Nuclear Import of αB-crystallin Is Phosphorylation-dependent and Hampered by Hyperphosphorylation of the Myopathy-related Mutant R120G*

Phosphorylation modulates the functioning of αB-crystallin as a molecular chaperone. We here explore the role of phosphorylation in the nuclear import and cellular localization of αB-crystallin in HeLa cells. Inhibition of nuclear export demonstrated that phosphorylation of αB-crystallin is required for import into the nucleus. As revealed by mutant analysis, phosphorylation at Ser-59 is crucial for nuclear import, and phosphorylation at Ser-45 is required for speckle localization. Co-immunoprecipitation experiments suggested that the import of αB-crystallin is possibly regulated by its phosphorylation-dependent interaction with the survival motor neuron (SMN) protein, an important factor in small nuclear ribonucleoprotein nuclear import and assembly. This interaction was supported by co-localization of endogenous phosphorylated αB-crystallin with SMN in nuclear structures. The cardiomyopathy-causing αB-crystallin mutant R120G was found to be excessively phosphorylated, which disturbed SMN interaction and nuclear import, and resulted in the formation of cytoplasmic inclusions. Like for other protein aggregation disorders, hyperphosphorylation appears as an important aspect of the pathogenicity of αB-crystallin R120G.

The mutation R120G in ␣B-crystallin is associated with a desminrelated myopathy, characterized by the presence of aggregates containing ␣B-crystallin and desmin (26). This mutant causes cardiac hypertrophy and heart failure upon cardiac-restricted transgenic expression in mice (27). ␣B-Crystallin R120G has altered structural properties and a diminished chaperoning activity, both in vitro (28 -31) and in vivo (32). The inclusion bodies in which ␣B-crystallin R120G accumulates upon transfection (32,33) and transgenesis (34) have the characteristics of aggresomes, cellular structures to sequester aggregated proteins. ␣B-Crystallin R120G might be responsible for aggresome formation in cardiomyocytes as a result of its defective chaperone activity, by binding tightly to the nascent contractile proteins, preventing correct folding and formation of viable sarcomeres (34).
The deleterious effects of the R120G mutation might well interfere with the phosphorylation-dependent trafficking and localization of ␣B-crystallin in the cell. Investigating these relations may contribute to the insight in the normal cellular functioning of ␣B-crystallin and in the pathogenicity of the R120G mutant. In that context we studied the nuclear import of ␣B-crystallin, and explored the distribution of phosphorylated ␣B-crystallin during interphase and in dividing cells. It is shown here that nuclear import of ␣B-crystallin is phosphorylation-dependent, and might be regulated by its interaction with survival motor neuron (SMN) 2 protein, which is involved in the assembly and nuclear import of snRNPs (35,36). Phosphorylation was also associated with the localization of ␣B-crystallin during interphase in nuclear splicing speckles and SMN-positive structures, and during cell division in mitotic interchromatin granule clusters (MIGs), which are the mitotic counterparts of nuclear speckles (25). ␣B-Crystallin R120G was found to be hyperphosphorylated, which disturbed the normal interactions with binding partners such as SMN and splicing components, resulting in impaired nuclear import and accumulation in cytoplasmic inclusions.
Cell Fractioning and Immunoblotting-To obtain detergent-soluble and -insoluble protein fractions, HeLa cells (1 ϫ 10 6 ) were transfected with 1 g of the appropriate DNA and harvested after 2 days by trypsinization. Cells were washed once with Dulbecco's modified Eagle's medium containing 10% fetal calf serum, and twice with phosphate-buffered saline. About 10 6 cells were resuspended in 50 l of ice-cold lysis buffer (10 mM Tris-HCl, pH 7.5, 100 mM KCl, 1 mM dithiothreitol, 1 mM EDTA, 5 mM MgCl 2 , 1 mM phenylmethanesulfonyl fluoride, and 0.5% Nonidet P-40) and kept on ice for 15 min. The cell extract was centrifuged for 15 min at 3,000 rpm and 4°C. The supernatant was supplemented with 50 l of 2ϫ SDS sample buffer (2% SDS, 0.125 M Tris-HCl, pH 6.8, 20% glycerol, 0.02% ␤-mercaptoethanol, 0.05% bromphenol blue) heated for 5 min at 95°C and used as the detergent-soluble fraction. The remaining pellet was washed once with 500 l of lysis buffer, resuspended in 50 l of lysis buffer, and 50 l of 2ϫ SDS sample buffer, heated for 5 min at 95°C and used as the detergentinsoluble fraction. The detergent-soluble and -insoluble fractions were separated by SDS-PAGE.
