A Mechanism Regulating Proteolysis of Specific Proteins during Renal Tubular Cell Growth*

Harold A. FranchDagger §, Sira Sooparb, Jie DuDagger With the technical assistance of Li Ling Shen, and Nikia S. Brown

From the  Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia and the Dagger  Atlanta Veterans Affairs Medical Center, Decatur, Georgia 30033

Received for publication, February 26, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Growth factors suppress the degradation of cellular proteins in lysosomes in renal epithelial cells. Whether this process also involves specific classes of proteins that influence growth processes is unknown. We investigated chaperone-mediated autophagy, a lysosomal import pathway that depends on the 73-kDa heat shock cognate protein and allows the degradation of proteins containing a specific lysosomal import consensus sequence (KFERQ motif). Epidermal growth factor (EGF) or ammonia, but not transforming growth factor beta 1, suppresses total protein breakdown in cultured NRK-52E renal epithelial cells. EGF or ammonia prolonged the half-life of glyceraldehyde-3-phosphate dehydrogenase, a classic substrate for chaperone-mediated autophagy, by more than 90%, whereas transforming growth factor beta 1 did not. EGF caused a similar increase in the half-life of the KFERQ-containing paired box-related transcription factor, Pax2. The increase in half-life was accompanied by an increased accumulation of proteins with a KFERQ motif including glyceraldehyde-3-phosphate dehydrogenase and Pax2. Ammonia also increased the level of the Pax2 protein. Lysosomal import of KFERQ proteins depends on the abundance of the 96-kDa lysosomal glycoprotein protein (lgp96), and we found that EGF caused a significant decrease in lgp96 in cellular homogenates and associated with lysosomes. We conclude that EGF in cultured renal cells regulates the breakdown of proteins targeted for destruction by chaperone-mediated autophagy. Because suppression of this pathway results in an increase in Pax2, these results suggest a novel mechanism for the regulation of cell growth.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A major response of cells to growth factors is a generalized increase in protein synthesis including the synthesis of specific classes of proteins (1). In addition to controlling synthesis, growth factors can suppress the bulk degradation of proteins (2). For example, in renal tubular epithelial cells we found that EGF1 suppresses the breakdown of the mass of intracellular proteins (3). The suppression of proteolysis in response to growth factors involved decreased lysosomal degradation rather than decreased proteasomal or calcium-sensitive proteases (3). Despite reports that proteolysis is regulated, no one has determined if specific classes of proteins are being regulated by growth factors.

Lysosomes degrade extracellular proteins (via endocytosis), membrane proteins, and organelles (via autophagy) and can degrade cytosolic proteins via direct import through the lysosomal membrane (4, 5). Dice and Terlecky (6) showed that there is a specific import pathway involving the 73-kDa heat shock cognate protein (hsc73) called chaperone-mediated autophagy. Hsc73 binds to a penta-peptide motif (consensus sequence, KFERQ) on the target protein and, acting as a chaperone, unfolds the target protein (7). Hsc73 bound to the substrate protein then interacts with an intrinsic lysosomal membrane protein, the 96-kDa lysosomal glycoprotein (lgp96, also called lysosomal membrane protein 2a) (8). After recruiting other accessory proteins, the target protein is transported through the lysosomal membrane and degraded (9). Dice and co-workers (10) also showed that chaperone-mediated autophagy can be regulated by calorie deprivation, which accelerates the proteolysis of proteins with KFERQ motifs in the lysosomes from liver. In kidney and liver, up to 30% of proteins contains the KFERQ motif, including many of the proteins involved in glycolysis. Because most glycolytic proteins have long half-lives, an increase in degradation could function to down-regulate their abundance.

