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

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 is dependent on the 73 kDa heat shock cognate protein (hsc73) and allows the degradation of proteins containing a specific lysosomal import consensus sequence (KFERQ motif). Epidermal growth factor (EGF 1 ) or ammonia, but not transforming growth factor b 1 (TGF b 1), suppresses total protein breakdown in cultured NRK-52E renal epithelial cells. EGF or ammonia prolonged the half-life of glyceraldehyde-3-phosphate, a classic substrate for chaperone mediated autophagy, by more than 90% , while TGF b 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 and Pax2. Ammonia also increased the level of 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. Since suppression of this pathway results in an increase in Pax2, these results suggest a novel mechanism for regulation of cell growth.


Introduction
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 EGF suppresses the breakdown of the mass of intracellular proteins (3). 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 colleagues showed that there is a specific import pathway involving the 73 kDa heat shock cognate protein (hsc-73) 1 , called chaperone-mediated autophagy (6). 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 (Lamp2a)) (8). After recruiting other accessory proteins, the target protein is transported through the lysosomal membrane and degraded (9). Dice and colleagues also showed that chaperone mediated autophagy can be regulated by calorie deprivation which accelerates the proteolysis of proteins with KFERQ motifs in lysosomes from liver (10). In kidney and liver, up to 30% of proteins contain the KFERQ motif, including many of the proteins involved in glycolysis. Since most glycolytic proteins have long half-lives, an increase in degradation could function to down regulate their abundance. by guest on July 10, 2020 http://www.jbc.org/ Downloaded from Since we found that growth factors suppress lysosomal proteolysis in renal cells, we wanted to determine if growth factors regulate the half-life of proteins which are substrates for chaperonemediated autophagy. In pursuing this question, we uncovered a novel mechanism that leads to accumulation of specific proteins involved in the regulation of cellular growth. Measurements of growth and protein turnover.

Experimental procedures
After exposure to an experimental variable, cells were washed with PBS, incubated with 0.05% trypsin/0.5 mM EDTA for 5 minutes, centrifuged at 1500 x g for 5 minutes, and washed with PBS. The final pellet was resuspended in 1 ml of 50 mM Na 2 PO 4 (pH 7.4) and lysed on ice by repeated passage though a 27 gauge needle. The lysate was divided and stored at -70 o C for protein and DNA determination as described (12).
Protein degradation was measured as the release of L-[U-14 C] phenylalanine from cells prelabeled as described (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). Briefly, 5 mM of unlabeled phenylalanine was added to the media to minimize reuse of phenylalanine released by protein breakdown and an initial 4 hour washout period was used to eliminate short-lived proteins and unincorporated L-[ 14 C] phenylalanine. Aliquots of media were removed at intervals, treated with TCA to remove protein, and radioactivity determined.
At the end of the experiment, cell protein was solubilized in 1 ml/well of 1% sodium dodecyl sulfate (SDS) and the remaining radioactivity was measured. The protein degradation rate was calculated as the slope of the logarithm of [ 14 C] phenylalanine remaining in cell protein versus time. mg/ml leupeptin, and 2 mg/ml pepstatin. One mg/ml anti-GAPDH or Pax2 anti-sera was added to equal amounts of cellular protein which was precipitated with by protein G sepherose beads. After 8 Cells in 60 mM tissue culture dishes were washed twice in ice cold PBS and lysed in a buffer containing 100 mg/ml PMSF, 2 mM sodium EDTA, 2 mg/ml aprotinin, 2 mg/ml leupeptin, and 2 mg/ml pepstatin. After centrifugation, protein in the supernatant was determined and the supernatant was boiled in buffer containing 1% SDS, 0.5% b-mercaptoethanol, proteins separated by SDS-PAGE, transfered to nitrocellulose filters, and 5% fat free milk protein or 3% BSA was used as blocking reagents. Antibodies were detected using the ECL system (Amersham, Arlington Heights, IL) and Kodak BCL film.

Results
We treated NRK-52E cells with growth factors and found different growth properties ( figure   1): EGF causes hyperplasia (increased DNA content), and increases (~30%) the half-life of long lived proteins. TGFb1 increases in the protein to DNA ratio (hypertrophy), but does not significantly suppress proteolysis. The combination of EGF plus TGFb1 causes hypertrophy with suppression of proteolysis, while ammonia causes less hypertrophy, even though there is even greater suppression of proteolysis. We have previously shown that EGF, TGFb1 and EGF plus TGFb1 increase protein synthesis, while ammonia does not affect synthesis (12,13,16).
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has a KFERQ consensus sequence (17) and is a classic substrate for chaperone mediated autophagy (15,18). As shown in figure 2 and 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 TGFb1, which does not affect proteolysis, did not increase GAPDH. We also examined the M 2 isoform of pyruvate kinase because it is a glycolytic enzyme with a KFERQ consensus sequences (19), and it binds hsc73 and is imported into lysosomes (6). In contrast, hexokinase is a glycolytic enzyme that lacks a KFERQ sequence (15). Thus different stimuli causing growth also increase Pax2 abundance.
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 (figure 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 control and EGF treated cells as assayed by hexosaminidase activity (Table 2). Isolated lysosomes exhibited immunostaining for lgp96 ( figure   5a): the level was seven-fold higher than in whole cell lysates (Figures 5b). Lgp96 was not detected in the mitochondrial fractions. In whole cell lysates, lgp96 abundance decreased by 30-40% after 24 or 96 hours of treatment with stimuli that suppress proteolysis (figure 5c, e). In contrast, TGFb1, which did not affect proteolysis, did not affect lgp96 levels. Since lysosomal associated lgp96, correlates more closely with activity of chaperone-mediated autophagy than total cellular lgp96 (21), we examined lysosomal associated lgp96 with EGF treatment and found a 48+10% (p<0.05, n=3) decrease compared to lysosomes isolated from control cells (Figure 5d, e).

Discussion
In the early 1980's, 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, nor 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 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 12 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)(24)(25). Thus, by acting in the opposite fashion as calorie deprivation, growth factors could suppress of 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. Firstly, 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, TGFb1 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, since the accumulation of KFERQ proteins that occurred with TGFb treatment almost certainly reflects increased synthesis (figures 3b and 3c). 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 5mM 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 accumulation of cytosolic proteins (16,29). However, our results suggest that ammonia also acts by suppressing degradation of specific signaling proteins such as Pax2. The upregulation of transcription factors could allow 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 ( figure 5). This finding is consistent with the close correlation between lgp96 associated with lysosomes and the activity of chaperone-mediated autophagy (30), (14,21).
Although there are similarities between the effects of EGF and ammonia on proteolysis of KFERQ containing proteins and lgp96 levels, there are differences in their actions on lysosomes.
We found that pharmacologic agents that specifically inhibits 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  (35)), and signaling molecules such as Pax2 (20). The KFERQ sequence is present in the Pax isoforms expressed in the urinary tract (Pax 2, 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 IkB have been shown to have their abundance regulated by this pathway (30,38).   Table 2. Hexosaminidase activity in isolated lysosomes. NRK-52E cells were grown as in figure   1 and lysosomes and mitochondria isolated by metrizamide density gradient centrifugation.
There are no significant differences between control and EGF. B p<0.05 vs. control. Autoradiogram shown is representative of 5 repeats. B. Density of radioactivity