Nuclear and cytoplasmic fractions were isolated from HeLa cells, either non-treated or treated with leptomycin B. Cells were harvested by trypsinization, washed once with Dulbecco's modified Eagle's medium containing 10% fetal calf serum, and twice with phosphate-buffered saline. The pelleted cells were taken up in 100 ml of buffer 1 (10 mM Hepes, pH 8.0, 10 mM KCl, 1 mM dithiothreitol, 1.5 mM MgCl 2 and supplemented with a protease inhibitor mixture from Roche Applied Science) and incubated on ice for 20 min. The cell suspension was passed 5 times through a 21-gauge needle, and the nuclei were pelleted by centrifugation for 5 min at 1,500 rpm to separate them from the cytoplasmic fraction. The cytoplasmic fraction was collected, and the remaining nuclei were washed twice with buffer 1. All fractions were taken up in 2ϫ SDS sample buffer without ␤-mercaptoethanol and bromphenol blue, and protein concentrations were determined with the BCA kit (Bio-Rad). Equal amounts of proteins were analyzed by SDS-PAGE and Western blotting.
For analysis of phosphorylated ␣B-crystallin, HeLa cells (1 ϫ 10 6 ) were transfected with 1 g of wild type or R120G ␣B-crystallin cDNA. Cells were harvested and washed as above, lysed in 2ϫ SDS sample buffer, without 2-mercaptoethanol and bromphenol blue, and heated for 5 min at 95°C. Protein concentrations were determined using the BCA kit, and 30 g of total protein was brought into 20 l of 2ϫ SDS sample buffer, heated for 5 min at 95°C, and separated by SDS-PAGE. To analyze phosphorylated ␣B-crystallin in hearts of non-transgenic and transgenic ␣B-crystallin R120G mice (described in Ref. 27), ϳ30 mg of heart tissue of 4-month-old animals was lysed by homogenization in 300 l of 2ϫ SDS sample buffer, without 2-mercaptoethanol and bromphenol blue and heated for 15 min at 95°C. Insoluble material was removed by centrifugation at 13,000 rpm for 30 min at 4°C, and protein concentrations were determined with the BCA kit. Cell extracts containing approximately equal amounts of ␣B-crystallin were separated by SDS-PAGE.
Immunoprecipitations-For co-immunoprecipitations, HeLa cells (1 ϫ 10 6 ) were transfected with 1 g of the appropriate DNA. Cells were harvested and washed as above and suspended in 1 ml of lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 0.5% Nonidet P-40) at 4°C. Cell lysates were cleared by centrifugation at 13,000 rpm for 30 min at 4°C and subsequently incubated with protein G-agarose beads (Roche Applied Science) coupled to the appropriate antibodies. After incubation at 4°C, beads were washed three times with buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 0.05% Nonidet P-40) and analyzed on Western blot.
Glycerol Gradient Centrifugation-For sedimentation analysis, HeLa cells (7.5 ϫ 10 6 ) were transfected with 7.5 g of DNA. Cells were harvested and washed as above and suspended in 750 l of lysis buffer (50 mM Tris-HCl, pH 7.5, 50 mM NaCl, and 0.5% Nonidet P-40). The cell extracts were kept on ice for 15 min and centrifuged for 15 min at 3,000 rpm and 4°C prior to application on top of 5-40% glycerol gradients in lysis buffer, generated in 12-ml tubes with the Gradient Master (Bio-Comp). The gradients were centrifuged in a Sorval Discovery 100 ultracentrifuge with a Th 641 rotor at 40,000 rpm for 16 h at 4°C. After centrifugation, 0.5-ml fractions were collected and 40 l of each fraction was analyzed by SDS-PAGE and Western blotting. The molecular mass standards were albumin (66 kDa), aldolase (158 kDa), catalase (232 kDa), and thyroglobulin (669 kDa).

