Hyperthermia adversely affects cell structure and function, but also induces adaptive responses that allow cells to tolerate these stressful conditions. For example, heat-induced expression of the molecular chaperone protein HSP70 can prevent stress-induced cell death by inhibiting signaling pathways that lead to apoptosis. In this study, we used high-resolution two-dimensional gel electrophoresis and phosphoprotein staining to identify signaling pathways that are altered by hyperthermia and modulated by HSP70 expression. We found that in heat-shocked cells, the actin-severing protein cofilin acquires inhibitory Ser3 phosphorylation, which is associated with an inhibition of chemokine-stimulated cell migration. Cofilin phosphorylation appeared to occur as a result of the heat-induced insolubilization of the cofilin phosphatase slingshot (SSH1-L). Overexpression of HSP70 reduced the extent of SSH1-L insolubilization and accelerated its resolubilization when cells were returned to 37°C after exposure to hyperthermia, resulting in a more rapid dephosphorylation of cofilin. Cells overexpressing HSP70 also had an increased ability to undergo chemotaxis following exposure to hyperthermia. These results identify a critical heat-sensitive target controlling cell migration that is regulated by HSP70 and point to a role for HSP70 in immune cell functions that depend upon the proper control of actin dynamics.

Introduction

A fundamental problem faced by cells of all organisms is the ability to maintain vital functions when exposed to cellular stress. Protein-damaging stresses have the capacity to disrupt cell structure, metabolism and signaling pathways controlling cell proliferation and cell death (Morimoto, 2008). Mild heat stress, which occurs in humans during infection or heat stroke, enhances immunological function through regulation of immune cell migration, proliferation, maturation and cytokine secretion (Park et al., 2005). More extreme exposures to elevated temperature can initiate apoptosis as a means to remove irreversibly damaged cells. However, prior exposure to mild heat stress induces an adaptive response that allows cells to survive these normally lethal conditions. This adaptive response occurs in part through the induced synthesis of the heat-shock proteins, which include several families of molecular chaperones (Young et al., 2004). The major stress-inducible heat-shock protein, HSP70 is a potent anti-apoptotic factor (Mosser and Morimoto, 2004). For example, HSP70 can blunt the apoptotic signal by preventing the heat-induced activation of the pro-apoptotic protein Bax thereby blocking mitochondrial disruption and release of caspase-activating proteins from the intermembrane space (Stankiewicz et al., 2005). Stress kinases have been implicated in the signaling of apoptotic pathways. Hyperthermia, and other protein-damaging stresses activate JNK, and this activation is inhibited in cells that overexpress HSP70 (Gabai et al., 1997; Mosser et al., 1997). Inhibition by HSP70 expression is achieved by preventing the heat-induced inactivation of JNK phosphatases (Meriin et al., 1999). ERK is also activated in heat-stressed cells by the aggregation and insolubilization of ERK phosphatases (Yaglom et al., 2003). HSP70 prevents the activation of ERK by protecting the ERK phosphatases MKP-1 and MKP-3 from heat inactivation.

In this study, we attempted to identify additional signaling pathways that are altered by hyperthermia and modulated by HSP70 expression. We used high-resolution two-dimensional gel electrophoresis and phosphoprotein staining to identify proteins that are differentially phosphorylated in cells exposed to hyperthermia in the presence or absence of HSP70. From this analysis, we identified cofilin as a heat-induced phosphoprotein and found that its phosphorylation was diminished in cells overexpressing HSP70. Cofilin is an actin-binding protein that has an important role in the regulation of actin dynamics (Bernstein and Bamburg, 2010; Van Troys et al., 2008). Cofilin regulates actin severing, stabilization and nucleation in a concentration-dependent fashion. Cofilin-mediated severing of actin filaments at the leading edge of motile cells controls the formation of lamellipodia, which is essential for T-cell migration and cancer cell metastasis (Burkhardt et al., 2008; Wang et al., 2007). Cofilin activity is spatially regulated within the cell through the activity of kinases including LIM kinase (LIMK) that inactivates it by phosphorylation of Ser3, resulting in the stabilization of actin filaments (Bernard, 2007; Nishita et al., 2005; Scott and Olson, 2007). Cofilin reactivation by specific (slingshot, chronophin) and general phosphatases (PP2A) cause F-actin severing and depolymerization (Huang et al., 2006). We found that hyperthermia caused an insolubilization of slingshot, which correlated with an increased level of cofilin phosphorylation and a reduced capacity for chemokine-stimulated cell migration. Cells expressing HSP70 maintained higher levels of soluble slingshot, had reduced levels of cofilin phosphorylation and retained the ability to migrate when exposed to hyperthermia.

