(Received for publication, July 20, 1995; and in revised form, September 11, 1995)
From the
Among the molecular mechanisms that control the cell division
cycle, proteolysis has emerged as a key regulatory process enabling
cells to pass critical check points. Such proteolysis involves a
cascade of enzymes including a multisubunit complex termed 26S
proteasome. Here we report on the analysis of a novel mouse cDNA
encoding the puromycin-sensitive aminopeptidase (PSA) and on its
expression in COS cells and 3T3 fibroblasts. PSA is 27-40%
homologous to several known Zn-binding
aminopeptidases including aminopeptidase N. Immunohistochemical
analysis revealed that PSA is localized to the cytoplasm and to the
nucleus and associates with microtubules of the spindle apparatus
during mitosis. Furthermore, puromycin and bestatin both arrested the
cell cycle, leading to an accumulation of cells in G
/M
phase, and ultimately induced cells to undergo apoptosis at
concentrations that inhibit PSA. Control experiments including
cycloheximide further suggested that the induction of apoptosis by
puromycin was not attributable to inhibition of protein synthesis.
Taken together, these data favor the novel idea that PSA participates
in proteolytic events essential for cell growth and viability.
Under basal metabolic conditions, the turnover of intracellular
proteins is regulated mainly by non-lysosomal mechanisms involving 26S
proteasomes. These multisubunit complexes are localized to the
cytoplasm and to the
nucleus(1, 2, 3, 4, 5) ,
and they associate with the mitotic spindle
apparatus(4, 5, 6) . Polyubiquitinated
proteins and some other proteins are degraded by proteasomes in an
ATP-dependent
manner(7, 8, 9, 10, 11) .
Interestingly, in yeast, mutations in the proteasome subunit genes mts2, cim-3/SUG1, or cim-5/MSS1 arrest the
cell division cycle during G/M phase(12, 13) suggesting essential roles for proteasomes during mitosis
such as the ubiquitin-dependent degradation of
cyclins(14, 15, 16) , the latter being
required at the onset of anaphase to inactivate maturation promoting
factor (17, 18) and to complete mitosis(19) .
Additionally, proteasome-mediated proteolysis promotes sister chromatid
segregation (20) and must further persist until early
G
phase to allow G
cyclins to become activated (21) . Taken together, these findings suggest that proteolysis
is of fundamental importance for cell cycle regulation.
However, the mechanism by which the substrates of proteasomes are degraded into small peptides and amino acids is not yet fully understood. Possibly, serine or cysteine proteases are involved since cyclin degradation in oocyte extracts is inhibited by tosyl-lysine chloromethyl ketone(22) . Tests with model peptides further indicated that proteasomes exhibit several distinct hydrolytic activities designated as chymotrypsin-like, trypsin-like, peptidyl-glutamyl peptide-hydrolyzing, branched chain amino acid-preferring, and small neutral amino acid-preferring activities, respectively(23) . More recently, a novel proteolytic mechanism was identified in proteasomes of Thermoplasma acidophilum, which involves a threonine residue(24, 25) . Conservation of this threonine suggests that a similar mechanism may account for some of the activities of eukaryotic proteasomes. Finally, it has been assumed that exopeptidases act distally from endoproteinases to prevent accumulation of peptides and to allow recycling of amino acids(10) .
Aminopeptidases hydrolyze N-terminal amino acids of oligopeptides.
Their widespread distribution in plant and animal tissues as well as in
bacteria and fungi suggest that they play important roles in various
biological processes (for review see (26) ). The observation
that aminopeptidase inhibitors can suppress cell
proliferation(27, 28) indicated that also cell growth
may depend on aminopeptidase activity. However, most of the available
data on aminopeptidases derive from studies on the metabolism of
neuropeptides (for review see (29) ) such as enkephalins, where
the Tyr-Gly
bond is susceptible to
aminopeptidase
cleavage(30, 31, 32, 33) . However,
the aminopeptidase that primarily is relevant for degrading enkephalins
and possibly other neuropeptides has not yet been identified
unequivocally. A candidate is the ectoenzyme aminopeptidase N, which in
brain was detected on synaptic membranes(34) , although at a
relatively low level. A puromycin-sensitive aminopeptidase (PSA) (
)has also been considered to play a
role(31, 35, 36) . PSA is approximately
100-fold more sensitive to puromycin than aminopeptidase N, and it is
present in most organs, including brain, where it is most abundant and
wherefrom it was purified(35, 36) . Moreover, PSA
efficiently degrades enkephalins in vitro(35) , and it
is present in brain in much higher amounts than aminopeptidase N.