Because we found that growth factors suppress lysosomal proteolysis in renal cells, we wanted to determine whether growth factors regulate the half-life of proteins that are substrates for chaperone-mediated autophagy. In pursuing this question, we uncovered a novel mechanism that leads to the accumulation of specific proteins involved in the regulation of cellular growth.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

All chemicals or reagents were purchased from Sigma, except Dulbecco's modified Eagle's medium, newborn calf serum, Trypsin-EDTA, and penicillin-streptomycin, which were obtained from Life Technologies, Inc. Recombinant human TGF-beta 1 and EGF were obtained from R&D Systems (Minneapolis, MN), and L-[U-14C]phenylalanine was obtained from PerkinElmer Life Sciences. Anti-hsc73 antiserum was purchased from Maine Biotechnology (Portland, ME), anti-M2 pyruvate kinase was purchased from Scebo-Tech, A.G. (Wettenburg, Germany), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purchased from Biodesign International (Kennebunk, ME). Anti-Pax2 serum was purchased from Zymed Laboratories Inc.. Affinity-purified anti-sera to the penta-peptide KFERQ and to lgp96 were a generous gift of J. F. Dice (Tufts University). The anti-hexokinase serum was a gift of E. Knecht (Universidad de Vallencia, Spain).

NRK-52E cells (a rat kidney epithelial cell line (11), passage 15) were obtained from ATCC (Manassas, VA), subcultured, and grown in high glucose Dulbecco's modified Eagle's medium supplemented with 25 mM HEPES, 25 mM glutamine, and 5% calf serum. Studies were performed on cells from passages 19-29. Cells in 6-well plates were grown to confluence and rendered quiescent by serum removal 48 h prior to experimental treatment. The cell culture medium was refreshed every 24 h to maintain a constant pH; it did not differ between control and treatment groups.

Recombinant human TGF-beta 1 was reconstituted in 4 mM HCl containing 0.1% heat-treated bovine serum albumin. Recombinant human EGF was reconstituted in PBS containing 0.1% heat-treated bovine serum albumin. In all studies, concentrations of 10-10 M (TGF-beta 1), 10-8 M (EGF), and 10 mM (NH4Cl) were used (12); the appropriate vehicle was added to control cells.

Measurements of Growth and Protein Turnover-- After exposure to an experimental variable, cells were washed with PBS, incubated with 0.05% trypsin/0.5 mM EDTA for 5 min, centrifuged at 1500 × g for 5 min, and washed with PBS. The final pellet was resuspended in 1 ml of 50 mM Na2PO4 (pH 7.4) and lysed on ice by repeated passage though a 27-gauge needle. The lysate was divided and stored at -70 °C for protein and DNA determination as described (12).

Protein degradation was measured as the release of L-[U-14C]phenylalanine from cells prelabeled as described (3-13). Briefly, 5 mM unlabeled phenylalanine was added to the medium to minimize reuse of the phenylalanine released by protein breakdown, and an initial 4-h washout period was used to eliminate short lived proteins and unincorporated L-[14C]phenylalanine. Aliquots of the medium were removed at intervals and treated with trichloroacetic acid to remove protein, and the radioactivity was determined. At the end of the experiment, cell protein was solubilized in 1 ml/well of 1% SDS, and the remaining radioactivity was measured. The protein degradation rate was calculated as the slope of the logarithm of the [14C]phenylalanine remaining in cell protein versus time.

Turnover of Specific Proteins-- Confluent cells in 100-mm dishes were incubated with 100 µCi of L-[35S]cystine/methionine (ICN, Costa Mesa, CA). For GAPDH, the labeling was performed in serum-free Dulbecco's modified Eagle's medium with cold cystine/methionine present for 72 h. For Pax2, cells were treated with EGF in cystine/methionine-free medium for 20 h to increase the labeling of Pax2 because its abundance is very low in quiescent cells. After two washes in serum-free medium, a 4-h washout in serum- and growth factor-free cystine/methionine-containing medium was performed prior to the addition of growth factors. Subsequently, cells were washed twice with serum-free medium before adding the experimental variable in the medium containing an excess of cold cystine and methionine. The culture medium was always changed daily with an additional wash. At time 0 and at various times up to 72 h for GAPDH and up to 24 h for Pax2, cells were lysed in a 1% Nonidet P-40 lysis buffer containing 100 µg/ml phenylmethylsulfonyl fluoride, 2 mM sodium EDTA, 4 µg/ml aprotinin, 2 µg/ml leupeptin, and 2 µg/ml pepstatin. One µg/ml anti-GAPDH or Pax2 anti-serum was added to equal amounts of cellular protein that was precipitated with protein G-Sepharose beads. After three washes with lysis buffer, the immunoprecipitate was separated by SDS-polyacrylamide gel electrophoresis, underwent autoradiography, and was quantitated by use of the Signmagel program. The protein half-life was calculated from the slope of the logarithmic transformation of the densitometry data plotted against time. We documented the completeness of recovery by performing Western blots on the supernatants after immunoprecipitation (data not shown).

