(Received for publication, October 26, 1995; and in revised form, December 28, 1995)
From the
The regulation of nuclear protein transport by phosphorylation plays a central role in gene expression in eukaryotic cells. We previously showed that nuclear import of SV40 large tumor antigen (T-ag) fusion proteins is regulated by the CcN motif, comprising phosphorylation sites for casein kinase II and the cyclin-dependent kinase cdc2, together with the nuclear localization signal. Regulation of nuclear uptake by CcN motif kinase sites also holds true for the yeast transcription factor SWI5 and the Xenopus nuclear phosphoprotein nucleoplasmin. To test directly whether a kinase site other than those of the CcN motif could regulate nuclear import of T-ag, the CcN motif casein kinase II site, which markedly increases the rate of T-ag nuclear import, was replaced by a consensus site for the cAMP-dependent protein kinase (PK-A) using site-directed mutagenesis. The resultant fusion protein could be specifically phosphorylated by PK-A in vitro and in cell extracts. Nuclear import of the fluorescently labeled protein was analyzed in the HTC rat hepatoma cell line both in vivo (microinjected cells) and in vitro (mechanically perforated cells) in the presence and the absence of cAMP and/or PK-A catalytic subunit using confocal laser scanning microscopy. In vitro PK-A-prephosphorylated protein was also tested. All results indicated that the rate of nuclear import was increased by phosphorylation at the PK-A site (2-5-fold), demonstrating that kinases other than those of the CcN motif can regulate nuclear import in response to stimulatory signals. The phosphorylation-regulated nuclear localization signal derived here represents an important first step toward developing a signal conferring inducible nuclear targeting of molecules of interest.
Although proteins such as histones appear to be constitutively
targeted to the nucleus, others are only translocated to the nucleus
under specific conditions, otherwise being predominantly cytoplasmic
(Nigg et al., 1991; Jans, 1995). The advantages of a
conditionally cytoplasmic location for a transcription factor (TF) ()include the potential to control its activity by
regulating its nuclear uptake and its direct accessibility to
cytoplasmic signal-transducing systems (Schmitz et al., 1991;
Jans, 1995). TFs able to undergo inducible nuclear import include the
glucocorticoid receptor (Picard and Yamamoto, 1987), the
-interferon-regulated factor interferon stimulated gene factor 3
(ISGF-3) (Schindler et al., 1992),
-interferon-activated
factor (GAF) (Shuai et al., 1992), the nuclear v-jun
oncogenic counterpart of the AP-1 transcription complex member c-jun (Chida and Vogt, 1992; Takagawa et al., 1995),
the Saccharomyces cerevisiae TF SWI5 (Moll et al.,
1991; Jans et al., 1995), the Drosophila melanogaster morphogen dorsal (Govind and Steward, 1991), and the
nuclear factor NF-
B (Schmitz et al., 1991; Shirakawa and
Mizel, 1989; Lenardo and Baltimore, 1989). The fact that the nuclear
translocation of various TFs, developmental morphogens, and oncogene
products accompanies changes in the differentiation or metabolic state
of eukaryotic cells indicates that nuclear protein import is a key
control point in the regulation of gene expression and signal
transduction.
Proteins larger than 45 kDa require a nuclear
localization signal (NLS) (see Jans, 1995; Jans and
Hübner, 1996) in order to be targeted to the
nucleus. In addition to the NLS, specific signals carried by the
transported proteins function in a regulatory fashion, whereby covalent
modifications such as phosphorylation play a central role (Jans, 1995;
Jans and Hübner, 1996). We have demonstrated that
nuclear import of SV40 large tumor antigen (T-ag) fusion proteins is
regulated by the CcN motif (Jans et al., 1991), comprising
phosphorylation sites for casein kinase II (CKII) and the
cyclin-dependent kinase cdc2 together with the NLS. Although
nuclear localization is entirely NLS-dependent (Rihs and Peters, 1989;
Rihs et al., 1991), the rate of nuclear import is regulated by
the CKII phosphorylation site (Ser) (Jans et
al., 1991; Rihs et al., 1991; Jans and Jans, 1994), and
phosphorylation at the cdc2 site (Thr
) adjacent
to the NLS (amino acids 126-132) determines the maximal extent of
nuclear accumulation (Jans et al., 1991). Regulation of
nuclear transport by CcN motif kinase sites also holds true for SWI5
(Moll et al., 1991; Jans et al., 1995) and the Xenopus nuclear phosphoprotein nucleoplasmin (Vancurova et
al., 1995). It is likely, however, that other kinases/kinase sites
function in analogous fashion to regulate nuclear protein import
specifically (Jans and Jans, 1994; Jans, 1995; Jans and
Hübner, 1996). The cAMP-dependent protein kinase
(PK-A), for example, has been implicated in enhancing nuclear import of
members of the rel/dorsal class of TFs, although
kinetic analyses have not been performed (Mosialos et al.,
1991; Norris and Manley, 1992).
