(Received for publication, November 18, 1996, and in revised form, January 21, 1997)
From the Department of Biology and Center for
Molecular Genetics, University of California at San Diego, La Jolla,
California 92093-0347 and the ¶ Division of Cellular Immunology,
La Jolla Institute for Allergy and Immunology, San Diego, California
92121
The product of the retinoblastoma susceptibility
gene, RB, is a negative regulator of cell proliferation. Inactivation
of RB does not interfere with embryonic growth or differentiation. However, Rb-deficient embryos show abnormal degeneration of
neurons and lens fiber cells through apoptosis, suggesting that RB may protect against programmed cell death. Consistent with this notion, RB
is found to be degraded in tumor necrosis factor (TNF)- and CD95-induced death. A consensus caspase cleavage site at the C terminus
of RB is cleaved in vitro and in vivo by
proteases related to CPP32 (caspase 3). Mutation of the consensus
cleavage site generates a cleavage-resistant RB which is not degraded
during cell death. Expression of this non-degradable RB is found to
antagonize the cytotoxic effects of TNF in
Rb/
3T3 cells, but this mutant RB cannot
attenuate the rapid death induced by anti-CD95 in Jurkat/T cells. These
results show that RB is a target of the caspase family of proteases
during cell death and suggest that the failure to degrade RB can
attenuate the death response toward some but not all death
inducers.
Higher eukaryotic cells have the capability to undergo active cell
death, and the programmed death process plays an important role in the
development as well as the homeostasis of multicellular organisms.
Execution of the death program, i.e. apoptosis, requires a
number of cysteine proteases (caspases), exemplified by the ced-3 death effector gene product of Caenorhabditis
elegans (1), and includes the vertebrate CED-3 homologs that
comprise the interleukin-1-converting enzyme
(ICE)1 family of proteases (2). Inhibitors
of the caspases, e.g. the cowpox virus CrmA protein, can
protect cells from apoptosis (2-5). A number of cellular proteins,
both nuclear and cytoplasmic, have been shown to be cleaved by the
caspases during apoptosis (6-8).
The retinoblastoma (RB) protein is a negative regulator of cell proliferation (9). Germ line heterozygous mutations of Rb predispose to the development of retinoblastoma in humans and pituitary tumors in mice (10-13). Homozygous mutation of Rb results in embryonic lethality at day 12-15 of gestation, accompanied by the abnormal degeneration of neurons, photoreceptor cells, and the ocular lens fiber cells (11-14). Inactivation of RB function, through the transgenic expression of the human papilloma virus E7 oncoprotein in the retina or the lens, leads to a similar death phenotype (14). These observations suggest that RB, in addition to its function in growth suppression, may also play a role in the suppression of the death program.
The caspases cleave polypeptides between an aspartic acid and a glycine in the consensus sequence DEXDG (6, 15). A caspase consensus cleavage site, DEADG, is found in the human RB sequence at amino acids 883 to 887, and this site is conserved in the mouse, chicken, and the Xenopus RB. We show in this report that RB is indeed cleaved by a caspase at this consensus site during the death response triggered by the ligation of CD95. Cleavage of RB is also observed in mouse 3T3 cells in TNF-induced death. Mutation at the consensus site blocked RB degradation in vitro and in vivo, and expression of this cleavage-resistant RB is found to antagonize the TNF cytotoxicity. Our findings show that RB is a target of the death effector proteases and suggest that degradation of RB may be required for cells to respond to TNF as a death signal.
Monoclonal antibody to human CD95 was obtained
from Kamiya Biomedical Co. Recombinant tumor necrosis factor (TNF-
) was obtained from Calbiochem-Novabiochem International.
Anti-RB polyclonal antibody 851 was described previously (16). GST and
GST-CrmA were purified from Escherichia coli. DEVD-CHO,
YVAD-CHO, and Z-VAD-cmk peptides were purchased from BACHEM Bioscience
Inc. All other chemicals were obtained from Sigma.
