Oncogene (1999) 18, 6129 ± 6134
ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $15.00
http://www.stockton-press.co.uk/onc
Regulated nuclear localization of stress-responsive factors: how the nuclear
tracking of protein kinases and transcription factors contributes to cell
survival
Paul Ferrigno1,2 and Pamela A Silver*,1,2
1
2
Department of Cancer Biology, The Dana-Farber Cancer Institute, 44 Binney Street, Boston, Massachusetts, MA 02115, USA;
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA
The details of nuclear transport mechanisms are
emerging rapidly, largely through work with model
organisms. Here, we brie¯y describe these advances,
with an emphasis on the remaining challenges. We then
address the nuclear transport of some high pro®le
cellular regulators, including p53 and the proto-oncogene
PKB/Akt. We discuss the mechanisms that contribute to
the di€erential subcellular localization of these proteins.
Finally, we analyse the provocative patterns that emerge
from our overview.
Keywords: nuclear transport; protein phosphorylation;
p53; Akt/PKB; NF-AT; STATs
Introduction
By forming a controlled frontier between the nucleus
and the cytoplasm, the nuclear envelope allows a level
of regulation whose importance has long been
suspected but is only now being elucidated. The bidirectional tracking of proteins and other macromolecules across the nuclear envelope is tightly
regulated, yet remarkably dynamic. For many macromolecules, nuclear transport is an ongoing, constitutive
process. In contrast, many proteins ± including p53,
cyclin B1 and Cdc25 and a variety of protein kinases
and transcription factors ± localize di€erentially between the nucleus and the cytoplasm in response to
cell cycle progression, to growth signals, or to
environmental stimuli.
Here, we will ®rst provide a brief summary of our
knowledge of the mechanics of nuclear transport. We
will then discuss the nuclear-cytoplasmic tracking of
those cellular regulators that provide the most insights
into the molecular mechanisms of regulated nuclear
transport. `Stress' will be considered here in its
broadest biological sense, as any condition that can
lead to the death of a cell. This encompasses a range of
experimental conditions, as arti®cial as growth factor
withdrawal from tissue culture cells or as natural as an
ultraviolet lamp mimicking sunlight on a bare forearm.
The patterns that emerge from this discussion provide
new insights into the details of nuclear transport and
have major implications for future investigations of cell
regulation.
*Correspondence: PA Silver
Nuclear transport
Proteins destined for the nucleus contain a nuclear
localization signal (NLS) that is recognized in the
cytoplasm by one of a family of proteins for which the
founding members were importins (or karyopherins) a
and b (for recent reviews, see Cole and Hammell, 1998;
Corbett and Silver, 1997; Gorlich, 1998; Gorlich and
Mattaj, 1996; Hood and Silver, 1999; Izaurralde and
Adam, 1998; Mattaj and Englmeier, 1998; Moroianu,
1998; Nigg, 1997; Ohno et al., 1998; Pemberton et al.,
1998). Together with the small GTP binding protein
Ran, most likely in its GDP bound form, the cargo/
importin complex is targeted to the nuclear pore
complex. Following translocation across the nuclear
pore, the complex dissociates leaving the cargo in the
nucleus while the importin subunits are returned to the
cytoplasm. Dissociation of the complex is likely to
involve the exchange of GTP for GDP on the Ran
moiety. It is unclear whether the returning importins
carry a di€erent cargo back to the cytoplasm, but
nuclear export-speci®c importin b family members have
been identi®ed that mediate the transport from the
nucleus to the cytoplasm of, for example, tRNA (Arts
et al., 1998a,b; Hellmuth et al., 1998; Kutay et al.,
1998) or of proteins that contain a leucine-rich Nuclear
Export Signal (NES); (Fornerod et al., 1997; Fukuda et
al., 1997a; Neville et al., 1997; Ossareh-Nazari et al.,
1997; Stade et al., 1997). Again, release of the export
cargo at the cytoplasmic face of the nuclear pore is
likely to involve the hydrolysis of GTP that is bound to
the Ran component of the export complex (Bischo€
and Gorlich, 1997; Schlenstedt et al., 1997; Kehlenbach
et al., 1999). Although the vectorial nature of nuclear
transport (nucleus to cytoplasm or vice versa) has
clearly been shown to be determined by the guanine
nucleotide status of Ran (Gorlich et al., 1996; Koepp
et al., 1996), the only step at which GTP hydrolysis is
required for the nuclear transport cycle is at the end of
a nuclear export event. Thus, it remains unclear how
nuclear transport is powered (Koepp and Silver, 1996;
Melchior and Gerace, 1998). Thousands of rounds of
nuclear transport are estimated to occur per minute
(Gorlich and Mattaj, 1996), potentially causing a
considerable energy drain. An as yet uncharacterized
GTPase distinct from Ran (Sweet and Gerace, 1996;
Weis et al., 1996) may play a role in providing some of
this energy.
