From the 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
Department of Biochemistry, Medical College of Wisconsin,
Milwaukee, Wisconsin 53226
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ABSTRACT |
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Nerve growth factor (NGF)-induced neurite
outgrowth from rat PC12 cells was coincident with elevated ( 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.
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 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 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 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 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
(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
-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
-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
-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
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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
-lactoglobulin were iodinated by reaction with chloramine T as
described (26).
-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).
80 °C.
-lactoglobulin was
measured in assays (as above) containing 2.0 µg of
125I-labeled
-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
-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
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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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 -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
-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
-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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We next determined if enhanced protein ubiquitylation in NGF-treated
cells was reflected in enhanced rates of Ub-dependent proteolysis. We incubated 125I-labeled -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
-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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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 -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
-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
-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
-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
-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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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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DISCUSSION |
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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 -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
-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).
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ACKNOWLEDGEMENTS |
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We thank Dr. Art Tischler and members of his laboratory for invaluable discussions and assistance with PC12 cell culture.
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FOOTNOTES |
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* 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.
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ABBREVIATIONS |
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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.
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