Download Nuclear-cytoplasmic distribution of Inh2

Research Article
3739
Domains of type 1 protein phosphatase inhibitor-2
required for nuclear and cytoplasmic localization in
response to cell-cell contact
Craig Leach*, Masumi Eto and David L. Brautigan
Center for Cell Signaling, University of Virginia School of Medicine, Box 800577-MSB7225, Charlottesville VA 22908, USA
*Author for correspondence (e-mail: [email protected])
Accepted 10 July 2002
Journal of Cell Science 115, 3739-3745 © 2002 The Company of Biologists Ltd
doi:10.1242/jcs.00052
Summary
Inhibitor-2 of type 1 protein phosphatase is a
phosphoprotein conserved among all eukaryotes, and it
appears in both the nucleus and cytoplasm of tissue culture
cells. We discovered that endogenous inhibitor-2 is
concentrated in the nucleus of cells cultured at low density,
whereas cells growing at high density excluded inhibitor-2
from the nucleus. There was rapid redistribution of
inhibitor-2 when cells were replated at low or high density.
Localization of myc-tagged forms of inhibitor-2 showed
that residues 119-197 were required for nuclear
accumulation in low-density cells and residues 78-119 were
required for cytoplasmic localization in high-density cells.
Fusion of inhibitor-2 residues 78-119 to green fluorescent
protein was sufficient to produce cytoplasmic retention.
Inhibitor-2 fused to triple tandem green fluorescent protein
(100 kDa) was imported into the nucleus of low-density
cells but was not excluded from the nucleus when cells
reached high density, implying that inhibitor-2 was actively
imported into the nucleus but exited by passive diffusion
instead of active export. We conclude that inhibitor-2
contains two separate domains that control its localization
in the nucleus or cytoplasm. This change in inhibitor-2
localization may direct inhibitor-2 to different forms of
protein phosphatase 1 or change the localization of protein
phosphatase, as part of the cellular response to cell-cell
contacts at high density.
Introduction
Type 1 protein phosphatase (PP1) is an essential and highly
conserved enzyme, with over 80% amino-acid sequence
identity between baker’s yeast (called GLC7) and human
(Cannon et al., 1994) PP1. PP1 is the major enzyme that
dephosphorylates serine and threonine residues on proteins. It
has been implicated in the control of a range of cellular
processes, including the cell cycle, gene expression, cell
adhesion and glycogen metabolism (Cohen, 1989; Shenolikar,
1994). To achieve this wide range of effects, PP1 exists as a
series of holoenzymes, with its catalytic subunit, PP1C, bound
to a regulatory subunit, of which there are dozens. Evidence
suggests that the subcellular localization, activity and substrate
specificity of PP1C are controlled by its interaction with these
regulatory subunits (Bollen, 2001; Egloff et al., 1997).
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-cycledependent 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,
Key words: Nuclear import, Green fluorescent protein, Cell density
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Journal of Cell Science 115 (19)
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.
Materials and Methods
Tissue culture and general reagents
Tissue culture media and sera were obtained from Gibco BRL. PC3
cells were cultured in RPMI media supplemented with 10% FBS, and
HeLa cells were cultured in DMEM supplemented with 10% NCS.
Cells were cultured at 37°C in 5% CO2. Mouse antibodies to both the
myc (clone 9E10) and HA (clone 12CA5) epitopes were obtained
from the antibody core facility at the University of Virginia. The PP1δ
antibody was obtained from QCB. Chemicals were from Sigma unless
otherwise noted.
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 20×
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 2-5×106 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.
Results
Distribution of Inh2 and PP1δ in the nucleus and
cytoplasm of low- and high-density cells
Endogenous Inh2 in both HeLa and PC3 cells cultured at low
cell density was concentrated in the nucleus relative to the
cytoplasm (Fig. 1A,B). By contrast, Inh2 was cytoplasmic in
HeLa and PC3 cells grown to high cell density (Fig. 1B), with
no detectable staining of Inh2 in the nucleus. Subcellular
fractionation confirmed the Inh2 distribution seen by
immunofluorescence microscopy. HeLa cells cultured at low
and high density were permeabilized with 0.01% digitonin to
selectively disrupt the plasma membrane but not the nuclear
envelope. The cell suspension was centrifuged to yield a
supernatant consisting of the cytoplasm and a pellet
containing the intact nuclei and cytoskeleton that were
dissolved in 1% SDS. Western blotting of these fractions
is shown in Fig. 1C. In low-density cells, Inh2 was
predominantly in the nuclear fraction, with a much lower level
detected in the cytoplasmic fraction. On the other hand, in
high-density cells Inh2 was present mostly in the cytoplasmic
fraction, with much lower levels in the nuclear fraction.