Western Blotting-- Cells in 60-mm tissue culture dishes were washed twice in ice-cold PBS and lysed in a buffer containing 100 µg/ml phenylmethylsulfonyl fluoride, 2 mM sodium EDTA, 2 µg/ml aprotinin, 2 µg/ml leupeptin, and 2 µg/ml pepstatin. After centrifugation, the proteins in the supernatant were determined and boiled in buffer containing 1% SDS and 0.5% beta -mercaptoethanol, separated by SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose filters, and 5% fat-free milk protein or 3% bovine serum albumin was used as a blocking reagent. Antibodies were detected using the ECL system (Amersham Pharmacia Biotech) and Kodak BCL film.

Lysosome Isolation-- Lysosomes were isolated as described by Cuervo et al. (14). Briefly, cells in two 25-cm2 plates/group were washed in ice-cold PBS and then homogenized after scraping in ice-cold buffer (2.5 mM Tris (pH 7.2), 0.25 M sucrose) by 20 strokes of a Teflon Polytron homogenizer at 4 °C. Then 1 g of protein/7 ml of 0.25 M sucrose was centrifuged at 2500 × g for 10 min and the post-nuclear supernatant was placed on a discontinuous gradient of 35 and 17% metrizamide (pH 7.0) and 6% Percoll and centrifuged at 6800 × g for 25 min. The lysosome/mitochondrial fraction at the metrizamide/Percoll interface was resuspended to a final concentration of 57% metrizamide. On top of this fraction, there was a discontinuous metrizamide gradient of equal volumes of 35, 17, and 5%, metrizamide with a final 0.25% sucrose layer. This gradient was centrifuged for 1 h at 95,000 × g. The lysosomes sediment to the interface of 5-17% metrizamide and mitochondria at the 35-57% interface. Purity of the lysosomal or mitochondrial fractions was determined by the activity of beta -N-hexosaminidase (lysosome) and mitochondrial succinic dehydrogenase and by the presence of lgp96 (15).

Statistics-- Results are expressed as mean ± S.E. Because there was experiment-to-experiment variation in the magnitude of responses, results are presented as a percentage of the control value determined simultaneously. The differences between two groups were analyzed by the Student's t test, but multiple comparisons were analyzed by analysis of variance. Comparisons of slopes of lines representing the release of L-[U-14C]phenylalanine were done by analysis of co-variance.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We treated NRK-52E cells with growth factors and found different growth properties (Fig. 1): EGF causes hyperplasia (increased DNA content) and increases (~30%) the half-life of long lived proteins. TGF-beta 1 increases in the protein/DNA ratio (hypertrophy) but does not significantly suppress proteolysis. The combination of EGF plus TGF-beta 1 causes hypertrophy with the suppression of proteolysis, whereas ammonia causes less hypertrophy even though there is an even greater suppression of proteolysis. We have shown previously that EGF, TGF-beta 1, and EGF plus TGF-beta 1 increase protein synthesis, whereas ammonia does not affect synthesis (12, 13, 16).


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Fig. 1.   Growth factors exert different effects on growth and proteolysis. NRK-52E cells were treated with EGF (10-8 M), TGF-beta 1 (10-10 M), EGF + TGF-beta 1, and NH4Cl (10 mM). A, protein, DNA, and protein to DNA levels after 72 h of treatment are expressed as a percentage of the increase over the value measured in cells treated with the vehicle only. n = 14-18. B, protein degradation expressed as the slope of the log of the percentage of the counts remaining in cells at different times. The figure is a representative experiment of three repeats, (n = 6/group). The slopes of all lines are significantly different from each other (p < 0.05) except for control compared with TGF-beta 1 and EGF compared with EGF + TGF-beta 1.

GAPDH has a KFERQ consensus sequence (17) and is a classic substrate for chaperone-mediated autophagy (15, 18). As shown in Fig. 2 and Table I, the half-life of GAPDH measured in a pulse-chase experiment increased by ~ 90% in cells treated with either EGF or ammonia but did not significantly change in cells treated with TGF-beta 1 alone.