Results

Identification of cofilin as a phosphorylated protein in heat-shocked cells

Given that several kinases and phosphatases are modulated during heat shock, we set out to identify proteins that show altered phosphorylation in response to hyperthermia and for which HSP70 exerted some control over this change in phosphorylation status. PErTA70 cells, a human acute lymphoblastic T-cell line with tetracycline-regulated expression of HSP70, were induced with doxycycline to overexpress HSP70 (ON) and compared with non-induced cells following a 60 minute heat shock at 43°C and with control cells incubated at 37°C. Proteins were separated by high-resolution two-dimensional gel electrophoresis, stained with Pro-Q diamond phosphoprotein stain and imaged (Fig. 1). Gels were counterstained with SYPRO Ruby total protein stain to ensure that any increased spot intensity in the Pro-Q-stained gels was due to increased phosphorylation and not due to increased protein abundance. A number of spots could be seen that were more intensely stained in heat-shocked cells compared with control cells. These spots appeared to be less intensely stained following heat shock when HSP70 was expressed, suggesting that HSP70 could suppress the heat-induced phosphorylation of these proteins. We focused our attention on a protein with an approximate pI of 8.5 and a molecular mass of 20 kDa. This spot was consistently found to be highly phosphorylated after heat-shock exposure in the non-induced cells with a substantially reduced level of increased phosphorylation in the induced cells (Fig. 1A, circled spot in bottom right corner). This spot was excised from Coomassie-Blue-stained gels and protein identification was obtained by MALDI-TOF mass spectrometry analysis and peptide mass fingerprinting. Protein Prospector MS-Fit database search identified this spot to be non-muscle cofilin (sequence coverage was 42% with a MOWSE score of 360). To verify that the spot of interest was indeed cofilin and that heat shock affected its level of phosphorylation, control and heat-shocked (43°C for 1 hour) cells were subjected to 2D PAGE and immunoblotted using an anti-cofilin antibody (Fig. 1B). A spot corresponding to a pI value of ~8 was detected in extracts from control cells, whereas an acidic shift of spots was present in extracts from heat-shocked cells (indicative of phosphorylation).

We wished to know whether the heat-induced phosphorylation of cofilin also occurs in other cell types besides the acute lymphoblastic T-cell line PEER. Cofilin phosphorylation status was examined by western blotting of extracts from PEER, HCT116 (colorectal carcinoma) and HeLa (cervical carcinoma) cells that were exposed to 43°C for 30 minutes to 2 hours using antibodies against cofilin and phosphorylated cofilin (cofilin-P) (Fig. 1C). The anti-cofilin-P antibody detects inactive Ser3-phosphorylated cofilin. Heat-induced cofilin phosphorylation was seen in all three of the cell lines, indicating that it is a general phenomenon; however, the PEER cells appeared to be the most sensitive.

Analysis of heat-induced cofilin phosphorylation and its inhibition by HSP70

To determine the temperature profile of cofilin phosphorylation and to characterize the ability of HSP70 to inhibit this heat-induced phosphorylation, we exposed non-induced and HSP70-overexpressing PErTA70 cells to temperatures ranging from 39°C to 43°C for 1 hour and examined protein phosphorylation levels by immunoblotting (Fig. 2A). This temperature range represents the extremes of a mild febrile response to severe hyperthermia such as might occur during heat stroke. The lowest temperature at which increased cofilin phosphorylation could be detected was 40°C in the non-induced cells, with 42°C and 43°C producing significantly higher levels of phosphorylation. The level of cofilin phosphorylation was much less at each temperature in the HSP70-overexpressing cells compared with the non-induced cells. These results indicate that exposure to hyperthermia causes inhibitory phosphorylation of cofilin and that this is prevented in cells expressing HSP70. When the non-induced cells were exposed to 43°C for 1 hour and then incubated at 37°C, the level of cofilin phosphorylation decreased over time, but by 6 hours of recovery, remained at a level that was still 30% of the maximum (Fig. 2B). In the HSP70-overexpressing cells, the same heat treatment resulted in reduced levels of heat-induced cofilin phosphorylation and the rate of cofilin dephosphorylation was also significantly more rapid. We next examined whether extended exposure to the moderately elevated temperature of 39°C could affect cofilin phosphorylation (Fig. 2C). Exposure to 39°C for 2 hours or longer caused cofilin phosphorylation, which slowly diminished with increasing exposure time. However, unlike acute exposure to temperatures above 41°C, HSP70 overexpression did not prevent cofilin phosphorylation during continuous exposure to 39°C.

Fig. 1.

Global changes in protein phosphorylation in cells exposed to hyperthermia in the presence or absence of overexpressed HSP70. (A) Representative Pro-Q-stained 2D gels containing extracts from equivalent cell numbers of non-induced (OFF) and HSP70-overexpressing (ON) PErTA70 cells cultured at 37°C (control) or exposed to 43°C for 1 hour (heat shock). The circled spot represents a protein that shows increased levels of phosphorylation in heat-shocked cells and which has reduced levels of heat-induced phosphorylation in cells overexpressing HSP70. The identity of this protein was determined by MALDI-TOF-based peptide mass fingerprinting of duplicate gels stained with Coomassie Blue to be human non-muscle cofilin (Acc. no. P23528). (B) Immunoblot analysis of extracts from control (C) and heat-shocked (HS) cells separated by 2DE and probed with an anti-cofilin antibody. (C) Immunoblot analysis of extracts from PEER, HCT116 and HeLa cells exposed to 43°C for 30, 60, 90 or 120 minutes probed with antibodies against cofilin and phosphorylated cofilin (p-cofilin).

Fig. 1.

Global changes in protein phosphorylation in cells exposed to hyperthermia in the presence or absence of overexpressed HSP70. (A) Representative Pro-Q-stained 2D gels containing extracts from equivalent cell numbers of non-induced (OFF) and HSP70-overexpressing (ON) PErTA70 cells cultured at 37°C (control) or exposed to 43°C for 1 hour (heat shock). The circled spot represents a protein that shows increased levels of phosphorylation in heat-shocked cells and which has reduced levels of heat-induced phosphorylation in cells overexpressing HSP70. The identity of this protein was determined by MALDI-TOF-based peptide mass fingerprinting of duplicate gels stained with Coomassie Blue to be human non-muscle cofilin (Acc. no. P23528). (B) Immunoblot analysis of extracts from control (C) and heat-shocked (HS) cells separated by 2DE and probed with an anti-cofilin antibody. (C) Immunoblot analysis of extracts from PEER, HCT116 and HeLa cells exposed to 43°C for 30, 60, 90 or 120 minutes probed with antibodies against cofilin and phosphorylated cofilin (p-cofilin).

