FIGURE 2 - uploaded by Kim G Lieu
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SRY nuclear import is enhanced by hsc70 and CaM. Nuclear import of GFP-SRY-HMG-wild type (WT) and its mutant derivatives was reconstituted in vitro as per the legend to Fig. 1, with or without ( No Add. ) 1.5 M hsc70 and/or CaM. CLSM images of cells at 18 –20 min are shown ( top panels ), with TR70 indicating intact nuclei, for GFP-SRY-HMG-WT ( A ), the CaM-NLS ( B ), and  -NLS ( C ) mutant derivatives, with quantitative analysis of such images ( bottom panels ) as per the legend to Fig. 1. D and E , pooled data (mean Ϯ S.E., n ϭ 3) for percentage maximal nuclear accumulation (Fn/c max ) ( D ) and initial rate of nuclear accumulation ( E ) determined as per the legend to Fig. 1, in the presence and absence of the various additions for the indicated GFP-SRY-HMG derivatives. p values are shown where there were significant differences compared with in the absence of the addition of CaM and hsc70 proteins.
Source publication
We recently showed that the developmentally important family of SOX (SRY (sex determining region on the Y chromosome)-related high mobility group (HMG) box) proteins require the calcium-binding protein calmodulin (CaM) for optimal nuclear accumulation, with clinical mutations in SRY that specifically impair nuclear accumulation via this pathway res...
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... the nucleus, scanning the area 40 times at a rate of 10 s/pixel at 100% laser power. Cells were imaged immediately post-bleaching with images acquired at 20-s inter- vals over a period of 280 s to monitor fluorescence recovery, using settings prior to photobleaching. Image analysis was performed using ImageJ as described above. Results were expressed as the fractional recovery of Fn/c (Fn/c of respective time points divided by prebleach value), and data were fitted exponentially according to the formula y ϭ a (1 Ϫ e Ϫ bx ) as described (17–20) to determine the maximal recovery and half- time ( t 1 2 ). The initial rate was determined using results for the Fn/c between 0 and 100 s post-bleaching. For analysis of intranuclear mobility of SRY, results were expressed as the fractional recovery of the bleached area (Fn bleach ) relative to prebleach (Fn non-bleach ), and data were fitted exponentially as above (19). SRY —We previously reported CaM-dependent nuclear import for SRY, as well as a number of other related SOX proteins, independent of the classical mode of nuclear transport through Imps (2, 3). The heat shock cognate protein hsc70 has been previously reported to play a role in modulating the nuclear import of proteins such as SV40-T-ag and nucleoplasmin (9, 10), as well as being a CaM-binding protein (21, 22) able to associate with nucleoporins (7). As a first step in establishing the potential role of hsc70 in CaM-dependent SRY nuclear import, we employed an established system of SRY nuclear transport reconstituted in vitro in mechanically perforated HTC cells (see “Experimental Procedures” (3)). We have previously used this system to demonstrate that CaM-dependent nuclear accumulation of SRY is active, requires ATP hydrolysis, and nucleoporin (nuclear pore) function, but does not require Ran (3). We also used the system to demonstrate that CaM dependence of nuclear import does not apply to a number of other proteins, including the Imp  1-recognized telomere repeat factor binding protein (TRF)-1 (3). As described previously, wild type SRY in the form of the bacterially expressed GFP-SRY-HMG fusion protein accumulated strongly within intact nuclei (those excluding Texas Red dextran; see Fig. 1 A , top panel ), but significantly, showed reduced nuclear accumulation upon addition of anti-hsc70 antibody (Fig. 1 A ). Quantitative analysis (see “Experimental Procedures” and supplemental Fig. 1) to determine the extent of nuclear accumulation expressed in terms of the nuclear to cytoplasmic ratio (Fn/c), confirmed this observation, revealing a significant ( p ϭ 0.01) 40% reduction in the maximal level of SRY nuclear accumulation in the presence of anti-hsc70 antibody (Fig. 1, B and C ); because Imp  1-dependent transport accounts for ϳ 50% of SRY nuclear accumulation in this system (2–5), this equates to close to complete inhibition of CaM-dependent nuclear import of SRY. No such changes to SRY nuclear import were observed in the presence of antibodies to importin 2 (see Fig. 1 C ), as described previously (3), indicating that the results observed in the presence of anti-hsc70 were specific for SRY, supporting the idea of a role for hsc70 in SRY nuclear import. Mutant derivatives of GFP-SRY HMG with inactive CaM- or  -NLS, previously shown to retain only specific Imp  1- or CaM-dependent nuclear transport respectively (see Fig. 1 B ) (3), were similarly analyzed (see Fig. 1). Significant reductions in nuclear accumulation were observed for both the CaM (R76P,M64T)- and  (Y127C)-NLS mutant derivatives compared with wild type SRY in the absence of anti-hsc70 antibody (see Figs. 1 and 2) as described previously (3). In the case of the CaM-NLS mutant (M64T), which lacks CaM binding, no change was observed upon addition of anti-hsc70 antibody, but significantly ( p ϭ 0.02) reduced ( ϳ 20%) nuclear accumulation was observed for the  -NLS mutant (Y127C), which possesses wild type CaM binding (see Fig. 1). Significant ( p Ͻ 0.05) reductions in the initial rate of nuclear accumulation were also observed for the wild type SRY (Fn/c s of 0.23) and -NLS mutant (Y127C; Fn/c s Ϫ 1 of 0.20), in the presence of anti-hsc70 antibody (Fn/c s Ϫ 1 of 0.16 and 0.15, respectively), with no such changes observed for