RESULTS
Nuclear Import of ␣B-crystallin Is Phosphorylation-dependent-We previously showed that transfected pseudophosphorylated ␣B-crystallin as well as endogenous ␣B-crystallin phosphorylated at Ser-45 are found in nuclear speckles in interphase cells (24). This localization suggests that nuclear import of ␣B-crystallin is phosphorylation-dependent. To corroborate this suggestion, we transfected HeLa cells with plasmids coding for wild type ␣B-crystallin and its non-phosphorylat-able (S 3 A) and pseudophosphorylated (S 3 D) mutants. After 24 h nuclear export was inhibited by leptomycin B, and cells were stained for ␣B-crystallin (Fig. 1). The most conspicuous effect was the complete trapping of pseudophosphorylated ␣B-crystallin in the nucleus upon leptomycin B treatment (Fig. 1a, panels E and F). Wild type ␣B-crystallin accumulated to a lesser extent in the nucleus (Fig. 1a, panels A and B), and non-phosphorylatable ␣B-crystallin hardly or not (Fig. 1a, panels C and D). Because pseudophosphorylation readily allows import of FIGURE 1. Nuclear import of ␣B-crystallin is phosphorylation-dependent. a, HeLa cells were transfected with expression constructs coding for wild type ␣B-crystallin (A and B), ␣B-crystallin S19A/S45A/S59A (C and D), or ␣B-crystallin S19D/ S45D/S59D (E and F) and treated with Me 2 SO (ϪLMB; control) or leptomycin B (ϩLMB) for 20 h. Cells were subsequently fixed, permeabilized, and stained with the monoclonal anti-␣B-crystallin antibody. b, untransfected HeLa cells were treated with Me 2 SO (ϪLMB) or LMB (ϩLMB) for 20 h and subsequently fixed, permeabilized, and stained with the polyclonal phospho-specific anti-␣Bcrystallin S45p (A and B) and anti-␣B-crystallin S59p (C and D) antibodies. These antibodies are much more sensitive to detect ␣B-crystallin in the nucleus than the monoclonal antibody (cf. panel A in a with panels A and C in b) (24). c, untransfected HeLa cells were treated with Me 2 SO (ϪLMB) or LMB (ϩLMB) for 20 h, harvested by trypsinization, and subsequently separated in a cytoplasmic (cyt) and a nuclear fraction (nuc). Equal amounts of each fraction were analyzed by SDS-PAGE and Western blot, using polyclonal antibodies against ␣B-crystallin S59p (upper panel), the nuclear marker lamin A (middle panel), and the cytoplasmic marker peroxiredoxin 1 (bottom panel). d, HeLa cells were transfected with expression constructs coding for ␣B-crystallin S19A/S45A (A and B), ␣B-crystallin S45A/S59A (C and D), or ␣B-crystallin S19A/S59A (E and F) and treated with Me 2 SO (ϪLMB) or leptomycin B (ϩLMB) for 20 h. Cells were subsequently fixed, permeabilized, and stained with the monoclonal anti-␣B-crystallin antibody. e, HeLa cells were transfected with expression constructs coding for ␣B-crystallin S45A (A and B) or ␣B-crystallin S19A (C and D) and treated with Me 2 SO (ϪLMB; control) or leptomycin B (ϩLMB) for 20 h. Cells were subsequently fixed, permeabilized, and stained with the monoclonal anti-␣B-crystallin antibody. Insets show enlarged regions of the nucleus. f, HeLa cells were treated as for panel D in e, and stained with the monoclonal anti-␣B-crystallin antibody (A) and co-stained with anti-Sm (B). The merged image shows the extent of co-localization (C).

Phosphorylation-dependent Nuclear Import of ␣B-crystallin
␣B-crystallin into the nucleus (Fig. 1a, panels E and F), whereas nonphosphorylatability prevents it (Fig. 1a, panels C and D), the import of wild type ␣B-crystallin (Fig. 1a, panels A and B) must be dependent on phosphorylation.
The phosphorylation dependence of nuclear import was further supported by staining untransfected HeLa cells, with and without leptomycin B treatment, for endogenous ␣B-crystallin phosphorylated at Ser-45 and Ser-59 (Fig. 1b). Inhibition of nuclear export clearly resulted in accumulation of ␣B-crystallin S59p in the nucleus (Fig. 1b, panels C and D). The nuclear accumulation could be confirmed by cell fractionation of HeLa cells showing an increased amount of endogenous ␣B-crystallin S59p in the nuclear fraction upon leptomycin B treatment (Fig. 1c). No such effect was observed for ␣B-crystallin S45p, because its localization was already exclusively nuclear without inhibition of nuclear export (Fig. 1b, panels A and B).
Phosphorylation at Ser-59 Is Required for Nuclear Import-To further assess which serine(s) must be phosphorylated for nuclear import of ␣B-crystallin, we transfected HeLa cells with plasmids coding for ␣B-crystallin with double S 3 A substitutions (S19A/S45A, S45A/ S59A, and S19A/S59A). After 24 h, nuclear export was inhibited by leptomycin B, and cells were stained for ␣B-crystallin (Fig. 1d). Only ␣B-crystallin S19A/S45A clearly accumulated in the nucleus (Fig. 1d,  panels A and B), whereas ␣B-crystallin S45A/S59A (Fig. 1d, panels C and D) and ␣B-crystallin S19A/S59A (Fig. 1d, panels E and F) hardly did. These results imply that phosphorylation at Ser-59 is important for nuclear accumulation of ␣B-crystallin.