However, a function for PSA in neuropeptide metabolism has been
questioned since it is was found to be a cytoplasmic
protein(37) .
The present paper reports on the cloning and
expression of PSA, its subcellular localization, and inhibitor studies
that suggest a novel potential function of PSA. PSA is shown to
localize both to the nucleus and to the cytoplasm and to associate with
the spindle apparatus during mitosis. Finally, we show that a block of
the cell cycle by PSA inhibitors is associated with an accumulation of
cells in G/M phase and with the induction of DNA
fragmentation. These findings are discussed in the context of the novel
idea that PSA may be involved in cell cycle regulation.
To
quantitate protein synthesis, COS-7 cells were washed twice with PBS,
resuspended at a density of 10 ml
in
leucine-free RPMI medium containing 5% fetal calf serum, and mixed in
aliquots of 0.5 ml with equal volumes of the same medium containing
puromycin or cycloheximide at various concentrations. After
preincubating these suspensions at 37 °C for 45 min, 5 µCi of
[
H]leucine (144 Ci/mmol; Dupont NEN) was added to
each sample for 30 min. Incorporated radioactivity was precipitated by
adding 1 ml of ice-cold 10% trichloroacetic acid followed by incubation
on ice for 30 min. Precipitates were collected on Whatman GF/C filters
and washed 3 times with 3 ml of 5% trichloroacetic acid using a vacuum
manifold (Millipore Corp., Bedford, MA). After air-drying the filters,
bound radioactivity was quantitated by scintillation counting. Values
were corrected by subtracting background (7-8
10
cpm) as determined in the absence of cells. Values
represent means of duplicate measurements. Stock solutions of bestatin
(25 mg/ml), puromycin (25 mM), and cycloheximide (10 mg/ml)
were prepared in deionized water. All inhibitors were purchased from
Sigma.
Figure 1:
Nucleotide sequence and deduced protein
sequence of the puromycin-sensitive aminopeptidase cDNA. The residues
presumably involved in the coordination of a catalytically active zinc
ion are boxed. Clusters of basic amino acids representing
potential nuclear localization signals are marked by filled
squares. Residues labeled with open squares are part of a
potential complementary nuclear localization signal. The sequences that
resemble both known microtubule-binding sites and a motif conserved
among 26S proteasome subunits (for details see Fig. 3) are underlined. , potential N-glycosylation sites;
, potential casein kinase phosphorylation sites; triangle, potential phosphorylation site for
cAMP/cGMP-dependent kinase.
Figure 3: PSA is induced in transiently transfected COS cells as shown by Western blot analysis. Transfection of COS cells with PSA leads to the induction of a protein with a molecular mass of 100 kDa and reacting with an antiserum against purified rat PSA (lane 1) but not with a preimmune serum (lane 3). The same band is present in mock-transfected cells, although at a lower level (lane 2). The artifactual staining of protein bands that were not induced in PSA-transfected cells was due to the streptavidin/avidin detection system.
In the open reading frame (ORF), two potential translational start sites were identified at nucleotide positions 106 and 241, respectively, the former being immediately preceded by a stop codon (Fig. 1). Translational start at the first AUG is expected to give rise to a protein containing a hydrophobic N-terminal sequence of 26 residues, which is interrupted after 10 amino acids by two arginines. Separated from these two arginines by a spacer of 19 amino acids, a stretch of five additional basic residues is located further downstream. Together, these basic residues form a potential bipartite nuclear localization signal (NLS) (Fig. 1). Interestingly, four out of five amino acids at positions 727-731 are also basic in nature and thus may represent a second NLS. In addition to these two NLSs, one potential complementary NLS was identified at positions 111-115 consisting of a stretch of negatively charged residues. Furthermore, nine potential phosphorylation sites for casein kinase II and one for cAMP/cGMP-dependent kinase were identified, and two potential N-glycosylation sites are at positions 63 and 649, respectively (Fig. 1).
