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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 27, 2001 as doi:10.1096/fj.00-0680fje. |
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Department of Physiology, McGill University, Montreal, PQ H3G 1Y6, Canada
2Correspondence: Ursula Stochaj, Department of Physiology, McGill University, 3655 Drummond St., Montreal, PQ, H3G 1Y6, Canada. E-mail: achu@med.mcgill.ca or stochaj{at}med.mcgill.ca
SPECIFIC AIMS
Members of the hsc70 protein family concentrate in nuclei when cells are exposed to heat stress. The effect of cell density on heat-induced hsc70 nuclear accumulation was analyzed in HeLa cell cultures. We also studied the role of protein kinases and protein phosphatases in concentrating hsc70s in nuclei of heat-treated cells.
PRINCIPAL FINDINGS
1. Hsc70s fail to accumulate in nuclei of confluent cells
The density and senescence of cultured cells alter a variety of
cell properties. We have stressed low and high density cultures of HeLa
cells and monitored the distribution of hsc70s upon heat stress. In
cells of low density cultures, we observed hsc70 nuclear accumulation
whereas high density cells failed to concentrate hsc70s in nuclei.
Particularly in cultures of very high density, hsc70s remained
predominantly in the cytoplasm after exposure to heat (Fig. 1D
). At the margin of high density layers, where cells are
less dense, some of the cells were able to concentrate hsc70s in nuclei
(Fig. 1F
). By contrast, in cells that were part of the
confluent layer, hsc70s were restricted mostly to the cytoplasm (Fig. 1F
).
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2. Confluent cells support classical nuclear protein import, which
is inhibited upon heat shock
Confluent cultures may have general defects in nucleocytoplasmic
trafficking, including hsc70 nuclear import. To address this question,
we monitored the distribution of NLS-GFP, a fluorescent reporter
protein that carries a classical nuclear localization sequence (NLS).
Nuclear accumulation of NLS-GFP requires nuclear protein import to be
constitutively active, and inhibition of import localizes NLS-GFP to
the cytoplasm. Transiently transfected HeLa cells of high and low
density concentrated the reporter protein in nuclei, when cells were
unstressed. However, upon mild (1 h at 42°C) or severe heat shock (1
h at 45.5°C), NLS-GFP equilibrated between nucleus and cytoplasm in
both high and low density cultures. Thus, high density as well as low
density HeLa cell cultures support classical nuclear protein import.
They also mediate diffusion across the nuclear pore complex, as NLS-GFP
was able to exit the nucleus upon heat treatment.
3. Inhibitors of protein tyrosine and Ser/Thr kinases or p70 S6
kinase do not prevent hsc70 nuclear accumulation in heat-stressed cells
One of the effects of stress, including heat shock, is the
activation of signaling cascades, a process that depends on protein
kinase activation. Therefore, we determined whether protein kinase
inhibitors alter the heat-induced nuclear accumulation of hsc70s in low
density cell cultures. However, none of the inhibitors tested prevented
hsc70 nuclear accumulation in heat-stressed HeLa cells. This included
PD98059, a compound specific for MEK (the upstream kinase that
activates ERK1/2), genistein, a more general inhibitor of tyrosine
kinases, rapamycin, an inhibitor of p70 S6 kinase, and staurosporine,
which affects a broad spectrum of Ser/Thr protein kinases.
4. Inhibitors of protein phosphatases interfere with heat-induced
nuclear transport of hsc70s and classical nuclear protein import in
unstressed cells
Before heat exposure, we treated low density cultures with the
Ser/Thr phosphatase inhibitor okadaic acid or sodium orthovanadate,
which inhibits tyrosine phosphatases. Both components prevented hsc70
nuclear accumulation induced by heat shock (Fig. 2H
, L
), and hsc70s were cytoplasmic as well as nuclear, a
distribution similar to unstressed controls. Although classical nuclear
import was also affected by sodium orthovanadate and okadaic acid, much
higher concentrations were required to inhibit the classical transport
pathway.