Phosphorylation at Ser-45 Is Required for Nuclear Speckle
Localization-We noticed that ␣B-crystallin S19A/S45A gave a diffuse nuclear staining and was hardly detectable in speckles. Because ␣B-crystallin S45p is predominantly found in nuclear speckles (Fig. 1b) it is tempting to speculate that phosphorylation at Ser-45 is needed for nuclear speckle localization of ␣B-crystallin. To test this hypothesis we transfected HeLa cells with plasmids coding for ␣B-crystallin with single S 3 A substitutions (S19A and S45A). After 24 h nuclear export was inhibited by leptomycin B, and cells were stained for ␣B-crystallin (Fig.  1e). Cells transfected with ␣B-crystallin S45A revealed a nuclear accumulation upon leptomycin B treatment, but no speckle localization (Fig.  1e, panels A and B). Interestingly, cells transfected with ␣B-crystallin S19A showed besides a nuclear accumulation a presence in nuclear speckles (Fig. 1e, panels C and D). This was confirmed by co-localization with Sm proteins, a marker of nuclear speckles (Fig. 1f, panels A-C). These results suggest that phosphorylation at Ser-45 is needed for nuclear speckle localization.
The wild type ␣B-crystallin that has accumulated in the nucleus in Fig. 1a, panel B, upon leptomycin B treatment, seems not to be concentrated in speckles but is distributed more diffusely through the nucleoplasm. This suggests that the ␣B-crystallin trapped in the nucleus is predominantly phosphorylated at Ser-59 and less so at Ser-45 and, therefore, not detectably localized in nuclear speckles.
Mutation R120G Hampers Nuclear Entry of ␣B-crystallin and Causes Excessively Phosphorylated Cytoplasmic Inclusions-The desmin-related myopathy-causing mutation R120G has been reported to prevent FIGURE 2. Increased phosphorylation of myopathy-related ␣B-crystallin R120G. a, HeLa cells transfected with ␣B-crystallin R120G were treated with Me 2 SO (ϪLMB) or leptomycin B (ϩLMB) for 20 h, and subsequently fixed, permeabilized, and stained with monoclonal anti-␣B-crystallin. b, HeLa cells were transfected with wild type ␣B-crystallin (WT) or ␣B-crystallin R120G (R120G) and harvested after 2 days. Equal amounts of protein were analyzed on Western blot using the polyclonal phospho-specific anti-␣B-crystallin S19p, S45p, and S59p antibodies. As a loading control Western blots were stained with the monoclonal anti-␣B-crystallin antibody (Total). c, protein extracts of hearts of a non-transgenic mouse (NTG) and an ␣B-crystallin R120G transgenic mouse (TG-R120G) containing approximately equal amounts of ␣B-crystallin were analyzed on Western blot as in panel a. The level of total ␣B-crystallin was ϳ5 to 7 times higher in the transgenic than in the non-transgenic mouse. the localization of ␣B-crystallin in nuclear speckles (23). This raises the question whether ␣B-crystallin R120G can actually enter the nucleus. Inhibition of nuclear export in cells transfected with ␣B-crystallin R120G only resulted in a weak increase in nuclear staining (Fig. 2a,  panels A and B), suggesting an impaired nuclear import as compared with wild type ␣B-crystallin (Fig. 1a, panels A and B). To determine whether the impaired import was due to reduced phosphorylation, we compared the phosphorylation of transfected wild type and R120G ␣B-crystallin by immunoblotting of HeLa cell lysates with antibodies specific for S19p, S45p, and S59p (Fig. 2b). Whereas wild type ␣B-crystallin showed no detectable phosphorylation at any of these sites, ␣B-crystallin R120G was phosphorylated at Ser-45 and Ser-59. Relative to wild type, ␣B-crystallin R120G is actually not hypo-, but hyperphosphorylated.
It is important to know whether hyperphosphorylation of ␣B-crystallin R120G also occurs in a physiologically more relevant situation. We therefore analyzed ␣B-crystallin R120G in transgenic mouse hearts, where it forms dramatic inclusions leading to early death of the transgenic animal (27). Comparison of phosphorylated ␣B-crystallin in hearts of a non-transgenic mouse and a transgenic ␣B-crystallin R120G mouse showed some phosphorylation at Ser-19 and Ser-59 in the nontransgenic heart, but pronounced phosphorylation at all three sites in the transgenic heart (Fig. 2c). Enhanced phosphorylation thus is a prominent and universal feature of ␣B-crystallin R120G.
To localize the phosphorylated ␣B-crystallin in HeLa cells transfected with the R120G mutant, double-staining with anti-␣B-crystallin and anti-S45p or -S59p antibodies was performed (Fig. 2d). Anti-␣Bcrystallin highlighted the cytoplasmic inclusions (Fig. 2d, panels A and D; also seen in Fig. 2a, panels A and B), which are characteristic for ␣B-crystallin R120G (32,33,39). These inclusions were prominently stained by both anti-phospho-antibodies (Fig. 2d, panels B and E). The intense green color of the inclusions in the merged panels (Fig. 2d, panels C and F) indicates that the phosphorylated form of ␣B-crystallin R120G is enriched in the inclusions. The nuclear staining in Fig. 2d, especially clear for S45p (panel B), is largely due to the endogenous ␣B-crystallin, as is obvious from the staining of adjacent untransfected cells (arrow in panel B). Interestingly, the nuclear S45p staining is clearly diminished in the transfected cells (arrowhead in panel B), indicating that endogenous ␣B-crystallin is retained in the cytoplasm by binding to the excess of structurally perturbed ␣B-crystallin R120G.
