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* INSERM, Dijon, France;
INSERM U309, La Tronche, France;
Center for Translational and Advanced Animal Research on Human Diseases, Tohoku University, Miyagi, Japan; and
Division of Cell Biology, Medical Institute of Bioregulation, Kyushu University, Japan
2Correspondence: INSERM U517, IFR100, Faculty of Medicine, 7 Boulevard Jeanne d’Arc, Dijon 21000, France. E-mail: cganrido{at}u-bourgogne.fr
SPECIFIC AIMS
Hsp27 is a small heat shock protein induced by many different stresses that help the cells to cope with otherwise lethal stimuli. We have recently shown that HSP27 under stress conditions could favor the proteasomal degradation of certain proteins. This work demonstrates that one of these proteins is p27Kip1 and analyzes the role of HSP27 in the ubiquitination/proteasomal degradation of p27Kip1 and the repercussion on the cell cycle.
PRINCIPAL FINDINGS
1. HSP27 overexpression enhances p27Kip1 proteasomal degradation
We observed that, in response to serum depletion, p27Kip1 first accumulated then progressively decreased as cells began to die. In these stressful conditions, HSP27 favored p27Kip1 degradation. This degradation was abolished in the presence of a proteasome inhibitor. A similar observation was made in response to stress induced by the NO donor glyceryl trinitrate (GTN). This effect of HSP27 overexpression on the proteasomal degradation of p27Kip1 was observed in different cell lines.
2. p27Kip1 phosphorylation on Thr187 or Ser-10 is not required for interaction with HSP27
Coimmunoprecipitation experiments demonstrated that HSP27 interacted with p27Kip1, while other heat shock proteins such as HSP70 or HSP90 did not. Exposure of cells overexpressing HSP27 to GTN or serum starvation enhanced the HSP27/p27Kip1 interaction (Fig. 1
A). Phosphorylation of p27Kip1 has been reported as necessary for its ubiquitination/degradation either by the complex SKP2 ubiquitin ligase (Thr187) or by the KPC ubiquitin ligase (Ser-10). However, HSP27 did not interact with Thr187 or Ser-10 phosphorylated p27Kip1. In accordance with these observations, in response to GTN, HSP27 overexpression stimulates the degradation of nonphosphorylated p27Kip1 without affecting the degradation of the Ser-10 or Thr187 phosphorylated forms (Fig. 1B
). Immunofluorescence analysis of the subcellular localization of HSP27 and p27Kip1 demonstrated that the degradation of p27Kip1 observed in these cells under stressful conditions (Fig. 1C
) might occur in the cytosol.
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3. HSP27 favors p27Kip1 ubiquitination
Ubiquitination experiments in vitro and in vivo demonstrated that HSP27, under stress conditions, participated in the ubiquitination process of p27Kip1. In our in vitro experimental conditions, only mono-ubiquitinated p27Kip1 was detected (Fig. 2
A). However, as in the absence of MG132, this mono-ubiquitinated p27Kip1 was rapidly degraded (Fig. 2B
), most probably HSP27 induced mono-ubiquitination of p27Kip1 is the first step of a polyubiquitination process that either cannot be detected in our experimental conditions or requires additional proteins.
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4. HSP27 induces the degradation of p27Kip1 in the form of small oligomers
Gel filtration experiments have demonstrated that HSP27 forms oligomers of various sizes, ranging from dimers to 
1000 kDa large oligomers. This is a dynamic process that has been shown to modulate some functions of HSP27. By the use of a mutant of HSP27 that can form only small oligomers, we demonstrated that HSP27 activated the proteasome and favored Skp2-independent p27Kip1 degradation in the form of small oligomers. Further, gel filtration experiments, followed by coimmunoprecipiation, showed that p27Kip1 was found in a unique fraction in which HSP27 was in the form of small oligomers and interacted with these small oligomers of HSP27.
5. HSP27 affects the G0-/G1-phase of the cell cycle
Finally, we observed in two different cellular models (leukemic U937 and colon cancer REG cells) that in serum-starved cells, HSP27, by inducing p27Kip1 degradation, helped G0/G1 arrested cells to progress toward the S-phase. Accordingly with the above-mentioned results, the mutant of HSP27 that formed only small oligomers was more efficient than wild-type HSP27 in this G1- to S-phase cell cycle progression function of HSP27.
CONCLUSIONS AND SIGNIFICANCE
The present study indicates that HSP27 interacts with p27Kip1 and, in some stressful conditions such as prolonged serum depletion or exposure to GTN, enhances the proteolysis of the cyclin-dependent kinase inhibitor through the ubiquitin-proteasome pathway to promote progression from G0-/G1- to S-phase of the cell cycle. HSP27-mediated p27Kip1 proteolysis occurs in a Thr187 phosphorylation- and Skp2-independent fashion and does not affect the protein when phosphorylated on Ser-10. This finding indicates that, under stress conditions, HSP27 could favor p27Kip1 ubiquitination/degradation by a not-yet-described, phosphorylation-independent pathway.
It has been recently shown that the main function of stress proteins could be to prevent accumulation of denaturated and/or aggregated proteins, as an increase in ubiquitin-dependent degradation of proteins could prevent heat shock toxicity as efficiently as heat shock proteins. ATP-dependent chaperones HSP70 and HSP90 have been shown to have a function in the ubiquitin/proteasome system through their cochaperones CHIP, an ubiquitin ligase protein, and Bag1, an ubiquitin-like protein. In this work, we enforced our previous observations that demonstrated a direct role for the ATP-independent chaperone HSP27 in the ubiquitination/degradation of proteins. This role seems quite selective and takes place only under stressful conditions. In the absence of stress, interaction of HSP27 with p27Kip1 does not result in its degradation. Further, in vivo, both proteins do not share the same cellular compartment. In response to stress, HSP27 shifts to small oligomers and p27Kip translocates into the cytosol to be ubiquitinated and degraded.
A major expectation of our observations was that overexpressed HSP27 would promote cell-cycle progression by increasing p27Kip1 degradation. The HSP27-mediated, Skp2-independent degradation of p27Kip1 at the G0/G1 transition, identified in tumor cells under certain stress conditions, could account for a previously suggested role for HSP27 in cell proliferation. In addition, HSP27 has been reported to accumulate in tumor cells grown at confluence. The accumulated protein could facilitate the re-entry of the quiescent cells into the cell cycle. Therefore, part of the protective effect of this late stress-responsive protein would be to restart cell proliferation.
The present study suggests that HSP27 overexpression could prevent p27Kip1 from arresting cell proliferation in response to stresses, which, depending on the cell context, may favor cell death by apoptosis or prevent DNA damage repair and favor transformation. This latter effect may be enforced by the ability of HSP27 to prevent cell death. HSP27 is a powerful protective protein that increases cancer cell resistance to chemotherapeutic agents. In addition, HSP27 expression is often constitutively high in many human cancer cells. Whether high expression concentration of HSP27 correlates with low expression of p27Kip1 in human tumors has now to be determined. Such a correlation would suggest that overexpressed HSP27 may be a potential target for tumor cell eradication.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4184fje
1 These authors contributed equally to this work. 
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