There are several levels at which nuclear transport
may be regulated: globally, perhaps by modulating the
rate of GTP exchange and hydrolysis; or more locally,
Nuclear transport and stress
P Ferrigno and PA Silver
6130
by post-translationally in¯uencing the relative anities
of importin family members for di€erent cargoes.
However, the only regulatory mechanism that has so
far been shown involves the post-translational modi®cation of cargoes. Speci®c examples are given below.
Protein kinases
Survival and apoptosis: Akt/PKB, BAD and 14-3-3
Early studies of protein kinase B, also known as RAC
(Related to protein kinase A and protein kinase C) and
c-Akt (cellular homolog of the viral oncogene v-Akt
Bellacosa et al., 1991; Co€er and Woodgett, 1991;
Jones et al., 1991a) found that it was activated in a
number of tumor cells, including a primary gastric
adenocarcinoma (Staal, 1987), ovarian and pancreatic
cancers (Cheng et al., 1992, 1996) and MCF-7 breast
cancer cells (Jones et al., 1991b). Activation of Akt/
PKB/RAC-PK/allows tissue culture cells to resist
apoptosis induced by a variety of treatments: UV
irradiation (Kulik et al., 1997), IGF-1 withdrawal from
neuronal cells (Dudek et al., 1997), growth factor
deprivation of c-myc overexpressing cells (Kau€mannZeh et al., 1997) and detachment of cells from the
extracellular matrix (Khwaja et al., 1997; reviewed in
Alessi and Cohen, 1998). Insights into the mechanism
by which Akt activation may lead to increased survival
come from the study of events that follow Akt
activation. For example, Akt activation correlates
with the phosphorylation and inhibition of GSK3
(Burgering and Co€er, 1995) and the phosphorylation
and inhibition of the pro-apoptotic proteins BAD
(Datta et al., 1997; del Peso et al., 1997), caspase-9
(Cardone et al., 1998) and FKHRL1, a forkheadrelated transcription factor (Brunet et al., 1999; see
below). Several recent studies show that Akt and its
e€ectors play a key role in transducing information
between the cytoplasm and the nucleus.
Both Akt/PKB itself and several of its upstream
regulators and downstream targets have been shown to
partition between the nucleus and the cytoplasm in a
manner that suggests a high degree of regulation of this
process. For example, following their activation, two
Akt/PKB isoforms, PKBa and PKBb, have been
shown to translocate into the nucleus of growth factor
stimulated cells (Andjelkovic et al., 1997; Meier et al.,
1997). One putative activator of AKT/PKB is
MAPKAP kinase 2 (MAP kinase activated protein
kinase 2; (Alessi et al., 1996). MAPKAP kinase-2
(discussed more fully below) moves between the
nucleus and cytoplasm, and its subcellular localization
has been found to be regulated at the level of nuclear
export (Engel et al., 1998), suggesting that MAPKAP
kinase 2 and AKT could interact either in the nucleus
or in the cytoplasm.
Nucleo-cytoplasmic tracking may also play a role
in coordinating the interactions between Akt and at
least one of its downstream targets: the forkheadrelated transcription factor FKHRL1. FKHRL1 is
phosphorylated by Akt in vitro and in vivo; when
phosphorylated, FKHRL1 binds to a 14-3-3 protein
and localizes to the cytoplasm (Brunet et al., 1999).
