(Received for publication, June 27, 1995; and in revised form, October 19, 1995)
From the
Regulated degradation of ornithine decarboxylase (ODC) is
mediated by its association with the inducible protein antizyme. The N
terminus of antizyme (NAZ), although unneeded for the interaction with
ODC, must be present to induce degradation. We report here that
covalently grafting NAZ to ODC confers lability that normally results
from the non-covalent association of native antizyme and ODC. To
determine whether NAZ could act similarly as a modular functional
domain when grafted to other proteins, we fused it to a region of
cyclin B (amino acids 13-90) capable of undergoing degradation or
to cyclin B (amino acids 13-59), which is not subject to
degradation. The association with NAZ made both NAZ-cyclin
B and NAZ-cyclin B
unstable. Furthermore, NAZ and cyclin B 13-59 were together
able to induce in vitro degradation of Trypanosoma brucei ODC, a stable protein. The ODC-antizyme complex bound to the 26 S
protease but not the 20 S proteasome, consistent with the observation
that ODC degradation is mediated by the 26 S protease. The association
was shown to be independent of NAZ, suggesting that NAZ does not act as
a recognition signal.
Cellular polyamines are essential for cells to grow and
proliferate. Ornithine decarboxylase (ODC), ()the key enzyme
in the biosynthesis of the polyamines, is highly regulated. Its
activity is dramatically increased by stimulating cell growth and
decreased by excess polyamines. Feedback regulation of ODC activity by
polyamines occurs via induction of the protein
antizyme(1, 2, 3, 4, 5) .
Antizyme binds to ODC, inhibits its activity, and accelerates its
degradation. The determinants within each protein needed for their
association have been identified; one element is near the N terminus of
ODC(6) , and the other element is the C-terminal half of
antizyme(7) . Besides regions that bring the two proteins
together, two additional elements are necessary for antizyme-dependent
degradation to occur. Both the C-terminal degradation domain of ODC (8) and the N terminus of antizyme are required for proteolysis
of ODC. The C terminus of ODC has been characterized as a degradation
domain that is sufficient for polyamine-independent basal
degradation(8, 9) . Deletion forms of antizyme devoid
of the N-terminal half still interact with ODC, but accelerated ODC
degradation can be directed only by molecules in which an antizyme
N-terminal region is also present(7) .
ODC, like other
short-lived proteins, is degraded by the 26 S protease in an
ATP-dependent manner. Ubiquitination is a modification that triggers
proteolysis of many short-lived proteins (10, 11, 12, 13) , but ODC does not
utilize this mechanism. Instead, regulated degradation of ODC requires
association with antizyme(14, 15, 16) . The
means by which the N terminus of antizyme (NAZ) acts in place of
ubiquitin for ODC degradation is unclear. It might function as a signal
domain to promote recognition by the 26 S protease or as an activation
domain to stimulate degradation. Here we report that AZ contains a
module that can be grafted to other proteins to make them labile. We
show also that the association of the ODCAZ complex with 26 S
protease is independent of NAZ.
Figure 1:
In vitro degradation of MODC
induced by fusion to NAZ. The time course of in vitro degradation of [S]methionine-labeled
proteins was examined. The labeled proteins remaining undegraded after
the indicated period of incubation were examined by SDS-PAGE and
autoradiography. A, structure of AZ and fusion proteins. Black bar, NAZ; fine cross-hatched bar, CAZ; coarse cross-hatched bar, MODC. B, MODC fused to the
C-terminal half of AZ (CAZ
MODC) or to the N-terminal half of AZ
(NAZ
MODC) were subjected to degradation for 0, 1, and 2 h. The
position of migration of each is indicated by an arrow.
Figure 2:
TbODC degradation induced by NAZ and C
termini of mouse ODC. A, structure of TbODC and fusions to NAZ
and C-terminal regions of MODC (MODC amino acids 376-461 or
422-461). Open bar, TbODC; black bar, NAZ; cross-hatched bar, MODC C termini. B, TbODC, Tb376M,
NAZ-TbODC, and NAZ-Tb376M were translated in vitro. The
[S]methionine-labeled proteins were subjected to
degradation and analyzed as in Fig. 1. C, quantitation
of degradation of TbODC, Tb376M, NAZ-TbODC, and NAZ-Tb376M. D,
Tb422M and NAZ-Tb422M degradation.