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Fig. 2.   Growth factors change GAPDH protein half-life. A, autoradiogram of NRK-52E cell lysates after immunoprecipitation with anti-GAPDH in pulse-chase experiments. Cells were treated as in Fig. 1 except they were radiolabeled when quiescent. Lanes are labeled according to treatment (control, EGF, or TGF-beta ) and duration of treatment (hours) after removal of L-[35S]cystine/methionine. 0 represents initial labeling. The autoradiogram shown is representative of five repeats. B, densitometry of all repeats are plotted as in Fig. 1B (n = 5).

                              
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Table I
Growth factors change GAPDH protein half-life
NRK-52E cells were radiolabeled when quiescent growth factors were added during a cold-chase period, and then GAPDH was immunoprecipitated at different times. Protein half-life was calculated from the slope of the logarithmic transformation of the densitometry of autoradiograms plotted against time.

We also tested whether these agents change the abundance of specific proteins with the KFERQ lysosomal import sequence. GAPDH abundance increased with treatments that suppress proteolysis, but TGF-beta 1, which does not affect proteolysis, did not increase GAPDH. We also examined the M2 isoform of pyruvate kinase because it is a glycolytic enzyme with KFERQ consensus sequences (19), it binds hsc73, and it is imported into lysosomes (6). In contrast, hexokinase is a glycolytic enzyme that lacks a KFERQ sequence (15). Conditions that stimulate cell growth also increase the abundance of the M2 isoform of pyruvate kinase as well as other proteins recognized by anti-KFERQ affinity-purified sera (Fig. 3). Hexokinase abundance did not increase with any of the growth factors. Although the pattern of changes was similar between proteins recognized by anti-KFERQ serum and the M2 isoform of pyruvate kinase, the magnitude of increase in the M2 isoform of pyruvate kinase was much greater, suggesting that its synthesis is also stimulated (Fig. 3, B and C).


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Fig. 3.   Protein abundance of extracts of NRK-52E cells treated for 96 h as denoted in Fig. 1. A, Western analysis of 40 µg of protein/lane with M2 pyruvate kinase, GAPDH, hexokinase, and anti-KFERQ peptide antibodies or serum, representating a blot of four or more repeats. B, quantification by densitometry of Western blots for M2 pyruvate kinase. *, p < 0.05 versus control, n = 5. C, quantification by densitometry of Western blots for total KFERQ proteins. *, p < 0.05 versus control, n = 9. D, Western analysis of 50 µg of protein/lane with anti-Pax2 antibodies (representative blot of four repeats).

Glycolytic enzymes are not the only proteins containing KFERQ sequences, which may be important intermediates influencing renal cell growth. For example, the renal paired box-related transcription factor, Pax2, contains a conserved KFERQ sequence at amino acids 38-42 (20). We found that the half-life of Pax2 (Fig. 4) also increased with EGF treatment and that the abundance of Pax2 was increased by growth factors; the smallest rise occurred with TGF-beta 1. Interestingly, the Pax2 abundance also increased when lysosomal proteolysis was inhibited with NH4Cl (Fig. 3D). Thus different stimuli causing growth also increase Pax2 abundance.


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Fig. 4.   EGF changes Pax2 protein half-life. A, autoradiogram of NRK-52E cell lysates after immunoprecipitation with anti-Pax2 in a pulse-chase experiment. Labeling with L-[35S]cystine/methionine was performed with 10-8 M EGF present for 20 h in cold cystine/methionine-free medium followed by a 4-h washout in serum and EGF-free medium with cystine/methionine present before the addition of EGF. Otherwise, cells were grown as in Fig. 1. Lanes are labeled as the type and time of treatment with EGF. 0 represents initial labeling. The autoradiogram shown is representative of three repeats. B, densitometry of all repeats are plotted as in Fig. 1B (n = 3-5).

