Neurite Outgrowth in PC12 Cells
DISTINGUISHING THE ROLES OF UBIQUITYLATION AND UBIQUITIN-DEPENDENT PROTEOLYSIS*

Martin ObinDagger §, Eugene Mesco, Xin GongDagger , Arthur L. Haasparallel , James Joseph**, and Allen TaylorDagger

From the Dagger  Laboratory for Nutrition and Vision Research and ** Neuroscience Laboratory, Jean Mayer United States Department of Agriculture-Human Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts 02111,  Department of Biology and Life Science, Savannah State University, Savannah, Georgia 31404, and parallel  Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Nerve growth factor (NGF)-induced neurite outgrowth from rat PC12 cells was coincident with elevated (>= 2-fold) levels of endogenous ubiquitin (Ub) protein conjugates, elevated rates of formation of 125I-labeled Ub~E1 (Ub-activating enzyme) thiol esters and 125I-labeled Ub~E2 (Ub carrier protein) thiol esters in vitro, and enhanced capacity to synthesize 125I-labeled Ub-protein conjugates de novo. Activities of at least four E2s were increased in NGF-treated cells, including E2(14K), a component of the N-end rule pathway. Ubiquitylation of 125 I-labeled beta -lactoglobulin was up to 4-fold greater in supernatants from NGF-treated cells versus untreated cells and was selectively inhibited by the dipeptide Leu-Ala, an inhibitor of Ub isopeptide ligase (E3). However, Ub-dependent proteolysis of 125I-labeled beta -lactoglobulin was not increased in supernatants from NGF-treated cells, suggesting that neurite outgrowth is promoted by enhanced rates of synthesis (rather than degradation) of Ub-protein conjugates. Consistent with this observation, neurite outgrowth was induced by proteasome inhibitors (lactacystin and clasto-lactacystin beta -lactone) and was associated with elevated levels of ubiquitylated protein and stabilization of the Ub-dependent substrate, p53. Lactacystin-induced neurite outgrowth was blocked by the dipeptide Leu-Ala (2 mM) but not by His-Ala. These data 1) demonstrate that the enhanced pool of ubiquitylated protein observed during neuritogenesis in PC12 cells reflects coordinated up-regulation of Ub-conjugating activity, 2) suggest that Ub-dependent proteolysis is a negative regulator of neurite outgrowth in vitro, and 3) support a role for E2(14K)/E3-mediated protein ubiquitylation in PC12 cell neurite outgrowth.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Understanding the mechanisms that regulate neuronal differentiation is important to developmental biologists and to those seeking to repair damaged neurons by cell or tissue transplantation, such as in the treatment of Parkinson's disease and retinal degenerations. Our laboratory is currently investigating the role of the ubiquitin (Ub)1-proteasome pathway (UPP) in the development and maintenance of the neuronal phenotype. The UPP is a conserved pathway of selective protein modification and degradation that controls levels and activities of many highly regulated eukaryotic proteins, including cyclins, tumor suppressors, receptors, and transcription factors (reviewed in Refs. 1-4). Substrates of the pathway are covalently ligated to one or more monomers of ubiquitin, an 8.5-kDa protein, by the sequential activities of three families of enzymes: Ub-activating enzymes (E1s), Ub carrier proteins (E2s), and Ub-isopeptide ligases (E3s) (2-4). The protein moiety of the Ub-protein conjugate can be subsequently degraded by the 26 S proteasome, a multicatalytic, ATP-dependent protease (reviewed in Ref. 5). Alternatively, the Ub-protein conjugate can be deubiquitylated by Ub isopeptidases, releasing the protein intact (2). The best recognized function of ubiquitylation is selective targeting of proteins for rapid degradation (1-5); however, evidence for nonproteolytic functions of this process is rapidly emerging (1, 2, 6, 7).

