BSCB Autumn Meeting 2000
The Cell and Molecular Biology of Apoptosis
Andrew Gilmore and Rosemary Gibson
This year’s
BSCB Autumn meeting was held in Heriot Watt University, just outside Edinburgh.
This proved to be quite an appropriate location, being the city in which
Andrew Wyllie first thought of falling leaves and coined the term apoptosis.
It was a wonderful meeting, despite grey weather, an escalating petrol
crisis and street protests by truck drivers, making delegates nervous
about how they would get home! The organisers went to great lengths to
entertain the delegates, including a ceilidh, dinner in Edinburgh, a midnight
demonstration by the Edinburgh fire department (when the alarms went off
in the halls of residence), and a by now world famous impersonation of
the structure of laminin.
The science covered
all aspects of apoptosis, from the layers of regulation in different types
of cells through to phagocytosis of the apoptotic debris. Necessarily,
we have not been able to report on every talk; apologies to those not
included but great thanks to the scientific organisers, Tony Metcalfe,
Charles Streuli and Bill Earnshaw for putting together such a great meeting.
Stanley Korsmeyer (Harvard) set the scene for the forthcoming exciting days with his keynote
presentation. His talk on integrating the cell death pathway concentrated
on how diverse survival signalling pathways converge to regulate Bcl-2
family proteins, the ‘gate-keepers’ of apoptosis. This family
of related molecules ultimately control mitochondrial dysfunction, a key
theme for much that followed, and subsequent activation
of caspase proteases. One family member, Bad, is phosphorylated on at
least three different serine residues. Phosphorylation of Bad promotes
cell survival, and different signalling pathways may target each site.
That all roads
lead to the mitochondria was emphasised by work he presented on Bid. Bid
is cleaved by caspase 8 during FAS/TNFR mediated apoptosis, and subsequently
translocates from the cytosol to mitochondria. FAS induces caspase 8 activation
directly, but in Bid knockout mice it does not effectively induce apoptosis,
indicating a mitochondrial requirement. Bid appears to work at the mitochondrial
surface by inducing oligomerization of other Bcl-2 family proteins, which
then form pores to release cytochrome c, a process independent of the
mitochondrial permeability transition pore. Bax, another member of the
Bcl-2 family, progressively forms dimers and then tetramers which are
capable of forming pores large enough for the passage of cytochrome c.
The following
morning, the theme of Bcl-2 family genes continued with Barbara Conradt (Munich) presenting data on Egl-1, a Bad homologue in C. elegans which is expressed only in those cell lineages destined to undergo apoptosis.
Mutations in the egl-1 promoter that lead to its expression in other lineages
results in ectopic cell death. The somatic sex determining gene Tra-1
regulates egl-1, resulting in expression that controls sex specific patterns
of apoptosis.
 Richard
Youle (Bethesda) pursued the role of Bax and its subcellular localization.
He described the translocation of Bax from a cytosolic, soluble distribution
in healthy cells, to an insoluble mitochondrial one following an apoptotic
insult. His use of monoclonal antibodies to specific epitopes within the
N-terminus of Bax has demonstrated that this translocation is accompanied
by a conformational change. He presented the 3D solution structure of
Bax and showed it has a very similar structure to the two other members
of the family which have been determined, Bcl-X and Bik. The Bax structure
however, has been solved for the full-length molecule, including the hydrophobic
C-terminal tail and shows that the tail fits into a groove on the surface,
therefore masking hydrophobic residues and allowing Bax to be soluble.
This groove is homologous to the groove on Bcl-X previously shown to bind
the Bak BH3 domain.
Jean-Claude
Martinou (Geneva) presented data showing that Bax induces cytochrome
c release from isolated mitochondria without mitochondrial swelling, laying
down the argument again that the permeability transition pore (PTP) is
not involved. Moreover, he has not observed an interaction between Bax
and any of the PTP components. The mechanism by which Bax is translocated
to the mitochondria appears to require Bid, a close relative of Bad. The
caspase 8 cleavage of Bid discussed
by Korsmeyer appears to allow Bid to trans-iently interact with Bax, an
interaction which induced Bax to associate with mitochondria. Martinou
reported that Bax oligomerizes on the mitochondria and that this appeared
to require a factor present on the mitochondrial surface, and was therefore
an event independent of the initial translocation of Bax. Thus activation
and pore formation occur in a complex and multi-step process.