To determine the mechanisms that control suppression of proteolysis, we examined if the regulatory proteins of chaperone-mediated autophagy change in response to growth factors. Hsc73 did not change in abundance (Fig. 3A). We also examined the lysosomal membrane receptor for protein translocation, lgp96, in lysates and in association with isolated lysosomes using sera directed against the 12-amino acid cytoplasmic portion of lgp96 that binds to hsc73 (8). The quality of lysosomes isolated did not vary between the control and EGF-treated cells as assayed by hexosaminidase activity (Table II). Isolated lysosomes exhibited immunostaining for lgp96 (Fig. 5A); the level was 7-fold higher than in whole cell lysates (Fig. 5B). Lgp96 was not detected in the mitochondrial fractions. In whole cell lysates, lgp96 abundance decreased by 30-40% after 24 or 96 h of treatment with stimuli that suppress proteolysis (Fig. 5, C and E). In contrast, TGF-beta 1, which did not affect proteolysis, did not affect lgp96 levels. Because the lysosomal-associated lgp96 correlates more closely with the activity of chaperone-mediated autophagy than total cellular lgp96 (21), we examined lysosomal-associated lgp96 with EGF treatment and found a 48 ± 10% decrease (p < 0.05, n = 3) compared with lysosomes isolated from control cells (Fig. 5, D and E).

                              
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Table II
Hexosaminidase activity in isolated lysosomes
NRK-52E cells were grown as in Fig. 1, and lysosomes and mitochondria were isolated by metrizamide density gradient centrifugation. There are no significant differences between the control and EGF.


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Fig. 5.   Lgp96 abundance by Western analysis of protein abundance of radioimmune precipitation buffer extracts of NRK-52E cells and isolated lysosomes and mitochondria. Representative blots of 3-4 repeats are shown. A, lgp96 abundance in 10 µg of protein from isolated mitochondria (Mito) and lysosomes (Lyso). B, lgp96 abundance in 50 µg of protein from whole cell homogenates (Homo) and 7 µg of lysosomal protein. C, lgp96 abundance in 50 µg of protein from cells treated for 96 h as in Fig. 1. D, lgp96 abundance in 5 mg of protein from isolated lysosomes in cells treated for 24 h as in Fig. 1. E, densitometry of all repeats of the experiment shown in C (n = 4) and D (n = 4) expressed as a percentage of control values (dotted line). *, p < 0.05 versus control.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the early 1980s, it was recognized that specific growth factors and activated oncogenes could suppress protein degradation in certain cell types including epithelial cells (22). It was not known, however, which classes of proteins develop longer half-lives during growth or how this response was regulated. We found that EGF suppresses the breakdown of the bulk of proteins in NRK-52E cells by a mechanism that involved the suppression of lysosomal function but not proteolysis by proteasomal or calcium-activated proteases (3).

Physiologic conditions can regulate specific pathways of lysosomal proteolysis. For example, calorie deprivation increases the degradation of proteins with a KFERQ motif in liver and kidney lysosomes (10). How does this finding bear on growth factor-induced renal cell growth? Conditions stimulating renal cell growth increase glycolysis, and many glycolytic enzymes contain KFERQ motifs (17, 23-25). Thus, by acting in the opposite fashion as calorie deprivation, growth factors could suppress the degradation of glycolytic enzymes and contribute to the increase in glycolysis that accompanies renal growth. Our results confirm that EGF acts to prolong the half-life of the classic substrate for chaperone-mediated autophagy, GAPDH, and increase the abundance of KFERQ-containing proteins.

Our results provide additional insights into the relationship among growth factors, cell growth, and lysosomal protein degradation. First, only specific growth factors influence lysosomal function. For example, EGF clearly stimulates cell growth and suppresses total proteolysis and the proteolysis of substrates of chaperone-mediated autophagy. In contrast, TGF-beta 1 caused the smallest increase in growth and has almost no effect on proteolysis. We do not conclude that suppression of proteolysis is the sole mechanism causing KFERQ-containing protein accumulation, because the accumulation of KFERQ proteins that occurred with TGF-beta treatment almost certainly reflects increased synthesis (Figs. 3, B and C).

Second, our results show that regulation of this lysosomal pathway by growth factors leads to prolongation of the half-life of Pax2, which has been implicated in renal cell growth in development, cyst formation, and renal cell carcinoma (20, 26). Because there is also an increase in the abundance of Pax2 in cells treated with EGF and because EGF causes only trivial increases in Pax2 mRNA in renal tubular cells (27), the increase in half-life we found could be physiologically relevant. Because Pax2 acts as a transcription factor, these responses suggest a new mechanism by which growth factors regulate cell growth; not only do they suppress the degradation of the bulk of cytoplasmic proteins (3), but they increase the availability of at least one critical transcription factor.