In order to test directly whether a kinase site other than those of the CcN motif could regulate T-ag nuclear import, and as a first step toward developing a phosphorylation regulated NLS (prNLS) (Jans, 1995) capable of conferring inducible nuclear translocation on carrier molecules of interest, we set out to replace the CKII site of the CcN motif by a consensus site for PK-A using site-directed mutagenesis. The resultant fusion protein could be specifically phosphorylated both in vitro and in cell extracts. Its nuclear import was analyzed in the HTC rat hepatoma cell line both in vivo and in vitro in the absence and the presence of cAMP and/or PK-A catalytic subunit (C-subunit) using confocal laser scanning microscopy (CLSM). In vitro PK-A-prephosphorylated protein was also tested. All results indicated that the rate of nuclear import was increased by phosphorylation at the PK-A site, indicating that kinases other than those of the CcN motif can regulate nuclear import in response to stimulatory signals. prNLSs similar to that derived here have potential application in precisely cuing the transport of molecules of interest to the nucleus of relevant cell types.
Figure 1:
Sequence of the SV40 T-ag fusion
proteins used in this study (A) and nuclear import kinetics in vivo and in vitro (B). A, all
fusion proteins contain SV40 T-ag sequences fused N-terminal to E.
coli -galactosidase (amino acids 9-1023). The
single-letter amino acid code is used, whereby the NLS is double
underlined and the phosphorylation sites in bold, with
that for CKII underlined and that for PK-A underlined with a dotted line. Capital letters indicate
T-ag sequence. The cN-, Cc-, and CcN-
-Gal fusion proteins have
been previously described (Jans and Jans, 1994, Rihs et al.,
1991). B, nuclear transport was measured in microinjected (in vivo) or mechanically perforated (in vitro) HTC
cells using CLSM as described under ``Experimental
Procedures'' (Jans et al., 1991, 1995; Jans and Jans,
1994). The in vivo measurements represent the average of at
least two separate experiments, where each point represents the average
of 6-10 separate measurements for each of nuclear (Fn)
and cytoplasmic (Fc) fluorescence respectively, with
autofluorescence subtracted. The in vitro measurements are
from a single typical experiment (see also Table 1), each point
representing the average of at least 10 separate measurements for each
of nuclear (Fn) and cytoplasmic (Fc) fluorescence
respectively, with autofluorescence subtracted. Curves are fitted for
the function Fn/c (t) = Fn/c
(1 - e
) (Jans et al., 1991, 1995; Jans
and Jans, 1994), where t is time in
min.
To confirm that the
engineered phosphorylation site was functional, phosphorylation was
tested in vitro using purified CKII and PK-A C-subunit (Fig. 2). The AcN--Gal T-ag fusion protein was not
phosphorylated to a significant extent by CKII, in contrast to the wild
type CcN-
-Gal T-ag fusion protein, indicating that as expected,
the CKII site was no longer functional. The AcN-
-Gal T-ag fusion
protein was, however, specifically phosphorylated by PK-A (Fig. 2), indicating that the introduced PK-A site was
functional. The CcN-
-Gal T-ag fusion protein and
-galactosidase itself were not phosphorylated by PK-A, as
expected. The results indicated that we had been successful in
introducing a functional PK-A site in place of the CKII site, within
the T-ag CcN motif, in AcN-
-Gal.
Figure 2:
Specificity of kinase site phosphorylation
of T-ag fusion proteins in vitro. Incubation was for 1 h at 30
°C. The stoichiometry of phosphorylation was determined as
described under ``Experimental Procedures.'' CKII possessed
no kemptide phosphorylation activity (12 ± 3 pmol
P/min activity for the CKII specific peptide substrate
Arg-Arg-Arg-Asp-Asp-Asp-Ser-Asp-Asp-Asp), whereas the PK-A C-subunit
showed no CKII peptide phosphorylation (26.9 pmol P
/min
activity for kemptide).