Fig. 2.

Analysis of heat-induced cofilin phosphorylation and its inhibition by HSP70. (A) Immunoblots showing levels of phosphorylated cofilin, cofilin, HSP70 and actin in non-induced (OFF) and HSP70-overexpressing (ON) PErTA70 cells incubated for 1 hour at the indicated temperatures (°C). (B) Immunoblots from cells exposed to 43°C for 1 hour and collected either immediately (0) or after incubation at 37°C for 2, 4 or 6 hours. (C) Immunoblots from cells exposed to 39°C for 2, 4, 6 or 8 hours. For comparison, the effects of a 1 hour exposure to 43°C is shown for the non-induced and induced cells. Bar graphs show levels of phosphorylated cofilin in the non-induced and induced cells plotted relative to the maximum level + s.e.m. (n=3). *P<0.05 in comparison of OFF vs ON (One-tailed t-test).

Fig. 2.

Analysis of heat-induced cofilin phosphorylation and its inhibition by HSP70. (A) Immunoblots showing levels of phosphorylated cofilin, cofilin, HSP70 and actin in non-induced (OFF) and HSP70-overexpressing (ON) PErTA70 cells incubated for 1 hour at the indicated temperatures (°C). (B) Immunoblots from cells exposed to 43°C for 1 hour and collected either immediately (0) or after incubation at 37°C for 2, 4 or 6 hours. (C) Immunoblots from cells exposed to 39°C for 2, 4, 6 or 8 hours. For comparison, the effects of a 1 hour exposure to 43°C is shown for the non-induced and induced cells. Bar graphs show levels of phosphorylated cofilin in the non-induced and induced cells plotted relative to the maximum level + s.e.m. (n=3). *P<0.05 in comparison of OFF vs ON (One-tailed t-test).

Effect of hyperthermia on cofilin distribution in heat-shocked cells

We next examined whether the heat-induced phosphorylation of cofilin was associated with a change in the intracellular distribution of cofilin because this might provide insight into its effect on cell function. In the non-stressed cells, cofilin was distributed in a granular fashion throughout the cell with a prominent brightly staining accumulation at one end of the cell (Fig. 3A). Exposure to hyperthermia (43°C for 60–120 minutes) led to a loss of this polarized cofilin localization. Most cells retained the diffuse granular distribution, but the brightly staining accumulation at one end of the cell was no longer evident. The extent of this loss was not as severe in the cells overexpressing HSP70 that were exposed to hyperthermia. Using image sets consisting of 0.5-μm-thick CSLM serial sections, we compiled a three-dimensional reconstruction of the cell that demonstrates the spatially concentrated cofilin distribution (Fig. 3B). This analysis clearly shows that cofilin retains the polarized distribution in cells exposed to hyperthermia when HSP70 is present, but not when it is absent. A similar analysis in cells co-stained for cofilin and DAPI demonstrates that the polarized accumulation of cofilin is cytoplasmic (Fig. 3B). We quantified the percentage of cells with bright-staining polarized cofilin (Fig. 3C) and found a significant level of protection against the loss of these polarized cofilin structures in the HSP70-overexpressing cells (ON, 44% polarized vs OFF, 9% polarized).

Fig. 3.

Examination of cofilin localization. (A) Confocal microscopic images of non-induced (OFF) and HSP70-overexpressing (ON) PErTA70 cells cultured at 37°C (C) or exposed to 43°C for either 60 or 120 minutes (HS). Cells were cytospun onto glass slides, fixed and incubated with an anti-cofilin antibody followed by an Alexa-Fluor-594-conjugated secondary antibody. Arrowheads indicate regions of intense localized cofilin staining. Scale bar: 20 μm. (B) Digital z-stack reconstruction of cells immunostained for cofilin following 120 minute heat-shock treatment demonstrating either the loss of the spatially concentrated localization of cofilin to a more diffuse granular distribution in the non-induced cells (left), or maintenance of the spatially concentrated localization in the HSP70-overexpressing cells (right). Image sets were colored with higher intensity voxels represented as warmer colors, and lower intensity voxels as cooler colors. Cross-section analyses (z-plane is indicated by yellow line) for comparing stacks illustrate spatial concentration of cofilin at distinct regions near the cell periphery (arrows) in a subpopulation of cells. Digital reconstruction image series were acquired at a sample rate of 0.5 μm and were processed and analyzed using ImageJ software (National Institutes of Health), and were compiled using Adobe Photoshop CS3. (C) Quantification of cells containing brightly stained cofilin at one end of the cell (polarized). Shown is the mean + s.e.m. (n=4); P<0.005 for OFF-HS vs ON-HS and for C vs HS in both OFF and ON cells (One-tailed t-test). There is no significant difference between the control cells for OFF vs ON.

Fig. 3.

Examination of cofilin localization. (A) Confocal microscopic images of non-induced (OFF) and HSP70-overexpressing (ON) PErTA70 cells cultured at 37°C (C) or exposed to 43°C for either 60 or 120 minutes (HS). Cells were cytospun onto glass slides, fixed and incubated with an anti-cofilin antibody followed by an Alexa-Fluor-594-conjugated secondary antibody. Arrowheads indicate regions of intense localized cofilin staining. Scale bar: 20 μm. (B) Digital z-stack reconstruction of cells immunostained for cofilin following 120 minute heat-shock treatment demonstrating either the loss of the spatially concentrated localization of cofilin to a more diffuse granular distribution in the non-induced cells (left), or maintenance of the spatially concentrated localization in the HSP70-overexpressing cells (right). Image sets were colored with higher intensity voxels represented as warmer colors, and lower intensity voxels as cooler colors. Cross-section analyses (z-plane is indicated by yellow line) for comparing stacks illustrate spatial concentration of cofilin at distinct regions near the cell periphery (arrows) in a subpopulation of cells. Digital reconstruction image series were acquired at a sample rate of 0.5 μm and were processed and analyzed using ImageJ software (National Institutes of Health), and were compiled using Adobe Photoshop CS3. (C) Quantification of cells containing brightly stained cofilin at one end of the cell (polarized). Shown is the mean + s.e.m. (n=4); P<0.005 for OFF-HS vs ON-HS and for C vs HS in both OFF and ON cells (One-tailed t-test). There is no significant difference between the control cells for OFF vs ON.