Roles for the UPP in neuronal differentiation are suggested by developmental regulation of ubiquitin and/or UPP enzyme expression in differentiating neurons (8-11), by localization of UPP components within nerve processes (9, 11), and by changes in levels of free and conjugated Ub during nerve growth factor (NGF)-induced neurodifferentiation (12). In Drosophila, determination of photoreceptor cell fate and the guidance of neurite growth cones to synaptic targets are regulated by a deubiquitylating enzyme and a Ub carrier protein (E2), respectively (13-15). Ub-dependent proteolysis may be required for growth factor-induced neurite outgrowth in vertebrates. This function of the UPP is suggested by studies that implicate the N-end rule pathway in neuritogenesis (16-18). The N-end rule is a set of N-terminal amino acids that govern the susceptibility of certain proteins to Ub-dependent proteolysis (reviewed in Ref. 19). In the mammalian N-end rule, destabilizing residues include bulky hydrophobic (e.g. Leu, Phe, Trp) or basic amino acids (e.g. Arg, Lys, His). These amino acids are selectively recognized by a bifunctional isopeptide ligase, E3 (20). E3 facilitates the transfer of Ub from a specific Ub carrier protein, E2(14K), to the substrate (21, 22). Dipeptides bearing destabilizing N-terminal residues inhibit E3-catalyzed ubiquitylation, thereby inhibiting Ub-dependent degradation of N-end rule substrates (23). A biological function of the N-end rule has been verified in yeast (24).

NGF-induced neurite outgrowth from rat pheochromocytoma (PC12) cells provides a well characterized model of sympathetic neuron differentiation (reviewed in Ref. 25). Neurite outgrowth in this model is believed to require UPP proteolytic activity (16), a notion that is consistent with increased levels of Ub-protein conjugates and coincident reductions in levels of free Ub that are observed in NGF-treated PC12 cells (12). However, nothing is currently known about alterations in either UPP enzyme activity or capacities for ubiquitylation and Ub-dependent protein degradation associated with neurite outgrowth in these cells. Novel data presented here demonstrate that neurite outgrowth is coincident with up-regulated activities of Ub-conjugating enzymes and capacity for de novo protein ubiquitylation but not with enhanced capacity for Ub-dependent proteolysis in vitro. Moreover, neurite outgrowth is accelerated by reagents that block Ub-dependent proteolysis, and such induced neurite outgrowth is inhibited by a dipeptide inhibitor of E3- dependent ubiquitylation. These data implicate ubiquitylation and Ub-dependent proteolysis as respective positive and negative regulators of neurite outgrowth.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Cell culture medium and sera were from Life Technologies, Inc.. Materials for electrophoresis were from Bio-Rad. Polyvinylidene difluoride membrane was from Millipore (Bedford, MA). Coomassie Plus protein assay reagent was purchased from Pierce. Na125I and 125I-labeled protein A were supplied by NEN Life Science Products. The ECL chemiluminescence kit was from Amersham Pharmacia Biotech. Lactacystin and MG132 were from Calbiochem. Ubiquitin aldehyde (Ubal) and clasto-lactacystin beta -lactone were purchased from Boston Biochem Inc. (Cambridge, MA). Dipeptides were obtained from Bachem Bioscience (King of Prussia, PA) and from Sigma. Sheep polyclonal serum against recombinant p53, control sheep serum, amd rabbit anti-sheep IgG were obtained from Oncogene Research products (Cambridge, MA). 7S NGF and all other materials were purchased from Sigma and were the highest grade available. Ubiquitin and beta -lactoglobulin were iodinated by reaction with chloramine T as described (26).

PC12 Cell Culture-- PC12 cells obtained from Dr. Arthur Tischler (Department of Pathology, Tufts University School of Medicine) were grown in 100-mm2 tissue culture dishes as described previously (25). Briefly, cells (3 × 104 cells/cm2) were plated on collagen-coated dishes and maintained at 37 °C in 95% air, 5% CO2 in either RPMI 1640 containing 10% heat-denatured horse serum, 5% fetal bovine serum, and antibiotics (complete medium) or in medium containing 1% horse serum, 0.5% fetal bovine serum and 100 ng/ml NGF. Medium was changed every 48 h.