the CaM-NLS (M64T) mutant (see Fig. 1 D ). These results clearly indicate that hsc70 contributes to CaM- rather than Imp  1-dependent nuclear import of SRY, modulating both the rate and maximal level of CaM-dependent nuclear accumulation. Binding of hsc70 to the SRY CaM Complex —To confirm the contribution of hsc70 to SRY nuclear import, the effect of add- ing purified hsc70 and/or CaM to the transport assay was assessed. No significant changes to wild type SRY nuclear import were observed in the presence of purified hsc70 or CaM alone (Fig. 2, A and D ), in stark contrast to in the presence of both CaM and hsc70, where significantly ( p Ͻ 0.05) ϳ 40% increased nuclear accumulation of SRY was observed (see Fig. 2, A and D ). No enhancement of nuclear accumulation was observed for the CaM-NLS mutant (R76P) in the presence of hsc70 and CaM (see Fig. 2, B and D ), whereas significantly ( p Ͻ 0.002) ϳ 70% increased maximal nuclear accumulation was observed for the  -NLS mutant (Y127C) derivative. Significant increases ( p Ͻ 0.05) in the initial rate of transport were also observed for the wild type protein (Fn/c s Ϫ 1 of 0.23) and  -NLS mutant (Y127C; Fn/c s Ϫ 1 of 0.18) derivative in the presence of both hsc70 and CaM (Fn/c s Ϫ 1 of 0.39 and 0.26, respectively), with no such changes observed for the CaM-NLS mutant (R76P) derivative. These results clearly imply that in combina- tion with CaM, hsc70 enhances SRY nuclear import dependent on the CaM-NLS. Hsp70 has been previously shown to bind to CaM in a Ca 2 ϩ dependent manner (21). The ability of hsc70 to bind to SRY and the SRY ⅐ CaM complex was assessed using native PAGE. A marked shift in mobility was observed for the wild type GFP- SRY-HMG protein in the presence of CaM as described previously (3), indicating complex formation (Fig. 3 A ), with no shift observed for SRY in the presence of hsc70 alone, indicating lack of complexation between hsc70 and SRY. Intriguingly, in the presence of hsc70 and CaM, a supershifted band was observed distant from that of the SRY ⅐ CaM complex. The clear implica- tion is that hsc70 can bind to SRY in the presence of CaM in a trimeric complex. CaM binding to SRY is known to be Ca 2 ϩ -dependent (3). To extend the observations, immunoprecipitation assays using GFP-TRAP TM were performed to assess association of hsc70 to GFP-SRY and its mutant derivatives. hsc70 was pulled down with wild type SRY in the presence of 2 M Ca 2 ϩ but not with the GFP control protein (see Fig. 3 B ). Only low levels of association of hsc70 with SRY were observed in the absence of Ca 2 ϩ . As expected, significantly ( p Ͻ 0.02), ϳ 70% reduced binding was observed of hsc70 to the CaM-NLS mutant derivative (R76P), in contrast to the  -NLS mutant derivative (Y127C) retaining wild type CaM binding (see Fig. 3 C ), which showed hsc70 binding comparable with that of wild type SRY. Taken together, these results indicate that hsc70 binds to SRY ⅐ CaM dependent upon Ca 2 ϩ and that this is required for enhanced CaM-dependent nuclear import of SRY. Import of SRY in Living Cells —To confirm that the above results have relevance to intact cell systems, HeLa cells were treated with hsc70-specific small interfering RNA duplexes (hsc70 siRNA) to knock down endogenous hsc70 protein expression prior to transfection to express full-length SRY- GFP-fusion proteins. Treatment led to an ϳ 65% reduction in hsc70 protein levels at 72 h compared with the untransfected control cells (Fig. 4 A ). In contrast to wild type full-length SRY, which showed exclusively nuclear localization as described previously (2), the protein showed increased cytoplasmic localiza- tion in the presence of siRNA to hsc70 (see Fig. 4 B ). Quantitative analysis (Fig. 4 C ) indicated significantly ( p Ͻ 0.003) ϳ 40% reduced nuclear accumulation of SRY upon knockdown of hsc70 expression. Reduced nuclear accumulation was observed for the NLS mutants compared with wild type in the absence of siRNA to hsc70 as described previously (2). Although no reduction to nuclear accumulation was observed for the CaM-NLS mutant (R76P) possessing intact Imp-dependent but impaired CaM-dependent nuclear import upon knockdown of hsc70, a significant ( p ϭ 0.0046) ϳ 40% reduction was observed for the  -NLS mutant (R133W) that possesses intact CaM-dependent but impaired Imp  1-dependent nuclear import in the presence of siRNA to hsc70. The control proteins (Fig. 4, B and C ), GFP alone, and TRF1, imported into the nucleus through the specific action of Imp  1, showed no changes to subcellular localization in the presence of siRNA to hsc70, indicating that reduction of hsc70 protein levels specifically inhibits SRY nuclear import and does not impact upon other nuclear transport pathways. Taken together, these results indicate that hsc70 is required for CaM-dependent nuclear import of SRY in intact cells. The role of hsc70 in enhancing SRY nuclear import in living cells was assessed using the established FRAP technique (18). Briefly, an area corresponding to 50% of the nucleus of cells transfected to express full length GFP-SRY fusion protein in the absence or presence of hsc70 siRNA was photobleached, and the return of nuclear fluorescence from the translocation of unbleached, cytoplasmic fluorescent protein into the nucleus monitored by CLSM for up to 280 s (see Fig. 5 A and Table 1; also see “Experimental Procedures”). The fractional recovery of the nuclear to cytoplasmic fluorescence ratio (Fn/c) was calculated by expressing the postbleach Fn/c value at each time point relative to the initial (prebleach) fluorescence value; this also enabled the determination of the time taken to achieve 50% recovery ( t 1 ...