Pseudophosphorylation Enhances Inclusion Body Formation, Detergent-insolubility, and Complex Size of ␣B-crystallin R120G-The phosphorylation-dependent localization of ␣B-crystallin R120G was further analyzed by transfection of its pseudophosphorylated and non-phosphorylatable mutants (Fig. 3a). In contrast to pseudophosphorylated wild type ␣B-crystallin, the pseudophosphorylated form of ␣B-crystallin R120G was only weakly detected in nuclear speckles (Fig. 3a, panels C and F), again showing that the R120G mutation impairs nuclear entry. Pseudophosphorylation strongly enhances the tendency of ␣B-crystallin R120G to form cytoplasmic aggregates (cf. Fig. 3a, panels D and F). Most importantly, in cells expressing the non-phosphorylatable ␣B-crystallin R120G no such cytoplasmic aggregates could be observed FIGURE 3. Cytoplasmic inclusions and insolubility of ␣B-crystallin R120G are phosphorylation-dependent. a, HeLa cells were transfected with wild type ␣B-crystallin and its non-phosphorylatable and pseudophosphorylated mutants (A-C) and ␣B-crystallin R120G and corresponding mutants (D-F). After 2 days the cells were fixed and permeabilized, and ␣B-crystallin was visualized with the monoclonal anti-␣B-crystallin antibody. b, HeLa cells were transfected with wild type ␣B-crystallin and the indicated mutants. A fixed number of transfected cells were separated into detergent-insoluble (ins) and detergent-soluble (sol) fractions, and analyzed by Western blotting using the monoclonal anti-␣B-crystallin antibody. c, HeLa cells transfected with expression constructs coding for wild type, R120G/S19A/S45A/S59A and R120G/S19D/S45D/S59D ␣B-crystallin were fractionated by centrifugation on a glycerol gradient (5-40%) and analyzed by Western blot using the monoclonal antibody against ␣B-crystallin. Arrowheads indicate the molecular masses (kilodaltons) of marker proteins. NOVEMBER 4, 2005 • VOLUME 280 • NUMBER 44 (Fig. 3a, panels E). These findings clearly demonstrate that phosphorylation is essential for the generation of the cytoplasmic inclusions.

Phosphorylation-dependent Nuclear Import of ␣B-crystallin
Western blot analysis of detergent-insoluble and -soluble fractions of transfected HeLa cells (Fig. 3b) showed that ␣B-crystallin R120G is partially in the insoluble fraction, whereas the pseudophosphorylated and non-phosphorylatable mutants are largely insoluble and completely soluble, respectively, in perfect agreement with their different tendencies to form inclusions. In the case of wild type ␣B-crystallin, only the pseudophosphorylated form is partially insoluble, reflecting the pronounced nuclear speckle formation seen in Fig. 3a, panel C. Also the complex size of pseudophosphorylated ␣B-crystallin R120G in the HeLa cell extract, as estimated by gradient centrifugation, is much larger than that of its non-phosphorylatable homologue, which is ϳ650 kDa, similar to that of wild type ␣B-crystallin (Fig. 3c). The fact that nonphosphorylatable ␣B-crystallin R120G forms no cytoplasmic inclusions, and has similar solubility and complex size as wild type ␣B-crystallin, indicates that the intracellular aggregation tendency of ␣B-crystallin R120G is not directly related to the R120G mutation but caused by excessive phosphorylation.
␣B-crystallin Phosphorylated at Ser-45 and ␣B-crystallin R120G Localize in Mitotic Interchromatin Granules-The finding that the mutant R120G displays enhanced phosphorylation, thereby hampering nuclear entry and speckle localization of ␣B-crystallin, makes it of interest to compare the localization of wild type and R120G ␣B-crystallin during mitosis, during which phosphorylation at Ser-19 and Ser-45 is increased (15). The subcellular localizations of aB-crystallin during metaphase in HeLa cells transfected with wild type and R120G ␣B-crystallin and their non-phosphorylatable and pseudophosphorylated mutants are shown in Fig. 4a. Pseudophosphorylated wild type and R120G ␣B-crystallin (panels G and P), but also ␣B-crystallin R120G itself (panel J), displayed a pronounced concentration in mitotic structures that are reminiscent of mitotic interchromatin granule clusters (MIGs). MIGs are formed by components of the nuclear speckles, which redistribute during the cell cycle (25,40). Double immunofluorescence staining showed that in these mitotic structures ␣B-crystallin co-localized with Sm protein (Fig. 4a, panels G-I, J-L, and P-R), which is a characteristic component of MIGs (40). Sm-stained MIGs were also observed in cells expressing wild type or non-phosphorylatable ␣B-crystallin, but not co-localizing with ␣B-crystallin (Fig. 4a, panels A-F). Interestingly, MIGs appeared less numerous and larger upon overexpression of pseudophosphorylated ␣B-crystallin (Fig. 4a, panels G-I), but also in the case of ␣B-crystallin R120G (Fig. 4a, panels J-L) and its pseudophosphorylated form (Fig. 4a, panels P-R). The fact that both pseudophosphorylation and the mutation R120G, now known to be hyperphosphorylated, interfere with proper MIG formation suggests that the normal interaction of ␣B-crystallin with MIG components is phosphorylation-dependent.