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5. Ethanol, oxidants, and osmotic stress do not trigger rapid
nuclear accumulation of hsc70
Ethanol, oxidants, and osmotic stress are other forms of insults
that can effectively alter the nucleocytoplasmic distribution of
proteins. However, when compared with heat, these stresses were not
efficient in concentrating hsc70s in nuclei.
CONCLUSIONS
As a particular form of stress, heat shock induces the rapid
accumulation of hsc70s in nuclei. We now show that nuclear
translocation of hsc70s depends on the density of the culture. In
confluent HeLa cells, hsc70s were restricted to the cytoplasm, and
cells were unable to concentrate hsc70s in nuclei upon heat shock.
Confluent cells failed to respond properly to mild as well as severe
heat shock, indicating that their physiological state prevents them
from undertaking proper repair of nuclear damage. The effect of cell
density on nuclear import of hsc70s is not a general defect in
nucleocytoplasmic trafficking, as confluent cells carry out classical
nuclear protein transport and diffusion across the nuclear envelope. We
therefore conclude that high cell density specifically inhibits hsc70
nuclear transport in response to heat stress. We propose that
cell–cell contacts may affect heat-induced hsc70 nuclear accumulation
and possibly other forms of nucleocytoplasmic trafficking. This is
supported by the observation that some of cells at the margin of high
density layers, which have fewer contacts to their neighbors,
concentrated hsc70s in nuclei.
Furthermore, our results demonstrate that protein phosphorylation plays a critical role in nuclear concentration of hsc70s after exposure to heat. About 2000 kinases are encoded by the mammalian genome, and members of the MAP kinase family or the p70 S6 kinase can be activated by heat stress. However, the activities of MAP kinases or p70 S6 kinase were not required to concentrate hsc70s in nuclei of heat-shocked cells.
By contrast, inhibitors of protein phosphatases strongly affected hsc70 concentration in nuclei of heat-treated cells. Our data imply that dephosphorylation of Ser/Thr and Tyr residues is a prerequisite to promote heat-induced nuclear accumulation.
On the basis of our data for low density cultures, we propose that upon
heat shock, phosphorylated cellular component(s), denoted X
P in
Fig. 3
, have to be dephosphorylated to trigger hsc70 concentration in nuclei.
Specific factors of the hsc70 nuclear transport apparatus in stressed
cells yet to be identified are possible targets for this modification.
Protein dephosphorylation may be achieved by inhibition of protein
kinases, activation of protein phosphatases, or a combination thereof.
As inhibitors of protein phosphatases abolish hsc70 nuclear
concentration, the activity of protein phosphatases is a prerequisite
to accumulate hsc70s in nuclei of stressed cells. Ser/Thr and Tyr
phosphorylation both prevented hsc70 nuclear accumulation in
heat-shocked cells. Future experiments will have to determine whether
the dephosphorylation involves dual specific phosphatases or a
combination of Ser/Thr and Tyr phosphatases.
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Although we have demonstrated that phosphatases play a crucial role in the heat shock response, we have not excluded the possibility that inactivation of particular protein kinases helps to concentrate hsc70s in the nuclei of heat-treated cells. However, in high density cultures, inhibitors of Ser/Thr or Tyr kinases were unable to restore heat-induced hsc70 nuclear accumulation. This might suggest that protein kinases relevant to the inhibition of hsc70 nuclear accumulation were insensitive to the inhibitors used. Alternatively, factors other than protein kinases could also contribute to the cytoplasmic retention of hsc70s in high density cultures.
Like heat shock, other forms of stress, including ethanol, oxidants or osmolytes, can relocate proteins within the cell, but these agents were less effective in triggering hsc70 nuclear accumulation. Nuclear concentration of hsc70 is believed to help repair heat-damaged proteins, particularly those of the nucleoli. It is possible that ethanol, osmotic, or oxidative stress damage to a larger extent cytosolic components or proteins of the various membranes. If this is the case, it would be advantageous to keep a greater proportion of hsc70s in the cytoplasm.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0680fje ; to cite this
article, use FASEB J. (April 27, 2001) 10.1096/fj.00-0680fje 
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