Zurich Open Repository and Archive. The opossum kidney cell type IIa Na/P(i) cotransporter is a phosphoprotein

Transcription

1 University of Zurich Zurich Open Repository and Archive Winterthurerstr. 190 CH-8057 Zurich Year: 2001 The opossum kidney cell type IIa Na/P(i) cotransporter is a phosphoprotein Jankowski, M; Hilfiker, H; Biber, J; Murer, H Jankowski, M; Hilfiker, H; Biber, J; Murer, H. The opossum kidney cell type IIa Na/P(i) cotransporter is a phosphoprotein. Kidney Blood Press. Res. 2001, 24(1):1-4. Postprint available at: Posted at the Zurich Open Repository and Archive, University of Zurich. Originally published at: Kidney Blood Press. Res. 2001, 24(1):1-4.

2 The opossum kidney cell type IIa Na/P(i) cotransporter is a phosphoprotein Abstract BACKGROUND/AIM: Parathyroid hormone (PTH)-dependent inhibition of proximal tubular P(i) reabsorption is mediated by protein kinase A and/or C and is associated with reduced border membrane expression of the type IIa Na/P(i) cotransporter. The aim of this study was to analyze phosphorylation of the type IIa cotransporter protein. METHODS: Opossum kidney cells were used as a 'proximal tubular' cell model. Protein phosphorylation was determined by immunoprecipitation of the type IIa Na/P(i) cotransporter, followed by autoradiography. The transporter protein content was evaluated by Western blotting and transport activity by tracer P(i) uptake. RESULTS: Under control conditions (no PTH) the transporter was phosphorylated; upon treatment with PTH, a decrease in phosphorylation was observed. A protein phosphatase inhibitor (okadaic acid) was unable to prevent PTH-induced Na/P(i) cotransporter inhibition but reduced transporter degradation. CONCLUSION: The type IIa Na/P(i) cotransporter is a phosphoprotein, but alterations in its phosphorylation seem not to be involved in P(i) transport inhibition.

3 Original Paper Kidney Blood Press Res 2001;24:1 4 Accepted: August 11, 2000 The Opossum Kidney Cell Type IIa Na/P i Cotransporter Is a Phosphoprotein Maciej Jankowski Helene Hilfiker Jürg Biber Heini Murer Institute of Physiology, University of Zurich, Zurich, Switzerland Key Words Type IIa Na/P i Cotransporter W Phosphoprotein W Phosphorylation Abstract Background/Aim: Parathyroid hormone (PTH)-dependent inhibition of proximal tubular P i reabsorption is mediated by protein kinase A and/or C and is associated with reduced border membrane expression of the type IIa Na/P i cotransporter. The aim of this study was to analyze phosphorylation of the type IIa cotransporter protein. Methods: Opossum kidney cells were used as a proximal tubular cell model. Protein phosphorylation was determined by immunoprecipitation of the type IIa Na/P i cotransporter, followed by autoradiography. The transporter protein content was evaluated by Western blotting and transport activity by tracer P i uptake. Results: Under control conditions (no PTH) the transporter was phosphorylated; upon treatment with PTH, a decrease in phosphorylation was observed. A protein phosphatase inhibitor (okadaic acid) was unable to prevent PTH-induced Na/P i cotransporter inhibition but reduced transporter degradation. Conclusion: The type IIa Na/P i cotransporter is a phosphoprotein, but alterations in its phosphorylation seem not to be involved in P i transport inhibition. Copyright 2001 S. Karger AG, Basel Introduction Physiological alterations in renal P i handling are associated with changes in brush border expression of a type IIa Na/P i cotransporter [see ref. 1 for a review]. For example, parathyroid hormone (PTH) decreases the brush border content of this transport protein by its specific membrane retrieval and subsequent lysosomal degradation [2]. Opossum kidney (OK) cells can be used to characterize PTH effects on P i transport function and on type IIa Na/ P i cotransporter protein content/expression; protein kinases C and A are involved in these regulations and lead to Na/P i cotransport inhibition, membrane retrieval and lysosomal degradation of the transporter protein [1, 3 7]. In studies on the type IIa transporter expressed in oocytes, kinase C-induced membrane retrieval has been observed [8]; however, this was independent of known protein kinase C and A consensus sites [9]. In a recent study on isolated perfused mice proximal tubules, we were also able to document the participation of protein kinases A and C in PTH-induced brush border membrane retrieval of the cotransporter [10]. The target proteins of PTH-activated protein kinase A and/or C have not been defined; PTH treatment of OK cells was shown to induce phosphorylation of proteins present in an enriched apical membrane fraction [7]. ABC Fax karger@karger.ch S. Karger AG, Basel /01/ $17.50/0 Accessible online at: Heini Murer Institute of Physiology, University of Zurich Winterthurerstrasse 190 CH 8057 Zurich (Switzerland) Tel , Fax