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... to photobleaching using 3% total laser power with excitation at 488 nm, scanning at a rate of 12.5 s/pixel. Bleaching was performed in an area corresponding to ϳ 50% of the nucleus, scanning the area 40 times at a rate of 10 s/pixel at 100% laser power. Cells were imaged immediately post-bleaching with images acquired at 20-s inter- vals over a period of 280 s to monitor fluorescence recovery, using settings prior to photobleaching. Image analysis was performed using ImageJ as described above. Results were expressed as the fractional recovery of Fn/c (Fn/c of respective time points divided by prebleach value), and data were fitted exponentially according to the formula y ϭ a (1 Ϫ e Ϫ bx ) as described (17–20) to determine the maximal recovery and half- time ( t 1 2 ). The initial rate was determined using results for the Fn/c between 0 and 100 s post-bleaching. For analysis of intranuclear mobility of SRY, results were expressed as the fractional recovery of the bleached area (Fn bleach ) relative to prebleach (Fn non-bleach ), and data were fitted exponentially as above (19). SRY —We previously reported CaM-dependent nuclear import for SRY, as well as a number of other related SOX proteins, independent of the classical mode of nuclear transport through Imps (2, 3). The heat shock cognate protein hsc70 has been previously reported to play a role in modulating the nuclear import of proteins such as SV40-T-ag and nucleoplasmin (9, 10), as well as being a CaM-binding protein (21, 22) able to associate with nucleoporins (7). As a first step in establishing the potential role of hsc70 in CaM-dependent SRY nuclear import, we employed an established system of SRY nuclear transport reconstituted in vitro in mechanically perforated HTC cells (see “Experimental Procedures” (3)). We have previously used this system to demonstrate that CaM-dependent nuclear accumulation of SRY is active, requires ATP hydrolysis, and nucleoporin (nuclear pore) function, but does not require Ran (3). We also used the system to demonstrate that CaM dependence of nuclear import does not apply to a number of other proteins, including the Imp  1-recognized telomere repeat factor binding protein (TRF)-1 (3). As described previously, wild type SRY in the form of the bacterially expressed GFP-SRY-HMG fusion protein accumulated strongly within intact nuclei (those excluding Texas Red dextran; see Fig. 1 A , top panel ), but significantly, showed reduced nuclear accumulation upon addition of anti-hsc70 antibody (Fig. 1 A ). Quantitative analysis (see “Experimental Procedures” and supplemental Fig. 1) to determine the extent of nuclear accumulation expressed in terms of the nuclear to cytoplasmic ratio (Fn/c), confirmed this observation, revealing a significant ( p ϭ 0.01) 40% reduction in the maximal level of SRY nuclear accumulation in the presence of anti-hsc70 antibody (Fig. 1, B and C ); because Imp  1-dependent transport accounts for ϳ 50% of SRY nuclear accumulation in this system (2–5), this equates to close to complete inhibition of CaM-dependent nuclear import of SRY. No such changes to SRY nuclear import were observed in the presence of antibodies to importin 2 (see Fig. 1 C ), as described previously (3), indicating that the results observed in the presence of anti-hsc70 were specific for SRY, supporting the idea of a role for hsc70 in SRY nuclear import. Mutant derivatives of GFP-SRY HMG with inactive CaM- or  -NLS, previously shown to retain only specific Imp  1- or CaM-dependent nuclear transport respectively (see Fig. 1 B ) (3), were similarly analyzed (see Fig. 1). Significant reductions in nuclear accumulation were observed for both the CaM (R76P,M64T)- and  (Y127C)-NLS mutant derivatives compared with wild type SRY in the absence of anti-hsc70 antibody (see Figs. 1 and 2) as described previously (3). In the case of the CaM-NLS mutant (M64T), which lacks CaM binding, no change was observed upon addition of anti-hsc70 antibody, but significantly ( p ϭ 0.02) reduced ( ϳ 20%) nuclear accumulation was observed for the  -NLS mutant (Y127C), which possesses wild type CaM binding (see Fig. 1). Significant ( p Ͻ 0.05) reductions in the initial rate of nuclear accumulation were also observed for the wild type SRY (Fn/c s of 0.23) and -NLS mutant (Y127C; Fn/c s Ϫ 1 of 0.20), in the presence of anti-hsc70 antibody (Fn/c s Ϫ 1 of 0.16 and 0.15, respectively), with no such changes observed for the CaM-NLS (M64T) mutant (see Fig. 1 D ). These results clearly indicate that hsc70 contributes to CaM- rather than Imp  1-dependent nuclear import of SRY, modulating both the rate and maximal level of CaM-dependent nuclear accumulation. Binding of hsc70 to the SRY CaM Complex —To confirm the contribution of hsc70 to SRY nuclear import, the effect of add- ing purified hsc70 and/or CaM to the transport assay was assessed. No significant changes to wild type SRY nuclear import were observed in the presence of purified hsc70 or CaM alone (Fig. 2, A and D ), in stark contrast to in the presence of both CaM and hsc70, where significantly ( p Ͻ 0.05) ϳ 40% increased nuclear accumulation of SRY was observed (see Fig. 2, A and D ). No enhancement of nuclear accumulation was observed for the CaM-NLS mutant (R76P) in the presence of hsc70 and CaM (see Fig. 2, B and D ), whereas significantly ( p Ͻ 0.002) ϳ 70% increased maximal nuclear accumulation was observed for the  -NLS mutant (Y127C) derivative. Significant increases ( p Ͻ 0.05) in the initial rate of transport were also observed for the wild type protein (Fn/c s Ϫ 1 of 0.23) and  -NLS mutant (Y127C; Fn/c s Ϫ 1 of 0.18) derivative in the presence of both hsc70 and CaM (Fn/c s Ϫ 1 of 0.39 and 0.26, respectively), with no such changes observed for the CaM-NLS mutant (R76P) derivative. These results clearly imply that in combina- tion with CaM, hsc70 enhances SRY nuclear import dependent on the CaM-NLS. Hsp70 has been previously shown to bind to CaM in a Ca 2 ϩ dependent manner (21). The ability of hsc70 to bind to SRY and the SRY ⅐ CaM complex was assessed