Further analyses of the predicted amino acid sequence revealed that the residues 269-285 (termed PSA I) and 683-701 (PSA II) were 72 and 40% similar, respectively, to the proteasome motif described by Zwickl et al.(43) (Fig. 2b). In addition, these sequences were found to resemble also the microtubule binding sites in tau protein and two other microtubule binding proteins, MAP-2 and MAP-4 (44, 45, 46, 47) (Fig. 2a). Note that within PSA I and II, the only residues that are not similar to either the proteasome motif or the microtubule binding sites or to both are the two glutamates and the second proline residue within PSA II.
Figure 2: Sequences within PSA align either with known microtubule-binding sites and with a proteasome motif or with the N-terminal amino acids of purified rat PSA, respectively. a, alignment of the sequence motifs PSA I and II to the microtubule-binding sites of tau protein and microtubule-associated protein-2 and -4. b, alignment of PSA I and II to the proteasome motif sequences of multiple proteasome subunits. c, alignment of PSA residues 47-64 to the N-terminal sequence of rat PSA(37) . Note that mouse PSA differs only in a single residue (Ala instead of Thr) from the N terminus of the purified rat PSA.
Interestingly, at positions 47-64 of the ORF the residues
were almost identical to the N-terminal sequence of the purified rat
puromycin-sensitive aminopeptidase(37) . The only difference
observed in the mouse sequence was Ala, which is a
threonine in position 9 of rat PSA (Fig. 2c). In
addition, at position 46 of the mouse ORF, a methionine is present,
which on the mRNA level is flanked by a Kozak consensus-like sequence.
This suggests that translation may be initiated at the second AUG of
the ORF, giving rise to a protein with an N terminus corresponding to
that of rat PSA. That the mouse cDNA is in fact the homologue of the
rat PSA became clearly evident from a comparison of the mouse cDNA
sequence to the sequence of nine tryptic peptides isolated from rat
PSA. As shown in Table 2, there is only one amino acid difference
among the nine tryptic peptides containing 118 amino acids (His
in the mouse sequence is Tyr in the rat sequence). These data,
coupled with the N-terminal sequence data, indicate a greater than 98%
sequence identity between the rat and mouse PSA.
Figure 4: Induction of aminopeptidase activity in extracts of transfected COS cells. Crude extracts of PSA-transfected (PSA) or mock-transfected (SVL) COS cells were incubated with various aminoacyl p-nitroanilide substrates to assess peptidase activity. The release of p-nitroaniline was measured spectrophotometrically.
Figure 5:
Tissue distribution of PSA mRNA expression
as determined by Northern blot analysis. A multiple tissue Northern
blot (1 µg/ml poly(A) mRNA per lane) was hybridized with a PSA cDNA
probe and exposed to x-ray film for 3 days (upper panel).
Equal loading of RNA was controlled by rehybridizing the same filter to
a human -actin cDNA probe (lower
panel).
Figure 6: Immunocytochemical localization of PSA in COS cells. a, transfected cells (arrows) are readily identified by increased PSA staining of the Golgi-cytoplasmic microtubule complex region as compared with cells resisting transfection (arrowheads). Fixation was performed in a mixture of acetone and ethanol. b, inhibition of PSA staining by preabsorbing the anti-PSA IgG with purified rat PSA. c, in cells entering M phase of the cell cycle, endogenous PSA is detected in association with arising asters. d, the same cell as in panel c is shown under Nomarsky optics to reveal the presence of microtubules and to identify the two most intensely stained spots as microtubule organizing centers (arrows). e, in metaphase cells, PSA is associated with the spindle apparatus. In panels c-e, the cells were extracted with detergent prior to fixation in formalin in order to substantiate that the association of PSA and microtubules is specific. Note that some unidentified microsomes also stained positive for PSA even after extraction by detergent (arrows in panel e). Size bars, 20 µm.