4 Fig. 1. Phosphorylation of the type IIa Na/P i cotransporter protein. Cells were metabolically labeled with [ 32 P]-orthophosphate in the presence of leupeptin and treated with PTH (10 8 M ) for 5 and 30 min. Immunoprecipitation and further analysis of the cotransporter protein (Western blot, autoradiography) was performed as described in Methods. Quantification of phosphorylation by phosphoimager counting indicated dephosphorylation after PTH treatment (5 and 30 min); quantification of autoradiographs and corresponding Western blots (Image quant software) resulted in an approximately 4-fold decrease in phosphorylation for the 30 min PTH exposure. Similar data were obtained in two additional experiments. Ca 2+ - and camp-dependent protein phosphorylationhas been observed in studies using isolated renal brush border membranes [11 13]. However, phosphorylation of the type IIa Na/P i cotransporter protein has not been reported; such modification of the transporter may be involved in its PTH-induced inhibition, internalization and lysosomal degradation. In the present study, the OK cell type IIa Na/P i cotransporter was found to be phosphorylated in the absence of PTH; upon treatment with the hormone, dephosphorylation was observed. A phosphatase inhibitor did not prevent Na/P i cotransporter inhibition, but did reduce its PTH-induced degradation. Thus, altered phosphorylation of the cotransporter may not be involved in P i transport inhibition. Methods OK cells were grown in 35-mm plastic dishes, and P i transport ( 32 P i ) was measured as previously described [4 7]. Confluent OK cells (3 dishes per experimental condition) were washed three times and then incubated in 1 ml of phosphate-free DMEM/Ham s F12, 22 mm NaHCO 3, 20 mm HEPES and 5% CO 2 (ph 7.4) for 30 min (at 37 C) in the presence of the lysosomal inhibitor leupeptin (100 Ìg/ml); after addition of 32 P i (0.25 mci/ml), the cells were kept for 2 h at 37 C and PTH (10 8 M ) was added during this time period as indicated. Subsequently, the cells were washed four times with ice-cold Tris-buffered saline and harvested by scraping in lysis buffer [RIPA: 150 mm NaCl, 5 mm EDTA, 50 mm Tris, 1% Triton X-100, 0.5% sodium deoxycholate, 1% SDS and 1 mm phenylmethyl sulfonylfluoride (PMSF)] containing also 1 mm okadaic acid (Calbiochem) and a 1/100 diluted protease inhibitor mix (Sigma) and 10 mm sodium pyrophosphate, 50 mm NaF, 0.2 mm sodium vanadate. After 30 min at 4 C, the suspension was centrifuged for 5 min at 10,000 g. Protein A-Sepharose (50 Ìl; 50% v/v in RIPA buffer) was used for preabsorption (30 min at 4 C) and then collected by centrifugation for 5 min at 10,000 g. The type IIa Na/P i cotransporter was immunoprecipitated from the supernatants by adding a transporter-specific antibody [5, 6] bound to protein A- Sepharose (40 Ìl, 50% v/v) and incubating for 2 h at 4 C. Finally, the protein A-Sepharose beads were collected by centrifugation and washed four times with 500 Ìl of RIPA buffer. Twenty microliters of 2! electrophoresis loading buffer were added to the final pellets to dissociate the proteins from the protein A-Sepharose (at 95 C). After centrifugation, proteins in the supernatant were analyzed by SDS- PAGE (9%) and by electrotransfer of the proteins onto nitrocellulose. To remove incorporated phosphate, the protein A-Sepharose beads were exposed for 90 min at 37 C to 50 Ìl of buffer [50 mm Tris-HCl, ph 8.5, 2 mm PMSF, 8 mm MgCl 2, 0.1% ß-mercaptoethanol (v/v)] containing 20 units of calf intestinal alkaline phosphatase. After this procedure, proteins were separated by SDS-PAGE as indicated. Phosphorylated proteins were visualized by exposing the dried nitrocellulose membranes to Kodak X-Omat films at 70 C for various time periods. Immunodetection of the cotransporter protein (same nitrocellulose membrane as used for the 32 P autoradiography) was performed as previously described [5, 6]. Results and Discussion Protein kinase A- and/or C-mediated phosphorylation of the OK cell type IIa Na/P i cotransporter might be involved in PTH-induced P i transport inhibition and may also be the initial event in its subsequent internalization and degradation [5, 6] (see Introduction). To prevent PTH-induced degradation of the transporter [5, 6, 10], the present experiments on protein phosphorylation had to be performed in the presence of an inhibitor of lysosomal degradation (leupeptin). Furthermore, to detect the initial event, we exposed cells for only 5 or 30 min to PTH or vehicle alone (fig. 1). In previous studies [6], we have shown that the transporter is under control conditions (no PTH, no lysosomal inhibitor) mainly present at the apical cell surface; only in the presence of the inhibitor can it be detected in lysosomal compartments. For technical reasons (amount of 32 P i required to obtain sufficient labeling; see Methods), in the present study we were only able to use small amounts of 2 Kidney Blood Press Res 2001;24:1 4 Jankowski/Hilfiker/Biber/Murer