Finally, our results provide unexpected information about a potential mechanism by which ammonia could increase cell growth. The growth of renal cells characteristically found in response to metabolic acidosis is attributed to ammonia, which can reach concentrations as high as 5 mM in the cortex of the kidney (28). Ammonia had been thought to act only by changing lysosomal pH and nonspecifically suppressing lysosomal proteolysis leading to the accumulation of cytosolic proteins (16, 29). However, our results suggest that ammonia also acts by suppressing the degradation of specific signaling proteins such as Pax2. The up-regulation of transcription factors could allow the expression of particular proteins important for growth without an increase in global protein synthesis.

Regarding the mechanism involved in changing lysosomal degradation, we and others find that the abundance of hsc73 does not change even when activity of this pathway changes (9, 14). Curiously, hsc73 contains KFERQ sequences but is resistant to degradation within hepatic lysosomes responding to starvation (9). On the other hand, we did observe a decrease in the abundance of lgp96 including a sharp decrease in the amount of lgp96 specifically associated with lysosomes (Fig. 5). This finding is consistent with the close correlation between the lgp96 associated with lysosomes and the activity of chaperone-mediated autophagy (14, 21, 30).

Although there are similarities between the effects of EGF and ammonia on the proteolysis of KFERQ-containing proteins and lgp96 levels, there are differences in their actions on lysosomes. We found that pharmacologic agents that specifically inhibit lysosomal proteolysis (ammonia, methylamine, bafilomycin A1, or leupeptin plus the protease inhibitor, E64) convert the cellular proliferation in response to EGF into hypertrophy (31). The change in lgp96 abundance may be a common pathway increasing growth-promoting proteins such as glycolytic enzymes and Pax2, and an additional influence of lysosomal inhibitors may account for the conversion of hyperplasia to hypertrophy.

Besides the regulation of chaperone-mediated autophagy, EGF could affect the function of other pathways of lysosomal proteolysis. Autophagy may also be regulated by growth factors (32), leading to slower degradation of organelles and membranes. EGF acts through phosphoinositide 3-kinase as it suppresses proteolysis in renal cells,2 and phosphoinositide 3-kinase has been reported to regulate autophagy in cultured liver cells (33).

One practical prediction of these results is that a KFERQ sequence may be used to identify proteins that are up-regulated during renal cell growth. Besides glycolytic enzymes, there are a large number of proteins in the National Center for Biotechnology Information data base that contain conserved KFERQ sequences and are important for renal tubule cell growth. These proteins include enzymes involved in phospholipid metabolism (choline kinase (GenBankTM accession number 139962) and phosphorylcholine transferase (34)), ion transporters (beta  subunit of the sodium/potassium ATPase (35)), and signaling molecules such as Pax2 (20). The KFERQ sequence is present in the Pax isoforms expressed in the urinary tract (Pax2, -5, and -8) but not in other Pax isoforms, suggesting that the link between Pax proteins and this proteolytic pathway may be specific to the urinary tract (36, 37). Finally, the signaling proteins MARKS and Ikappa B have been shown to have their abundance regulated by this pathway (30, 38).

    ACKNOWLEDGEMENTS

We thank Drs. J. Fred Dice and Ana Maria Cuervo for help with reagents and techniques and Drs. William Mitch and Russ Price for advice and critical reading of the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health K08 DK02496 a Young Investigator Research Grant from the National Kidney Foundation, a Veterans Administration Merit Review Award (to H. F.), an American Heart Association Scientist Development Award (to J. D.), and a fellowship grant from the National Kidney Foundation of Georgia (to S. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Renal Division, Emory University School of Medicine, W.M.B., Rm. 338, 1639 Pierce Dr., N. E., Atlanta, GA 30322. Tel.: 404-727-9217; Fax: 404-727-3425; E-mail: hfranch@emory.edu.

Published, JBC Papers in Press, March 21, 2001, DOI 10.1074/jbc.M101777200

2 H. A. Franch and J. Du, unpublished observation.

    ABBREVIATIONS

The abbreviations used are: EGF, epidermal growth factor; hsc73, 73-kDa heat shock cognate protein; lgp96, 96-kDa lysosomal glycoprotein; TGF-beta 1, transforming growth factor beta 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PBS, phosphate-buffered saline.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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