Center for Cell Signaling, University of Virginia School of Medicine, Box 800577-MSB7225, Charlottesville VA 22908, USA
* Author for correspondence (e-mail: cal4a{at}virginia.edu)
Accepted 10 July 2002
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Summary |
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Key words: Nuclear import, Green fluorescent protein, Cell density
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Introduction |
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Inhibitor-2 (Inh2) was discovered as a heat stable protein, purified from
rabbit skeletal muscle, which could inhibit PP1C activity in vitro
(Huang and Glinsmann, 1976).
Inh2, like PP1, is conserved among eukaryotes, with Inh2 homologues identified
in baker's yeast (GLC8), Drosophilia, rats, rabbits and humans
(Cannon et al., 1994
;
Helps and Cohen, 1999
;
Osawa et al., 1996
;
Sanseau et al., 1994
;
Zhang et al., 1992
). Inh2
forms a 1:1 complex with PP1C and inhibits the activity of PP1 towards several
substrates in vitro; it also binds to and inhibits the activity of the PP1G
holoenzyme (Resink et al.,
1983
; Yang et al.,
1981
). Previous studies have suggested that there are multiple
sites in Inh2 that interact with the catalytic subunit of PP1
(Huang et al., 1999
;
Yang et al., 2000
). It is
unclear if these different sites target Inh2 to different PP1 holoenzymes in
order to respond to changes in cell signaling.
Inh2 is a 23 kDa phosphoprotein, small enough to passively diffuse through
nuclear pores, yet it contains both a putative nuclear localization signal
(137KKRQFEMKRK147) and a sequence resembling a
leucine-rich nuclear export signal (155LNIKLARQLI165)
(Gerace, 1995;
Quimby and Corbett, 2001
).
Previous reports have shown that Inh2 can differentially localize to the
cytoplasm or nucleus, depending on the phase of the cell cycle, in human HS68
fibroblast cells (Kakinoki et al.,
1997
). Mutation of two lysine residues present in the putative
bipartite NLS domain diminished the cell-cycle-dependent nuclear accumulation
of Inh2, suggesting that Inh2 utilized an active import mechanism
(Kakinoki et al., 1997
). Inh2
levels fluctuate during the cell cycle, starting from a low level and
increasing during G1, peaking during S phase, dropping during G2 and then
reaching its highest peak during mitosis
(Brautigan et al., 1990
;
Kakinoki et al., 1997
).
Progression through the cell cycle can be halted by the formation of cell-cell contacts in a process known as contact inhibition (Bunge, 1979). This process is poorly understood and in some instances, such as tissue formation, cells must overcome contact inhibition. On the other hand, aberrant control of this process may contribute to the type of uncontrolled growth seen in cancer.
In this study we discovered that in both HeLa and PC3 cells, the subcellular localization of Inh2 changes in response to cell density and is independent of the cell cycle. We mapped the regions of Inh2 involved in its subcellular distribution in response to cell density and have identified a domain capable of causing the cytoplasmic retention of a heterologous protein.
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Materials and Methods |
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Immunofluorescence microscopy
PC3 and HeLa cells were cultured to confluency and then replated at
different cell densities onto coverslips coated with fibronectin. After 12-18
hours, or other times as specified in the figure legends, the samples were
fixed with 3% paraformaldehyde [2% for green fluorescent protein (GFP)
fluorescence] and permeabilized with 0.1% Triton-X100. Specimens were blocked
with 3% bovine serum albumin/1% ovalbumin and incubated with a mouse antibody
to Inh2 (Transduction Laboratories). A sheep antibody to mouse IgG conjugated
with fluorescein (Amersham) was used for immunofluorescence. Coverslips were
mounted onto glass slides with Vectashield mounting medium (Vector
Laboratories). Slides were visualized using a Nikon Microphot-5A microscope
with 20x objective and were captured with a Hamamatsu C4742 digital
camera operated by OpenLab software (Improvision) and processed in Adobe
Photoshop for printing.