Effects of Proteasome Inhibitors and Dipeptides on Neurite Outgrowth-- Cells that were plated 24 h previously were treated in 3 ml of complete medium containing either lactacystin (10 µM final concentration), the biologically active analogue, clasto-lactacystin beta -lactone (5 µM final concentration), or Me2SO carrier (<= 0.2% final concentration). After 2 or 4 h, cultures received bestatin (200 µM final concentration) followed by dipeptides (Leu-Ala or His-Ala, 2 mM final concentration). When dipeptides were added after 2 h of proteasome inhibition, neurite outgrowth was quantified after an additional 4 h of incubation as the proportion of cells with one or more neurites. When dipeptides were added after 4 h of proteasome inhibition, neurite outgrowth was quantified after an additional 20 h of incubation as the proportion of cells with neurites greater than or equal to the length of one cell body. These determinations were made with a Zeiss inverted microscope using phase-contrast objectives. Counts were made in at least three randomly selected microscopic fields (containing >50 cells), one each from one of the four quadrants of the culture dish. Each experiment was conducted in duplicate or triplicate. Data were analyzed for statistical significance using the general linear models procedures of SYSTAT (Evanston, IL).

Preparation of Cell Lysates and Supernatants-- Cell lysates were prepared by washing cells 2× with phosphate-buffered saline followed by scraping into 150 µl of lysis buffer (5 mM Tris-HCl, 4% SDS and 10 mM iodoacetate or 50 mM N-ethylmaleimide, pH 7.6) and immediate boiling. Insoluble material was removed by centrifugation (15,000 × g, 10 min). To prepare supernatants containing an active UPP, cells were gently scraped into ice-cold phosphate-buffered saline, washed and pelleted (800 × g) twice, resuspended in cold 5 mM Tris-HCl, pH 7.8, containing 0.5 mM dithiothreitol, and homogenized by hand (26). Supernatants (85,000 × g, 20 min, 2 °C) were collected, and protein concentrations (10-25 mg/ml) were determined. Lysates and supernatants were aliquoted and stored at -80 °C.

Protein Electrophoresis and Immunoblotting-- Cell lysates and aliquots of UPP activity assays (see below) were diluted (1:1) and boiled with SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer containing 10% 2-mercaptoethanol or with thiol ester sample buffer lacking reductant (27). Proteins were separated by SDS-PAGE. For immunoblotting, electrophoresed proteins were transferred to polyvinylidene difluoride membrane as described previously (27). Blots were probed either with rabbit anti-serum that specifically recognizes Ub-protein conjugates (27), sheep anti-serum against p53 (see above), or with appropriate preimmune sera. Specific binding was detected by either 125I-labeled protein A or ECL, visualized by autoradiography, and quantified by laser densitometry (Molecular Dynamics).

UPP Activity Assays-- Capacity for de novo synthesis of 125I-labeled Ub-protein conjugates and 125I-labeled Ub~E1 and 125I-labeled Ub~E2 thiol esters was measured as described previously (27). Assays (25 µl) contained 50 mM Tris-HCl, pH 7.8, 150 µg of PC12 supernatant, 2 mM ATP and an ATP-regenerating system, 80 µM MG132, 2 µM Ubal and 2.0 µg of 125I-labeled Ub. Some assays contained 0.8 µM recombinant E2(14K). Assays were incubated at 37 °C for the times indicated in the figure legends and were terminated by boiling with gel loading buffer in the presence or absence of 2-mercaptoethanol. Samples electrophoresed in the absence of reductant retain Ub thiol esters of E1s and E2s. Following SDS-PAGE, gels were stained, dried, and subjected to autoradiography, and radiolabeled adducts were quantified by densitometry. Ub~E1s and Ub~E2s were distinguished from Ub-protein conjugates by comparing autoradiograms of samples electrophoresed in the presence or absence of 2-mercaptoethanol.

The formation of Ub-125I-labeled beta -lactoglobulin was measured in assays (as above) containing 2.0 µg of 125I-labeled beta -lactoglobulin (~5 × 105 cpm/µg) instead of radiolabeled Ub. Assays additionally contained 200 µM bestatin and either Leu-Ala or Ala-Leu (20 mM final concentration). Ub-dependent degradation of 125I-labeled beta -lactoglobulin was assayed as described previously (26, 27). ATP-dependent proteolysis was determined to be exclusively UPP-dependent, because all ATP-dependent proteolysis was blocked by the proteasome inhibitor, MG132 (80 µM final concentration).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The transition to neuronal morphology in PC12 cells exposed to NGF occurred as described previously (25). Briefly, cells became flattened within 24 h, and neurite outgrowth was apparent after 48 h. The frequency and length of neurites increased with the duration of NGF treatment such that by 5 days of NGF treatment >50% of cells exhibited at least 1 neurite that extended more than 1 cell body length from the cell periphery. Coincident with these NGF-induced morphological changes, levels of high mass Ub-protein conjugates increased >2-fold after 24 h of NGF treatment (Fig. 1A, compare lanes 1 and 2), 4-fold after 3 days (Fig. 1A, compare lanes 3 and 4) and attained maximal levels (>= 5-fold increase) after 5 days (Fig. 1A, compare lanes 5 and 6). Levels of lower mass Ub-protein conjugates also increased in NGF-treated cells, with the exception of a ~42-kDa species that was more abundant in the absence of NGF (data not shown). In concert with these increases in Ub-protein conjugates, levels of free (nonconjugated) Ub declined up to 3-fold within 48 h of NGF treatment (data not shown). These results confirm that NGF induces neurite outgrowth coincident with the redistribution of cellular Ub from the free to the conjugated pool (12).