Conformational
changes are a common feature of the activation of Bcl-2 family proteins,
and Caroline Dive (Manchester) presented data that the Bax homologue,
Bak, undergoes similar modifications following a number of diverse apoptotic
insults. Use of a panel of monoclonal antibodies indicated that Bak undergoes
a two step activation. Initially the N-terminus becomes exposed, and this
precedes morphological changes associated with apoptosis. However, a second
site within Bak, the BH1 domain becomes exposed later, concomitant with
morphological changes within the cell. This echoed the multistage activation
of Bax discussed by Martinou.
A number of speakers
dealt with the plethora of survival signals and the mechanism by which
these regulate apoptosis. Many diverse survival signals appear to feed
into the very same parts of the apoptosis machinery, the Bcl-2 proteins. Renato Baserga (Philadelphia) talked about the role of IGF-1 signalling
in haematopoietic cells. As well as its role as a mitogenic signal, IGF-1
also acts as a potent survival signal in many cells. IGF-1 induced cell
survival through regulating the phosphorylation of Bad. This occurred
through a mechanism independent of IRS-1
and the mitogenic role of IGF, and did not occur following insulin receptor
activation. It appears that the survival signal initiated by IGF-1 activates
MAP kinase, and requires the translocation of Raf to mitochondria, an
event without which the survival effect of IGF-1 was not seen.
John Blenis (Harvard) showed that the signals from IL-3 in haematopoietic cells also
regulate survival through Bad. He has been looking at the kinase Rsk-1,
a downstream target of the MEK-MAP kinase pathway activated by IL-3. Dominant
negative MEK blocked IL-3 survival signals, and constitutively activated
Rsk-1 restored survival. This appeared to be through the phosphorylation
of Bad on serine 112 by Rsk-1. Bad can also be phosphorylated on serine
136 by the kinase Akt. Phosphorylation on both sites was found to provide
more potent protection from apoptosis than phosphorylation on either alone.
Furthermore, phosphorylation on these sites appeared to promote Bad phosphory-lation
on serine 155, providing even stronger inhibition of its pro-apoptotic
function.
Charles Streuli (Manchester) had also examined Bad phosphorylation, this time in mammary
epithelia where both growth factors and the extracellular matrix (ECM)
provide independent survival signals. He presented data demonstrating
that Bad became dephosphorylated in the mammary gland following weaning,
just prior to the massive increase in apoptosis associated with the involution
of the gland. A number of growth factors regulated Bad phosphorylation
in mammary epithelial cells, including EGF and IGF.
IGF has a potential
role in regulating involution, and Christine Watson (Cambridge)
showed up-regulation of IGF-BP5, a negative regulator of IGF signalling,
at the start of involution, and a role for STAT transcription factors.
ECM appears to control epithelial cell survival through a different mechanism,
and adhesion did not regulate Bad phosphorylation. Charles Streuli showed
that this occurs via focal adhesion kinase (FAK) and PI3-kinase. In the
absence of ECM and FAK activation, Bax rapidly redistributes to the mitochondria
where it induces apoptosis, and cells from Bax knockout mice were found
to survive for longer in suspension.
The ECM connection
was pursued in two further presentations, which indicated that the signalling
pathways used are very different depending upon cell type. Caroline
Damsky (sans Francisco) has looked at synovial fibroblasts. Like epithelial
cells, FAK was the primary signalling molecule, but unlike them it appeared
to work through JNK and not PI3-kinase. She presented data showing that
phosphorylated JNK was found in the focal adhesions of cells, but not
if FAK signalling was blocked with a dominant negative construct. In contrast,
growth factor induced survival did work through PI3-kinase. This indicated
that not only did different pathways work in one cell in response to different
signals, but that the same signals could work through different pathways
in different cell types.
Julian Downward (ICRF) was interested in how oncogenes regulate a cell’s susceptibility
to apoptosis. Ras can have a role in both protecting cells from apoptosis,
and also sensitising them, activating multiple downstream pathways. Ras
transformation of MDCK cells made them insensitive to detachment induced
apoptosis. Detachment of cells from ECM appeared to activate the death
receptor pathway, resulting in caspase 8 activation and Bax translocation
to mitochondria. The kinase Akt appeared to be in the pathway, and activated
constructs protected from death. Ras activation of Akt may protect cells
from apoptosis through transcriptional regulation, via the forkhead transcription
factor. He is currently using gene chips to investigate which genes may
be regulated following Akt activation.
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