Mimicking phosphorylation of the small heat-shock protein aB-crystallin recruits the F-box protein FBX4 to nuclear SC35 speckles John den Engelsman 1 , Erik J. Bennink 1 , Linda Doerwald 1 , Carla Onnekink 1 , Lisa Wunderink 1 , Usha P. Andley 2 , Kanefusa Kato 3 , Wilfried W. de Jong 1 and Wilbert C. Boelens 1 1 Department of Biochemistry 161, Nijmegen Center for Molecular Life Sciences, University of Nijmegen, the Netherlands; 2 Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, MO, USA; 3 Institute for Developmental Research, Aichi Human Service Center, Kasugai, Aichi, Japan The mammalian small heat shock protein aB-crystallin can be phosphorylated at three different sites, Ser19, Ser45 and Ser59. We compared the intracellular distribution of wild- type, nonphosphorylatable and all possible pseudophos- phorylation mutants of aB-crystallin by immunoblot and immunocytochemical analyses of stable and transiently transfected cells. We observed that pseudophosphorylation at t wo (especially S19D/S45D) or all three (S19D/S45D/ S59D) sites induced the partial translocation of aB-c rystallin from the detergent-soluble to the detergent-insoluble frac- tion. Double immunofluorescence studies showed that the pseudophosphorylation mutants localized in nuclear speck- les containing the splicing factor SC35. The aB-c rystallin mutants in these speckles were resistant to mild detergent treatment,andalsotoDNaseIorRNaseAdigestion, indicating a stable i nteraction with on e or more s peckle proteins, not dependent on intact DNA or RNA. We further found that FBX4, an adaptor protein of the ubiquitin-pro- tein isopeptide ligase SKP1/CUL1/F-box known to interact with pseudophosphorylated aB-crystallin, was also recruited to SC35 speckles when cotransfected with the pseudo- phosphorylation mutants. Because SC35 s peckles also react with an antibody against aB-crystallin endogenously phos- phorylated at Ser45, o ur findings suggest that aB-crystallin has a phosphorylation-dependent role in the ubiquitination of a component of SC35 speckles. Keywords: desmin-related myopathy; phosphorylation; SC35; small heat-shock p rotein; ubiquitin isopeptide ligase. aB-crystallin is a member of the family of small heat-shock proteins [1–3]. In mammals, aB-crystallin is present in m any cell types, but the highest expression is found in e ye lens and muscle cells [4]. It occurs in polydisperse hetero-o ligomeric complexes with masses of up to 800 kDa, which may comprise various other small heat-shock proteins, such as aA-crystallin in the eye lens, and HSP27 and HSP20 in muscle cells [5,6]. Phosphorylation of aB-crystallin mainly occurs at three s erine residues: Ser19, for which the kinase is not known, and Ser45 and Ser59, which can be phosphor- ylated by p44/42 mitogen-activated protein kinase and MAP k inase-activated protein kinase-2, respectively [ 7,8]. The differential phosphorylation of t hese serines s uggests specific functional i mplications for each of t hem [ 9,10]. Under stress conditions a ll three sites become phosphoryl- ated to some extent, but after proteasomal inhibition and i n disused soleus muscle Ser59 is most prominently phosphor- ylated [7,11]. Biochemical and i mmunofluorescence analyses of mitotic cells revealed that phosphorylation a t Ser19 and Ser45, b ut not at Ser59, is increased during the mitotic phase of the cell cycle [8]. Different functions for aB-crystallin have been described. The protein shows in vitro chape rone-like activity, which i s reduced upon phosphorylation [12]. In vivo, aB-crystallin is important for the maintenance and control of the cytoske- leton [13,14]. It can interact in a phosphorylation-independ- ent manner with type III intermediate filaments, in this way modulating the assembly of these filaments [15], and probably protects the cytoskeleton during stress [16,17]. aB-crystallin is able to confer resistance to differen t kinds of stress, as well as to apoptosis [18]. It inhibits apoptosis by preventing the a ctivation of procaspase 3, in w hich proce ss phosphorylation of Ser59 is essential [19–21]. Ample evidence indicates the involvement of aB-crystallin in the ubiquitin proteasome system [17,22–25], and in the aggre- somal response to misfolded proteins in degenerative neuro- and myopathies [26–33]. Recently, we reported that aB-crystallin with mimicked phosphorylation at two or three serines (S19D/S45D and S19D/S45D/S59D), as well as aB-crystallin R120G, a mutant found to be causative for a desmin-related myo- pathy [34], interact with the F-box protein FBX4 [25]. FBX4 is an adaptor molecule of the ubiquitin-protein isopeptide ligase SKP1/CUL1/F-box (SCF). The mutant aB-crystal- lins translocate FBX4 t o the de tergent-insoluble fraction and promote the ubiquitination of an as yet uniden tified Correspondence to W. C. Boelens, Department of Biochemistry 161, NCMLS, University of Nijmegen, PO B ox 9101, 6500 HB Nijmegen, the Netherlands. Fax: +31 24 3540525, Tel.: +31 24 3616753, E-mail: W.Boelens@ncmls.kun.nl Abbreviation: SCF, SKP1/CUL1/F-box; FBS, fetal bovine serum; GFP, green fluorescent protein. (Received 19 January 2004, revised 18 August 2004, accepted 6 September 2004) Eur. J. Biochem. 