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Fig. 1.   PC12 cells stimulated with NGF exhibit increased steady-state levels of Ub-protein conjugates, enhanced ubiquitylation of endogenous proteins de novo, and elevated rates of Ub~E1 and Ub~E2 thiol ester formation. A, endogenous high mass Ub-protein conjugates detected by Western blotting of lysates (30 µg) of PC12 cells cultured in the presence (lanes 1, 3, and 5) or absence (lanes 2, 4, and 6) of NGF (100 ng/ml) for 1, 3, or 5 days (d). Visualization was by ECL. Molecular mass markers (kDa) are at the right. One of four independent experiments is shown. B, autoradiogram demonstrating enhanced capacity to conjugate 125I-labeled Ub to endogenous proteins in supernatant from PC12 cells cultured (5 days) with NGF. Reactions were incubated for 60 s. One of four independent experiments is shown. C, autoradiogram following nonreducing SDS-PAGE showing higher levels of 125I-labeled Ub incorporated into thiol esters of E1s (125I-Ub~E1s) and E2s (125I-Ub~E2s) by supernatant from NGF-treated (5d) cells. Thiol esters were confirmed by destruction with 2-mercaptoethanol (BME, lane 4). Lane 3 is empty to prevent contamination of lane 2 with 2-mercaptoethanol. Reactions were incubated for 3 min. *, putative 125I-labeled Ub~E2(14K) thiol ester. One of three independent experiments is shown.

Increased levels of Ub-protein conjugates in NGF-treated cells could be due either to enhanced rates of ubiquitylation or to decreased rates of conjugate degradation or disassembly. To determine whether neurite outgrowth was coincident with enhanced rates of ubiquitylation, supernatant obtained from PC12 cells cultured for 5 days in the presence or absence of NGF was incubated with saturating levels of 125I-labeled Ub, ATP, and inhibitors of Ub-dependent proteolysis (MG132) and Ub-protein conjugate deubiquitylation (Ubal). Autoradiography of SDS gels indicates that the radiographic intensity of high mass 125I-labeled Ub-protein conjugates was >= 2-fold greater in supernatants from NGF-treated cells (Fig. 1B, compare lanes 1 and 2). Because the incubation time was within the interval during which formation of high mass conjugates was linear (data not shown), these results indicate that the rate of formation of high mass conjugates is elevated in supernatants of NGF-treated PC12 cells.

Enhanced rates of ubiquitylation can reflect elevated activities of Ub-conjugating enzymes (E1s, E2s, E3s) as well as increased availability of protein substrates. An in vitro measure of E1 and E2 activities is their capacity to form thiol esters with saturating levels of 125I-labeled Ub (28). Because these esters are formed on active-site cysteines, they are labile in the presence of reductants such as mercaptoethanol. We determined that levels of 125I-labeled Ub~E1s thiol esters (~125 kDa) and at least four 125I-labeled Ub~E2s thiol esters (24-40 kDa) were elevated an average of 40-50% in supernatants from NGF-treated cells (Fig. 1C, compare lanes 1 and 2). It is unlikely that these results reflect decreased ability of E2s to transfer Ub to E3s, because rates of de novo protein ubiquitylation were elevated after NGF treatment (Fig. 1B). We therefore conclude that activities of Ub-conjugating enzymes are elevated in NGF-stimulated PC12 cells. This NGF-associated enhancement of Ub-conjugating enzyme activities provides one mechanism to account for the enhanced pool of Ub-protein conjugates and the increased capacity for de novo protein ubiquitylation in PC12 cells during growth factor induced neuritogenesis (Figs. 1, A and B).