The finding that in COS cells the subcellular localization of PSA was heterogeneous led to the hypothesis that it may be regulated in a cell cycle-specific manner. To further evaluate this possibility, endogenous PSA was localized within cells of the nontransformed murine fibroblast cell line Swiss 3T3. Like all other cell lines examined so far, 3T3 fibroblasts express PSA mRNA (data not shown). As shown in Fig. 7, 3T3 cells exhibited similar staining patterns as observed in untransfected COS cells, a difference being the nuclear staining observed in a subpopulation of 3T3 cells (Fig. 7a). Such nuclear staining appeared to be associated with cells that were in prophase of the cell cycle (Fig. 7c). During metaphase, PSA staining was associated with the spindle apparatus (Fig. 7d), and it became concentrated around the chromosomes during anaphase (Fig. 7e). Most intense staining was observed in the newly formed nuclei and in the cytoplasm during telophase (Fig. 7f). As in COS cells (Fig. 6, c-e), in 3T3 cells the microtubule-associated staining resisted extraction with detergent (not shown). Taken together, these data support the conclusion that PSA binds specifically to mitotic spindles and may play a role in proteolysis during mitosis.
Figure 7: Immunocytochemical localization of endogenous PSA in Swiss 3T3 fibroblasts. a, exponentially growing cells exhibiting cytoplasmic (arrowhead) and, in a subpopulation, also a punctate nuclear staining (boldface arrows). Most intense staining is seen in M phase cells (open arrows). The bridge connecting cells during cytokinesis is also stained (thin arrow). b, background staining after preabsorption of the anti-PSA IgG with purified rat PSA. c, some cells exhibiting nuclear staining appeared to be in prophase as based on signs of nuclear membrane disintegration and chromatin condensation (arrowhead). In a neighboring early prophase cell (short arrow), nuclear staining is concentrated in the center of a growing aster (long arrow). d, during metaphase, PSA is detected in association with the spindle apparatus and in the cytoplasm. e, PSA staining during anaphase. f, late telophase cells (arrowheads) undergoing cytokinesis. The bridge is out of focus. Size bars, 20 µm.
Figure 8:
Inhibitors of PSA induce an accumulation
of cells in G/M phase. Flow cytometric analysis (dotted
line) of the DNA content of COS-7 cells stimulated with puromycin,
bestatin, or the etoposide VP16 (positive control) is shown. Shaded
areas represent the fraction of cells that are in
G
/G
, S, and G
/M phase,
respectively, as calculated by the program
Multicycle.
Figure 9:
Puromycin, but not cycloheximide, inhibits
DNA synthesis at concentrations significantly below those required to
inhibit protein synthesis. a, COS cells were incubated for 40
h with puromycin (PM), cycloheximide (CHX), or
bestatin before adding [H]thymidine (
H-Thd) to assess proliferation relative to that
in the absence of inhibitors (100%). b, dose-dependent
inhibition of protein synthesis by puromycin or
cycloheximide.
Interestingly, cells that were treated with puromycin or bestatin showed the morphological characteristics of apoptosis such as chromatin condensation around the margin of the nuclei, membrane blebbing and DNA fragmentation (Fig. 10, B and C). Fragmentation of DNA was assessed by in situ DNA end-labeling in COS cells that were treated with bestatin (100 µg/ml) or puromycin (5 µM); DNA breaks were detected in 76 (±5)% and 81 (±3)% of the cells, respectively. This contrasts with cycloheximide (5 µg/ml), which ultimately leads to cell death without inducing more than 3% of the cells to undergo DNA fragmentation. These data were confirmed by gel electrophoretic analysis showing significant DNA fragmentation in puromycin- and bestatin-treated cells but not in cells exposed to cycloheximide (Fig. 10E). These findings indicate that aminopeptidase inhibitors can induce cells to die via apoptosis.