RB-MI and RB-I were prepared by
a polymerase chain reaction-based strategy. The nucleotide sequence of
each polymerase chain reaction-generated construct was determined in
its entirety to verify the mutations. In RB-MI, amino acids Asp-886 and
Gly-887 in the ICE/CED-3 cleavage site (DEADG) were substituted with
Ala and Glu, respectively. In RB-
I, amino acids from Gly-887 to the end were deleted, mimicking the caspase-cleaved product. Wild type RB
and the two mutants, MI and
I, were cloned into the pCMV vector (17)
to make pCMV-RB, pCMV-RB-MI, and pCMV-RB-
I. Wild type RB and the two
mutants, MI and
I, were also cloned into the pEBB vector (18) to
make pEBB-RB-WT, pEBB-RB-MI, and pEBB-RB-
I.
Cell lines used in this study
were cultured under standard conditions. Jurkat/T cells were
co-transfected with pBABE-puro (19) plus pEBB, pEBB-RB-WT, pEBB-RB-MI,
or pEBB-RB-I by electroporation. Polyclonal populations of
transfected cells were obtained by selection with 0.5 µg/ml puromycin
for 2 days. Rb
/
3T3 cells were
co-transfected with pCMV-
-galactosidase and pCMV-RB-WT, pCMV-RB-MI,
or pCMV-RB-
I by the LipofectAMINE method (Life Technologies, Inc.).
Human Jurkat and CEM cells (1 × 106 cells/ml) were treated with 50 ng/ml anti-CD95
antibody, respectively, and collected at the indicated times. To
inhibit apoptosis, Jurkat cells were preincubated with 10 µM Z-VAD-cmk for 1 h or with 50 ng/ml TPA plus 1 µg/ml PHA for 38 h and then treated with anti-CD95 antibody. At
30 h post-transfection, Rb/
3T3 cells
were seeded at a density of 1 × 105 cells per well of
a six-well plate and treated with various amounts of TNF-
plus 100 ng/ml actinomycin D for 14 h or treated with various amounts of
TNF-
alone for 45 h.
For Jurkat and Jurkat/T cells, dead cells
were assessed by staining with 4 µg/ml acridine orange. Apoptotic
nuclei stained with acridine orange showed margination and condensation
of the dye. Apoptosis was also verified by DNA degradation and the
generation of nucleosomal ladders. Live cells were counted by the
exclusion of trypan blue. To determine the death of transfected
Rb/
3T3 cells, the
-galactosidase
activity in the attached live cells and the floating dead cells was
measured. The percent
-galactosidase activity in the floating cells
relative to the total activity was given as the percent dead cells.
Apoptosis of the floating cells was verified by staining with Hoechst
dye to reveal the formation of pyknotic nuclei.
Reaction mixtures containing CL
granules with CEM cell extracts (20 µg) or Saos-2 extracts (30 µg)
expressing either RB, RB-MI, or RB-I were incubated at 37 °C for
90 min to 3 h. Inhibitors used were GST (10 µM),
GST-CrmA (0.00033-10 µM), DEVD-CHO (0.0001-1 µM), or YVAD-CHO (1 µM). CEM and Saos-2
cell-free extracts were prepared as described previously (20, 21).
The human T cell lines, Jurkat and CEM, express CD95 (also
known as Fas) and can be killed rapidly by treatment with anti-CD95 (22, 23). CD95-induced cell death is blocked by CrmA, suggesting that
the activation of the caspase family of proteases is essential in the
apoptotic response (4, 5). Jurkat cells contained a high level of
phosphorylated RB (ppRB), which was degraded within 2 h of
treatment with anti-CD95 (Fig. 1a). In Jurkat
cells, the ppRB bands were first converted to a series of lower bands
(lanes 3 and 4 in Fig. 1a) and then to
a predominant RB band (lane 7) which is about 5 kDa
smaller than unphosphorylated full length pRB (compare lanes
9 and 10). The total amount of RB also decreased, indicating that
RB was further degraded. Treatment of CEM with anti-CD95 also induced apoptosis and the rapid degradation of RB (not
shown). In both cell types,
RB was a detectable intermediate in the
degradation process. RB degradation did not reflect a general loss of
total cellular proteins, as exemplified by the constant levels of
cyclin A throughout the course of cell killing (Fig. 1a).
CD95-induced apoptosis was prevented when Jurkat cells were
pre-treated with TPA/PHA or with Val-Ala-Asp-chloromethyl ketone (Z-VAD-cmk), an inhibitor of caspases. Addition of Z-VAD-cmk completely blocked RB degradation (Fig. 1b, lanes 3,
5, and 7) and prevented cell death. TPA/PHA
treatment induced a partial dephosphorylation of RB (even
lanes in Fig. 1c); further treatment of these cells with anti-CD95 did not cause death, and no RB was detected in the
experimental time course (compare odd and even
lanes in Fig. 1c). These results show a tight
correlation of RB degradation and cell death.