Subcellular fractionation
HeLa cells were cultured at either low density (LD), 60 cells per
mm2 or high density (HD),
600 cells per mm2, and
permeabilized by scraping with 0.25 M sucrose, 10 mM Tris-HCl (pH 7.5), 5 mM
MgCl2 and 0.01% digitonin. Samples were incubated on ice for 10
minutes and then centrifuged at 5000 g for 5 minutes. The pellets
containing cell nuclei were washed once in the same buffer and then dissolved
in buffer containing 1% SDS. Samples of the supernatant (cytosol, C) and the
dissolved pellets (nuclear, N) were normalized for total protein by a
Coomassie-dye-binding assay from BioRad. Samples were resolved by SDS-PAGE and
analyzed by western blotting using antibodies to Inh2 (Transduction
Laboratories) and RCC1 (Santa Cruz). HRP-conjugated secondary antibodies from
Pierce were used in conjunction with Supersignal reagent (Pierce) and used to
expose X-ray film.
Replating assay and quantification
HeLa cells growing at either low or high density were trypsinized for 5
minutes, centrifuged for 2 minutes at 1000 g and replated to the
opposite condition onto fibronectin-coated coverslips for either 20, 40 or 60
additional minutes. Adherent cells were washed with PBS, fixed with 2%
paraformaldehyde, permeabilized with 0.1% Triton-X100 and immunostained for
Inh2 using an antibody from Transduction Laboratories. On the basis of
microscopic examination, cells were divided into three categories: (1) nucleus
brighter than cytoplasm; (2) cytoplasm brighter than nucleus; and (3)
indistinguishable (owing to cell shape or equivalent staining). A total of 100
cells for each time point was counted for each of three separate
experiments.
Electroporation
HeLa cells were trypsinized, pelleted and resuspended in DMEM supplemented
with 10% neonatal calf serum and 15 mM HEPES to a concentration of
2-5x106 cells/ml. This suspension was electroporated with
20-40 µg of the plasmid diluted in 210 mM NaCl using a BioRad Genepulser
set on 300 mV and 960 µF. For electroporation of myctagged Inh2, cells were
replated to low density for 12-18 hours and then replated to low or high
density onto fibronectin-coated coverslips for an additional 4 hours. To
compare Inh2-GFP3 with the NLS mutant, electroporated cells were
replated to low density on fibronectin-coated coverslips for 6-10 hours.
Analysis of Inh2HA3 and Inh2GFP3 at high cell density
was done by initially plating electroporated cells to low density overnight
and then replating to high density for 4 hours.
Calculation of import index
Total cell fluorescence intensity and nuclear fluorescence were determined
by a digital CCD camera using Openlab software. Cytoplasmic staining was
determined by subtracting the nuclear fluorescence from the total
fluorescence. The import index was calculated by the equation
[(Ci-Ni)/Ni],
which subtracted the average cytoplasmic pixel intensity from the average
nuclear pixel intensity to create a value that represented how much brighter
the nucleus was than the cytoplasm. This number was then divided by the
average nuclear pixel intensity to normalize for differences in protein
expression between cells.
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Results |
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A constitutively nuclear protein, RCC1, was used as a marker and as expected was only present in the nuclear fraction. The same amounts of total protein were analyzed, and indeed samples from both low- and high-density cells displayed the same level of RCC1 staining. We noted thatInh2 staining was more intense in the low-density cells. This result showed that low-density cells had a higher concentration of Inh2 than high-density cells, consistent with the difference in intensity seen by immunofluorescent staining (Fig. 1).
As Inh2 binds to the catalytic subunit of PP1, we investigated the
localization of PP1 by immunofluorescence. PP1
localized mostly
to the nucleus of low-density HeLa cells
(Fig. 2A) and was excluded from
the nucleus of high-density cells (Fig.
2B) in a manner similar to Inh2.
|
Kakinoki et al. reported nuclear accumulation of Inh2 in cells during the S
phase of the cell cycle, so we wanted to determine if low- and high-density
cells had obvious differences in cell cycle distribution
(Kakinoki et al., 1997). FACS
analysis of propidium-iodide-stained low- and high-density HeLa cells showed
similar distribution of cells in the G1/S/G2-M phases of the cell cycle
(Fig. 3). Therefore high cell
density did not arrest HeLa cell cycle progression nor did the different
localization of Inh2 in HeLa cells grown to low or high density seem to depend
on the phase of the cell cycle.
|
Redistribution of Inh2 in response to replating at different cell
densities
The nuclear-cytoplasmic distribution of Inh2 was reversed within minutes in
high-density cells replated at low density and low-density cells replated at
high density. HeLa cells cultured at high density were trypsinized and
replated within 10 minutes at low density onto coverslips and examined by
immunofluorescence for Inh2 at 20 (Fig.