The possible phosphorylation-dependent localization of ␣B-crystallin in MIGs was assessed by localizing endogenous phosphorylated ␣B-crystallin in untransfected mitotic cells by indirect immunofluorescence, using the highly sensitive anti-S45p and -S59p antibodies (Fig. 4b). Co-staining with anti-SC35, another marker of MIGs (41), clearly revealed co-localization of ␣B-crystallin S45p in MIGs in metaphase cells (Fig. 4b, panels A-C). In contrast, ␣B-crystallin S59p did not co-localize with MIGs, but appeared to be located in the centrosome, as observed before (17) (Fig. 4b, panels D-F). It thus appears that phosphorylation at Ser-45 is an essential parameter for the localization of ␣B-crystallin during the cell cycle: in nuclear speckles during interphase (24) (Fig. 1b, panels A) and in MIGs during mitosis.
The snRNP-specific Import Factor SMN Is Involved in the Nuclear Import of Phosphorylated ␣B-crystallin-The phosphorylationdependent nuclear entry and cell cycling of ␣B-crystallin raises the question of which interacting proteins might be involved. While analyzing the co-localization of ␣B-crystallin and Sm proteins in mitotic cells (Fig. 4a) we noticed in the corresponding interphase cells that nonphosphorylatable ␣B-crystallin strongly increased the cytoplasmic  Sm (B, E, and H), and examined at interphase. b, co-immunoprecipitation of ␣B-crystallin with SMN from lysates of HeLa cells transfected with the empty pIRES vector, non-phosphorylatable, and pseudophosphorylated ␣B-crystallin. SMN was immunoprecipitated using a monoclonal anti-SMN antibody coupled to protein G-agarose beads. Immunoprecipitates were analyzed on Western blots by staining with a polyclonal antibody against ␣B-crystallin. c, ␣B-crystallin was immunoprecipitated using a monoclonal anti-␣B-crystallin antibody coupled to protein G-agarose beads. Immunoprecipitates were analyzed on Western blots by staining with a polyclonal antibody against SMN. d, co-immunoprecipitation of ␣B-crystallin R120G with SMN. HeLa cells were transfected with the empty vector pIRES, wild type ␣B-crystallin, ␣B-crystallin R120G, and its non-phosphorylatable form. Co-immunoprecipitation with SMN and staining were as for panel b. e, control co-immunoprecipitation of ␣B-crystallin R120G with SC35. HeLa cells were transfected with the empty vector pIRES or ␣B-crystallin R120G. SC35 was immunoprecipitated using a monoclonal anti-SC35 antibody coupled to protein G-agarose beads. Immunoprecipitates were analyzed on Western blots by staining with a polyclonal antibody against ␣B-crystallin and a monoclonal antibody against SC35. f, untransfected HeLa cells were stained with polyclonal anti-␣B-crystallin S45p (A) and S59p (D), recognizing endogenous phosphorylated ␣B-crystallin, and co-stained with anti-SMN (B and E). Arrows indicate co-localization, as also shown in the merged pictures (C and F). Cells were examined at interphase. g, HeLa cells were transfected with ␣B-crystallin R120G. After 2 days the cells were fixed, permeabilized, and stained with the polyclonal anti-␣Bcrystallin S59p (A and D), and co-stained with anti-SMN or anti-SC35 (B and E). The merged image shows the extent of co-localization (C and F).
Phosphorylation-dependent Nuclear Import of ␣B-crystallin NOVEMBER 4, 2005 • VOLUME 280 • NUMBER 44 staining of Sm proteins (Fig. 5a, panels G-I). Overexpression of wild type ␣B-crystallin led to a lesser cytoplasmic accumulation of Sm (Fig.  5a, panels D-F; compare untransfected with transfected cells in panel E), whereas Sm staining was exclusively nuclear in cells transfected with an empty vector (Fig. 5a, panels A-C).