using native PAGE. A marked shift in mobility was observed for the wild type GFP- SRY-HMG protein in the presence of CaM as described previously (3), indicating complex formation (Fig. 3 A ), with no shift observed for SRY in the presence of hsc70 alone, indicating lack of complexation between hsc70 and SRY. Intriguingly, in the presence of hsc70 and CaM, a supershifted band was observed distant from that of the SRY ⅐ CaM complex. The clear implica- tion is that hsc70 can bind to SRY in the presence of CaM in a trimeric complex. CaM binding to SRY is known to be Ca 2 ϩ -dependent (3). To extend the observations, immunoprecipitation assays using GFP-TRAP TM were performed to assess association of hsc70 to GFP-SRY and its mutant derivatives. hsc70 was pulled down with wild type SRY in the presence of 2 M Ca 2 ϩ but not with the GFP control protein (see Fig. 3 B ). Only low levels of association of hsc70 with SRY were observed in the absence of Ca 2 ϩ . As expected, significantly ( p Ͻ 0.02), ϳ 70% reduced binding was observed of hsc70 to the CaM-NLS mutant derivative (R76P), in contrast to the  -NLS mutant derivative (Y127C) retaining wild type CaM binding (see Fig. 3 C ), which showed hsc70 binding comparable with that of wild type SRY. Taken together, these results indicate that hsc70 binds to SRY ⅐ CaM dependent upon Ca 2 ϩ and that this is required for enhanced CaM-dependent nuclear import of SRY. Import of SRY in Living Cells —To confirm that the above results have relevance to intact cell systems, HeLa cells were treated with hsc70-specific small interfering RNA duplexes (hsc70 siRNA) to knock down endogenous hsc70 protein expression prior to transfection to express full-length SRY- GFP-fusion proteins. Treatment led to an ϳ 65% reduction in hsc70 protein levels at 72 h compared with the untransfected control cells (Fig. 4 A ). In contrast to wild type full-length SRY, which showed exclusively nuclear localization as described previously (2), the protein showed increased cytoplasmic localiza- tion in the presence of siRNA to hsc70 (see Fig. 4 B ). Quantitative analysis (Fig. 4 C ) indicated significantly ( p Ͻ 0.003) ϳ 40% reduced nuclear accumulation of SRY upon knockdown of hsc70 expression. Reduced nuclear accumulation was observed for the NLS mutants compared with wild type in the absence of siRNA to hsc70 as described previously (2). Although no reduction to nuclear accumulation was observed for the CaM-NLS mutant (R76P) possessing intact Imp-dependent but impaired CaM-dependent nuclear import upon knockdown of hsc70, a significant ( p ϭ 0.0046) ϳ 40% reduction was observed for the  -NLS mutant (R133W) that possesses intact CaM-dependent but impaired Imp  1-dependent nuclear import in the presence of siRNA to hsc70. The control proteins (Fig. 4, B and C ), GFP alone, and TRF1, imported into the nucleus through the specific action of Imp  1, showed no changes to subcellular localization in the presence of siRNA to hsc70, indicating that reduction of hsc70 protein levels specifically inhibits SRY nuclear import and does not impact upon other nuclear transport pathways. Taken together, these results indicate that hsc70 is required for CaM-dependent nuclear import of SRY in intact cells. The role of hsc70 in enhancing SRY nuclear import in living cells was assessed using the established FRAP technique (18). Briefly, an area corresponding to 50% of the nucleus of cells transfected to express full length GFP-SRY fusion protein in the absence or presence of hsc70 siRNA was photobleached, and the return of nuclear fluorescence from the translocation of unbleached, cytoplasmic fluorescent protein into the nucleus monitored by CLSM for up to 280 s (see Fig. 5 A and Table 1; also see “Experimental Procedures”). The fractional recovery of the nuclear to cytoplasmic fluorescence ratio (Fn/c) was calculated by expressing the ...
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... images acquired at 20-s inter- vals over a period of 280 s to monitor fluorescence recovery, using settings prior to photobleaching. Image analysis was performed using ImageJ as described above. Results were expressed as the fractional recovery of Fn/c (Fn/c of respective time points divided by prebleach value), and data were fitted exponentially according to the formula y ϭ a (1 Ϫ e Ϫ bx ) as described (17–20) to determine the maximal recovery and half- time ( t 1 2 ). The initial rate was determined using results for the Fn/c between 0 and 100 s post-bleaching. For analysis of intranuclear mobility of SRY, results were expressed as the fractional recovery of the bleached area (Fn bleach ) relative to prebleach (Fn non-bleach ), and data were fitted exponentially as above (19). SRY —We previously reported CaM-dependent nuclear import for SRY, as well as a number of other related SOX proteins, independent of the classical mode of nuclear transport through Imps (2, 3). The heat shock cognate protein hsc70 has been previously reported to play a role in modulating the nuclear import of proteins such as SV40-T-ag and nucleoplasmin (9, 10), as well as being a CaM-binding protein (21, 22) able to associate with nucleoporins (7). As a first step in establishing the potential role of hsc70 in CaM-dependent SRY nuclear import, we employed an established system of SRY nuclear transport reconstituted in vitro in mechanically perforated HTC cells (see “Experimental Procedures” (3)). We have previously used this system to demonstrate that CaM-dependent nuclear accumulation of SRY is active, requires ATP hydrolysis, and nucleoporin (nuclear pore) function, but does not require Ran (3). We also used the system to demonstrate that CaM dependence of nuclear import does not apply to a number of other proteins, including the Imp  1-recognized telomere repeat factor binding protein (TRF)-1 (3). As described previously, wild type SRY in the form of the bacterially expressed GFP-SRY-HMG fusion protein accumulated strongly within intact nuclei (those excluding Texas Red dextran; see Fig. 1 A , top panel ), but significantly, showed reduced nuclear accumulation