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Title: Recruitment of phosphorylated small heat shock protein Hsp27 to nuclear speckles without stress

Abstract

During stress, the mammalian small heat shock protein Hsp27 enters cell nuclei. The present study examines the requirements for entry of Hsp27 into nuclei of normal rat kidney (NRK) renal epithelial cells, and for its interactions with specific nuclear structures. We find that phosphorylation of Hsp27 is necessary for the efficient entry into nuclei during heat shock but not sufficient for efficient nuclear entry under control conditions. We further report that Hsp27 is recruited to an RNAse sensitive fraction of SC35 positive nuclear speckles, but not other intranuclear structures, in response to heat shock. Intriguingly, Hsp27 phosphorylation, in the absence of stress, is sufficient for recruitment to speckles found in post-anaphase stage mitotic cells. Additionally, pseudophosphorylated Hsp27 fused to a nuclear localization peptide (NLS) is recruited to nuclear speckles in unstressed interphase cells, but wildtype and nonphosphorylatable Hsp27 NLS fusion proteins are not. The expression of NLS-Hsp27 mutants does not enhance colony forming abilities of cells subjected to severe heat shock, but does regulate nuclear speckle morphology. These data demonstrate that phosphorylation, but not stress, mediates Hsp27 recruitment to an RNAse soluble fraction of nuclear speckles and support a site-specific role for Hsp27 within the nucleus.

Authors:
 [1];  [1];  [2]
  1. School of Molecular Biosciences, Washington State University, Pullman, WA (United States)
  2. School of Molecular Biosciences, Washington State University, Pullman, WA (United States) and Center for Reproductive Biology, Washington State University, Pullman, WA (United States). E-mail: eshelden@wsu.edu
Publication Date:
OSTI Identifier:
20858071
Resource Type:
Journal Article
Resource Relation:
Journal Name: Experimental Cell Research; Journal Volume: 313; Journal Issue: 1; Other Information: DOI: 10.1016/j.yexcr.2006.10.004; PII: S0014-4827(06)00422-8; Copyright (c) 2006 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; BIOLOGICAL STRESS; CELL NUCLEI; HEAT-SHOCK PROTEINS; KIDNEYS; MITOSIS; MORPHOLOGY; MUTANTS; NUCLEAR STRUCTURE; PEPTIDES; PHOSPHORYLATION; RATS