The physiological consequence of this phosphorylation
is that the FKHRL1-mediated expression of pro-
apoptotic genes is prevented. Clearly, for Akt to be
able to phosphorylate FKHRL1, they both need to be
in the same compartment (nucleus or cytoplasm), and
it is easy to envisage a scenario where cytoplasmic
retention of Akt, and nuclear sequestration of
FKHRL1, would be a critical event in apoptotic
progression. Finally, a recent study links Akt to
another stress/survival element, the transcription
factor NF-kB (Kane et al., 1999), which is discussed
below.
It is of more than passing interest that activation of
Akt/PKB leads to the phosphorylation of BAD with
two consequences that may have separate e€ects on
BAD function and cell survival. First, phosphorylated
BAD dissociates from BclXL. This is important
because the BAD/Bcl complex is pro-apoptotic in
some cells (Zha et al., 1996). Secondly, the liberated,
phosphorylated BAD binds to a member of the 14-3-3
family (Zha et al., 1996). 14-3-3 proteins have recently
been shown to lead to decreased nuclear presence of
proteins to which they bind, although whether at the
level of increased nuclear export or decreased nuclear
import remains controversial (Lopez-Girona et al.,
1999; Yang et al., 1999). Although it seems likely that
one role of 14-3-3 proteins is to anchor their partners
in the cytoplasm, preventing their import into
organelles such as the nucleus or the mitochondrion,
it will be of great interest to know whether BAD ever
enters the nucleus and whether Akt/PKB itself binds to
a 14-3-3 protein in manner that regulates its subcellular
localization ultimately a€ecting cellular survival.
MAP kinases: the road HOG travels
The transduction of signals that originate at the plasma
membrane into the nucleus, where they impinge upon
gene expression, is best understood for the MAP kinase
pathway. Entry into the nucleus of the prototypical
MAP kinases, ERK1 and ERK2, requires their
phosphorylation and activation as protein kinases as
well as their dimerization (Khokhlatchev et al., 1998),
while dephosphorylated, inactive ERKs are returned to
the cytoplasm. In both budding and in ®ssion yeast,
the stress activated MAP kinase homolog Hog1/Spc1
has been shown to be exported from the nucleus in a
manner that requires the activity of the export receptor
Crm1/Xpo1p (Ferrigno et al., 1998; Gaits et al., 1998;
Gaits and Russell, 1999; Reiser et al., 1999). In
budding yeast, the import of Hog1 requires the
activity of the importin homolog Nmd5p (Ferrigno et
al., 1998), whose closest known vertebrate homologs
are human RanBP7 and Xenopus RanBP8 (Gorlich et
al., 1997). It will of great interest to see whether the
transport of ERKs and related protein kinases (JNKs,
p38 family members) involves the vertebrate homologs
of Nmd5p, and whether distinct MAP kinase family
members use di€erent pathways.
Up and down the MAP kinase pathway: MAPKAP
kinase-2 and MAP kinase kinases
The MAP kinase activating protein MAP kinase kinase
(MAPKK) is excluded from the nucleus during MAPK
pathway activation (Fukuda et al., 1997b). Furthermore, the cytoplasmic localization of MAPKK has
been proposed to serve to simultaneously tether
Nuclear transport and stress
P Ferrigno and PA Silver
MAPK in the cytoplasm (Fukuda et al., 1997c).
However, MP1 (Schae€er et al., 1998), a MAPK
kinase module sca€old protein that promotes the
interaction between MAPK and the MAPKK actually
increases expression from a reporter gene downstream
of MAP kinase activation. Thus, MAPKK may enter
the nucleus together with MAPK and MP1, perhaps
leading to sustained activation of the MAPK by
protecting it from inactivation by nuclear protein
phosphatases (e.g. CL100, Lewis et al., 1995), as
proposed for the Hog1 stress-activated protein kinase
in yeast (Ferrigno et al., 1998). The exact contribution
of the many proposed MAP kinase sca€old proteins to
the regulation of MAP kinase activity, and their
interplay with nuclear transport, remains to be
elucidated.