upon addition of anti-hsc70 antibody (Fig. 1 A ). Quantitative analysis (see “Experimental Procedures” and supplemental Fig. 1) to determine the extent of nuclear accumulation expressed in terms of the nuclear to cytoplasmic ratio (Fn/c), confirmed this observation, revealing a significant ( p ϭ 0.01) 40% reduction in the maximal level of SRY nuclear accumulation in the presence of anti-hsc70 antibody (Fig. 1, B and C ); because Imp  1-dependent transport accounts for ϳ 50% of SRY nuclear accumulation in this system (2–5), this equates to close to complete inhibition of CaM-dependent nuclear import of SRY. No such changes to SRY nuclear import were observed in the presence of antibodies to importin 2 (see Fig. 1 C ), as described previously (3), indicating that the results observed in the presence of anti-hsc70 were specific for SRY, supporting the idea of a role for hsc70 in SRY nuclear import. Mutant derivatives of GFP-SRY HMG with inactive CaM- or  -NLS, previously shown to retain only specific Imp  1- or CaM-dependent nuclear transport respectively (see Fig. 1 B ) (3), were similarly analyzed (see Fig. 1). Significant reductions in nuclear accumulation were observed for both the CaM (R76P,M64T)- and  (Y127C)-NLS mutant derivatives compared with wild type SRY in the absence of anti-hsc70 antibody (see Figs. 1 and 2) as described previously (3). In the case of the CaM-NLS mutant (M64T), which lacks CaM binding, no change was observed upon addition of anti-hsc70 antibody, but significantly ( p ϭ 0.02) reduced ( ϳ 20%) nuclear accumulation was observed for the  -NLS mutant (Y127C), which possesses wild type CaM binding (see Fig. 1). Significant ( p Ͻ 0.05) reductions in the initial rate of nuclear accumulation were also observed for the wild type SRY (Fn/c s of 0.23) and -NLS mutant (Y127C; Fn/c s Ϫ 1 of 0.20), in the presence of anti-hsc70 antibody (Fn/c s Ϫ 1 of 0.16 and 0.15, respectively), with no such changes observed for the CaM-NLS (M64T) mutant (see Fig. 1 D ). These results clearly indicate that hsc70 contributes to CaM- rather than Imp  1-dependent nuclear import of SRY, modulating both the rate and maximal level of CaM-dependent nuclear accumulation. Binding of hsc70 to the SRY CaM Complex —To confirm the contribution of hsc70 to SRY nuclear import, the effect of add- ing purified hsc70 and/or CaM to the transport assay was assessed. No significant changes to wild type SRY nuclear import were observed in the presence of purified hsc70 or CaM alone (Fig. 2, A and D ), in stark contrast to in the presence of both CaM and hsc70, where significantly ( p Ͻ 0.05) ϳ 40% increased nuclear accumulation of SRY was observed (see Fig. 2, A and D ). No enhancement of nuclear accumulation was observed for the CaM-NLS mutant (R76P) in the presence of hsc70 and CaM (see Fig. 2, B and D ), whereas significantly ( p Ͻ 0.002) ϳ 70% increased maximal nuclear accumulation was observed for the  -NLS mutant (Y127C) derivative. Significant increases ( p Ͻ 0.05) in the initial rate of transport were also observed for the wild type protein (Fn/c s Ϫ 1 of 0.23) and  -NLS mutant (Y127C; Fn/c s Ϫ 1 of 0.18) derivative in the presence of both hsc70 and CaM (Fn/c s Ϫ 1 of 0.39 and 0.26, respectively), with no such changes observed for the CaM-NLS mutant (R76P) derivative. These results clearly imply that in combina- tion with CaM, hsc70 enhances SRY nuclear import dependent on the CaM-NLS. Hsp70 has been previously shown to bind to CaM in a Ca 2 ϩ dependent manner (21). The ability of hsc70 to bind to SRY and the SRY ⅐ CaM complex was assessed using native PAGE. A marked shift in mobility was observed for the wild type GFP- SRY-HMG protein in the presence of CaM as described previously (3), indicating complex formation (Fig. 3 A ), with no shift observed for SRY in the presence of hsc70 alone, indicating lack of complexation between hsc70 and SRY. Intriguingly, in the presence of hsc70 and CaM, a supershifted band was observed distant from that of the SRY ⅐ CaM complex. The clear implica- tion is that hsc70 can bind to SRY in the presence of CaM in a trimeric complex. CaM binding to SRY is known to be Ca 2 ϩ -dependent (3). To extend the observations, immunoprecipitation assays using GFP-TRAP TM were performed to assess association of hsc70 to GFP-SRY and its mutant derivatives. hsc70 was pulled down with wild type SRY in the presence of 2 M Ca 2 ϩ but not with the GFP control protein (see Fig. 3 B ). Only low levels of association of hsc70 with SRY were observed in the absence of Ca 2 ϩ . As expected, significantly ( p Ͻ 0.02), ϳ 70% reduced binding was observed of hsc70 to the CaM-NLS mutant derivative (R76P), in contrast to the  -NLS mutant derivative (Y127C) retaining wild type CaM binding (see Fig. 3 C ), which showed hsc70 binding comparable with that of wild type SRY. Taken together, these results indicate that hsc70 binds to SRY ⅐ CaM dependent upon Ca 2 ϩ and that this is required for enhanced CaM-dependent nuclear import of SRY. Import of SRY in Living Cells —To confirm that the above results have relevance to intact cell systems, HeLa cells were treated with hsc70-specific small interfering RNA duplexes (hsc70 siRNA) to knock down endogenous hsc70 protein expression prior to transfection to express full-length SRY- GFP-fusion proteins. Treatment led to an ϳ 65% reduction in hsc70 protein levels at 72 h compared with the untransfected control cells (Fig. 4 A ). In contrast to wild type full-length SRY, which showed exclusively nuclear localization as described previously (2), the protein showed increased cytoplasmic localiza- tion in the presence of siRNA to hsc70 (see Fig. 4 B ). Quantitative analysis (Fig. 4 C ) indicated significantly ( p Ͻ 0.003) ϳ 40% reduced nuclear accumulation of SRY upon knockdown of hsc70 expression. Reduced nuclear accumulation was observed for the NLS mutants compared with wild type in the absence of siRNA to hsc70 as described previously (2). Although no reduction to nuclear accumulation was observed for the CaM-NLS mutant (R76P) possessing intact Imp-dependent but impaired CaM-dependent