosphorylation of GS-peptide by the renal cytosol and solubilized membrane fractions was linear from O-5 pg of protein and O-20 min of time. Therefore, all subsequent PKC assays were performed at 2 pug of protein for 10 min at 3O“C. Enzyme activity was not stimulated by Ca2+ alone in the absence of phosphatidyl serine and diacylglycerol, which are specifically required for PKC activity. No measurable endogenous protein phosphorylation was detected in the absence of GS-peptide. Effect of PTH
These studies demonstrate that the tandem chromatographic column method can be usedto study the hormonal regulation of protein kinases in tissue samples. The method offers several advantages over the existing methods. First, since the method has a low radioactive background (15-20 cpm), it is very sensitive in detecting pro-
on PKC
The effect of PTH on renal PKC activity was studied using renal cortical slices. The protein kinase basal activity in control (no PTH) cytosol and membrane fractions was 80 + 3 and 83 f 1 pmol/min/mg protein and the activity with Ca2+/phospholipids was 255 f 6 and 151
0.0
I 0
Ib-11
lo-'0 ’
10-a PTH (M)
I
10-e
I
10-7
I
2.5x10-’
FIG. 2. The dose-response curve of PKA activation by PTH. Thin slices of rat renal cortex prepared as described in the legend to Fig. 1 were incubated at 37’C in Krebs-Ringer bicarbonate buffer. PKA activation was measured 5 min after exposure to PTH at different concentrations in the presence of 1 mM MIX as described under Materials and Methods. At the end of 5 min, the slices were quickly homogenized in the ice-cold homogenization buffer and centrifuged. The capacity of the supernate to phosphorylate Kemptide was determined as described under Materials and Methods. Each datum represents the mean of three separate slice incubations using 2.5 pg protein incubated for 15 min. There was significant stimulation by PTH at all concentrations. Standard error was smaller than symbols.
NEMANI,
WONGSURAWAT,
AND
ARMBRECHT
2.0
I
0
15
30
60
120
240
0
I 0
I 15
I 30
50
FIG. 3. Effect of PTH on the time course of renal PKC activity in cytosol and membranes. Thin slices of rat renal cortex prepared as described under Materials and Methods were preincubated at 37°C for 15 min in 1 ml of Krebs-Ringer bicarbonate buffer as described in the legend to Fig. 1. At the end of the second incubation, PTH (2.5 X 10-r M) was added to the slice incubation medium and the slices were exposed for the indicated time. At the end of the indicated time, the slices were quickly homogenized (in 20 mM Tris-HCl buffer (pH 7.5) containing 0.25 M sucrose, 1 mM EDTA, 0.5 mM PMSF, 2 mM EGTA, 0.1 mM DTT, 20 pg/ml leupeptin) and the homogenate was centrifuged for 30 min at 35,000g at 4°C. The supernatant was carefully removed. The pellet (particulate fraction) was homogenized in the original volume (1 ml) of homogenizing buffer containing 0.025% Triton X-100. After sonication, the suspension was left on ice for 1 h and then centrifuged as described above. The resulting supernatant was used as the membrane fraction. Just before assay, the cytosolic and membrane fractions were diluted with the homogenizing buffer. The enzyme activities were determined as described under Materials and Methods, using 2 pg of protein incubated for 10 min.
tein kinase activity. Using previous methods, it has been reported that PKC activities were undetectable in kidney (20), but with the present method we could detect significant activity in renal tissue. A second advantage is that only a small amount of protein (2-5 pg) is needed for the assay of PKA and PKC. The current methods require a high amount of protein (40-100 pg) (16, 21) to overcome the high background. This small protein sample requirement is especially useful when measuring the protein kinase activity of subcellular fractions or cultured cells. We have used this technique to measure protein kinase activity in rat adrenal glands, renal mitochondria, and cultured renal cortical cells (data not shown). A third advantage of this method is the selectivity gained by using synthetic peptides as specific substrates. In a recent study, Giembycz and Diamond (22) have shown that Kemptide is a suitable phosphate acceptor for the measurement of soluble PKA activity in bovine tracheal smooth muscle and in guinea pig lung-parenchyma. With Kemptide as the substrate for PKA, it was found that non-PKA activities accounted for ~5% of the total Kemptide phosphorylating activity (11, 22). In terms of hormonal stimulation, the tandem chromatographic column method with synthetic peptide substrate allowed a more sensitive characterization of the
1 240
120
Time (Set)
Time
(Sac)
Effect of PTH on PKC activity ratio. FIG. 4. of membrane to cytosol was plotted as function Fig. 3.
Ratio of specific activity of time using data from
effect of PTH on renal PKA compared to previous studies (14). In the present study, the unstimulated PKA ratio was 0.18 (Fig. 2). This compares with an unstimulated protein kinase ratio of 0.48 obtained with histone as a substrate and precipitation as the method of collection (14). Since PTH stimulated the PKA ratio to a maximum of about 1.0 regardless of method, use of the synthetic substrate resulted in a four- to fivefold increase in response to PTH compared to the onefold increase seen using histone. In terms of the dose response to PTH, both methods reported a large increase in PKA activity between 1 and 10 nM, although the fold increase was much larger when the synthetic substrate was used (Fig. 2). In addition, the use of the synthetic substrate combined with the columns detected a significant increase in PKA activity even at 0.01 nM PTH (Fig. 2).
LL ’
0
16”
16’O
10-a
10
PTH
-9
IO
-7
2.5X10-’
(M)
FIG. 5. The dose-response curve of PKC activity ratio. Thin rat renal cortex slices were prepared as described under Materials and Methods, incubated in Krebs-Ringer bicarbonate buffer at 37°C. PKC activities were measured 30 s after exposure to PTH at different concentrations. At the end of 30 s incubation, the slices were quickly homogenized in ice-cold buffer. PKC activities were determined in the supernatant from the tissue homogenate and soluble membrane fraction, as described under Materials and Methods. Each datum represents the mean + SE for three separate slice incubations. Significant stimulation occurred at PTH concentrations of 10-s M and greater.
EFFECT
OF
PARATHYROID
HORMONE
In terms of PKC activity, these techniques demonstrated that PTH clearly increases the PKC activity from cytosol to membrane in renal tissue (Fig. 4). Previously, this effect of PTH had only been demonstrated in cultured cell lines (6, 7). PTH treatment resulted in a threefold increase in the activity ratio (Fig. 4) and was effective at a concentration of 10 nM (Fig. 5). Similar results have been reported in renal cell lines (7). In contrast to PKA, which remained activated for at least 5 min (Fig. l), PKC peaked at 30 s and then declined rapidly (Fig. 4). These studies demonstrate that PTH activates PKC in renal tissue in addition to its well-known effect on PKA. The development of a sensitive assay for renal PKA and PKC activity will help to elucidate their specific roles in mediating the actions of PTH in the kidney. This will be of considerable interest in cases where the action of PTH in the kidney appears to be blunted, such as aging (23), diabetes (24), and hypophosphatemia (25). In these pathophysiological conditions, the effect of PTH on PKA and PKC in terms of dose and time response may be especially significant. ACKNOWLEDGMENTS This investigation was supported by USPHS Grant AM32158 (H.J.A.) and by the Medical Research Service of the Department of Veterans Affairs (N.W. and H.J.A.), and the American Federation for Aging Research, Inc., (R.N.). We thank Dr. R. Gopalakrishna, University of Southern California, and Drs. T. V. Zenser and M. L. Chen, St. Louis VA Medical Center, for helpful discussions. We also acknowledge Monica Boltz for skillful technical assistance, and Carol McCleary for typing the manuscript.
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