The ability of caspases to cleave RB was further examined in a
cell-free system of granzyme B-induced apoptosis. Granzyme B, a serine
protease derived from the granules of cytotoxic lymphocytes (CL), has
been shown to activate at least one caspase, CPP32 (caspase 3), which
is found in a wide variety of cell extracts in a latent, inactive form
(24, 25). We incubated CEM extracts with CL granules in
vitro and examined the status of the endogenous RB (Fig.
2a). RB was converted to the unphosphorylated
form in the extracts (lane 2). Incubation with CL granules
induced the cleavage of RB (lane 3), and the in
vitro-cleaved RB migrated identically to that of the RB
detected in anti-CD95-treated Jurkat cells (Fig. 2a, compare
lane 1 to lane 3). The in vitro
cleavage of RB was inhibited by CrmA (lane 5), which blocks
the proteolytic activity of granzyme B (25), and by the peptide
Ac-Asp-Glu-Val-Asp-aldehyde (DEVD-CHO) (lane 6), which does
not inhibit granzyme B but does block CPP32 (caspase 3) and related
proteases (25). In contrast, Ac-Tyr-Val-Ala-Asp-aldehyde (YVAD-CHO),
which is more specific for proteases in the ICE (caspase 1) subfamily,
did not inhibit RB cleavage (lane 7). The effects of CrmA
and DEVD-CHO on RB cleavage were dependent on the concentrations of
these inhibitors (Fig. 2b). These results suggested that RB
cleavage may be mediated by CPP32 (caspase 3) or a CPP32 (caspase 3)
subfamily protease in this system.
A consensus caspase family cleavage site,
Asp883-Glu-Ala-Asp-Gly887 (DEADG), is found in
RB and is situated about 5 kDa from the C terminus of RB (Fig.
2d). To determine if RB was indeed cleaved at this site
during apoptosis, we constructed two mutants: RB-MI, with substitutions
of Asp886 and Gly887 to Ala and Glu,
respectively, and RB-I with a truncation at the putative cleavage
site (Fig. 2d). The RB-MI mutant lacking the consensus site
was completely resistant to cleavage by granzyme B-activated proteases
(Fig. 2c, lanes 5-7). As expected, the in vitro cleaved RB migrated identically to the RB-
I mutant (Fig. 2c). To determine if RB-MI was also resistant to cleavage
in vivo, it was expressed in a line of Jurkat cells that
also expressed the SV40 T-antigen (Jurkat/T cells, Fig. 2e).
Expression of exogenous RB could not be achieved in Jurkat cells but
was possible with the Jurkat/T cells, most likely because T-antigen can
inactivate the growth suppression function of RB. Overproduction of
RB-WT or RB-MI was indicated by the increased levels of ppRB (Fig.
2e, compare lanes 2 and 3 to
1). In cells transfected with RB-
I, several new bands,
most likely corresponding to phosphorylated RB-
I (pp
RB), were
detected (lane 4). When treated with anti-CD95, the
exogenous RB-WT was cleaved to
RB indicated by the increased intensity of this band (lane 6), and the exogenous RB-
I
bands were converted to a tight
RB consistent with dephosphorylation (lane 8). Interestingly, in cells expressing RB-MI, no
increase in the
RB band was observed, instead, anti-CD95 treatment
led to the partial dephosphorylation of RB-MI and the generation of pRB
(lane 7). Taken together, these results demonstrate that RB is indeed cleaved at the consensus site by caspase family proteases in vitro as well as in anti-CD95-treated cells.
Expression of RB-MI did not have a detectable effect on the
anti-CD95-induced death in Jurkat/T cells (Fig. 2e, percent
apoptotic cells was identical in all samples). To further examine the
effect of RB-MI on the death response, we chose TNF- as the death
inducer because the cytotoxic activity of TNF is dependent on the
activation of caspases (26). With normal 3T3 cells, TNF-
treatment
alone induced an inefficient and protracted death which could be
enhanced by actinomycin D (27). A Rb
/
3T3 line,
however, underwent efficient apoptosis by treatment with TNF-
alone,
although actinomycin D also accelerated the death response (Fig.