These observations suggest that the nuclear import of Sm proteins is affected by the presence of especially the non-phosphorylatable ␣B-crystallin. Because this mutant cannot be imported into the nucleus (Fig. 1a, panels C and D), it might have a dominant negative effect on Sm import by binding to nuclear import factors, which are common for both proteins. So ␣B-crystallin might partially use a similar import route as Sm proteins. Sm proteins are imported into the nucleus as almost fully assembled snRNP complexes (42). An essential factor for snRNP import is the survival motor neuron (SMN) protein (36). Because ␣B-crystallin does not detectably interact with Sm proteins (data not shown), we wondered whether ␣B-crystallin is able to interact with the SMN protein, possibly in a phosphorylation-dependent manner. As shown in Fig. 5 (b and c), this is indeed the case; pseudophosphorylated ␣B-crystallin could readily be co-immunoprecipitated with SMN, in contrast to nonphosphorylatable ␣B-crystallin, and SMN could specifically be coimmunoprecipitated with pseudophosphorylated ␣B-crystallin. If phosphorylation of ␣B-crystallin is important for interaction with SMN, the hyperphosphorylated ␣B-crystallin R120G might be expected to interact much stronger than wild type ␣B-crystallin. This assumption is again fully supported by the co-immunoprecipitation results in Fig. 5d, which also show that non-phosphorylatable ␣B-crystallin R120G does not interact with SMN, as should be expected. To show that soluble ␣B-crystallin R120G does not bind non-specifically to SMN we also performed a co-immunoprecipitation with the splicing factor SC35. As can be seen in Fig. 5e, SC35 could be precipitated, whereas ␣B-crystallin R120G was not detectable.
The phosphorylation-dependent interaction of ␣B-crystallin and SMN is evident from immunocytochemical analyses, too. We co-stained non-transfected cells for endogenous phosphorylated ␣B-crystallin and SMN (Fig. 5f). Part of the S45p and S59p staining perfectly co-localized with conspicuous SMN-positive nuclear bodies (arrows in Fig. 5f). These nuclear structures enriched with SMN, called Gems and Cajal bodies (43), have been reported for HeLa cells (44). Because ␣B-crystallin R120G strongly interacts with SMN (Fig. 5d), it is no surprise that SMN co-localized with the phosphorylated cytoplasmic inclusions in cells transfected with ␣B-crystallin R120G (Fig. 5g, panels  A-C). The SC35 control antibody does not stain cytoplasmic aggregates (Fig. 5g, panels D--F), indicating that SMN localization of the cytoplasmic inclusions is specific.

DISCUSSION
Phosphorylation of ␣B-crystallin occurs differentially at serines 19, 45, and 59, depending on cell cycle and cellular status (14,15) and has an important effect on the functioning of the protein in apoptosis (9). Here we show that the phosphorylation of ␣B-crystallin affects its subcellular localization. Both S45p and S59p are associated with SMN in nuclear structures called Gems and Cajal bodies (Fig. 5f), whereas S45p also specifically occurs in nuclear speckles (Fig. 1b, panel A) and in MIGs (Fig. 4b), their analogous mitotic structures. Gems and Cajal bodies have related functions and play a role in snRNP biogenesis and their trafficking in the nucleus (45), while nuclear speckles have an important function as storage sites for pre-mRNA splicing factors (25,46). Our findings imply completely novel roles for ␣B-crystallin, but why its dif-ferentially phosphorylated forms localize with these specific structures is not clear at the moment.
In interphase cells ␣B-crystallin is imported into the nucleus in a multistep process (see Fig. 6). We demonstrate here for the first time that this import is dependent on the phosphorylation of Ser-59, whereas phosphorylation at Ser-45 is important for nuclear speckle localization (Fig. 1). However, the nuclear import of the myopathyrelated ␣B-crystallin R120G is hampered despite (or because of) its abundant phosphorylation (Fig. 2). The excessive phosphorylation of ␣B-crystallin R120G results in the formation of cytoplasmic inclusions (Fig. 3) and affects the localization of nuclear speckle components during the cell cycle (Fig. 4a, panels J-K). Our data (Fig. 5) may suggest that the nuclear import of ␣B-crystallin requires a phosphorylation-dependent interaction with the SMN protein, which is disturbed in the case of the R120G mutant, contributing to the formation of the cytoplasmic aggregates. Besides SMN also other factors might be needed or competing for nuclear import of ␣B-crystallin, and their interactions could likewise be hampered by the R120G mutation.