Therefore the results of immunofluorescence and subcellular
fractionation were consistent, showing nuclear versus
cytoplasmic localization of Inh2 in response to cell density.
The results reinforce each other and exclude possible artifacts
of immunofluorescence, such as masking of epitopes or
interference by fixation.
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
Nuclear-cytoplasmic distribution of Inh2
3741
Fig. 2. Endogenous PP1δ has a similar localization to Inh2. HeLa
cells were grown to either low (A) or high density (B) on coverslips
and examined by immunofluorescence using an antibody against
PP1δ.
Fig. 1. Inh2 localization corresponding to cell density. PC3 (A,B)
and HeLa (C,D) cells were grown to either low (A,C) or high density
(B,D) on fibronectin-coated coverslips and examined by
immunofluorescence using an antibody against human Inh2. To
confirm Inh2 distribution by western blotting, high-density (HD) or
low-density (LD) HeLa cells were permeabilized with 0.01%
digitonin to yield a cytosolic fraction, labeled C, and a nuclear
fraction, labeled N. The top panel shows western blotting using an
antibody to Inh2 and the bottom panel shows blotting with an
antibody to RCC1 the Ran activator protein, a constitutively nuclear
protein.
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 PP1δ by immunofluorescence.
PP1δ 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 highdensity 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 Crm1mediated 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[0119] 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 Nterminal domain and the C-terminal domain, we created a
minimal protein containing both the central domain and the
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Journal of Cell Science 115 (19)
Fig. 3. FACS analysis of cells
cultured at different densities.
HeLa cells growing at low or high
density were trypsinized and
stained with propidium iodide as
described in Materials and
Methods. The phase of the cell
cycle was determined on the basis
of the intensity of the propidium
iodide staining using CellQuest
software.
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[78197] 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).
Fig. 4. Inh2 relocalization occurs within minutes of altering cell density. HeLa cells
growing at high density were trypsinized and replated at low density on fibronectincoated coverslips. After 20 (A,B,C), 40 or 60 minutes (D,E,F), cells were fixed with
3% paraformaldehyde and examined by immunofluorescence microscopy using an
antibody to Inh2. Panels A and D show the staining of endogenous Inh2 in green.
Panels B and E show the DAPI staining of DNA in red. Panels C and F show the
merged image of the Inh2 staining with DAPI staining in red with the overlay of the
two stains yielding yellow. Cells were scored as having nuclear staining brighter than,
equal to, or dimmer than cytoplasmic staining. The bottom graph shows the combined
result of three independent experiments with more than 100 cells being tallied in each
experiment. The dashed line shows the percentage of cells with predominant nuclear
staining and the solid line shows the percentage of cells with predominant cytoplasmic
staining.
Nuclear-cytoplasmic distribution of Inh2
3743
Fig. 6. Residues 78-119 are sufficient for cytoplasmic retention of a
heterologous protein. HeLa cells cultured at low density and
expressing either GFP (A) or a fusion of residues 78-119 of Inh2
with GFP (B) were visualized for GFP fluorescence.
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.
Discussion
This study shows that endogenous Inh2 as well as
overexpressed Inh2 accumulates in the nucleus of both HeLa
and PC3 cells when cultured at low density. When a higher cell
density is reached, Inh2 is retained in the cytoplasm and
Fig. 5. Separate domains of Inh2 are required for cytoplasmic
retention and nuclear import. HeLa cells expressing myc-tagged Inh2
proteins were cultured at either low (B,D,F,H,J) or high density
(A,C,E,G, I) on fibronectin-coated coverslips. A and B show a phase
image for I and J and are representative of all panels. C and D show a
near full-length Inh2 [0-197]; E and F are an N-terminally truncated
form (residues 78-197); G and H are a C-terminal truncation
(residues 0-119) and I and J are of a central construct consisting of
residues 78-150. K shows a schematic of the truncations used.
Fig. 7. Inhibitor-2 is actively imported into the nucleus. A and B
show low-density HeLa cells expressing either an Inh2GFP3 fusion
(A) or an Inh2GFP3 fusion in which lysines 142 and 144 have been
mutated to alanine, termed NLS mutGFP3 (B). Cells were visualized
with the fluorescence of GFP and the nuclear, cytoplasmic and total
fluorescence were quantified. The graph shows the import index for
GFP3, Inh2GFP3 fusion, NLS MutGFP3 or Inh2[Thr72Ala]GFP3
fusion.