nuclear import upon knockdown of hsc70, a significant ( p ϭ 0.0046) ϳ 40% reduction was observed for the  -NLS mutant (R133W) that possesses intact CaM-dependent but impaired Imp  1-dependent nuclear import in the presence of siRNA to hsc70. The control proteins (Fig. 4, B and C ), GFP alone, and TRF1, imported into the nucleus through the specific action of Imp  1, showed no changes to subcellular localization in the presence of siRNA to hsc70, indicating that reduction of hsc70 protein levels specifically inhibits SRY nuclear import and does not impact upon other nuclear transport pathways. Taken together, these results indicate that hsc70 is required for CaM-dependent nuclear import of SRY in intact cells. The role of hsc70 in enhancing SRY nuclear import in living cells was assessed using the established FRAP technique (18). Briefly, an area corresponding to 50% of the nucleus of cells transfected to express full length GFP-SRY fusion protein in the absence or presence of hsc70 siRNA was photobleached, and the return of nuclear fluorescence from the translocation of unbleached, cytoplasmic fluorescent protein into the nucleus monitored by CLSM for up to 280 s (see Fig. 5 A and Table 1; also see “Experimental Procedures”). The fractional recovery of the nuclear to cytoplasmic fluorescence ratio (Fn/c) was calculated by expressing the postbleach Fn/c value at each time point relative to the initial (prebleach) fluorescence value; this also enabled the determination of the time taken to achieve 50% recovery ( t 1 2 ) (see Fig. 5, B and C ). Upon the addition of siRNA to hsc70, a significant ( p Ͻ 0.05) reduction of ϳ 25% was observed for Fn/c ...
Citations
... HSC70 is a regulator of HSF1 (heat-shock factor 1), which plays an essential role in mediating the appropriate cellular response to physiological stresses (Ahn et al., 2005). In humans, hsc70 is also a mediator of the CaM-dependent nuclear import of male-specific SRY (Kaur et al., 2013). A previous study showed that heat shock protein 70 kDa (Hsp70/Hsc70) family proteins interact with steroid hormone receptors through BAG (Bcl-2 associated athanogene), and that Bag1L can enhance the transcriptional activity of the androgen receptor (Knee et al., 2001). ...
The sex of Chinese tongue sole (Cynoglossus semilaevis) is determined by both genetic sex determination (GSD) and environmental sex determination (ESD), making it an ideal model to study the relationship between sex-determination and temperature. In the present study, transcriptomes of undifferentiated gonads from genetic females and males, as well as differentiated gonads from males, females, and pseudomales under high and normal temperature treatments were generated for comparative transcriptomic analysis. A mean of 68.24 M high-quality clean reads was obtained for each library. Differentially expressed genes (DEGs) between different sexes and environmental treatments were identified, revealing that the heat shock protein gene family was involved in the high temperature induced sex reversal. The Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that were enriched in pseudomale and genetic female comparison included neuroactive ligand-receptor interaction, cortisol synthesis and secretion, and steroid hormone biosynthesis. Furthermore, weighted gene co-expression network analyses were conducted on all samples, and two modules were positive correlated with pseudomale under high temperature. An illustrated protein-protein interaction map of the module identified a hub gene, hsc70. These findings provide insights into the genetic network that is involved in sex determination and sexual differentiation, and improve our understanding of genes involved in sex reversal under high temperature.
... Interestingly, 10 large molecular weight Hsps, especially Hsc70, were up-regulated at the mRNA level, while 3 small Hsps, with a low molecular weight ranging between 15 and 42 kDa, were down-regulated at the protein level. Heat shock congnate 70 kDa protein (Hsc70), a member of the Hsp70 family, is constitutively expressed and involved in protein folding and translocation, which protects proteins from denaturation and dysfunction [50]. The up-regulated expression of Hsc70 genes is required to refold damaged proteins and to carry irreversibly denatured proteins to the proteasome. ...
Harvested banana ripening is a complex physiological and biochemical process, and there are existing differences in the regulation of ripening between the pulp and peel. However, the underlying molecular mechanisms governing peel ripening are still not well understood. In this study, we performed a combination of transcriptomic, proteomic, and metabolomics analysis on peel during banana fruit ripening. It was found that 5784 genes, 94 proteins, and 133 metabolites were differentially expressed or accumulated in peel during banana ripening. Those genes and proteins were linked to ripening-related processes, including transcriptional regulation, hormone signaling, cell wall modification, aroma synthesis, protein modification, and energy metabolism. The differentially expressed transcriptional factors were mainly ethylene response factor (ERF) and basic helix-loop-helix (bHLH) family members. Moreover, a great number of auxin signaling-related genes were up-regulated, and exogenous 3-indoleacetic acid (IAA) treatment accelerated banana fruit ripening and up-regulated the expression of many ripening-related genes, suggesting that auxin participates in the regulation of banana peel ripening. In addition, xyloglucan endotransglucosylase/hydrolase (XTH) family members play an important role in peel softening. Both heat shock proteins (Hsps) mediated-protein modification, and ubiqutin-protesome system-mediated protein degradation was involved in peel ripening. Furthermore, anaerobic respiration might predominate in energy metabolism in peel during banana ripening. Taken together, our study highlights a better understanding of the mechanism underlying banana peel ripening and provides a new clue for further dissection of specific gene functions.