The electrophoretic mobility of the 24-kDa 125I-labeled Ub~E2 thiol ester (Fig. 1C, *) suggested that it contained E2(14K). This was confirmed based on comigration of this 24-kDa 125I-labeled Ub thiol ester with the 125I-labeled Ub thiol ester formed with recombinant E2(14K) (Fig. 2, compare lanes 1 and 2, arrow). The higher mass radiolabeled band in the sample containing recombinant E2(14K) (Fig. 2, lane 1) is a partially unfolded electrophoretic variant of 125I-labeled Ub~E2(14K) (29). Loss of radiolabel in the presence of reductant (Fig. 2, lane 4) confirms that these radiolabeled species are thiol esters. In conjunction with the demonstration of up-regulated E2 activities in NGF-treated cells (Fig. 1C), verification that the 24-kDa 125I-labeled Ub~E2 thiol ester contains E2(14K) provides evidence that E2(14K) activity is enhanced in supernatant from NGF-treated PC12 cells.


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Fig. 2.   The 24-kDa 125I-labeled Ub~E2 thiol ester generated in PC12 cell supernatant contains E2(14K). Thiol ester assays were conducted as above with supernatant from PC12 cells cultured without NGF. Samples were electrophoresed in the absence (lanes 1 and 2) or presence (lane 4) of reductant (2-mercaptoethanol (BME)). Lane 1, 125I-labeled Ub~E2(14K) formed in the presence of supplemental (recombinant) E2(14K). The higher mass species of 125I-labeled Ub~E2(14K) (*) is an electrophoretic variant with a slightly greater stokes radius. The ~16-kDa radiolabeled band is 125I-labeled di-Ub. Lane 2, endogenous 125I-labeled Ub~E2 thiol ester ([125I]Ub~E2(14K). Lane 3, contains no sample (as in Fig. 1C). Lane 4, destruction of 125I-labeled Ub~E2(14K) by reductant confirms the [125I]Ub~E2 thiol ester linkage. The molecular mass markers are at right. The representative experiment shown is one of three.

To assess whether enhanced E2(14K) activity associated with PC12 cell neurite outgrowth is sufficient to up-regulate E3-dependent protein ubiquitylation, we incubated the N-end rule substrate,125I-labeled beta -lactoglobulin (16, 20), with PC12 supernatants and determined the level of ubiquitylated substrate at steady state. To confirm that ubiquitylation was E3-dependent in these experiments, some assays included either a dipeptide (Leu-Ala) to block E3 binding to the bulky hydrophobic N terminus (leucine) of the substrate (20, 24) or a control dipeptide (Ala-Leu). SDS-PAGE and autoradiography verify the formation of ubiquitylated 125I-labeled beta -lactoglobulin, evident as a ladder of radiolabel migrating above the substrate at progressively higher 8.5-kDa increments (~28 kDa, ~36 kDa) and as a smear of higher mass radiolabel in overexposed autoradiograms (Fig. 3, lanes 2-5). When adjusted for total substrate (cpm) per assay tube, the intensity of these radiolabeled bands was 2-4 times greater in supernatants from NGF-treated cells, indicating that these supernatants had an enhanced capacity to ubiquitylate the exogenous substrate (Fig. 3, compare lanes 2 and 3). Consistent with an E3-dependent mechanism, the dipeptide Leu-Ala inhibited the formation of ubiquitylated 125I-labeled beta -lactoglobulin by 70% in supernatant from NGF-stimulated cells (Fig. 3, compare lane 5 with lanes 3 and 4) and nonstimulated cells (data not shown). The control dipeptide Ala-Leu was also slightly (10-15%) inhibitory (Fig. 3, compare lanes 3 and 4).


