(Received for publication, June 27, 1995; and in revised form, October 19, 1995)
From the
Degradation provides one means for controlling the cellular level of the p53 tumor suppressor. Here we have determined a structural element of p53 required for degradation. To create a substrate amenable to in vitro analysis of proteolysis, we appended to p53 the N terminus of antizyme, a protein that binds to and induces degradation of mammalian ornithine decarboxylase (ODC). We found using deletion analysis that an element within amino acids 100-150 is required for degradation of the fusion protein. A monoclonal antibody (PAb246) that binds close to this region prevents the degradation induced by human papillomavirus 16 E6 protein. Furthermore, we found that amino acids 100-150 of p53 can function as an independent domain to induce Trypanosoma brucei ODC, a stable protein, to be degraded in vivo or, by cooperating with an antizyme binding domain of ODC, to confer polyamine-dependent regulation.
Tumor suppressor p53 imposes negative regulation on the cell
cycle, and abnormal inactivation of the protein has been shown to be
related to cell transformation and tumorigenesis(1) .
Elimination of p53 function takes place by multiple means, including
degradation, interaction with other proteins, interaction with mutant
forms of p53 itself, or somatic or germline mutation(1) .
Degradation of p53 is influenced by protein-protein interactions. A
variety of viral oncoproteins have been shown to act on p53. The
association of p53 with SV40 large T antigen or with adenovirus 2
E1B-55K protein extends its half-life(2, 3) , but the
E6 protein of human papillomavirus (HPV) ()16 shortens
it(4, 5) . Mutation of p53 can change its function and
stability(6) . The multiple influences that can play on p53
stability make its degradation an interesting subject for analysis.
Degradation is mediated by the ATP-dependent and nonlysosomal large 26 S protease complex. Many short-lived proteins need to be modified by ubiquitination in order to be degraded(7, 8, 9, 10) , but some labile proteins such as ornithine decarboxylase (ODC) are not ubiquitinated(11, 12, 13) . Ubiquitination is required for p53 degradation(5, 14) . Studies from Howley and his colleagues (15) demonstrated that E6 and the associated host cell protein E6-AP act as a ubiquitin ligase E3, which leads to ubiquitination of p53 and its degradation. The structural elements of p53 needed for recognition and destruction by the proteolytic machinery remain unclear, as is true for most short-lived proteins. We propose that for the 26 S protease to act, substrate proteins must contain a structural motif, a degradation domain. This domain is required for proteins to undergo degradation via both ubiquitin-dependent and -independent pathways.
ODC, the key enzyme in the biosynthesis of polyamines, is a short-lived protein. Two types of degradation occur: polyamine-dependent and polyamine-independent(16) . The C terminus of ODC suffices as a degradation domain to confer polyamine-independent degradation(16, 17, 18) , but it is insufficient for polyamine-dependent degradation(16) . Polyamines regulate ODC activity via induction of antizyme(19, 20) . Antizyme binds to ODC, inhibits its activity, and promotes its degradation(16, 21, 22) . Antizyme binding depends on an element near the N terminus of ODC(21) . For polyamine-dependent degradation to occur, two elements are necessary within ODC: the antizyme binding domain and the C-terminal degradation domain(16) . Furthermore, we showed that the C terminus of antizyme interacts with ODC and that the N-terminal half of antizyme is not involved in the antizyme/ODC interaction, but it is required for promoting ODC degradation(23) . The N terminus of antizyme (NAZ), when directly coupled to diverse short-lived proteins, can direct their degradation(24) . Because NAZ is a module that must function in collaboration with a degradation domain, a simple strategy can in principle be applied to identify such domains within a labile protein: appended NAZ and carry out deletion analysis.
Here we report that we have identified a degradation domain of p53 by fusing it to NAZ and making deletions within the p53 moiety. The degradation domain of p53 so identified was able to function, like the degradation domain of mouse ODC, to induce degradation of trypanosome ODC, a stable protein.
Figure 1: Structure of NAZ-p53 and deletions of p53 moiety. Solid bars, NAZ; hatched bars, p53.