Effect of hyperthermia on cell migration

Because exposure to hyperthermia resulted in cofilin phosphorylation, which was associated with a loss of polarized cofilin distribution, we next investigated whether heat-shock treatment altered the ability of cells to migrate towards a chemokine source. Chemotaxis was measured using Transwell chambers containing 10−8 M stromal cell derived factor-1 α (SDF-1α) in the lower chamber (Fig. 4A). Control and heat-shocked cells (43°C for 1 hour) were added to the upper wells of individual Transwell chambers and incubated at 37°C for 2 hours. The migration index (number of cells in the lower chamber as a percentage of the total number of cells added to the upper chamber) was severely reduced in the non-induced cells that were exposed to hyperthermia. However, similarly to the results observed for cofilin distribution, HSP70-overexpressing cells displayed a significant level of protection against the heat-induced inhibition of cell migration (ON, 19% vs OFF, 5.5%). These results suggest that HSP70 is able to maintain a migration-competent state in cells exposed to hyperthermia and that this competency is correlated with the maintenance of a polarized distribution of cofilin.

We next examined the effect of exposure to 39°C on cell migration because this treatment was found to increase the level of cofilin phosphorylation in both control and HSP70-overexpressing cells (Fig. 2C). In this experiment, the non-induced and induced cells were mixed at a 1:1 ratio, exposed to hyperthermia and then assayed for their ability to undergo chemotaxis. For the 39°C treatment, cells were incubated at 39°C for 2 hours and then the cells were allowed to migrate through Transwell chambers for 2 hours at 39°C. This was compared with cells that were incubated at 43°C for 1 hour and assayed for migration at 37°C for 2 hours and to control cells in which non-treated cells were assayed for migration at 37°C for 2 hours (Fig. 4B). The results show that lymphocyte migration is significantly impaired at 39°C relative to the 37°C control cells. To determine whether HSP70 overexpression protected the cells from this impairment, we next determined the percentage of GFP-positive cells before and after migration (Fig. 4C). The induced cells express both HSP70 and GFP from an IRES-containing transcript and were 96% GFP positive before mixing with the non-induced GFP-negative (and HSP70-negative) cells. Although HSP70-overexpressing cells had an increased ability to migrate at 37°C after a 1 hour exposure to 43°C, these cells showed no increased ability to migrate when exposed to 39°C. This provides further evidence that heat-induced cofilin phosphorylation impairs lymphocyte cell chemotaxis because this was seen after exposure to either 39°C or 43°C. In addition, resistance to this impairment occurs only under conditions in which HSP70 is able to prevent heat-induced cofilin phosphorylation.

Insolubilization of slingshot in heat-shocked cells

To investigate the mechanism of heat-induced cofilin phosphorylation, we initially examined whether levels of phosphorylated LIMK (LIMK-P) were altered in cells exposed to 43°C. Surprisingly, we found that heat shock did not affect the level of LIMK-P in either the non-induced or HSP70-expressing cells. Because HSP70 is known to prevent heat-induced protein aggregation, we next considered whether hyperthermia altered the solubility of LIMK or the cofilin phosphatases slingshot (SSH1-L), chronophin or PP2A. Cells were exposed to 43°C for 30–120 minutes and were then divided into two aliquots. One sample was lysed in 1% Triton X-100 and insoluble proteins were removed by centrifugation (Fig. 5A, soluble). The pellets were resolubilized in an equal volume of Laemmli buffer (Fig. 5B, pellet). The other sample was lysed in buffer containing 2% SDS to recover total cellular proteins (Fig. 5A, total). Exposure to hyperthermia caused a depletion of soluble SSH1-L, which is indicative of aggregation (Fig. 5A,B). LIMK was also subject to heat-induced insolubilization; however, this was less extensive in comparison with that of SSH1-L, and importantly, the amount of active LIMK-P remained relatively constant in the soluble fraction. Whereas SSH1-L was very sensitive to heat-induced insolubilization, chronophin and PP2A were not affected. In HSP70-overexpressing cells, the extent of SSH1-L (and LIMK) insolubilization was greatly reduced compared with that in non-induced cells (Fig. 5C). These results suggest that the heat-induced phosphorylation of cofilin occurs as a consequence of the inactivation of SSH1-L and that HSP70 prevents cofilin phosphorylation in heat-shocked cells by protecting SSH1-L from insolubilization.

Fig. 4.