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Fig. 3.   Ubiquitylation of 125I-labeled beta -lactoglobulin is up-regulated in supernatant from NGF-treated PC12 cells. 125I-Labeled beta -lactoglobulin (4 µg, 7 × 105 cpm/µg) (lane 1) was incubated with supernatant from PC12 cells cultured in the absence (lane 2) or presence (lanes 3-5) of NGF (100 ng/ml, 5d). The dipeptides Ala-Leu (lane 4) or Leu-Ala (lane 5) were added to some assays (20 mM final concentration) (see "Experimental Procedures"). The boxed area of the autoradiogram is overexposed to reveal higher mass bands. The radiolabeled band at ~38 kDa is a contaminant or aggregated form of the substrate comprising <= 2% of the substrate (lane 1). Molecular mass markers are at the left. Ub1-[125I]beta-lactoglobulin, monubiquitylated substrate; Ub2-[125I]beta-lactoglobulin, diubiquitylated substrate; Ubn-[125I]beta-lactoglobulin, multiubiquitylated substrate. One of two independent experiments is shown.

We next determined if enhanced protein ubiquitylation in NGF-treated cells was reflected in enhanced rates of Ub-dependent proteolysis. We incubated 125I-labeled beta -lactoglobulin in ATP- and Ub-supplemented supernatants from PC12 cells that had been cultured in the presence or absence of NGF, and we determined the proportion of substrate that was degraded to acid-soluble cpm. The percent of 125I-labeled beta -lactoglobulin that was degraded by the UPP in vitro was not significantly different in supernatants from NGF-treated cells relative to nontreated cells (Fig. 4). Thus, the capacity for substrate degradation by the UPP did not increase in NGF-stimulated PC12 cells commensurate with enhanced rates of substrate ubiquitylation (Fig. 3).


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Fig. 4.   Ub-dependent proteolysis of 125I-labeled beta -lactoglobulin is not up-regulated in supernatant from NGF-treated PC12 cells. 125I-Labeled beta -lactoglobulin was incubated in PC12 cell supernatants as in Fig. 3. Substrate degradation was determined after 30 min as acid-soluble radioactivity. Rates of proteolysis remained linear during the incubation period. Ub-dependent proteolysis was determined as the proportion of total degradation that was ATP-dependent and inhibitable by MG132. One of two independent experiments performed in triplicate is shown.

Subsequent experiments assessed the roles of ubiquitylation and Ub-dependent proteolysis in neurite outgrowth. First, we induced neurite outgrowth with the proteasome inhibitor, clasto-lactacystin beta -lactone (30, 31). Within 2 h of exposure to the inhibitor, most PC12 cells became flattened, and neuritogenesis was observed as the extension of small neurite buds (data not shown). After 6-12 h of treatment, >50% of cells treated with clasto-lactacystin beta -lactone had extended one or more neurites, whereas <0.5% of control cells extended neurites (Fig. 5A, compare panels a and b). Cells treated with clasto-lactacystin beta -lactone remained viable for at least 48 h, during which time neurites increased in frequency and length. Significantly, neurite outgrowth was coincident with inhibition of Ub-dependent proteolysis, as evidenced by the accumulation in cells treated with clasto-lactacystin beta -lactone of endogenous Ub-protein conjugates (Fig. 5B, compare lanes 1 and 2) and the UPP substrate, p53 (32) (Fig. 5B, compare lanes 3 and 4). Accumulation of Ub-protein conjugates was maximal in the presence of 5 µM clasto-lactacystin beta -lactone. These results demonstrate that neurite outgrowth can be rapidly induced in PC12 cells coincident with (at least partial) inhibition of Ub-dependent proteolysis.


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Fig. 5.   Induction of neurite outgrowth by proteasome inhibitors is coincident with inhibition of Ub-dependent proteolysis. A, induction of neurite outgrowth by clasto-lactacystin beta -lactone. PC12 cells were incubated (12 h) in complete medium in the absence (top panel) or presence (bottom panel) of 5 µM clasto-lactacystin beta -lactone. Note neurite outgrowth in cells treated with inhibitor. Arrowheads point to representative cells with neurites >= 1 cell body length. One of seven representative assays is shown. B, inhibition of Ub-dependent proteolysis by clasto-lactacystin beta -lactone. PC12 cells were incubated in complete medium in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of clasto-lactacystin beta -lactone. Cells were harvested in SDS-PAGE sample buffer after 6 h, at which time >50% of the cells had extended neurites (as in Fig. 5A, bottom panel). Cell lysates were analyzed by SDS-PAGE and immunoblotting using antibodies against Ub-protein conjugates (lanes 1 and 2) or against p53 (lanes 3 and 4; see "Experimental Procedures"). Molecular mass markers are at right. One of two representative experiments is presented.