Figure 2:
NAZ-induced degradation. A,
NAZ-induced degradation of p53; B, the C-terminal deletions of
p53: NAZ-p53 1-304, NAZ-p53 1-202, and NAZ-p53 1-103; C, the N-terminal deletions of p53: NAZ-p53 101-393,
NAZ-p53 201-393, and NAZ-p53 301-393; D, NAZ-p53
1-150, NAZ-p53 1-200
100-139.
Figure 3:
Effect of ATPS on p53 degradation.
Proteolysis was induced by non-covalent association of p53 with E6 (left three lanes) or by fusion of NAZ to p53 amino acids
1-150 (right three lanes), and the products of the
reaction were analyzed by SDS-polyacrylamide gel electrophoresis and
autoradiography. In both cases, time-dependent proteolysis occurred in
the presence of ATP. When ATP
S was used in place of ATP,
proteolysis was inhibited in both cases. In the presence of ATP
S,
high molecular weight forms of p53 accumulated in the presence of E6
(but not in its absence, result not shown). Under identical conditions
of incubation, no high molecular weight forms of NAZ-p53 1-150
were detected.
Figure 4:
Effect of monoclonal antibody PAb246
specific for p53 amino acids 88-109 on E6-mediated degradation of
mouse p53. [S]Methionine-labeled mouse p53 was
subjected to degradation in the absence of HPV16 E6 (lane 1)
and in the presence of E6 (lanes 2-4). p53 was
preincubated with control monoclonal antibody PAb421 (lane 3)
or with monoclonal antibody PAb246 (lane
4).
Figure 5: Effect of the p53 degradation domain (amino acids 100-150) on in vivo degradation of trypanosome ODC (TbODC). Open symbols, indicate components of TbODC chimeric proteins; hatched symbols, indicate components of p53 chimeric proteins. ODC stability in cells expressing TbODC or Tb-p53 100-150 was assessed by inhibiting protein synthesis with cycloheximide and determining residual enzymatic activity immediately and after 1, 2, or 3 h. Control cells were similarly incubated, harvested, and analyzed but were not treated with cycloheximide. ODC activities are represented as a percentage of initial activity; TbODC and TbODC-p53 100-150 initial activities were 51.9 and 15 pmol/mg/min, respectively. Triangle represents TbODC activity, circles represent TbODC-p53 100-150 activity, solid symbols represent treated cells, and open symbols represent untreated control cells.
Figure 6: Effect of the p53 degradation domain (amino acids 100-150) on polyamine-dependent degradation of chimeric mouse/trypanosome ODC (M314T). Open symbols, indicate components of TbODC chimeric proteins; cross-hatched symbols, indicate components of mouse ODC chimeric proteins; hatched symbols, indicate components of p53 chimeric proteins. To elicit polyamine-mediated regulation and induce endogenous AZ, putrescine was added to the culture medium of cells expressing M314Tb or M314Tb-p53 100-150 to a final concentration of 100 µM. Cell lysates were prepared after the indicated time of treatment and assayed for ODC activity as described(16, 28) . Control cells were similarly incubated, harvested, and analyzed but were not treated with putrescine. ODC activities are represented as the percentage of initial activity: M314Tb and M314Tb-p53 100-150 initial activities were 72.9 and 6.6 pmol/mg/min, respectively. Circles, M314T; triangles, M314T-p53 100-150; solid symbols, treated cells; open symbols, untreated control cells.
p53 is among the most labile of proteins. Its degradation is
required for cells to enter S phase. Overexpression of the functioning
protein or reduced degradation of the protein via DNA damage arrests
cell cycle progression at the G/S
boundary(33, 34) . Ubiquitination has been
demonstrated both in vivo and in vitro to be
associated with p53 degradation(5, 14) . In ts 20
cells, with a temperature-sensitive mutation of ubiquitin-activating
enzyme (E1), degradation of p53 is blocked at the non-permissive
temperature(14) . Howley and his colleagues(35, 36) have shown that HPV16 E6 protein activates p53 degradation in vitro, and the activation requires its associated protein,
E6-AP, a ubiquitous host cell protein co-factor. Their recent studies
have indicated that these two proteins, E6 and E6-AP, together function
as a ubiquitin-protein ligase(15) . In addition, E6-mediated
p53 degradation involves a novel ubiquitin carrier protein (E2) for p53
ubiquitination (15, 37, 38) . This E2 is
distinguishable by its chromatographic properties from those previously
characterized. The novel E2 may participate in the ubiquitination of
multiple endogenous substrates.