5 cell material per experimental condition, insufficient for cell fractionation experiments. We assume that a significant amount of the transporter protein recovered from the cell lysates was at the apical cell surface prior to addition of PTH [6]. Therefore, our data on protein phosphorylation should allow conclusions on the hypothetical role of phosphorylation in the control of apical expression and function of this transporter. In immunological detections, the cotransporter protein appeared in the immunoprecipitates as strong stainings at kd [5, 6]. In the autoradiographs, phosphorylation signals were obtained at the corresponding molecular weight ( kd; fig. 1). The phosphorylation could be removed by exposing the precipitated proteins to alkaline phosphatase treatment prior to SDS-PAGE and autoradiography (data not shown; see Methods). As phosphorylation was observed in the absence of PTH, it can be concluded that the cotransporter is phosphorylated under basal conditions. PTH did not induce an increase but rather a decrease in phosphorylation of the cotransporter after 5 or 30 min of exposure to the hormone (fig. 1). Phosphoimager counting and quantification of autoradiographs and Western blots (Image quant software) resulted in 3 experimental repetitions in dephosphorylations (2- to 4-fold) of the cotransporter in response to the above shortterm exposure to PTH. As PTH effects are mimicked by pharmacological activators or prevented by inhibitors of protein kinase A and/or C [5, 10], it seems likely that the observed PTH-independent phoshorylation is not a result of such kinase activation. Obviously, these kinases also cannot be directly involved in the observed dephosphorylations. The above-mentioned dephosphorylation might be explained by at least two separate processes. Firstly, under PTH treatment, a protein phosphatase activity can be activated. Secondly, PTH shifts the transporter to the lysosomes [2, 5, 6], where it can undergo dephosphorylation. The involvement of a specific dephosphorylation reaction in PTH-induced P i transport inhibition was studied by an analysis of the effect of inhibitors of protein phosphatase activities. We used okadaic acid (10 7 M), as a widely used inhibitor of protein phosphatases, in cells treated (data not shown) or not treated with leupeptin (fig. 2). Okadaic acid had no effect on Na/P i cotransport activity or its PTH-dependent inhibition in the presence (data not shown) and absence of leupeptin (fig. 2, upper panel). In contrast, in cells not treated with leupeptin, PTH-induced transporter degradation was found to be reduced in the presence of okadaic acid (fig. 2, lower panel; approximately 60 vs. 35%). We also used calyculin Fig. 2. Na/P i cotransport activity (upper panel) and expression of the type IIa NaP i cotransporter (lower panel) in the presence or absence of okadaic acid (OA): effect of PTH. Cells were incubated with PTH (10 8 M ) in the absence of leupeptin and with or without okadaic acid (10 7 M ) for 1 h. Then, Na/P i cotransport activity was measured and transport protein was determined by Western blotting. Transport activity in control and PTH-exposed cells was unaffected by the presence of okadaic acid during the 1-hour incubation in the presence or absence of the hormone. Quantification of the Western blots indicated an approximately 60% decrease in response to PTH, which was reduced to an approximately 35% decrease in the presence of okadaic acid. Similar data were obtained in two additional experiments. A (10 8 M) as proteinphosphatase inhibitor and observed similar results (data not shown). These data suggest that PTH-induced dephosphorylation does not participate in the mechanisms leading to transport inhibition. The (weak) effect of phosphatase inhibitors on the PTHinduced degradation of the transporter might involve specific or unspecific actions of the inhibitors at a stage beyond transport inhibition, i.e. membrane internalization and/or delivery to the lysosomes, including effects on the cytoskeleton. In conclusion, the OK cell type IIa Na/P i -cotransporter is a phosphoprotein, and PTH does not increase, but rather decreases its phosphorylation state. Thus, a phosphorylation/dephosphorylation step seems not to be involved in Type IIa Na/P i Cotransporter Is a Phosphoprotein Kidney Blood Press Res 2001;24:1 4 3