Effect of hyperthermia on cell migration. (A) Inhibition of cell migration following exposure to 43°C for 1 hour. Cells were allowed to migrate for 2 hours through 5 μm pore polycarbonate Transwell chambers into medium containing 10−8 M SDF. The migration index was calculated by counting the number of cells in the bottom chamber and dividing this by the number of cells in the original suspension. Migration was examined at 37°C in non-induced and HSP70-expressing cells that were either maintained at 37°C (C) or exposed to 43°C for 1 hour (HS). Shown is the mean + s.e.m. (n=5), P<0.015 for OFF-HS vs ON-HS, P<0.002 for OFF C vs OFF HS, P<0.01 for ON C vs ON HS (One-tailed t-test). There is no significant difference between the control cells for OFF vs ON. (B) Non-induced and HSP70-expressing cells were mixed 1:1 and assayed for cell migration at 37°C (37), at 39°C after a 2 hour incubation at 39°C (39) or at 37°C after a 1 hour incubation at 43°C (43). In each case, the cells were allowed to migrate for 2 hours. Shown is the mean + s.e.m. (n=3). (C) The percentage of GFP-positive cells (induced cells co-express GFP) was measured by flow cytometry for the experiment shown in B. Shown is the mean + s.e.m. (n=3). P<0.05 for 37 vs 39 or 43 in B and P<0.005 for total vs migrated for cells exposed to 43°C in C (One-tailed t-test).

Fig. 4.

Effect of hyperthermia on cell migration. (A) Inhibition of cell migration following exposure to 43°C for 1 hour. Cells were allowed to migrate for 2 hours through 5 μm pore polycarbonate Transwell chambers into medium containing 10−8 M SDF. The migration index was calculated by counting the number of cells in the bottom chamber and dividing this by the number of cells in the original suspension. Migration was examined at 37°C in non-induced and HSP70-expressing cells that were either maintained at 37°C (C) or exposed to 43°C for 1 hour (HS). Shown is the mean + s.e.m. (n=5), P<0.015 for OFF-HS vs ON-HS, P<0.002 for OFF C vs OFF HS, P<0.01 for ON C vs ON HS (One-tailed t-test). There is no significant difference between the control cells for OFF vs ON. (B) Non-induced and HSP70-expressing cells were mixed 1:1 and assayed for cell migration at 37°C (37), at 39°C after a 2 hour incubation at 39°C (39) or at 37°C after a 1 hour incubation at 43°C (43). In each case, the cells were allowed to migrate for 2 hours. Shown is the mean + s.e.m. (n=3). (C) The percentage of GFP-positive cells (induced cells co-express GFP) was measured by flow cytometry for the experiment shown in B. Shown is the mean + s.e.m. (n=3). P<0.05 for 37 vs 39 or 43 in B and P<0.005 for total vs migrated for cells exposed to 43°C in C (One-tailed t-test).

Fig. 5.

Hyperthermia causes a loss of soluble LIMK and SSH1-L. Non-induced (OFF) and HSP70-overexpressing (ON) PErTA70 cells were exposed to 43°C for 30, 60, 90 or 120 minutes and then lysed in either 1% Triton X-100 buffer or Laemmli buffer. Control cells ‘C’ remained at 37°C. (A) Immunoblots showing levels of the indicated proteins in Triton-soluble fractions and in total cell lysates (Laemmli extract). (B) Comparison of the Triton-soluble and -insoluble fractions. (C) Quantification of the percentage of LIMK and SSH1-L that remained soluble in cells exposed to hyperthermia compared with control non-stressed cells. Shown is the mean + s.e.m. (n=3). *P<0.05 for comparison of OFF vs ON (One-tailed t-test).

Fig. 5.

Hyperthermia causes a loss of soluble LIMK and SSH1-L. Non-induced (OFF) and HSP70-overexpressing (ON) PErTA70 cells were exposed to 43°C for 30, 60, 90 or 120 minutes and then lysed in either 1% Triton X-100 buffer or Laemmli buffer. Control cells ‘C’ remained at 37°C. (A) Immunoblots showing levels of the indicated proteins in Triton-soluble fractions and in total cell lysates (Laemmli extract). (B) Comparison of the Triton-soluble and -insoluble fractions. (C) Quantification of the percentage of LIMK and SSH1-L that remained soluble in cells exposed to hyperthermia compared with control non-stressed cells. Shown is the mean + s.e.m. (n=3). *P<0.05 for comparison of OFF vs ON (One-tailed t-test).

We also examined SSH1-L insolubilization in cells that were exposed to 43°C for 1 hour and then incubated at 37°C for 2–6 hours in an attempt to correlate changes in cofilin phosphorylation with the resolubilization of SSH1-L (Fig. 6A). These results show a resolubilization of SSH1-L in the HSP70-overexpressing cells, which correlates with the rapid dephosphorylation of cofilin. However, only a minimal extent of SSH1-L resolubilization occurred in the non-induced cells, reflecting the reduced ability of these cells to dephosphorylate cofilin during recovery from hyperthermia. Total and LIMK-P levels decreased during incubation at 37°C in the non-induced cells. This loss of LIMK most likely results from caspase-3-mediated cleavage of LIMK (Tomiyoshi et al., 2004). LIMK levels remained intact in the HSP70-overexpressing cells because HSP70 inhibits heat-induced caspase activation (Mosser et al., 1997).

Discussion

Heat shock can affect the phosphorylation status of many proteins, although the significance of these changes to cell function at elevated temperatures has been documented for only a few of them (Kim et al., 2002). Our phosphoprotein analysis led to the finding that the actin-binding protein cofilin becomes phosphorylated and inactivated in cells exposed to hyperthermia. This inactivation of cofilin resulted in an altered cellular distribution of cofilin and an inhibition of chemokine-stimulated cell migration. The mechanism responsible for heat-induced cofilin phosphorylation appears to be a temperature-dependent insolubilization of the cofilin phosphatase slingshot (Fig. 7). Overexpression of HSP70 prevented the heat-induced phosphorylation of cofilin by reducing the extent of slingshot insolubilization. This was accompanied by improved polarized cellular distribution of cofilin and hence increased chemotaxis ability.