By utilizing the NAZ-p53 protein, we
carried out deletion analysis of the p53 moiety and found such
constructs to be labile only if they contain amino acids 100-150
of p53. Having identified amino acids 100-150 as a putative
degradation domain in p53, we tested its modularity by placing it in an
ODC context. It was able to destabilize trypanosome ODC, as does the
degradation domain of mouse ODC. Last, we showed that in mouse p53,
E6-mediated degradation could be blocked by an antibody specific for
this region. Except for the degradation domain here identified, the
structural determinants of p53 degradation such as the binding site for
the E6E6-AP complex and the ubiquitinated lysine(s) have not
determined. Such information will facilitate our understanding of p53
degradation.
Recent x-ray crystallographic studies (39) of a
complex of p53 core and DNA indicate that the protease-resistant core
(amino acids 94-312) consists of a sandwich hydrophobic
core and a loop-sheet-helix motif. A conserved region in amino acids
117-142 contains part of the L1 loop, the paired
S2-S2`
sheets and their short connecting loop, and a part of
strand 3.
The back of the
S2-S2` hairpin interacts with a pocket formed by
the S1, S3, and S8
sheets, and the pocket leaves the hairpin
buried. We hypothesize that the S2-S2` hairpin may constitute an
important part of the degradation domain and that ubiquitination
exposes the buried structure. The PAb246 may stabilize a native
conformation, preventing exposure of the structure and thereby blocking
E6-mediated p53 degradation. Deletion of amino acids 203-304 from
NAZ-p53 1-304 (to make NAZ-p53 1-202) destroys the pocket,
which can be functionally accessible for the degradation machinery by
NAZ. This could also explain the increased lability of NAZ-p53
1-202 compared with NAZ-p53 1-304.
Assignment of the degradation domain in p53 to amino acids 100-150 may illuminate two observations. First it could explain regulation of the half-life of p53 by large T antigen of the SV40 virus. The half-life of p53 protein is regulated by its associated viral oncoproteins. Among the oncoproteins, association with the SV40 large T antigen has been shown to extend the p53 half-life. The minimal binding region of p53 for large T antigen targeting has been identified as amino acids 94-293(40) . The degradation domain of p53, which we have identified here, is located within this binding region. Therefore, the association of p53 with large T antigen could cover the degradation domain and prevent p53 degradation. Second, our observations are consistent with the distribution of mutations that increase the half-life of p53. Mutations in p53 are commonly found in tumors, and the mutated protein often has a much longer half-life than wild type p53(6) . The mutations are widely distributed within the protein but are mainly located in four regions(41) . One of the regions lies in the amino acid 100-150 degradation domain. Mutations in the region may either directly change the degradation domain itself to prevent protease targeting or change protein conformation to prevent ubiquitination. Although we not have experimentally tested this, we speculate that most p53 mutations outside the degradation domain extend half-life by reducing the efficiency of ubiquitination. Conversely, we expect mutations in the 100-150 region to alter stability without preventing ubiquitination. One such temperature-sensitive mutation, murine p53 A135V, originally found in a mouse tumor, appears to have that property(37) . It is important to remember that p53 mutations that cause tumors are likely to constitute a highly unrepresentative subset of those that can stabilize; because p53 is a tumor suppressor, oncogenic mutations that stabilize must also act as dominant negative mutations. Site-directed deletion and substitution mutations within the amino acid 100-150 region are therefore required to test these predictions without selection bias.