3). In separate experiments, we have found that RB,
RB-MI, and RB-
I all have growth suppression
function.2 In
Rb
/
3T3 cells, reintroduction of RB induced
G1 increase but not a cell cycle arrest.3
The RB-WT, RB-MI, or RB-
I was each expressed in
Rb
/
cells, and the RB level was determined
after treatment with TNF alone (Fig. 3b) or TNF plus
actinomycin D (Fig. 3d). TNF treatment (200 ng/ml, 45 h) led to a decrease of RB-WT and RB-
I (Fig. 3b, compare
lanes 3 and 4 or lanes 7 and
8), but did not reduce the level of RB-MI (compare
lanes 5 and 6). The
RB band was not prominent possibly because the further degradation of
RB was more efficient in
these Rb
/
3T3 cells than it was in Jurkat
cells. When these cells were treated with TNF (3 ng/ml) plus
actinomycin D for 14 h (Fig. 3d),
RB was detected in
cells expressing RB-WT (compare lane 6 to lane
2), the level of RB-
I decreased (compare lane 8 to
lane 4), but the level of RB-MI was found to increase
(compare lane 7 to lane 3) suggesting that cells
expressing RB-MI might be selectively preserved. The cleavage-resistant
RB-MI was stable at all TNF concentrations tested (Fig. 3 and data not
shown). This finding suggests that cleavage at the caspase consensus
site is a prerequisite for the further degradation of RB in TNF-treated
cells.
Cells expressing RB-WT or RB-I were found to be less sensitive to
TNF than the parental RB-deficient cells (Fig. 3, a and c). In contrast, cells expressing RB-MI were completely
resistant to TNF-
at concentrations that killed cells expressing
RB-WT or RB-
I (Fig. 3a). Even at a higher concentration
of TNF (200 ng/ml), when 50% of the RB-deficient cells and 25% of the
RB-WT or RB-
I expressing cells were killed, only 5% of the RB-MI
cells were dead (Fig. 3a). Thus, the degradation of RB,
induced by cleavage at the caspase consensus site, appears to be
required for TNF to induce death. The death protection function of
RB-MI is not absolute. When death was accelerated by treatment with TNF
plus actinomycin D, RB-MI expressing cells were still more resistant than RB-WT cells, but they were killed at a TNF concentration of 30 ng/ml (Fig. 3c). Moreover, the dead cells were found to contain undegraded RB-MI (not shown). This result suggests that RB
cannot inhibit the death process per se; however, the
preservation of RB can attenuate the death response to TNF.
In addition to TNF and CD95, treatment with cisplatin and the withdrawal of survival factors also induce the cleavage of RB at the C-terminal caspase consensus site. An and Dou (28) have described the degradation of RB to fragments of 68 kDa and 48 kDa in cancer cells exposed to chemotherapeutic agents. In our hands, smaller fragments of RB could also be detected in anti-CD95 and TNF-treated cells but the sizes were variable (not shown). These observations suggest that RB is first cleaved at the C-terminal caspase site, and the cleaved product is then further degraded by other types of proteases.
The induced degradation of RB during apoptosis supports the notion that RB plays an active role in antagonizing the death response. Removal of RB is expected to release E2F-1, which can promote S phase entry and induce apoptosis when overproduced (29) and has been implicated as a physiological regulator of thymocyte apoptosis (30). Thus, the degradation of RB may unleash the apoptotic function of E2F-1. Alternatively, preservation of RB may protect against death by promoting a growth arrest response to TNF signal.
The caspase family of proteases are thought to be executioners, and
they kill cells by a rapid disruption of the cell integrity (31). RB is
not a protease inhibitor because its death protection function is not
absolute. Rather, degradation of RB by a caspase-related protease may
sensitize cells to the toxic effect of TNF-. These findings suggest
that one or more of the caspases, which may be activated early by
TNF-
signal, can play a regulatory role by cleaving RB to direct the
cell toward the choice of death. This would expand the biological
functions of the caspases to include both the modulation and the
execution of apoptosis.
We thank Dr. Tony Hunter for
anti-cyclin A antibodies, Dr. Arnold H. Greenberg for CL granules, and
Drs. Juan Zalvide and J. A. DeCaprio for Rb/
3T3 cells. We also thank Drs. Laura L. Whitaker, Heyun Su, Rajasekaran Baskaran, and Eric Knudsen for helpful comments on the
manuscripts.