To test whether NAZ was capable of inducing Tb376M degradation in vitro, we coupled NAZ in front of the protein, to make NAZ-Tb376M, as we did for NAZ-MODC (Fig. 2A). In vitro degradation analysis showed that NAZ-Tb376M was degraded (Fig. 2B). Both NAZ and the C terminus of mouse ODC were needed to induce efficient degradation of TbODC; neither Tb376M nor NAZ-TbODC alone was capable of changing the stability of TbODC (Fig. 2B). Densitometric analysis of the data of Fig. 2B is displayed as Fig. 2C. NAZ also stimulated the degradation of Tb422M (Fig. 2D). NAZ-Tb422M contains the entire open reading frame of trypanosome ODC except for the last 2 amino acids, sandwiched between NAZ and the C terminus (amino acids 422-461) of mouse ODC. Therefore, these two functional domains are together necessary and sufficient to cause in vitro degradation of a stable protein, trypanosomal ODC.
We made control fusion proteins consisting of NAZ-TbODC extended at its C terminus by either full-length human papillomavirus 16 E6 (151 amino acids) or the N-terminal 90 amino acids of that protein to determine whether any extension, regardless of sequence specificity, can induce degradation of NAZ-TbODC. Both fusion proteins were subjected to the degradation assay. During the 2-h incubation, the proteins remained undegraded (data not shown). Therefore, the C terminus of mouse ODC must contain a specific functional sequence motif that is able to cooperate with NAZ.
Antizyme-mediated degradation of ODC by the 26 S
proteasome is independent of ubiquitination(16) . To examine
whether degradation induced by appending NAZ involves ubiquitin
modification, we incubated NAZ-Tb376M and NAZ-Tb422M as for in
vitro degradation but substituting ATPS in place of ATP.
ATP
S has been shown to block degradation but not
ubiquitination(25) , thereby leading to the accumulation of
high molecular weight forms of a target protein decorated by multiple
ubiquitin chains. Both NAZ-Tb376M and NAZ-Tb422M were degraded in the
presence of ATP (Fig. 3A). Substitution of ATP
S
for ATP blocked degradation, but high molecular weight conjugates of
NAZ-Tb376M and NAZ-Tb422M did not appear. As a positive control, we
used HPV16 E6-mediated degradation of p53, which is
ubiquitin-dependent. In the presence of E6 and ATP, p53 was degraded (Fig. 3A). When ATP
S was used in place of ATP,
degradation was blocked and high molecular weight forms of p53
accumulated. Immunoprecipitation with a monoclonal antibody to p53
(PAb421) followed by Western blot analysis using anti-ubiquitin
antiserum confirmed that the high molecular weight proteins enhanced in
the presence of ATP
S consisted of polyubiquitinated p53 (Fig. 3B). Therefore, NAZ-induced degradation, like
antizyme-mediated degradation of ODC, is ubiquitin-independent.
Figure 3:
Effect of ATPS on degradation of
NAZ-Tb376M and NAZ-Tb422M. A, proteolysis was induced by
co-incubation of oncoprotein p53 with E6 (left three lanes) or
by fusion of NAZ to Tb376M and Tb422M (right six lanes).
Samples were analyzed at time 0 or after 2 h of incubation. As
indicated, reactions were performed with ATP or replacing ATP with
ATP
S. B, the composition of conjugates produced as in A was tested by immunoprecipitation with anti-p53 antibody
PAb421, separation on SDS-PAGE, and (lanes 1 and 2)
autoradiography or (lanes 3 and 4) transfer to
nitrocellulose paper and immunodetection with anti-ubiquitin antibody. Lanes 1 and 3, p53 without E6; lanes 2 and 4, p53 incubated with E6 and ATP
S. Indicated is the
position of high molecular weight ubiquitin conjugates and of IgG heavy
chains reactive with the secondary
antibody.
To test whether the cyclin B degradation
domain within amino acids 13-90 can confer lability on the
otherwise stable TbODC, we fused this region to the C terminus of TbODC (Fig. 4A). As shown in Fig. 4B, the
TbODC-cyclin B fusion protein was stable in the
reticulocyte lysate. The result agreed with the conclusion that this
portion of cyclin alone cannot promote degradation of protein
A(27) . However, it became unstable after NAZ was appended to
form NAZ-TbODC-cyclin B
. Furthermore, deleting
the lysine residues that are putative sites of ubiquitination from
NAZ-TbODC-cyclin B
to form NAZ-TbODC-cyclin
B
did not alter its degradation. These results
suggested that cyclin B amino acids 13-59 contain a degradation
domain that can cooperate with NAZ to promote trypanosome ODC
degradation in vitro. Furthermore, the degradation is
independent of the presence of lysine residues (contained within amino
acids 60-90), which are the normal targets for ubiquitination,
suggesting that NAZ can provide an alternate signal for degradation
that bypasses the requirement for ubiquitin modification.