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Journal of Cell Science 115 (19)
Fig. 8. Inhibitor-2 exits the nucleus by passive diffusion. HeLa cells
were electroporated with either Inh2GFP3 or Inh2HA and grown at
low density for 6 hours. The cells were then trypsinized and plated
onto fibronectin-coated coverslips for 4 hours. The coverslips were
then fixed and visualized either for GFP fluorescence or by indirect
immunofluorescence techniques directed against the triple HA tag
(orange). A shows the GFP fluorescence and B shows a merge of the
HA immunofluorescence (orange) and DAPI staining of the DNA
(Blue).
excluded from the nucleus. Truncation of the C-terminal 80
amino acids of Inh2 prevented the nuclear accumulation of
Inh2. Within this region, which is required for import, there are
two clusters of basic amino acids between residues 137-146,
similar to known bipartite NLSs (Quimby and Corbett, 2001).
HeLa cells expressing an Inh2GFP3 fusion in which K144 and
K146 were mutated to alanines did not show nuclear import in
low-density cells. The results indicate that Inh2 contains a
functional NLS and enters the nucleus by active import.
Nuclear pores allow passive diffusion of proteins with a mass
up to 70 kDa, which raises the question of why would Inh2, a
23 kDa protein, contain a functional NLS? One possible
explanation is that Inh2 exists in the cell as part of a protein
complex and that the NLS of Inh2 is responsible for mediating
the nuclear translocation of the entire complex. PP1 is present
as holoenzymes consisting of the catalytic subunit bound to
various targeting subunits, such as GM or neurabin. Inh2 is
capable of binding and inhibiting the activity of at least one
such PP1 holoenzyme (Strålfors et al., 1985). Thus, Inh2 could
be controlling the localization of one or more PP1
holoenzymes in response to changes in cell density.
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 lowdensity 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.
This work was supported by grant GM 56362 from the National
Institute of General Medical Sciences. C.A.L. was supported by
training grant GF-10461 to the University of Virginia Cell and
Molecular Biology Program. M.E. was supported by a post-doctoral
fellowship from the American Heart Association Mid-Atlantic
Affiliate.
References
Baba, M., Hirai, S., Kawakami, S., Kishida, T., Sakai, N., Kaneko, S., Yao,
M., Shuin, T., Kubota, Y., Hosaka, M. et al. (2001). Tumor suppressor
protein VHL is induced at high cell density and mediates contact inhibition
of cell growth. Oncogene J. 20, 2727-2736.
Bollen, M. (2001). Combinatorial control of protein phosphatase-1. Trends
Biochem. Sci. 26, 426-431.
Brautigan, D. L., Sunwoo, J., Labbe, J. C., Fernandez, A. and Lamb, N.
J. C. (1990). Cell cycle oscillation of phosphatase inhibitor-2 in rat
fibroblasts coincident with p34CDC2 restriction. Nature 344, 74-78.
Nuclear-cytoplasmic distribution of Inh2
Bunge, R. G. L., Lieberman, M., Raben, D., Salzer, J., Whittenberger, B.
and Woolsey, T. (1979). Growth control by cell to cell contact. J. Supramol.
Struct. 11, 175-187.
Cannon, J. F., Pringle, J. R., Fiechter, A. and Khalil, M. (1994).
Characterization of glycogen-deficient glc mutants of Saccharomyces
cerevisiae. Genetics J. 136, 485-503.
Cohen, P. (1989). The structure and regulation of protein phosphatases. Annu.
Rev. Biochem. 58, 453-508.
Daniel, S., Zhang, S., DePaoli-Roach, A. A. and Kim, K. H. (1996).
Dephosphorylation of Sp1 by protein phosphatase 1 is involved in the
glucose-mediated activation of the acetyl-CoA carboxylase gene. J. Biol.
Chem. 271, 14692-14697.
Egloff, M., Johnson, D. F., Moorhead, G., Cohen, P. T. W., Cohen, P. and
Barford, D. (1997). Structural basis for the recognition of regulatory
subunits by the catalytic subunit of protein phosphatase 1. EMBO J. 16,
1876-1887.
Gerace, L. (1995). Nuclear export signals and the fast track to the cytoplasm.