Figure 3:
Visualization of T-ag fusion protein
nuclear accumulation in vivo (A) and in vitro (B). CLSM images of cN--Gal, CcN-
-Gal, and
AcN-
-Gal (in the presence and the absence of exogenous 25
µM cAMP, lower panels) are from in vivo (after 5 min at 37 °C, 40
magnification with water
immersion objective) and in vitro (after 10-12 min at
room temperature, 60
magnification with oil immersion
objective) nuclear transport assays as indicated. In the case of B, one nucleus is shown in the panel for cN-
-Gal, whereas
two nuclei are shown in the other panels. Scale bars are as
indicated.
Figure 4:
PK-A C-subunit activity in HTC cells
subsequent to fusion with or without polyethylene glycol and with or
without pretreatment with the adenylate cyclase activator forskolin
(0.1 mM) and the phosphodiesterase inhibitor
isobutylmethylxanthine (0.5 M). Total C-subunit activity was
3.0 ± 0.2 and 3.2 ± 0.2 units/mg for unfused and fused
HTC cells, respectively. Under the same experimental conditions,
purified PK-A C-subunit transferred 11.2 pmol
P/min.
Figure 5:
Nuclear import of the AcN--Gal T-ag
fusion protein in the absence or the presence of exogenous cAMP (25
µM) with or without the addition of the PK-A C-subunit
inhibitor peptide PK-I 5-24 (2.5 µM). Measurements
were performed in microinjected HTC cells (in vivo) and
mechanically perforated HTC cells (in vitro) using CLSM as
described in the legend to Fig. 1(see ``Experimental
Procedures''). The results are shown for a single typical
experiment (see also Table 1), where each point represents the
average of at least eight separate measurements for each of nuclear (Fn) and cytoplasmic (Fc) fluorescence, respectively,
with autofluorescence subtracted.
More detailed
examination of the nuclear import kinetics showed that the effect of
cAMP appeared to be exerted at the level of the initial rate of nuclear
import (Table 2). The initial rate of import of AcN--Gal was
increased by about 2-fold in response to cAMP, the effect being
abrogated by PK-I 5-24 (Table 2). As expected, cAMP had no
effect on the rate of nuclear import of the CcN-
-Gal fusion
protein that lacks the PK-A site (Table 2), indicating that the
enhancement of nuclear uptake by cAMP was specific to AcN-
-Gal.
The above results closely parallelled results for the
phosphorylation of AcN--Gal in cytosolic extracts (Fig. 6,
and not shown). As observed previously, the wild type CcN-
-Gal
fusion protein, which contains the CKII site, was strongly
phosphorylated due to the presence of cytosolic CKII (Jans and Jans,
1994). The basal level of phosphorylation of AcN-
-Gal in the
absence of cAMP was about 20% that of CcN-
-Gal (Fig. 6).
This basal phosphorylation activity is presumably sufficient to support
the nuclear transport of AcN-
-Gal in vitro at the rate
observed in the absence of exogenous cAMP (Fig. 1B and Table 1). The addition of cAMP increased the phosphorylation of
AcN-
-Gal over 2-fold above this basal level (Fig. 6). Both
basal and cAMP-induced phosphorylation of AcN-
-Gal was inhibited
markedly by PK-I 5-24 (Fig. 6), demonstrating that PK-A
was indeed the kinase responsible for AcN-
-Gal phosphorylation in
reticulocyte lysate. Consistent with this, the PK-A C-subunit was
demonstrated to be present in reticulocyte lysate by Western blot
analysis, as well as in cytosolic extracts from HTC cells (not shown).
Figure 6:
Phosphorylation of T-ag fusion proteins in
the absence or the presence of cAMP and PK-I 5-24 in cytosolic
extract. Subsequent to the incubation of fusion proteins in
reticulocyte lysate (60 min at 30 °C), affinity chromatography, and
SDS gel electrophoresis (7.5% 30:1 acrylamide:bis-acrylamide), the
stoichiometry of phosphorylation was determined as described under
``Experimental Procedures'' using phosphor imaging of the
dried gel. The standard used was the CcN--Gal protein
prephosphorylated by purified CKII to a stoichiometry of 0.54 mol
P
/mol tetramer.
Similar results to those above for nuclear transport were obtained
in in vitro experiments in which the PK-A C-subunit was
included together with AcN--Gal, the initial rate of nuclear
transport being increased over 2-fold (Table 2, in
vitro). PK-I 5-24 abrogated the effect of the inclusion of
C-subunit, which did not affect nuclear import of the control
CcN-
-Gal fusion protein (Table 2, in vitro).