Exposure to hyperthermia has long been known to affect cytoskeletal structures (Welch and Suhan, 1985). Many studies have shown that heat-shock proteins participate in the synthesis and assembly of cytoskeletal components and can protect from heat-induced damage (Brown et al., 1996; Liang and MacRae, 1997; Mounier and Arrigo, 2002). Our findings show that HSP70 provides protection of the actin cytoskeleton by preventing the heat-induced inactivation of cofilin and that this prevented the inhibitory effect of hyperthermia on cell migration. Our immunofluorescence studies showed that cofilin is spatially concentrated within non-stressed cells and that exposure to hyperthermia leads to a loss of this spatial concentration, which was correlated with an impaired chemotactic response. Both the polarized localization of cofilin and the ability to undergo chemotaxis were protected from the effects of hyperthermia in cells overexpressing HSP70. These results suggest that heat shock affects cofilin localization and migration competency by disrupting the localized control of cofilin phosphorylation. Interestingly, cofilin phosphorylation and cell migration could be affected by both mild (39°C) and extreme (43°C) exposures to hyperthermia; however, HSP70 overexpression only provided protection to cells exposed to 43°C. It should be noted that migration was measured at 37°C after a 1 hour exposure to hyperthermia for the cells exposed to 43°C, whereas for the cells exposed to 39°C, cell migration was measured at 39°C after a 2 hour pre-incubation at 39°C. Therefore, continuous exposure to 39°C impairs additional components of the cell migration apparatus in addition to affecting cofilin phosphorylation. The inhibitory effect of mild hyperthermia on cell migration has also been reported in human mononuclear leukocytes exposed to 38.5°C (Roberts and Sandberg, 1979).

Exposure to mild or severe hyperthermia has been shown to result in activation of multiple signaling pathways (Park et al., 2005). In some cases, the activation occurs through the direct stimulation of these pathways by upstream signaling events, whereas in others, kinase activation occurs as a result of phosphatase inactivation (Meriin et al., 1999; Yaglom et al., 2003). For example, the heat-induced activation of ERK has been shown to be dependent upon inactivation of the dual-specificity phosphatases MKP1 and MKP3 (Yaglom et al., 2003), whereas JNK activation results from inactivation of JNK phosphatase M3/6 (Palacios et al., 2001). Aggregation and inactivation of these phosphatases resulted in the accumulation of the phosphorylated substrate through the activity of the constitutively active upstream kinases. We propose that a similar mechanism exists for cofilin phosphorylation. We found that although there was no increase in the level of LIMK phosphorylation in cells exposed to hyperthermia, there was a loss in the total levels of both soluble SSH1-L and soluble LIMK, although SSH1-L was clearly more heat sensitive than LIMK. In addition, LIMK can be dephosphorylated and inactivated by SSH1-L (Soosairajah et al., 2005) and therefore the insoluble LIMK could potentially represent the pool of LIMK that was associated with SSH1-L and subject to insolubilization along with the aggregating SSH1-L (see Fig. 5). Nevertheless, in cells exposed to hyperthermia, sufficient LIMK-P remained available to phosphorylate cofilin and in conjunction with depleting levels of SSH1-L, led to the overall accumulation of cofilin-P. This effect appeared to be specific to SSH1-L because chronophin and PP2A remained soluble.

Fig. 6.

Recovery of soluble LIMK and SSH1-L in HSP70-expressing cells. Non-induced (OFF) and HSP70-expressing (ON) PErTA70 cells were exposed to 43°C for 60 minutes and collected immediately ‘0’ or incubated at 37°C for 2, 4 or 6 hours. Control cells (C) remained at 37°C. Cells were lysed in either 1% Triton X-100 buffer or Laemmli buffer. (A) Immunoblots showing levels of the indicated proteins in Triton-soluble and -insoluble (pellet) fractions and in the total cell lysate (Laemmli extract) in the OFF and ON cells. (B) Quantification of the percentage of LIMK and SSH1-L that remained soluble in cells exposed to hyperthermia compared to control non-stressed cells. Shown is the mean + s.e.m. (n=3). *P<0.05 for comparison of OFF vs ON (One-tailed t-test).

Fig. 6.

Recovery of soluble LIMK and SSH1-L in HSP70-expressing cells. Non-induced (OFF) and HSP70-expressing (ON) PErTA70 cells were exposed to 43°C for 60 minutes and collected immediately ‘0’ or incubated at 37°C for 2, 4 or 6 hours. Control cells (C) remained at 37°C. Cells were lysed in either 1% Triton X-100 buffer or Laemmli buffer. (A) Immunoblots showing levels of the indicated proteins in Triton-soluble and -insoluble (pellet) fractions and in the total cell lysate (Laemmli extract) in the OFF and ON cells. (B) Quantification of the percentage of LIMK and SSH1-L that remained soluble in cells exposed to hyperthermia compared to control non-stressed cells. Shown is the mean + s.e.m. (n=3). *P<0.05 for comparison of OFF vs ON (One-tailed t-test).

The overexpression of HSP70 not only reduced the extent of SSH1-L insolubilization in cells exposed to hyperthermia, it also increased the rate of recovery of soluble SSH1-L when heat-stressed cells were returned to 37°C. This enhanced recovery of soluble SSH1-L is probably responsible for the elevated rate of cofilin-P dephosphorylation in the HSP70-overexpressing cells. During this recovery period, there was a loss of total LIMK, which is subject to caspase-3-mediated cleavage (Tomiyoshi et al., 2004). Inhibition of caspase-3 activity by HSP70 (Mosser et al., 1997) prevented this loss of LIMK. Consequently, the combined loss of SSH1-L activity and the eventual decline in LIMK levels led to an inability to regulate actin dynamics in heat-stressed cells in the absence of HSP70, whereas in its presence, both SSH1-L and LIMK retained their ability to act on cofilin. HSP70 could also have a direct role in stabilizing LIMK activity. Both HSP70 and HSP90 have been shown to interact with LIMK1, and in the case of HSP90, this interaction promotes LIMK1 homo-dimerization leading to an increase in its activity (Li et al., 2006; Lim et al., 2007).