... In yeast, CaM binds to Hsp75 (also denoted Ssb1) in a Ca 2 + -dependent manner at residues 260-277, but based on a phylogenetic analysis of the amino acid sequences of these proteins, it was shown that a branch of eukaryotic Hsp70 proteins does not bind CaM [139]. Interesting, in HeLa cells Hsc70 facilitates the nuclear translocation of the sex-determining factor SRY in a CaM-dependent manner by allowing the passage of an Hsp70/CaM/SRY complex through the nuclear pore [140]. As SRY is also a CaM-binding protein itself, it is likely that the role of CaM in this complex is to bridge the chaperone to its cargo. ...
Calmodulin (CaM) is a universal regulator for a huge number of proteins in all eukaryotic cells. Best known is its function as a calcium-dependent modulator of the activity of enzymes, such as protein kinases and phosphatases, as well as other signaling proteins including membrane receptors, channels and structural proteins. However, less well known is the fact that CaM can also function as a Ca2+-dependent adaptor protein, either by bridging between different domains of the same protein or by linking two identical or different target proteins together. These activities are possible due to the fact that CaM contains two independently-folded Ca2+ binding lobes that are able to interact differentially and to some degree separately with targets proteins. In addition, CaM can interact with and regulates several proteins that function exclusively as adaptors. This review provides an overview over our present knowledge concerning the structural and functional aspects of the role of CaM as an adaptor protein and as a regulator of known adaptor proteins.
... Hsp70 can also play an important role in nuclear protein import by regulating the export of importin-β back to the cytoplasm from the nucleus [32]. In addition, both Hsp70 and Hsp90 have been implicated in the nucleocytoplasmic transport of specific cargo proteins [33,34]. In the present study, none of these other heat shock proteins could have confounded our results since their expression levels were not altered by an overexpression of Hsp60 in primary VSMCs. ...
Aim:
Heat shock protein 60 (Hsp60) is a mediator of stress-induced vascular smooth muscle cell (VSMC) proliferation. This study will determine, first, if the mitochondrial or cytoplasmic localization of Hsp60 is critical to VSMC proliferation and, second, the mechanism of Hsp60 induction of VSMC proliferation with a focus on modification of nucleocytoplasmic trafficking.
Methods and results:
Hsp60 was overexpressed in primary rabbit VSMCs with or without a mitochondrial targeting sequence (AdHsp60mito-). Both interventions induced an increase in VSMC PCNA expression and proliferation. The increase in VSMC PCNA expression and growth was not observed after siRNA-mediated knockdown of Hsp60 expression. Nuclear protein import in VSMC was measured by fluorescent microscopy using a microinjected fluorescent import substrate. Nuclear protein import was stimulated by both AdHsp60 and AdHsp60mito-treatments. AdHsp60 treatment also induced increases in nucleoporin (Nup) 62, Nup153, importin-α, importin-β and Ran expression as well as cellular ATP levels compared to control. AdHsp60mito-treatment induced an up-regulation in importin-α, importin-β and Ran expression compared to control. Hsp60 knockdown did not change nuclear protein import nor the expression of any nuclear transport receptors or nucleoporins. Both heat shock treatment and Hsp60 overexpression promoted the interaction of Ran with Hsp60.
Conclusions:
VSMC proliferation can be modulated via an Hsp60 dependent, cytosol localized mechanism that in part involves a stimulation of nuclear protein import through an interaction with Ran. This novel cellular signaling role for Hsp60 may be important in growth-based vascular pathologies like atherosclerosis and hypertension.
... Some studies reported that Hsps induced by various postharvest treatments such as heat treatment, and salicylate and jasmonate treatments, are involved in the chilling tolerance in fruits (), suggesting a central role of Hsps in acquired tolerance to chilling stress. Heat shock congnate 70 kDa protein (Hsc70) is a member of heat shock protein 70 (Hsp70) family, which is located in both the nuclear and cytoplasmic compartments and plays a role in protein folding and translocation (Kaur et al., 2013). Unlike canonical heat shock proteins, Hsc70 is constitutively expressed and is related to normal cellular process. ...
... Some studies reported that Hsps induced by various postharvest treatments such as heat treatment, and salicylate and jasmonate treatments, are involved in the chilling tolerance in fruits , suggesting a central role of Hsps in acquired tolerance to chilling stress. Heat shock congnate 70 kDa protein (Hsc70) is a member of heat shock protein 70 (Hsp70) family, which is located in both the nuclear and cytoplasmic compartments and plays a role in protein folding and translocation (Kaur et al., 2013). Unlike canonical heat shock proteins, Hsc70 is constitutively expressed and is related to normal cellular process. ...
To better understand the mechanism involved in ethylene-induced chilling tolerance in harvested banana fruit, a gel-based proteomic study followed by MALDI-TOF-TOF MS was carried out. Banana fruit were treated with 500 ppm ethylene for 12 h and then stored at 6°C. During cold storage, the chilling tolerance was assessed and the proteins from the peel were extracted for proteomic analysis. It was observed that ethylene pretreatment significantly induced the chilling tolerance in harvested banana fruit, manifesting as increases in maximal chlorophyll fluorescence (Fv/Fm) and decreased electrolyte leakage. Sixty-four proteins spots with significant differences in abundance were identified, most of which were induced by ethylene pretreatment during cold storage. The up-regulated proteins induced by ethylene pretreatment were mainly related to energy metabolism, stress response and defense, methionine salvage cycle and protein metabolism. These proteins were involved in ATP synthesis, ROS scavenging, protective compounds synthesis, protein refolding and degradation, and polyamine biosynthesis. It is suggested that these up-regulated proteins might play a role in the ethylene-induced chilling tolerance in harvested banana fruit.
... Hsp70 is a molecular chaperone essential to protein transport during cellular stress, including inflammation and autoimmune conditions (Jolesch et al., 2012). Hsp70 has been implicated in the nuclear import of multiple proteins, including NF-κB (Fujihara and Nadler, 1999), the temperature sensitive p53 mutant (Akakura et al., 2001), p38 MAPK (Gong et al., 2012), Simian virus 40 large tumor antigen (Yang and DeFranco, 1994), nucleoplasmin (Imamoto et al., 1992) and the sexdetermining factor SRY (Kaur et al., 2013). Hsp70 also facilitates the nuclear export of importins (Kose et al., 2005;Miyamoto et al., 2004), T cells stimulated with anti-CD3 abs and Jagged1, or anti-CD3 and Delta-like1. ...