Next, we tested the effect of dipeptides on lactacystin-induced neurite outgrowth. The dipeptides used were Leu-Ala, which competes for the E3 hydrophobic site, and His-Ala, directed aginst the E3 basic site. In the initial experiments, cells pretreated with lactacystin (10 µM, 2 h) were incubated with either Leu-Ala (2 mM) or vehicle, and the proportion of cells that failed to extend neurites was measured after an additional 4 h of incubation (See "Experimental Procedures"). Incubation with Leu-Ala increased the proportion of cells that failed to extend neurites by almost 2-fold (32% ± 1% versus 17% ± 1%, p < 0.003, n = 2 experiments performed in quadruplicate). Subsequent experiments assessed the effect of dipeptides on the lengthening of elaborated neurites. Neurite outgrowth was induced by a 4-h pretreatment of cells with lactacystin followed by the addition of dipeptides, incubation for 20 h, and the quantification of cells with neurites greater than or equal to the length of the cell body. In these studies, the proportion of cells exhibiting neurites greater than or equal to the length of the cell body was reduced by 3-fold (15% versus 5%) in cultures treated with Leu-Ala as compared with control cultures (no dipeptide) (p < 0.001; Fig. 6, A and B). In contrast, the proportion of cells with neurites greater than or equal to the length of the cell body was not reduced in the presence of His-Ala (p > 0.05; Fig. 6, A and C). The selective inhibitory effect of Leu-Ala suggests that lactacystin-induced neurite outgrowth requires ubiquitylation of a subset of N-end rule substrate(s).


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Fig. 6.   The dipeptide Leu-Ala selectively inhibits neurite outgrowth induced by proteasome inhibition. A, neurite outgrowth was induced by exposing PC12 cells to 10 µM lactacystin for 4 h, followed by the addition of either buffer sham (panel a), 2 mM Leu-Ala (panel b), or 2 mM His-Ala (panel c). After an additional 20 h of incubation, the proportion PC12 cells (mean ±S.E.) with neurites greater than or equal to cell-body length was counted (arrows). B, histogram of neurite outgrowth data. Each bar represents counts from three microscopic fields of one culture dish. One of three representative experiments is presented.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Ubiquitylation is best understood as a protein degradation signal (1-5). In this work we demonstrate that growth factor-induced neurite outgrowth is associated with up-regulated activity of E2(14K) (Figs. 1C and 2) and enhanced capacity for E2(14K)/E3-dependent protein ubiquitylation (Fig. 3) but not with increased capacity for Ub-dependent proteolysis of E2(14K)/E3-dependent substrates (Fig. 4). We propose that the enhanced pool of ubiquitylated protein observed in NGF-stimulated cells (Fig. 1A; Ref. 12) results from elevated rates of protein ubiquitylation in the absence of comparable increases in rates of Ub-dependent proteolysis or rates of conjugate disassembly by isopeptidases.

The enhanced capacity for protein ubiquitylation in NGF-stimulated cells (Fig. 1, A and B) appears to reflect globally up-regulated activities of Ub-conjugating enzymes (E1s and E2s) (Fig. 1C). Coordinated regulation of Ub-conjugating enzymes characterizes other developmental transitions, including erythroid maturation (33) and insect metamorphosis (28). The molecular mechanism(s) underlying the coordinated regulation of Ub-conjugating enzymes during development are unknown. Preliminary studies suggest that protein levels of E1s and at least some E2s (E2(14K) and E2(25K)) are not increased in NGF-stimulated cells.2 A potential alternative mechanism to account for up-regulation of E1 and E2 activities is the 2-fold increase in reduced GSH reported for NGF-stimulated PC12 cells (34), because we previously demonstrated that E1 and E2 activities are posttranslationally up-regulated by increases in the cellular GSH:GSSG ratio (35, 36). Our data do not rule out the possibility that the enhanced capacity for ubiquitylation in NGF-stimulated PC12 cells also reflects increased availability of UPP substrates.