Figure 10: Aminopeptidase inhibitors, but not cycloheximide, induce apoptosis. COS cells were treated for 40 h (A-D) or 12 h (E) with cycloheximide (5 µg/ml), puromycin (5 µM), or bestatin (100 µg/ml). DNA fragmentation was assessed by in situ DNA end labeling (A-D) and by agarose gel electrophoresis (E). DNA breaks were detected in cells treated with puromycin (B) or bestatin (C) but not in untreated (A) or cycloheximide-treated cells (D). Likewise, significant DNA fragmentation was observed in puromycin- and bestatin-treated cells (E, lanes 3 and 5) but not in cells incubated in control medium (E, lanes 1 and 4) or in cycloheximide (E, lane 2).
Recently, we have isolated from a human fetal brain cDNA
library a novel cDNA that was assumed to encode for a
Zn-binding aminopeptidase as based on sequence
information.
To allow subsequent characterization of this
novel putative aminopeptidase in the mouse, the corresponding murine
cDNA was isolated and sequenced. Here we report that the deduced
protein sequence is similar to known aminopeptidases such as
aminopeptidase N, this similarity being most concentrated in a region
that contains the sequence motif
HEXXHX
E. Similar motifs have been
identified in several metallopeptidases where they coordinate a zinc
ion at the active site(48) . Thus, the HELAH sequence encoded
by our novel clone presumably has the same function. Interestingly,
both the N terminus and a number of tryptic peptides of rat PSA were
essentially identical to this clone. Therefore, the novel clone was
concluded to encode the murine homologue of rat PSA. Consistently, the
mouse protein cross-reacted with an anti-rat PSA antiserum if expressed
in COS cells, and it had the expected molecular mass of 100 kDa.
However, the mouse ORF appeared to be extended by 46 amino acids as
compared with purified rat PSA(37) . This indicates either that
rat PSA is derived from a precursor or that its N terminus results from
translational initiation at an internal AUG. In fact, translational
initiation at the second AUG in the mouse ORF would give rise to an N
terminus corresponding precisely to that of rat PSA. The observation
that only the second but not the first AUG codon of the ORF is flanked
by a Kozak consensus-like sequence further supports the idea that
translation at least under certain conditions is initiated
preferentially at the second AUG. It is unlikely that the N terminus of
rat PSA is generated by cleavage of a precursor since the in vitro translated mouse protein is not susceptible to cleavage by
microsomal membranes. (
)Likewise, the finding that the
N-terminal extension is conserved also in the human ORF
suggests that it does not simply reflect a species difference.
Two potential NLS are present in the PSA sequence, which may account for the observed nuclear localization of PSA in a subpopulation of cells. Furthermore, a potential complementary NLS was identified. In proteasomes, such complementary NLS have been speculated to account for the inhibition of nuclear translocation since several proteasome subunits harbor NLS and cNLS motifs in cis(49) . According to this model, tyrosine phosphorylation of proteasome subunits may cause a conformational change, which in turn disrupts NLS-cNLS interactions and allows proteasomes to be translocated to the nucleus(49) . Interestingly, PSA also harbors several potential phosphorylation sites. It will be important to determine whether PSA undergoes phosphorylation and whether this is a mechanism to regulate its subcellular distribution. Also, it should be addressed whether the localization of PSA within a cell may be regulated by alternative translational initiation since one of the putative NLS resides at the N terminus, which apparently is not necessarily translated.
With
respect to the potential function of PSA, two other motifs, termed PSA
I and II, appeared to be interesting. PSA I and II show significant
similarity to the microtubule binding sites of MAP-2, MAP-4, and tau
protein (44, 45, 46, 47) as well as
to a sequence that is highly conserved among -type proteasome
subunits of various species(43) . PSA I more closely resembled
the proteasome motif than did PSA II, whereas PSA II C-terminally
contained the residues PGEG, which aligned better to microtubule
binding sites than did the corresponding residues of PSA I. Given these
similarities with microtubule binding sites, PSA I and PSA II are
candidates to mediate the observed association of PSA with
microtubules. Likewise, it is tempting to speculate, whether PSA may
functionally interact with proteasomes given that PSA I and II resemble
a proteasome motif that is thought to play a role in the assembly of
the proteasome multisubunit complex.