4A,B,C), 40 or 60 (Fig.
4D,E,F) minutes after replating. Nuclear accumulation of Inh2 was
evidently completed within 60 minutes (Fig.
4F), as seen by the appearance of overlap (yellow) of Inh2
staining (green) and DAPI staining (red) that were initially separate
(Fig. 4C). The graph in
Fig. 4 displays the kinetics of
Inh2 import into the nucleus. In a reciprocal experiment, HeLa cells cultured
at low density were trypsinized, collected by centrifugation and replated at
high density onto coverslips. The kinetics of Inh2 redistribution from the
nucleus to the cytoplasm was the same as import into the nucleus (data not
shown). Addition of Leptomycin B, an inhibitor of Crm1-mediated export, did
not prevent the translocation of Inh2 from the nucleus to the cytoplasm when
low-density cells were replated at high density (data not shown). The data
show that the redistribution of Inh2 from the nuclear to cytoplasmic
compartments occurs rapidly and does not depend on active nuclear export.
|
Separate domains of Inh2 control subcellular distribution
We prepared a series of Inh2 proteins that were myc-tagged at the
N-terminus. These defined four major domains of Inh2, a C-terminal region, an
N-terminal region, a central domain and a NLS-containing region
(Fig. 5K). The Inh2 proteins
were transiently expressed in HeLa cells and visualized by anti-myc
immunofluorescence (Fig. 5).
The myc-Inh2[0-197] behaved exactly like the endogenous full-length Inh2 (205
residues), showing accumulation in the nucleus of low-density cells
(Fig. 5D) and exclusion from
the nucleus of high density cells (Fig.
5C). The myc-Inh2 displayed the same localization when expressed
in HEK293, COS7 and NIH3T3 cells grown at low or high density (data not
shown). Without the N-terminal domain (residues 0-77), myc-Inh2[78-197] showed
the same cell-density-dependent nuclear localization as full-length Inh2
(Fig. 5E,F). By contrast,
deletion of both the C-terminal domain (residues 150-197) and the
NLS-containing domain (residues 120-150) yielded a truncated form of
myc-Inh2[0-119] that appeared in the cytoplasm but not the nucleus, regardless
of cell density (Fig. 5G,H).
The results suggest that the region 120-197 was required for accumulation of
Inh2 in the nucleus of low-density cells. By truncation of both the N-terminal
domain and the C-terminal domain, we created a minimal protein containing both
the central domain and the NLS domain: myc-Inh2[78-150]. This truncated form
exhibited both nuclear accumulation in low-density cells and cytoplasmic
retention in high-density cells in a manner identical to the full-length
protein (Fig. 5I,J).
|
Residues 78-119 are sufficient for cytoplasmic retention
On the basis of size, both myc-Inh2[0-119] and myc-Inh2[78-197] would be
expected to pass through nuclear pores and be uniformly distributed throughout
the cell. Because these proteins were both predominantly localized to the
cytoplasm in high-density cells, we propose that residues 78-119, common to
both proteins, are involved in retention of these proteins in the cytoplasm.
We created a fusion protein of Inh2[78-119] with GFP. Transient expression of
this protein in HeLa cells revealed that whereas GFP alone was localized to
both the nucleus and cytoplasm of low-density cells
(Fig. 6A), the fusion protein
containing Inh2 residues 78-119 was retained in the cytoplasm
(Fig. 6B). We conclude that
residues 78-119 are sufficient to retain a heterologous protein in the
cytoplasm.
|
Localization of Inh2 fusion proteins
To examine transit in and out of the nucleus, Inh2 was produced as two
different fusion proteins; one with a triple HA (HA3) tag resulting
in a protein small enough (<30 kDa) for diffusion and the other with three
tandem GFPs (GFP3) to create a protein of >100 kDa, which is too
large to traverse nuclear pores by passive diffusion. HeLa cells expressing
these fusion proteins were plated at low density for 12 hours and then
replated at either low (Fig. 7)
or high density (Fig. 8). After
allowing for cell spreading, the cells were fixed for microscopy and the
nuclear and cytoplasmic fluorescence (anti-HA or GFP) quantified. In
low-density cells that have endogenous Inh2 in the nucleus, we found that both
HA3- and GFP3-Inh2 fusion proteins also accumulated in
the nucleus (Fig. 7A). When two
lysines (K144, K146) in the putative NLS sequence were mutated to alanine in
Inh2-GFP3, nuclear accumulation was abrogated
(Fig. 7B). Mutation of a highly
conserved phosphorylation site (Thr72) to alanine was previously reported to
abrogate the nuclear accumulation of Inh2
(Kakinoki et al., 1997). We
tested this mutation in the context of the Inh2-GFP3 protein but
did not see a significant reduction in nuclear import
(Fig. 7).