This study is the first report of the ability of proteasome inhibitors to induce neurite outgrowth (30, 31, 37-39) coincident with stabilization of UPP substrates and their ubiquitylated forms (40-43). The data demonstrate that lactacystin and clasto-lactacystin beta -lactone can induce neuritogenesis in PC12 cells, in contrast with a prior study (31) that reported the inability of these inhibitors to induce differentiation in PC12 cells. This discrepancy is likely to reflect the narrow dose range (<= 1 log) over which the neuritogenic effects of proteasome inhibitors are manifest (38).2 Neurite outgrowth that is induced in PC12 cells by lactacystin, clasto-lactacystin beta -lactone, or by the peptidyl aldehyde MG132 (0.1 µM)2 is associated with inhibition of Ub-dependent proteolysis (stabilization of p53) and concommitant increases in Ub-protein conjugates (Fig. 5, A and B). Induction of neurite outgrowth coincident with inhibition of Ub-dependent proteolysis allows us to reconcile our inability to detect increased rates of proteolysis in preparations of NGF-treated cells, and argues against the hypothesized requirement for Ub-dependent proteolysis in neurite outgrowth (16-18). In fact, the data suggest that Ub-dependent proteolysis is a negative regulator of this process. Ub-independent proteolytic activities of the proteasome have also been proposed to negatively regulate neurite outgrowth (31, 38, 39).

Ub-dependent proteolysis of regulatory proteins is firmly established as a mechanism by which the UPP controls key cellular events (1-4). Thus, negative regulation of neurite outgrowth by the UPP is likely to involve selective, rapid degradation of neurite-promoting protein(s) in nondifferentiated PC12 cells. Lactacystin and other proteasome inhibitors induce neurite outgrowth in the absence of growth factors by stabilizing levels of these putative neuritogenic substrates. Proteasome inhibitors may also induce neurite outgrowth by stabilizing levels of neuritogenic Ub-protein conjugates. Ub-protein conjugates are generally viewed as biologically inactive proteolytic intermediates awaiting degradation. However, disruption of lactacystin-induced neurite outgrowth with a dipeptide inhibitor of E3 (Fig. 5C) suggests the intriguing possibility that (some) E3-dependent Ub-protein conjugates possess neurite-promoting biological activity. A caveat to these arguments is that despite the well documented inhibitory effect of dipeptides on E3 in cell-free preparations (Refs. 19 and 20 and present study) and in yeast in vivo (23), it is not established with certainty that dipeptides selectively inhibit E3 in cultured mammalian cells. Other molecular targets of dipeptides (44) could therefore modulate neurite outgrowth as well.

The postulated neurite-promoting activity of E3-generated Ub-protein conjugates is consistent with four features of physiologically (i.e. NGF)-induced neurite outgrowth in PC12 cells: (i) enhanced Ub-conjugating activity and levels of ubiquitylated protein, (ii) up-regulated activity of E2(14K) and increased capacity for E3-dependent ubiquitylation, (iii) no coincident up-regulation of proteolysis of E3-dependent Ub-protein conjugates, and (iv) inhibition by E3-directed dipeptides (16, 17). Future studies will determine how increases in the cellular pool of Ub-protein conjugates promote neurite outgrowth and other physiological responses of neurons in which protein flux through the UPP is altered (45).

    ACKNOWLEDGEMENTS

We thank Dr. Art Tischler and members of his laboratory for invaluable discussions and assistance with PC12 cell culture.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants EY11703 (to M. O.) and GM34009 (to A. L. H.), United States Department of Agriculture (USDA) Contract 53-3K06-0-1 (to A. T.), and USDA intramural grant funds (to A. T. and J. J.).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: JMUSDA-HNRCA at Tufts University, 711 Washington St., Boston, MA 02111. Fax: 617-556-3344; E-mail: Obin_c1{at}HNRC.tufts.edu.

2 M. Obin, X. Gong, and A. Taylor, unpublished data.

    ABBREVIATIONS

The abbreviations used are: Ub, ubiquitin; Ubal, ubiquitin aldehyde; UPP, ubiquitin-proteasome pathway; E1, ubiquitin-activating enzyme; E2, ubiquitin-carrier protein; E2(14K), ~14-kilodalton E2; E2(25K), 25-kilodalton E2; E3, ubiquitin isopeptide ligase; Ub~E1, thiol ester of ubiquitin and E1; Ub~E2, thiol ester of ubiquitin and E2; NGF, nerve growth factor; PC12 cells, rat pheochromocytoma cells; ECL, enhanced chemiluminescence; MG132, carbobenzoxyl-leucinyl-leucinyl-leucinal; PAGE, polyacrylamide gel electrophoresis.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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