The ability of cofilin to affect actin dynamics endows it with the ability to control numerous cellular functions including not only cell migration, but also the regulation of apoptosis, lipid metabolism and gene expression (Bernstein and Bamburg, 2010). Not surprisingly, interference with its normal function is linked to diseases such as cancer (Bamburg and Wiggan, 2002). Our results suggest that the ability of HSP70 to modulate cofilin activity could have effects on cofilin-dependent processes, such as T-cell activation and migration, that could contribute to disease. HSP70 is often overexpressed in tumor cells, and evidence suggests that its overexpression contributes to tumorigenesis (Calderwood et al., 2006; Mosser and Morimoto, 2004). In the case of bladder cancer, overexpression of the testis-specific protein HSP70-2 was associated with increased malignancy because specific knockdown resulted in reduced migration and invasion abilities in vitro and suppressed tumor growth in a xenograft assay (Garg et al., 2010). Clinically, hyperthermia increases cancer patient survival when used in combination with radiation or chemotherapy. Our results suggest that this benefit might be due in part to the suppressive effects of hyperthermia on cancer cell migration. However, this beneficial effect would be limited in tumor cells with elevated levels of HSP70, and therefore therapies targeting HSP70 might need to be used in combination with hyperthermia to be effective.

Fig. 7.

Model illustrating the effect of hyperthermia on cofilin activity. Cofilin is inactivated by phosphorylation of Ser3 by LIMK and activated by dephosphorylation of this residue by the cofilin phosphatase slingshot (SSH1-L). LIMK is activated by kinases downstream from Rac, Cdc42 and Rho and inactivated by slingshot. The spatially coordinated activity of LIMK and SSH1-L is required for regulating actin dynamics and controlling actin-dependent cellular processes, including cell migration. Heat shock (HS) causes the insolubilization of SSH1-L, leading to the accumulation of phosphorylated cofilin and inhibition of cell migration.

Fig. 7.

Model illustrating the effect of hyperthermia on cofilin activity. Cofilin is inactivated by phosphorylation of Ser3 by LIMK and activated by dephosphorylation of this residue by the cofilin phosphatase slingshot (SSH1-L). LIMK is activated by kinases downstream from Rac, Cdc42 and Rho and inactivated by slingshot. The spatially coordinated activity of LIMK and SSH1-L is required for regulating actin dynamics and controlling actin-dependent cellular processes, including cell migration. Heat shock (HS) causes the insolubilization of SSH1-L, leading to the accumulation of phosphorylated cofilin and inhibition of cell migration.

Materials and Methods

Cells and treatments

The effects of HSP70 (NM_005345) overexpression were examined using a human acute lymphoblastic T-cell line (PEER) with tetracycline-regulated expression of human HSP70 (PErTA70) (Mosser et al., 2000). HSP70 was induced by incubation with 1.0 μg/ml doxycycline for 24 hours before each experiment. Cells were maintained at 37°C in a humidified 5% CO2 incubator in RPMI medium with 10% fetal bovine serum (Invitrogen, Burlington, Canada). Cells were heat shocked in medium supplemented with 20 mM HEPES buffer (pH 7.2) by immersion of log-phase cells in a circulating water bath. A humidified 5% CO2 incubator was used for long-term exposure to 39°C.

Two-dimensional gel electrophoresis, phosphoprotein staining and identification

For two-dimensional gel analysis, 1.0×107 cells were collected, washed with PBS and then with 250 mM sucrose in 10 mM Tris-HCl (pH 7.5). Cell pellets were resuspended on ice in lysis solution (7 M Urea, 2 M Thiourea, 4% CHAPS, 2% IPG buffer (pH 4–7), 60 mM DTT, 200 mM sodium orthovanadate, 200 mM sodium fluoride and 200 mM aprotinin) and then sonicated. Cell debris was pelleted by centrifugation (14,000 g, 5 minutes, 4°C) and the supernatants were clarified by precipitation using a 2-D Clean-Up kit (GE Healthcare, Baie d'Urfe, Quebec, Canada). Precipitated proteins were resuspended in rehydration buffer (7 M Urea, 2 M Thiourea, 2% CHAPS, 0.5% IPG buffer (pH 4–7), 40 mM DTT) and applied to IPG strips (11 cm, pI 4–7) for rehydration and first-dimension focusing using an ETTAN IPGPhor apparatus (GE Healthcare). The first dimension was performed at 20°C for 45,000 V hours with a maximum current of 50 μA. Focused strips were equilibrated in 10 mg/ml DTT and 25 mg/ml iodoacetamide, rinsed in SDS electrophoresis buffer and loaded onto a precast 13 cm 10.5–14% Criterion gel (Bio-Rad, Mississauga, Canada) and resolved at constant power. SDS-PAGE gels were fixed overnight in 10% TCA, 50% methanol, rinsed with water and stained with Pro-Q diamond phosphoprotein stain (Invitrogen). Gels were destained in 50 mM sodium acetate (pH 4.0), 4% acetonitrile, rinsed in water and scanned using a Typbon 9400 imager using 532 nm excitation and 580 nm emission at a resolution of 200 μm and a PMT of 580 V (GE Healthsciences). Images were visualized using ImageQuant V1.4 software (GE Healthsciences). Pro-Q stained gels were subsequently post-stained with SYPRO Ruby total protein stain (Invitrogen), destained with 10% methanol, 7% acetic acid, rinsed with water and imaged using 532 nm excitation and 610 nm emission.