Figure 4: NAZ-induced degradation of TbODC-cyclin B 13-90 (with ubiquitination site) and TbODC-cyclin B 13-59 (without ubiquitination site). A, structure of Tb-cyclin B 13-90, NAZ-Tb-cyclin B 13-90, and NAZ-Tb-cyclin B 13-59 fusion proteins. The region of cyclin B 13-90-containing lysines that are the ubiquitination sites of cyclin B is marked K. Solid bar, NAZ; open bar, TbODC; hatched bar, cyclin B. B, Tb-cyclin B 13-90, NAZ-Tb-cyclin B 13-90 and NAZ-Tb-cyclin B 13-59 were subjected to degradation and the results analyzed as in Fig. 1.
To test whether NAZ was able directly to induce degradation of the cyclin B degradation region 13-90 in a rabbit reticulocyte lysate, we coupled NAZ in front of either amino acids 13-90 or amino acids 13-59 (Fig. 5A). Both fusion proteins were degraded (Fig. 5B). As expected for a metaphase-specific substrate subjected to degradation in the reticulocyte-based degradation assay system, the cyclin B degradation region 13-90 alone used as a control was relatively stable. NAZ therefore can alter the degradation properties of cyclin B amino acids 13-90 in two ways: promoting its lability in the absence of cycle-specific signals and diverting it to a pathway that does not require ubiquitination.
Figure 5: NAZ-induced degradation of cyclin B 13-90 (with ubiquitination site) and cyclin B 13-59 (without ubiquitination site). A, structure of NAZ-cyclin fusion proteins and unfused cyclin B 13-59 as control. Solid bar, NAZ; hatched bar, cyclin B. B, the proteins were subjected to degradation and the results analyzed as in Fig. 1. C, quantitation of degradation of above fusion proteins, mean of two experiments. (Triangles represent cyclin B 13-90, open circles NAZ-cyclin B 13-90, and solid circles NAZ-cyclin B 13-59.)
Figure 6:
Association of ODCAZ complex with
the 26 S protease. Mouse ODC was in vitro translated in the
presence of [
S]methionine and affinity-purified
on a matrix consisting of fusion proteins GAZ 55-212 or
106-212 affixed to glutathione-Sepharose 4B beads. The complexes
ODC
GAZ 55-212 and ODC
106-212 were eluted with
glutathione and mixed with 26 S protease, 20 S proteasome, or 11 S
activator. The mixture was then subjected to non-denaturing gel
electrophoresis. The 26 S protease and 20 S proteasome were identified
by the fluorogenic peptide overlay assay (not shown) and by Coomassie
Blue staining (A). B, the complexes were mixed with
26 S protease, 20 S proteasome, or 11 S activator as indicated, and the
ODC
AZ complex was visualized by
autoradiography.
To test whether each
component of the ODCAZ complex can separately associate with the
26 S proteasome, we produced
[
S]methionine-labeled ODC and AZ as GST fusion
proteins by in vitro translation. The fusion proteins,
purified by affinity chromatography with glutathione-Sepharose 4B, were
mixed with 20 S proteasome or 26 S protease for the association assay.
GST
ODC and GST
AZ fusion proteins were each able to
associate with the 26 S protease but not the 20 S proteasome (Fig. 7A). GST alone was unable to associate with
either of the proteases. Next, to test whether NAZ is necessary for the
association of AZ with 26 S, we analyzed both forms of AZ, GST
AZ
55-212 and GST
AZ 106-212. We found that both
truncated proteins were able to associate with the 26 S protease, again
indicating that NAZ is not required for association (Fig. 7B). Therefore, NAZ must serve some other
function in the destruction of ODC.
Figure 7:
Association of ODC and AZ with 26 S
protease. Constructs encoding GST or fusions of GST to AZ or to MODC
were translated in vitro in the presence of
[S]methionine and purified with
glutathione-Sepharose 4B beads. The purified proteins were eluted and
mixed with a preparation of 20 S proteasomes or the 26 S protease. The
mixtures were then loaded on non-denaturing gel for electrophoresis
separation. Associated ODC or AZ was determined as in Fig. 6. A, association of GST, GST
MODC, or GST
AZ with 20
S proteasome and 26 S protease. B, association of GAZ
55-212 or GAZ 106-212 with 26 S protease. C,
association of GST, GST
MODC, GST
M314T, and GST
TbODC
with 26 S protease.