Finally, the nuclear import kinetics of AcN-
-Gal prephosphorylated
by PK-A in vitro were also measured both in vivo and in vitro ( Table 1and Table 2). Prephosphorylated
AcN-
-Gal was accumulated at a 2-3-fold higher rate (Table 1), which was attributable to a 2-3-fold higher
initial rate of transport (Table 2). As a control, the
CN-
-Gal T-ag fusion protein lacking the PK-A site was incubated
with PK-A, and the nuclear import kinetics was subsequently measured ( Table 1and Table 2). Preincubation with PK-A had no
significant effect on the rate of import, meaning that the enhanced
import rate of prephosphorylated AcN-
-Gal could be directly
attributed to phosphorylation at the PK-A site.
This study constitutes the first report of a kinase site being replaced by a consensus site for another kinase in order to alter the regulation of the physiological effects of phosphorylation at the site in question. It shows that the introduction of a consensus site for PK-A in place of the CKII site of the T-ag CcN motif can confer PK-A-mediated regulation of the kinetics of nuclear import of T-ag fusion proteins. The addition of cAMP or PK-A C-subunit or prephosphorylation by PK-A at the site increases the rate of nuclear import 2-5-fold, largely through increasing the initial rate of nuclear uptake. The PK-A-specific inhibitor peptide PK-I 5-24 inhibits the cAMP-induced and PK-A C-subunit-induced enhancement of nuclear import, indicating that the effects are mediated by PK-A phosphorylation at the PK-A site. That PK-A may regulate nuclear protein import in the case of TFs such as those of the rel/dorsal family has been established by others (Mosialos et al., 1991; Norris and Manley, 1992); this, however, is the first time that a PK-A site has been engineered in place of a kinase site in a heterologous protein and shown to be capable of regulating nuclear import. The results here clearly demonstrate that kinases other than those of the CcN motif can regulate nuclear import of T-ag if the appropriate phosphorylation site is present.
The fact that PK-A regulates the rate rather than the
maximal extent of nuclear import of AcN--Gal is consistent with
the fact that the CKII site, replaced by the consensus PK-A site in
AcN-
-Gal, regulates the rate of nuclear import of the wild type
T-ag CcN-
-Gal fusion protein (Jans and Jans, 1994; Rihs et
al., 1991). Phosphorylation at the PK-A/CKII site thus modulates
the same parameter of nuclear protein import, but because the kinases
phosphorylating at the respective sites exhibit distinct regulation,
this results in differences in the stimuli enhancing nuclear import of
the respective fusion proteins. Although treatments activating PK-A
enhance nuclear import of AcN-
-Gal, CKII-mediated enhancement of
that of CcN-
-Gal appears to be largely constitutive (see Jans,
1995; Jans and Jans, 1994), due to the fact that CKII activity appears
to be largely constitutive in most cell types (Allende and Allende,
1995). The engineered AcN signal is thus a prNLS conferring inducible
nuclear import, hormonal, or other stimuli that activate PK-A able to
directly modulate the rate of nuclear entry of a carrier protein to
which it is attached.
prNLSs have an important potential application in targeting molecules of interest to the nucleus (Jans, 1994, 1995). Those such as the engineered prNLS described here where the PK-A site modulates the rate of nuclear import are of particular interest, because they potentially confer tightly regulated nuclear localization according to hormonal or other stimuli, thus enabling precise cuing of the nuclear localization of relevant proteins and other molecules according to need. This may have application in gene therapy through facilitating the directed transport of DNA molecules to the nucleus of mammalian or plant cells to increase transfection and/or homologous recombination efficiencies (see Jans, 1994; Rosenkranz et al., 1992). Alternatively, toxic molecules might be efficiently targeted to sensitive subcellular sites such as the nucleus in order to effect tumor cell killing (Akhlynina et al., 1993, 1995). The prNLS derived and characterized in this study represents an important first step toward developing a signal conferring inducible nuclear targeting of molecules of interest. Although the PK-A-T-ag-NLS prNLS did not show absolute dependence on induction of PK-A activity for nuclear translocation in HTC cells, presumably due to the relatively high basal PK-A activity (see Fig. 4, 5; Vandromme et al., 1994), it may, however, be useful for conditional inducible nuclear targeting of molecules of interest in other cell lines where PK-A activity is more tightly regulated. Apart from the derivation of further inducible variants of the T-ag CcN motif, future work in this laboratory will include investigation of the efficacy of this prNLS in various cell lines, including somatic cell PK-A mutants (Botterell et al., 1987), as well as the investigation of its use in conjunction with plasmid constructs encoding the cDNAs for the PK-A C-subunit and/or PK-A inhibitor PK-I (see Norris and Manley, 1992) expressed from inducible promoters. Our ultimate aim is to achieve fully inducible/conditional nuclear targeting of molecules of interest for use in a variety of cell types with widespread clinical and research applications.