Protein kinases that are downstream of MAP
kinase may include MAPKAP kinase 1 and MAPKAP kinase 2 (Stokoe et al., 1992). MAPKAP
kinase 1 contains a Nuclear Export Sequence that
appears to be regulated by protein phosphorylation in
stimulated cells (Tolwinski et al., 1999); in addition,
the nuclear localization of MAPKAP kinase 1 may
correlate with cell cycle phase (Tolwinski et al., 1999).
MAPKAP K-2 is nuclear in unstressed cells but
translocates to the cytoplasm following stress (Engel
et al., 1998). While the import of MAPKAP kinase 2
into the nucleus appears to be constitutive, its export
appears to be under protein phosphorylation-mediated
regulation whose details remain to be understood.
Finally, protein kinases are opposed by protein
phosphatases. The nucleo-cytoplasmic partioning of
both PP1 (Fernandez et al., 1992) and its regulatory
subunit I-2 (Brautigan et al., 1990) and PP2A
(Turowski et al., 1995) has been suggested to be
regulated in a cell-cycle dependent manner. The
relevance of these observations to stress, and to
cellular regulation as a whole, remains to be elucidated.
Transcription factors
p53
The nuclear localization of p53 has been known for some
time (e.g. Shaulsky et al., 1990) and has been shown to
depend on both import and export events (Middeler et
al., 1997). Nuclear export is likely to be mediated by
leucine-rich NESs present in the C-terminus of p53, that
may be masked when p53 tetramerizes and is thought to
involve Crm1 (Stommel et al., 1999). In non-stressed
cells, p53 moves from the nucleus to the cytoplasm (Roth
et al., 1998) where it is degraded by ubiquitin-mediated
proteolysis. This movement requires that p53 interact
with Mdm2 (Haupt et al., 1997; Kubbutat et al., 1997).
Stress-induced stabilization of p53 leads to either
apoptotic cell death or to cell cycle arrest, depending
on the nature of the stress and of the cell experiencing it.
The phosphorylation of p53 in DNA-damaged cells by
protein kinases including JNK (Fuchs et al., 1998) and
DNA-PK (Shieh et al., 1997) decreases its interaction
with Mdm2, presumably leading to increased nuclear
accumulation of p53 by decreasing Mdm2-mediated
export. Cells that accumulate high levels of DNA
damage die by p53-dependent apoptosis. Identifying
the nuclear transport factors that mediate the import of
newly synthesized p53, and the export of Mdm2/p53 may
open up new avenues for cancer therapy. Two
particulary novel approaches that would lead to
increased levels of p53 in the nucleus would be the
identi®cation of proteins or drugs that increased the rate
of import of p53 into the nucleus, or of factors that
inhibited the import of Mdm2. A peptide that disrupts
the interaction between p53 and Mdm2 and is able to
stabilize p53 in vivo has already been reported (Bottger et
al., 1997). Interestingly, the p53/Mdm2 interaction is
susceptible to the e€ects of the large (14 kDa) peptide
encoded by the alternative reading frame (ARF) of the
INK4a locus (Kamijo et al., 1998; Pomerantz et al., 1998;
Stott et al., 1998). ARF stabilizes p53 by preventing its
association with Mdm2, but rather than inducing the
apoptotic pathway, ARF enables the cell cycle arrest
function of p53.
NF-kB
NF-kB is a transcription factor linked to cell survival e.g.
(Heck et al., 1999). In unstressed cells, NF-kB is retained
in the cytoplasm as a heterodimer with IkB. Following
an appropriate signal, IkB is phosphorylated and
degraded, unmasking the NLS of the NF-kB subunit
(Beg et al., 1992; Ganchi et al., 1992; Henkel et al., 1992;
Zabel et al., 1993) which forms homodimers, or
heterodimers with another NF-kB family member.
These dimers accumulate in the nucleus where they
mediate the transcription of genes required to respond to
the signal. The localization of NF-kB in the nucleus can
be reversed by nuclear forms of IkB, which bind to NFkB to provide an NES. Newly synthesized IkB that is not
bound to NF-kB is imported into the nucleus in a
manner that requires the activity of importins a and b,
but also of other cytosolic factors that appear to bind to
the ankyrin repeats of IkB (Turpin et al., 1999).