Finally, we examined the
capacity of trypanosome ODC (TbODC) to associate with proteasomes.
Using a GSTTbODC fusion protein, we found that TbODC, unlike
MODC, did not associate with the 26 S protease. To find out whether the
C terminus of MODC, required for protein instability, is also necessary
for MODC association with 26 S, we made a GST
M314T fusion
chimera, containing the N terminus of MODC and the C terminus of TbODC,
with the junction at amino acid 314. This GST
M314T chimera was
able to associate with the 26 S protease (Fig. 7C).
This result suggests that the difference between TbODC and MODC in
their ability to associate with the 26 S protease does not depend on
the C terminus of mouse ODC but rather on sequence information
contained within its first 314 amino acids.
Vertebrate ODC is a labile protein with a half-life in cells
of less than an hour(28) . C-terminal deletions or mutations
can make it stable, and the C terminus appended to other proteins can
confer on these a short half-life(17, 24) . The C
terminus is therefore both necessary and sufficient to provide a
moderate degree of lability. ODC becomes still more labile in the
presence of AZ(28) . The AZ binding site within ODC is near its
N terminus. Mutations in that binding site destroy the regulatory
effect of AZ on ODC activity and abolish the regulatory effect of
polyamines on ODC in cells. The AZODC complex is an efficient
substrate for in vitro degradation. Normally, AZ binds to ODC
and is not itself consumed as rapidly as ODC but acts catalytically to
mediate ODC degradation(20, 21) . We have shown here,
by directly coupling it to ODC, that the N terminus of AZ is solely
responsible for its degradative role. The fusion construct
NAZ
MODC contains the two domains needed for degradation, the AZ N
terminus and the mouse ODC C terminus. These are enough to destabilize
the otherwise stable protein trypanosome ODC. This finding has
encouraged us to use NAZ fusions as a general means to identify and
analyze degradation domains of other proteins uncoupled from earlier
steps in the degradation process(29) .
NAZ could act either
as a bridge to bring together substrate ODC and 26 S protease or as a
protease activator. Our results are inconsistent with the first
possibility; the 26 S protease associates with ODCAZ
independently of the presence of NAZ. In fact, each protein alone can
associate with the protease, and this recognition process is dependent
on neither the N terminus of AZ nor the C terminus of mouse ODC. It is
therefore improbable that these degradative elements serve as
recognition signals.
Regulatory proteins, such as cyclins and oncoproteins, are usually short-lived in cells. Understanding the signals that direct their degradation is facilitated by mutagenesis and in vitro analysis of those signals. It may be difficult, however, to interpret the results of such experiments with proteins that require ubiquitination for turnover. Mutations of target proteins that interfere with degradation could do so by inhibiting ubiquitination or, alternatively, by impeding downstream steps. One can bypass the need for ubiquitination and thus simplify analysis by attaching a protein element that provides a functional alternative to ubiquitination to target proteins. NAZ has these properties. Its presence drives proteins down a proteasome-mediated degradation pathway with downstream elements common to ubiquitinated and non-ubiquitinated targets, thus bypassing the need for that modification. Furthermore, the NAZ domain is effective in promoting in vitro degradation. By conferring in vitro lability on natural substrates of in vivo degradation, it can be used to assist the analysis of cis-acting structural determinants of degradation. We here applied this form of analysis to establish that a region of cyclin B containing the destruction box acts as a degradation domain that can function independently of ubiquitin. This was further confirmed by demonstrating that the degradation domain of cyclin can effectively replace of the C-terminal degradation domain of mouse ODC.
Most short-lived proteins require polyubiquitination to be degraded. The exact role of ubiquitination in proteolysis is not well understood. It has been proposed that the modification is a direct recognition signal for the protease complex. Recently a specific protease subunit has been identified as the locus of interaction with polyubiquitin (30) . A degradation domain could act as a proteolysis site, which is made available for digestion by ubiquitin modification, or a site of association with other proteins required for proteolysis, e.g. a chaperone. The work described here supports the hypothesis that NAZ shares with polyubiquitination the need for collaboration with a degradation domain. Because NAZ can function as an independent module when appended to diverse proteins, it can be used as an analytic reagent for probing the structure of degradation domains. This method of analysis is applied to the tumor suppressor p53 in the accompanying paper(29) .