Citation Formats

Bryantsev, A.L., Chechenova, M.B., and Shelden, E.A. Recruitment of phosphorylated small heat shock protein Hsp27 to nuclear speckles without stress. United States: N. p., 2007. Web. doi:10.1016/j.yexcr.2006.10.004.
Bryantsev, A.L., Chechenova, M.B., & Shelden, E.A. Recruitment of phosphorylated small heat shock protein Hsp27 to nuclear speckles without stress. United States. doi:10.1016/j.yexcr.2006.10.004.
Bryantsev, A.L., Chechenova, M.B., and Shelden, E.A. Mon . "Recruitment of phosphorylated small heat shock protein Hsp27 to nuclear speckles without stress". United States. doi:10.1016/j.yexcr.2006.10.004.
@article{osti_20858071,
title = {Recruitment of phosphorylated small heat shock protein Hsp27 to nuclear speckles without stress},
author = {Bryantsev, A.L. and Chechenova, M.B. and Shelden, E.A.},
abstractNote = {During stress, the mammalian small heat shock protein Hsp27 enters cell nuclei. The present study examines the requirements for entry of Hsp27 into nuclei of normal rat kidney (NRK) renal epithelial cells, and for its interactions with specific nuclear structures. We find that phosphorylation of Hsp27 is necessary for the efficient entry into nuclei during heat shock but not sufficient for efficient nuclear entry under control conditions. We further report that Hsp27 is recruited to an RNAse sensitive fraction of SC35 positive nuclear speckles, but not other intranuclear structures, in response to heat shock. Intriguingly, Hsp27 phosphorylation, in the absence of stress, is sufficient for recruitment to speckles found in post-anaphase stage mitotic cells. Additionally, pseudophosphorylated Hsp27 fused to a nuclear localization peptide (NLS) is recruited to nuclear speckles in unstressed interphase cells, but wildtype and nonphosphorylatable Hsp27 NLS fusion proteins are not. The expression of NLS-Hsp27 mutants does not enhance colony forming abilities of cells subjected to severe heat shock, but does regulate nuclear speckle morphology. These data demonstrate that phosphorylation, but not stress, mediates Hsp27 recruitment to an RNAse soluble fraction of nuclear speckles and support a site-specific role for Hsp27 within the nucleus.},
doi = {10.1016/j.yexcr.2006.10.004},
journal = {Experimental Cell Research},
number = 1,
volume = 313,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}
  • Hsp27 is a small heat shock protein (shsp) regulating stress tolerance and increasingly thought to play roles in tissue homeostasis and differentiation. The zebrafish Danio rerio is an important model for the study of developmental processes, but little is known regarding shsps in this animal. Here, we report the sequence, expression, regulation, and function of a zebrafish protein (zfHsp27) homologous to human Hsp27. zfHsp27 contains three conserved phosphorylatable serines and a cysteine important for regulation of apoptosis, but it lacks much of a C-terminal tail domain and shows low homology in two putative actin interacting domains that are features ofmore » mammalian Hsp27. zfHsp27 mRNA is most abundant in adult skeletal muscle and heart and is upregulated during early embryogenesis. zfHsp27 expressed in mammalian fibroblasts was phosphorylated in response to heat stress and anisomycin, and this phosphorylation was prevented by treatment with SB202190, an inhibitor of p38 MAPK. Expression of zfHsp27 and human Hsp27 in mammalian fibroblasts promoted a similar degree of tolerance to heat stress. zfHsp27 fusion proteins entered the nucleus and associated with the cytoskeleton of heat stressed cells in vitro and in zebrafish embryos. These results reveal conservation in regulation and function of mammalian and teleost Hsp27 proteins and define zebrafish as a new model for the study of Hsp27 function.« less
  • Highlights: ► We examined effect of the quaternary structure of yeast sHsp on Aβ aggregation. ► Aβ aggregation was inhibited by the oligomeric form of sHsp, but not by dimeric sHsp. ► The fibrillar amyloids consisted of both Aβ and dimeric sHsp. ► They exhibited different inner structure and cytotoxicity from authentic Aβ amyloids. ► These results suggest the formation of new type fibrillar Aβ amyloid by sHsp. -- Abstract: Small heat shock protein (sHsp) is a molecular chaperone with a conserved alpha-crystallin domain that can prevent protein aggregation. It has been shown that sHsps exist as oligomers (12–40 mer)more » and their dissociation into small dimers or oligomers is functionally important. Since several sHsps are upregulated and co-localized with amyloid-β (Aβ) in senile plaques of patients with Alzheimer’s disease (AD), sHsps are thought to be involved in AD. Previous studies have also shown that sHsp can prevent Aβ aggregation in vitro. However, it remains unclear how the quaternary structure of sHsp influences Aβ aggregation. In this study, we report for the first time the effect of the quaternary structure of sHsp on Aβ aggregation using sHsp from the fission yeast Schizosaccharomyces pombe (SpHsp16.0) showing a clear temperature-dependent structural transition between an oligomer (30 °C) and dimer (50 °C) state. Aβ aggregation was inhibited by the oligomeric form of SpHsp16.0. In contrast, amyloid fibrils were formed in the presence of dimeric SpHsp16.0. Interestingly, these amyloid fibrils consisted of both Aβ and SpHsp16.0 and showed a low ThT intensity and low cytotoxicity due to their low binding affinity to the cell surface. These results suggest the formation of novel fibrillar Aβ amyloid with different characteristics from that of the authentic Aβ amyloid fibrils formed in the absence of sHsp. Our results also suggest the potential protective role of sHsp in AD under stress conditions.« less
  • Thrombin plays a critical role in platelet activation, hemostasis, and thrombosis. Cellular activation by thrombin leads to the phosphorylation of multiple proteins, most of which are unidentified. The authors have characterized several 29-kDa proteins that are rapidly phosphorylated following exposure of intact human platelets to thrombin. A murine monoclonal antibody raised to an unidentified estrogen receptor-related 29-kDa protein selectively recognized these proteins as well as a more basic, unphosphorylated 27-kDa protein. Cellular activation by thrombin led to a marked shift in the proportion of protein from the 27-kDa unphosphorylated form to the 29-kDa phosphoprotein species. Using this antibody, they isolatedmore » and sequenced a human cDNA clone encoding a protein that was identical to the mammalian 27-kDa heat shock protein (HSP27), a protein of uncertain function that is known to be phosphorylated to several forms and to be transcriptionally induced by estrogen. The 29-kDa proteins were confirmed to be phosphorylated forms of HSP27 by immunoprecipitation studies. Thus, the estrogen receptor-related protein is HSP27, and the three major 20-kDa proteins phosphorylated in thrombin-activated platelets are forms of HSP27. These data suggest a role for HSP27 in the signal transduction events of platelet activation.« less
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  • The patterns of radioactively labeled proteins from cultured chicken embryo cells stressed in the presence of either D2O or glycerol were analyzed by using one-dimensional polyacrylamide gel electrophoresis. These hyperthermic protectors blocked the induction of stress proteins during a 1-hour heat shock at 44 degrees C. The inhibitory effect of glycerol but not D2O on the induction of heat shock proteins could be overcome by increased temperature. By using transcriptional run-on assays of isolated nuclei and cDNA probes to detect hsp70- and hsp88-specific RNA transcripts, it was shown that the D2O and glycerol blocks occurred at or before transcriptional activationmore » of the hsp70 and hsp88 genes. After heat-stressed cells were returned to 37 degrees C and the protectors were removed, heat shock proteins were inducible by a second heating. This result and the fact that the chemical stressor sodium arsenite induced stress proteins in glycerol medium indicated that the treatments did not irreversibly inhibit the induction pathways and that the stress response could be triggered even in the presence of glycerol by a stressor other than heat. In principle then, cells incurring thermal damage during a 1-hour heat shock at 44 degrees C in D2O or glycerol medium should be competent to respond by inducing heat shock proteins during a subsequent recovery period at 37 degrees C in normal medium. We found that heat shock proteins were not induced in recovering cells, suggesting that glycerol and D2O protected heat-sensitive targets from thermal damage. Evidence that the heat-sensitive target(s) is likely to be a protein(s) is summarized. During heat shocks of up to 3 hours duration, neither D2O nor glycerol significantly altered hsp23 gene activity, a constitutively expressed chicken heat shock gene whose RNA transcripts and protein products are induced by stabilization (increased half-life).« less