Similarly, the import of NF-kB in human (Jurkat) T
cells has been suggested to be mediated by Rch1/
importin b (Torgerson et al., 1998). Because the import
of AP1, NF-AT and STAT1 can be competitively
inhibited by a peptide carrying the basic NLS of NFkB, the regulated import of these transcription factors
may also be mediated by Rch1/importin b (Torgerson et
al., 1998). However, in the absence of evidence for a
direct interaction between each transcription factor and
speci®c importins, one should be cautious in interpreting
these results. The observed competition could be due to
an indirect e€ect such as a requirement for importins a
and b for the recycling of each transcription factor's
nuclear importer from the nucleus back to the cytoplasm.
Indeed, in budding yeast the regulated transport of
transcription factors has been shown to be mediated by
distinct members of the importin b family. For example,
the starvation-activated transcription factor Pho4
translocates from the cytoplasm to the nucleus using
not the classical importin b but a homolog, Pse1/
Kap121p (Ka€man et al., 1998b). Similarly, the export
of Pho4 from the nucleus involves not the classical Xpo1/
Crm1 but a related protein, Msn5 (Ka€man et al.,
1998a).
NF-AT
Nuclear factor of activated T cells (NF-AT) mediates
the e€ects of Ca2+ signals on gene expression and
6131
Nuclear transport and stress
P Ferrigno and PA Silver
6132
cooperates with transcription factors such as AP-1 that
respond to other cellular signals to ensure cell growth
and survival (see Crabtree, 1999; Rao et al., 1997 for
review). Because NF-AT shuttles continuously between
the nucleus and the cytoplasm, and because the
cytoplasmic form is able to bind DNA and activate
transcription, this is an excellent example of the
importance of regulated nuclear transport to the
coordination of cellular events. Nuclear accumulation
of NF-AT only occurs following the activation of the
Ca2+dependent protein serine/threonine phosphatase
calcineurin, which dephosphorylates a number of
serine residues at the N terminus of NF-AT (Park et
al., 1995). The e€ect of this could be to displace
protein kinases such as MEKK1 and CKI that bind to
NF-AT4 and mask its NLS (Zhu et al., 1998).
However, it has also been suggested that phosphorylation would be required to unmask the NES, or to
promote the interaction between NF-AT and the
nuclear export machinery (Beals et al., 1997a,b) or
that calcineurin could itself sterically mask the NES
(Zhu and McKeon, 1999). Using permeabilized HeLa
cells, the export of NF-AT from the nucleus has been
found to be mediated by the importin b family member
exportin/Crm1 (Kehlenbach et al., 1998).
Conclusion
p53 itself has been famously called `the Guardian of
the genome' (Lane, 1992) but this term may be more
appropriately applied to the nuclear envelope, which
forms a physical barrier that limits the access of
proteins, including p53, to DNA (Silver, 1991). The
entry of a protein into the nucleus requires that at least
one of three conditions be met: (1) a positive signal
must be present, usually within the primary sequence
of the cargo, that serves to direct nuclear import, (2) an
appropriate regulatory signal must be given to drive an
interaction between the cargo and the importer; this
may involve dimerization and/or post-translational
modi®cation (e.g. phosphorylation, methylation) and
(3) the surmounting of the nuclear export obstacle. By
this last, we mean that each molecule to be imported
into the nucleus must not be an immediate candidate
for nuclear export. However, nuclear export exists not
simply in oppostion to nuclear protein import: by
allowing the constant cycling of proteins between the
two compartments, nuclear partitioning may allow
signal on/o€ switches to serve as cellular rheostats,
where the relative levels of active proteins on either
side of the nuclear envelope determines the magnitude
of the output. Additionally, nuclear export may itself
serve as a regulatory mechanism, for example in the
case of the inhibition of RNA export by heat shock
(Krebber et al., 1999; Saavedra et al., 1996).
Henceforth, the dissection of signal transduction
pathways cannot be considered complete until we
understand how each of the molecules that crosses
the nuclear envelope interacts with the nuclear
transport machinery and how those interactions are
regulated.
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