6 PTH-dependent control of the apical expression and transport activity of this transporter. The possibility has to be considered that PTH-induced phosphorylation might occur at the level of proteins interacting specifically with this transporter [14]. Acknowledgements We thank Ch. Gasser for assistance in preparing the figures and D. Neukom-Rossi and E. Müller for secretarial help. This work was supported by the Swiss National Science Foundation (grant to H.M.) and the International Society of Nephrology (fellowship for M.J.). We thank E. Lederer and M. Levi for NaP i -4-specific antibodies. References 1 Murer H, Biber J: A molecular view of proximal tubular inorganic phosphate (P i ) reabsorption and of its regulation. Pflügers Arch 1997; 433: Keusch I, Traebert M, Lötscher M, Kaissling B, Murer H, Biber J: Parathyroid hormone and dietary phosphate provoke a lysosomal routing of the proximal tubular Na/P i -cotransporter type II. Kidney Int 1998;54: Caverzasio J, Rizzoli R, Bonjour JP: Sodiumdependent phosphate transport inhibited by parathyroid hormone and cyclic AMP stimulation in an opossum kidney cell line. J Biol Chem 1986;261: Jankowski M, Biber J, Murer H: PTH-induced internalization of a type IIa Na/P i cotransporter in OK-cells. Pflügers Arch 1999;438: Pfister MF, Forgo J, Ziegler U, Biber J, Murer H: camp-dependent and -independent downregulation of type II Na-P i cotransporters by PTH. Am J Physiol 1999;276:F720 F Pfister MF, Ruf I, Stange G, Ziegler U, Lederer E, Biber J, Murer H: Parathyroid hormone leads to the lysosomal degradation of the renal type II Na/P i cotransporter. Proc Natl Acad Sci USA 1998;95: Reshkin SJ, Wuarin F, Biber J, Murer H: Parathyroid hormone-induced alteration of protein content and phosphorylation in enriched apical membranes of opossum kidney cells. J Biol Chem 1990;265: Forster IC, Traebert M, Jankowski M, Stange G, Biber J, Murer H: Protein kinase C activators induce membrane retrieval of type II Na + - phosphate cotransporters expressed in Xenopus oocytes. J Physiol (Lond) 1999;517: Hayes G, Busch AE, Lang F, Biber J, Murer H: Protein kinase C consensus sites and the regulation of renal Na/P i -cotransport (NaPi-2) expressed in Xenopus laevis oocytes. Pflügers Arch 1995;430: Traebert M, Volkl H, Biber J, Murer H, Kaissling B: Luminal and contraluminal action of 1-34 and 3-34 PTH peptides on renal type IIa Na/P i -cotransporter. Am J Physiol Renal Physiol 2000;278:F792 F Hammerman MR, Hruska KA: Cyclic AMPdependent protein phosphorylation in canine renal brush-border membrane vesicles is associated with decreased phosphate transport. J Biol Chem 1982;257: Malmstrom K, Murer H: Ca 2+ -dependent protein phosphorylation in brush border membranes of rat kidney proximal tubules. Pflügers Arch 1985;404: Takenawa T, Wada E, Tsumita T, Masaki T, Filburn CR, Sacktor B: Effect of parathyroid hormone, cyclic AMP and Ca 2+ on the phosphorylation of brush border membranes in rabbit kidney. Miner Electrolyte Metab 1984;10: Gisler S, Stagliar I, Biber J, Murer H: Type IIa Na/P i -cotransporter associated proteins: A two hybrid approach. J Am Soc Nephrol 1999;10: A Kidney Blood Press Res 2001;24:1 4 Jankowski/Hilfiker/Biber/Murer