For protein identification, the two-dimensional gels were stained with 0.1% Coomassie Blue G250, 40% methanol, 10% acetic acid and destained overnight in 5% methanol, 7% acetic acid. Protein spots of interest were selected based on differential spot staining of a duplicate set of gels with Pro-Q Diamond and Sypro Ruby. Spots were manually excised and washed in 50 mM ammonium bicarbonate. The slices were dehydrated using 50% acetonitrile, 25 mM ammonium bicarbonate and then reduced by treatment with 10 mM DTT for 30 minutes at 56°C followed by alkylation with 100 mM iodoacetamide at room temperature. The gel slices were again dehydrated and then rehydrated in 0.12 μg/μl trypsin (Trypsin Gold, Promega, Madison, WI) in 50 mM ammonium bicarbonate. Digestion was carried out at 37°C for 16 hours. Peptides were collected by first transferring the supernatants to new tubes followed by removal of residual digestion mixture from gel slices with 5% formic acid treatment and a wash with acetonitrile. The samples were evaporated and then purified using C18 Ziptips (Millipore) immediately prior to MALDI-TOF mass spectrometry analysis (performed by the Advanced Protein Technology Centre at the Hospital for Sick Children, Toronto, Canada).

Immunoblotting

Cell lysis and immunoblotting were performed as described previously (Stankiewicz et al., 2005). To examine protein solubility following exposure to hyperthermia, cells were lysed in Triton buffer (10 mM HEPES pH 7.4, 100 mM NaCl, 5 mM MgCl2, 1 mM EGTA, 1% Triton X-100, 10 μg/ml each of pepstatin A, leupeptin and aprotinin, 10 mM sodium fluoride, 1 mM sodium vanadate, 20 mM sodium phosphate, 3 mM β-glycerophosphate, 5 mM sodium pyrophosphate). Lysates were centrifuged at 15,000 g for 10 minutes at 4°C. The supernatants were mixed with 2× Laemmli buffer (100 mM Tris-HCl pH 6.8, 20% glycerol, 4% SDS, 10% β-mercaptoethanol) and heated to 95°C for 5 minutes before electrophoresis. The 1% Triton X-100 pellets were washed with lysis buffer and then resuspended in an equal volume of 1× Laemmli buffer. The following antibodies against the following proteins were used for immunoblotting: actin (ACTN05: NeoMarkers, Fremont, CA), Caspase-3 (SA-320-0100; BIOMOL International, Plymouth Meeting, PA), Chronophin/PDXP C85E3 (4686, Cell Signaling Technology), Cofilin (C8736, Sigma), Ser3-P Cofilin (3311S, Cell Signaling Technology), HSP70 (C92F3A-5: Stressgen/Assay Designs, Ann Arbor, MI), LIMK (3842, Cell Signaling Technology), LIMK-1/2 Thr508/505-P (sc-28409-R, Santa Cruz Biotechnology, Santa Cruz, CA), PP2A catalytic α-subunit (610555, Sigma), Slingshot SSH1 (ab46202, Abcam, Cambridge, MA).

Cell-migration analysis

Chemotaxis was measured essentially as described (Vicente-Manzanares et al., 1999). Cells were resuspended in RPMI medium containing 0.1% human serum albumin (HSA, Sigma) at a concentration of 5×106 cells/ml. One hundred microliters of the cell suspension was added to the upper chamber of a Costar Transwell cell culture chamber (6.5 mm diameter, 10 μm thickness, 5-μm-diameter pore size polycarbonate membrane). The lower chamber contained 600 μl RPMI, 0.1% HSA medium supplemented with 10−8 M recombinant human stromal cell derived factor-1α (SDF-1α, PeproTech, Dollard des Ormeaux, Quebec, Canada). Cells were allowed to migrate at 37°C for 2 hours in a humidified 5% CO2 incubator. The number of cells in the lower chamber and in the original cell suspension was counted using a hemocytometer and the migration index was calculated [(no. cells per ml in lower well/no. cells per ml in original cell suspension) ×100]. Flow cytometry (Beckman Coulter FC500) was used to measure the percentage of GFP-positive cells in migration assays containing a mixture of non-induced and induced cells (PErTA70 cells co-express GFP and HSP70 from a dicistronic expression cassette).

Immunofluorescence

Cells (100 μl of a 2×105 cells/ml suspension) were collected onto glass slides (Shandon cytoslide blue mask, Fisher Scientific, Markam, Ontario, Canada) in a cytocentrifuge (Cytospin 4, Shandon Products), air-dried, fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 in PBS and then blocked with 3% bovine serum albumin in PBS. The slides were incubated at room temperature for 1 hour with anti-cofilin antibody (1:1000 dilution, C8736, Sigma), washed with PBS and then incubated with an Alexa-Fluor-594-conjugated rabbit secondary antibody (Invitrogen, Burlington, Ontario, Canada). Fluorescent images were acquired using a Leica multiphoton laser-scanning confocal microscope (DM 6000B microscope connected to a TCS SP5 system). Images were processed using ImageJ software (National Institutes of Heath), and were compiled using Adobe Photoshop CS3.

Acknowledgements

We thank Julian Kwan and Alicia Soltys for their contribution of preliminary data and Mhairi Skinner for her comments on the manuscript. This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Canadian Institutes of Health Research (CIHR). G.S.T.S. is a recipient of a Doctoral Studentship from the Multiple Sclerosis Society of Canada.

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