Notch receptors (Notch1-4) are involved in the differentiation of CD4 T cells and the development of autoimmunity. Mechanisms regulating Notch signaling in CD4 T cells are not fully elucidated. In this study we investigated potential crosstalk between Notch pathway molecules and heat shock protein 70 (Hsp70), the major intracellular chaperone involved in the protein transport during immune responses and other stress conditions. Using Hsp70(-/-) mice we found that Hsp70 is critical for up-regulation of NICD1 and induction of Notch target genes in Jagged1- and Delta-like1-stimulated CD4 T cells. Co-immunoprecipitation analysis of wild-type CD4 T cells stimulated with either Jagged1 or Delta-like1 showed a direct interaction between NICD1 and Hsp70. Both molecules co-localized within the nucleus of CD4 T cells stimulated with Notch ligands. Molecular interaction and nuclear colocalization of NICD1 and Hsp70 were also detected in CD4 T cells reactive against myelin oligodendrocyte glycoprotein (MOG)35-55, which showed Hsp70-dependent up-regulation of both NICD1 and Notch target genes. In conclusion, we demonstrate for the first time that Hsp70 interacts with NICD1 and contributes to the activity of Notch signaling in CD4 T cells. Interaction between Hsp70 and NICD1 may represent a novel mechanism regulating Notch signaling in activated CD4 T cells.
... Some studies reported that Hsps induced by various postharvest treatments such as heat treatment, and salicylate and jasmonate treatments, are involved in the chilling tolerance in fruits , suggesting a central role of Hsps in acquired tolerance to chilling stress. Heat shock congnate 70 kDa protein (Hsc70) is a member of heat shock protein 70 (Hsp70) family, which is located in both the nuclear and cytoplasmic compartments and plays a role in protein folding and translocation (Kaur et al., 2013). Unlike canonical heat shock proteins, Hsc70 is constitutively expressed and is related to normal cellular process. ...
2015) Proteomic analysis of differentially expressed proteins involved in ethylene-induced chilling tolerance in harvested banana fruit. Front. Plant Sci. 6:845. To better understand the mechanism involved in ethylene-induced chilling tolerance in harvested banana fruit, a gel-based proteomic study followed by MALDI-TOF-TOF MS was carried out. Banana fruit were treated with 500 ppm ethylene for 12 h and then stored at 6 • C. During cold storage, the chilling tolerance was assessed and the proteins from the peel were extracted for proteomic analysis. It was observed that ethylene pretreatment significantly induced the chilling tolerance in harvested banana fruit, manifesting as increases in maximal chlorophyll fluorescence (Fv/Fm) and decreased electrolyte leakage. Sixty-four proteins spots with significant differences in abundance were identified, most of which were induced by ethylene pretreatment during cold storage. The up-regulated proteins induced by ethylene pretreatment were mainly related to energy metabolism, stress response and defense, methionine salvage cycle and protein metabolism. These proteins were involved in ATP synthesis, ROS scavenging, protective compounds synthesis, protein refolding and degradation, and polyamine biosynthesis. It is suggested that these up-regulated proteins might play a role in the ethylene-induced chilling tolerance in harvested banana fruit.
... M1 also appears to contribute to vRNP nuclear export through a NES that would appear to be recognized by an export protein other than XPO1 (Figure 2xii; see Table 1); mutation of the NES results in vRNPs nuclear accumulation (Cao et al., 2012). Additionally, two recent studies have identified additional cellular proteins as co-factors (Figure 2xiii) for vRNP-M1-NS2-XPO1 export ( Table 1); the heat shock protein Hsc70 , which is believed to play a key role in calcium-/calmodulindependent nuclear import of SOX proteins nuclear import (Kaur et al., 2013), and NXF1/TAP (Read and Digard, 2010), integrally involved in the nuclear export of cellular mRNA. These may also represent potential candidates for antiviral intervention. ...
The respiratory diseases caused by rhinovirus, respiratory syncytial virus, and influenza virus represent a large social and financial burden on healthcare worldwide. Although all three viruses have distinctly unique properties in terms of infection and replication, they share the ability to exploit/manipulate the host-cell nucleocytoplasmic transport system in order to replicate effectively and efficiently. This review outlines the various ways in which infection by these viruses impacts on the host nucleocytoplasmic transport system, and examples where inhibition thereof in turn decreases viral replication. The highly conserved nature of the nucleocytoplasmic transport system and the viral proteins that interact with it make this virus–host interface a prime candidate for the development of specific antiviral therapeutics in the future.
... Cells were imaged live by confocal laser scanning microscopy (CLSM; Bio Rad MRC-500, Richmond, CA, USA) using a 40Â water immersion objective as indicated, with image analysis of digitized confocal images subsequently performed for populations of cells using the Image J (National Institutes of Health, Bethesda, MD, USA) public domain software. Nuclear (Fn) and cytoplasmic (Fc) fluorescence were quantified subsequent to the subtraction of autofluorescence, and used to derive the nuclear to cytoplasmic ratio (Fn/c), as previously [6,20,23]. ...
We previously showed that increased intracellular calcium can modulate Importin (Imp)β1-dependent nuclear import of SRY-related chromatin remodeling proteins. Here we extend this work to show for the first time that high intracellular calcium inhibits Impα/β1- or Impβ1-dependent nuclear protein import generally. The basis of this relates to the mislocalisation of the transport factors Impβ1 and Ran, which show significantly higher nuclear localization in contrast to various other factors, and RCC1, which shows altered subnuclear localisation. The results here establish for the first time that intracellular calcium modulates conventional nuclear import through direct effects on the nuclear transport machinery.