Subcellular localization of PSA
by immunocytochemistry in COS-7 and in Swiss 3T3 fibroblasts further
revealed several interesting features. First, in both cell types the
staining of individual cells was heterogeneous, some cells showing PSA
staining exclusively in a cytoplasmic compartment where the origin of
the cytoplasmic microtubule complex and the Golgi complex are
localized, others exhibiting nuclear staining as well. A fraction of
those cells showing nuclear staining were identified to be in prophase
of the cell cycle as based on chromatin condensation and breakdown of
the nuclear membrane and on the identification of newly arising asters.
Furthermore, PSA was found to associate with the spindle apparatus
during mitosis and remained detectable at a high level in mitotic and
early G cells. Note that microtubule-bound PSA was
detectable even after extracting soluble PSA by detergent. In contrast
to microtubule-associated PSA, cytoplasmic PSA was eluted almost
completely by extraction with detergent, the residual staining being
associated with some unidentified vesicular structures. This
observation is consistent with the previous prediction that PSA may
belong to a class of amphitropic proteins that exist both in soluble
and membrane-associated forms(37) . Interestingly, 26S
proteasomes, like PSA, have been localized to the cytoplasm and to the
nucleus and to the spindle apparatus in a variety of cell types and by
several independent
laboratories(1, 2, 3, 4, 5) .
This is consistent with the idea that PSA may contribute to or act
downstream of proteolytic processes known to involve proteasomes.
In
an attempt to assess whether PSA may be essential for cell division,
COS cells that had been treated with the PSA inhibitors puromycin or
bestatin were analyzed by flow cytometry for their content of DNA. A
significant accumulation of cells in G/M phase was observed
within hours, indicating that puromycin-sensitive and aminopeptidase
activities are required to complete mitosis. The idea that PSA is
involved in cell cycle-regulating proteolysis is consistent with
previous reports demonstrating that the aminopeptidase inhibitor
bestatin, at concentrations similar to those used in the present study,
inhibits the proliferation of primary
hepatocytes(27, 28) . However, according to these
authors, bestatin blocked primary hepatocytes reversibly at the
G
/S transition upon their release from serum starvation,
and cell death was not reported. In contrast, we observed that growth
inhibition by bestatin is associated with profound and rapid induction
of apoptosis. A possible explanation for this discrepancy is that at
G
/S, additional aminopeptidase activities may be required
that are sensitive to bestatin but not to puromycin. Support for this
idea arises from the fact that bestatin, unlike puromycin,
significantly reduced the number of cells in S phase also in our
asynchronously growing cultures. Given the need for relatively high
concentrations of bestatin, though, we cannot fully rule out the
possibility that some nonspecific toxicity may account for this
reduction. On the other hand, the fact that bestatin like puromycin
induced a block preferentially during G
/M phase argues that
bestatin does act specifically and that pharmacological concentrations
of bestatin may rather be required due to its limited cellular uptake.
The observation that puromycin, but not cycloheximide, induced DNA fragmentation similar to that observed in the presence of bestatin further suggests that bestatin and puromycin may both affect cell viability by specifically inhibiting PSA activity or a similar aminopeptidase. Moreover, the finding that puromycin induces apoptosis is consistent with previous results reported by Davidoff and Mendelow (50, 51) for a human erythroid leukemia cell line. Although these authors did not discuss the possibility that puromycin-induced apoptosis might result from inhibition of PSA activity, this view is supported by the present data showing that the half-maximal effective dose of puromycin is on the order of 1 µM both for suppressing DNA synthesis and inhibiting PSA activity, whereas no detectable inhibition of protein synthesis is observed at 1 µM puromycin.
An interesting but maybe too simple explanation for the common effects of puromycin and bestatin to induce apoptosis would be that both of these inhibitors interfere with the proteolytic degradation of some important cell cycle regulators. For example, inhibition of cyclin degradation may result in excessive activation of the kinase p34cdc2, which in turn can induce apoptosis(52) . Likewise, accumulation of ornithine decarboxylase, which is normally degraded by proteasomes, can lead to the induction of apoptosis(53) . Alternatively, inhibition of PSA-like activity may result in the accumulation of a peptide that by itself may become toxic above a certain threshold concentration.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U35646[GenBank].