Cell 82, 341-344.
Helps, N. R. and Cohen, P. T. W. (1999). Drosophila melanogaster protein
phosphatase inhibitor-2: identification of a site important for PP1 inhibition.
FEBS Lett. 463, 72-76.
Huang, F. L. and Glinsmann, W. H. (1976). Separation and characterization
of two phosphorylase phosphatase inhibitors from rabbit skeletal muscle.
Euro. J. Biochem. 70, 419-426.
Huang, H.-B., Horichi, A., Watanabe, T., Shih, S.-R., Tsay, H.-J., Li, H.C., Greengard, P. and Nairn, A. C. (1999). Characterization of the
inhibition of protein phosphatase-1 by DARPP-32 and inhibitor-2. J. Biol.
Chem. 274, 7870-7878.
Kakinoki, Y., Somers, J. and Brautigan, D. L. (1997). Multisite
phosphorylation and the nuclear localization of phosphatase inhibitor 2green fluorescent protein (Inh2-GFP) fusion protein during S phase of the
cell growth cycle. J. Biol. Chem. 272, 32308-32314.
Osawa, Y., Nakagama, H., Shima, H., Sugimua, T. and Nagao, M. (1996).
Identification and characterization of three isotypes of protein phosphatase
inhibitor-2 and their expression profiles during testis maturation in rats. Eur.
J. Biochem. 242, 793-798.
Quimby, B. B. and Corbett, A. H. (2001). Nuclear transport mechanisms.
Cell. Mol. Life Sci. 58, 1766-1773.
3745
Resink, T. J., Hemmings, B. A., Tung, H. Y. L. and Cohen, P. (1983).
Characterization of a reconstituted Mg-ATP-dependent phosphatase. Eur. J.
Biochem. 133, 455-461.
Rodriguez, P., Vinuela, J. E., Alvarez-Fernandez, L., Buceta, M., Vidal,
A., Dominguez, F. and Gomez-Marquez, J. (1998). Overexpression of
prothymosin alpha accelerates proliferation and retards differentiation in
HL-60 cells. Biochem. J. 331.
Sanseau, P., Jackson, A., Alderton, R. P., Beck, S., Senger, G., Sheer, D.,
Kelly, A. and Trowsdale, J. (1994). Cloning and characterization of
human phosphatase inhibitor-2 (IPP-2) sequences. Mamm. Genome 5,
490-496.
Shenolikar, S. (1994). Protein serine/threonine phosphatases – new avenues
for cell regulation. Annu. Rev. Cell Biol. 10, 55-86.
Strålfors, P., Hiraga, A. and Cohen, P. (1985). The protein phosphatases
involved in cellular regulation. Purification and characterisation of the
glycogen-bound form of protein phosphatase-1 from rabbit skeletal muscle.
Eur. J. Biochem. 149, 295-303.
Trompeter, H., Schiermeyer, A., Blankenburg, G., Hennig, E. and
Soling, H. (1999). Factors involved in the cell density-dependent
regulation of nuclear/cytoplasmic distribution of the 11.5-kDa Zn2+binding protein (parathymosin-alpha) in rat hepatocytes. J. Cell Sci. 112,
4113-4122.
Trompeter, H.-L., Brankd, I. A. and Soling, H.-D. (1996). Variable nuclear
cytoplasmic distribution of the 11.5 kDa zinc-binding protein
(parathymosin-alpha) and identification of a bipartite nuclear localization
signal. J. Biol. Chem. 271, 1187-1193.
Wolthuis, A., Boes, A. and Grond, J. (1993). Cell density modulates growth,
extracellular matrix, and protein synthesis of cultured rat mesangial cells.
Am. J. Pathol. 143, 1209-1219.
Yang, J., Hurley, T. D. and DePaoli-Roach, A. (2000). Interaction of
inhibitor-2 with the catalytic subunit of type 1 protein phosphatase. J. Biol.
Chem. 275, 22635-22644.
Yang, S.-D., Vandenheede, J. R. and Merlevede, W. (1981). Identification
of inhibitor-2 as the ATP-Mg-dependent protein phosphatase modulator. J.
Biol. Chem. 256, 10231-10234.
Zhang, Z., Bai, G. and Lee, E. Y. C. (1992). PCR cloning of the cDNA of
rabbit skeletal muscle protein phosphatase inhibitor-2. Biochem. Biophys.
Res. Commun. 186, 1168-1170.