|
|
On the other hand, in high-density cells only Inh2-HA3 (Fig. 8B) behaved in a manner identical to endogenous Inh2, becoming mostly cytoplasmic, whereas Inh2-GFP3 (Fig. 8A) remained concentrated in the nucleus. The results imply that Inh2 passively diffuses out of the nucleus and that the size of the GFP3 fusion prevented this diffusion, but the much smaller HA tag did not.
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Discussion |
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Kakinoki et al. described a nuclear-cytoplasmic redistribution of
overexpressed Inh2 correlating to the phase of the cell cycle in HS68 cells
that depended on an NLS and on Inh2 phosphorylation
(Kakinoki et al., 1997).
However, the localization of Inh2 discovered in this study, although it
requires an NLS, does not rely on the cell cycle or Inh2 phosphorylation.
Thus, there seem to be multiple mechanisms controlling Inh2 localization.
Western blotting of Inh2 showed that low-density cells had more Inh2 than high-density cells. This raises the question of whether or not protein degradation or synthesis is responsible for the changes in Inh2 localization. One possibility is that low-density cells produce more Inh2 and saturate the system responsible for cytoplasmic retention leading to nuclear import and accumulation. This is unlikely because overexpression of Inh2[0-197] does not result in nuclear accumulation. Protein degradation might control localization by degrading nuclear Inh2 in high density but not low-density cells. However, this does not appear to be the case, because Inh2-GFP3 expressed in high-density cells still accumulates in the nucleus.
We defined residues 78-150 as the minimal domain of Inh2 that is able to
undergo nuclear-cytoplasmic redistribution in response to changes in cell
density. Conspicuously absent from this domain are residues
10IKGI13, which interact with the catalytic subunit of
PP1 (Huang et al., 1999). We
further defined this region by demonstrating that residues 78-119 were
sufficient to cause cytoplasmic retention of GFP, which is present in both the
cytoplasm and nucleus when expressed in HeLa cells. Previous studies suggest
that this region of Inh2 does not bind to PP1
(Huang et al., 1999
). Although
PP1 binding is not required for Inh2 localization, there is the possibility
that PP1 may be bound to Inh2 when it redistributes between the nucleus and
cytoplasm in response to cell density.
There have been reports of other proteins altering their subcellular
distribution in response to cell density
(Baba et al., 2001;
Trompeter et al., 1996
). One
example is ZnBP, which localizes to the nucleus of low-density cells and is
excluded from the nucleus in high-density cells
(Trompeter et al., 1996
).
ZnBP, like Inh2, contains a bipartite NLS responsible for its nuclear
localization in low-density cells
(Trompeter et al., 1999
).
Another similarity is that the cytoplasmic retention domain of Inh2 [78-119]
contains a cluster of acidic residues (7/13); ZnBP also contains clusters of
acidic residues. It is possible that these acidic clusters interact with a
similar cytoplasmic protein that retains Inh2 and ZnBP in the cytoplasm.
Additional work with ZnBP has led to the proposal of a developmental
function of ZnBP. Overexpression of ZnBP in HL-60 cells leads to an increase
in cell proliferation accompanied by a decrease in differentiation
(Rodriguez et al., 1998). PP1
has previously been shown to activate transcription factors, such as Sp1, by
dephosphorylation (Daniel et al.,
1996
). There are several examples of genes whose transcription is
altered in response to cell density (e.g. VHL protein, fibronectin and
collagen) (Baba et al., 2001
;
Wolthuis et al., 1993
). One
potential model is that Inh2 transports a PP1 holoenzyme into the nucleus of
low-density cells to alter gene transcription by dephosphorylating certain
transcription factors.
Investigations of how cell density and gene transcription interrelate are fundamental for understanding both cancer and embryonic development. Cell-density-related control of Inh2 localization opens new avenues of study into the role of PP1 function in cell-cell signaling.
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Acknowledgments |
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References |
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