Our findings lead us to propose the following scenario for the role of phosphorylation in the nuclear import and functioning of ␣B-crystallin, and for the way in which this process is frustrated by the R120G mutant, resulting in hyperphosphorylated cytoplasmic inclusions. Although phosphorylation decreases the complex size of ␣B-crystallin from ϳ500 to 100 kDa (16), this still is too large to allow unassisted nuclear entrance. ␣B-crystallin does not contain a classic nuclear localization signal, and we therefore must assume that phosphorylation induces interaction with a specialized nuclear import factor. Because pseudophosphorylated ␣B-crystallin interacts with the SMN complex (cf. Fig. 5, b and c), a complex involved in the nuclear import (36) and biogenesis of snRNPs (35,47), it is likely that ␣B-crystallin uses a similar nuclear import route as snRNPs and is possibly involved in snRNP biogenesis. Such a role for ␣B-crystallin in snRNP import and biogenesis is supported by the finding that overexpression of ␣B-crystallin or its non-phosphorylatable mutant (Fig. 5a) alters the localization of Sm proteins.
Although ␣B-crystallin R120G can readily be phosphorylated and associate with SMN ( Figs. 2 and 5 (d and g)), its import into the nucleus appears to be impaired (Fig. 2a). Impaired nuclear import of ␣B-crystallin R120G has also been shown by van den IJssel et al. (23). Whether the reduced presence of ␣B-crystallin R120G in the nucleus also con-FIGURE 6. Putative model of nuclear import of ␣B-crystallin. Upon phosphorylation at Ser-59, ␣B-crystallin is imported into the interphase nucleus, possibly mediated by phosphorylation-dependent interaction with the SMN-complex. This process is disturbed by the R120G mutation, which causes hyperphosphorylation and subsequent aggregation of ␣B-crystallin. After entry into the nucleus phosphorylation at Ser-45 is required for localization of ␣B-crystallin in nuclear speckles. Release from nuclear speckles could simply be caused by dephosphorylation at Ser-45, but also other processes may play a role.
tributes to the development of the myopathy remains to be established. ␣B-crystallin R120G has an enhanced tendency to associate and aggregate with substrate proteins (31,39), which is likely to interfere with the proper transient interactions required for nuclear import of ␣B-crystallin. Nevertheless, just like wild type ␣B-crystallin, the R120G mutant localizes in MIGs in a phosphorylationdependent manner (Fig. 4a, panels J-R), indicating that it is still capable of having in situ interactions. However, similar to pseudophosphorylated ␣B-crystallin, MIGs containing ␣B-crystallin R120G are fewer and larger than in the case of wild type ␣B-crystallin, suggesting that the normal reversible interactions of aB-crystallin with MIG proteins are disturbed by the R120G or phospho-mimicking mutations. Such a functional disturbance is in agreement with the fact that both ␣B-crystallin R120G (28 -31) and pseudophosphorylated ␣B-crystallin (16,48) have perturbed structures and deviating interactions with substrate proteins. The adverse effects of ␣B-crystallin R120G are, however, much more severe, becoming trapped in cytoplasmic inclusions which in turn hamper nuclear import.
The greatly enhanced phosphorylation of ␣B-crystallin R120G is an important novel observation and could relate to its increased tendency to aggregate. The abnormal protein folding within aggregates might inflict a stress situation, resulting in the activation of kinases able to phosphorylate ␣B-crystallin, such as p44/42 MAPK and MAPKAP kinase-2 (15), whereas hyperphosphorylation may in turn enhance aggregation. However, the excessive phosphorylation might also be a consequence of the impaired nuclear import of ␣B-crystallin R120G, preventing contact with any nuclear-localized phosphatases. The fact that in interphase cells ␣B-crystallin phosphorylated at Ser-45 could only be detected in the nucleus and not in the cytoplasm (cf. Fig. 1b, panels A and B) indeed suggests that Ser-45 becomes dephosphorylated in the nucleus before its export to the cytoplasm. Enhanced phosphorylation and impairment of nuclear entry may thus be mutually reinforcing processes.
It now becomes clear that phosphorylation is not only important for specific cellular functions of ␣B-crystallin, but also plays a major role in the pathogenesis of the desmin-related myopathy caused by the mutation R120G (26,49), also referred to as ␣B-crystallinopathy (50). In forms of desmin-related myopathies caused by desmin mutations, the desmin is abnormally phosphorylated. Phosphorylated desmin represents the non-filamentous form, and abnormal phosphorylation may compromise desmin filament formation (51,52). It can be envisaged that the affinity of ␣B-crystallin for desmin is regulated by phosphorylation of both proteins. The in vitro affinity of ␣B-crystallin R120G for desmin is already high (30,39), and in combination with hyperphosphorylation dissociation of bound desmin might be strongly reduced, thereby stimulating aggregate formation. Similarly, in brains of patients with Alzheimer disease phosphorylated ␣B-crystallin is also accumulated in the insoluble fraction (18), and ␣B-crystallin is prominently associated with ubiquitinated taupositive inclusion bodies (53). Hyperphosphorylation thus appears as an intriguing recurrent theme, and possibly as an aggravating factor, in protein aggregation disorders in which small heat-shock proteins are involved.