271, 4195–4203 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04359.x protein. This suggests that during this process the aB- crystallin mutants interact with a detergent-insoluble sub- cellular structure [25]. T o study this phenomenon in more detail, we now determined the detergent-insolubility and cellular localization of a series of aB-c rystallin mutants containing all possible combinations of mimicked phos- phoserines. W e found that the increased detergent- insolubilization of pseudophosphorylated aB-crystallin is associated with its localization at SC35 speckles, a nuclear compartment involved in storage and r ecycling of splicing factors. Additionally, w e show that aB-crystallin S19D/ S45D and S19D/S45D/S59D recruit FBX4 to the SC35 speckles. The fact that SC35 speckles also contain aB- crystallin endogenously phosphorylated at Ser45 argues for the physiological relevance of our observations. Materials and methods Cell culture, plasmids and transfections HeLa cells were grown a t 3 7 °C i n D ulbecco’s modified Eagle’s medium (DMEM; Invitrogen, San Diego, CA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; PAA laboratories, Linz, Austria), 100 UÆmL )1 peni- cillin and 200 lgÆmL )1 streptomycin, in the presence of 5% (v/v) CO 2 . DNA fragments encoding the sequence of human aB-c rystallin and its mutants w ere cloned in the eukaryotic expression vector pIRES (Clontech, P alo Alto, CA, USA). FBX4 was cloned in the pGEX (Amersham B iosciences, Uppsala, Sweden), pIRES and pEGFP-C1 vector (Clon- tech). More details about cloning and mutagenesis can be foundindenEngelsmanet al. [25]. Transfections of plasmids into HeLa cells were performed by lipofection using the FuGENE TM 6 system (Roche Molecular Bio- chemicals, Basel, Switzerland), as described by the manu- facturer. To obtain stable cell lines, T -Rex TM HeLa cells expressing the Tet repressor (Invitrogen) were transfected with pcDNA4/TO (Invitrogen) containing the coding sequences for w ild type aB-crystallin, aB-crystallin S19D/S45D/S59D or aB-crystallin S19A/S45A/S59A using the F uGENE TM 6 system. As a vector control, T-Rex TM HeLa cells were transfected with p cDNA4/TO without insert. The cells were grown at 37 °C in Minimum Essential Medium E agle (Bio Whittaker Europe, Verviers, Belgium) supplemented with 10% (v/v) FBS, 100 UÆmL )1 penicillin, 200 lgÆmL )1 strep- tomycin, a nd 5 lgÆmL )1 blasticidine (ICN Biomedicals Inc., Irvine, CA, USA) in the presence of 5% (v/v) CO 2 .Stable transfectants were selected by adding 200 lgÆmL )1 zeocin (Invitrogen) to the culture medium. Stable cell lines were grownwith1lgÆmL )1 doxycyclin for 3 days to induce overexpression. Overexpression of the different aB-crystal- lin mutants w as assessed by indirect immunofluorescence and by immunoblotting, as described below. Immunocytochemistry HeLa cells were se eded on co verslips (18 · 18 mm 2 ) one day prior to transfection. Two days after transfection cells were either fixed in 3% (v/v) paraformaldehyde f or 15 min and permeabilized for 1 0 min in 0.2% (v/v) Triton i n NaCl/ P i or first permeabilized in 0.2% (v/v) Triton in NaCl/P i for 1 min and then fixed i n 3 % (v/v) paraformaldehyde for 10 min. For DNase I (Roche) and RNase A (Roche) treatment, T-Rex TM HeLa cells expressing aB-crystallin S19D/S45D/S59D were fixed in methanol for 2 min at 20 °CandtreatedwithDNaseI(400UÆmL )1 )orRNaseA (1 mgÆmL )1 ) for 1 h at 37 °C. A m onoclonal antibody to aB-crystallin (RIKEN Cell Bank, Shanghai, China) was primarily used in these studies. For immunocytochemical analysis, the antibody was added undiluted to the fixed cells. In addition another monoclonal antibody to aB-crystallin (2D2B6) [35], and a poly- clonal p eptide antibody to the N -terminal 1 0 residues of aB-c rystallin (NCL-ABCrys, Novoc astra, Newcastle upon Tyne, UK) were also u sed (undiluted and at 1 : 50 dilution, respectively), and gave the same results as the RIKEN antibody. W e f urther tested a polyclonal antiserum (K79) to the C-terminal 13 residues of aB-crystallin, as has been widely used in other studies. B ecause this a ntiserum was earlier s uggested to give nonspecific staining of nuclear bodies [36], we used primary cultures of lens epithelial cells derived f rom wild type and aB–/– mouse lenses [ 37] to assess the specificity of the K79 antiserum. Our analysis showed that this antibody diffusely stained the cytoplasm of wild type but not of aB–/– mouse lens epithelial cells. However, this antibody additionally gave a p ronounced staining of nuclear bodies, not only in wild type but also in aB–/– lens epithelial cells (data not shown). We therefore did not use the K79 antibod y further in o ur experiments. A polyclonal antibody against a phosphopeptide corresponding with the Ser45 phosphorylation site of aB-crystallin (S45p) [8] was used at 1 : 40 dilution. Monoclonal antibodies to SC35 (Sigma) were used a t 1 : 20 dilution, and Sm proteins were stained with a human autoimmune serum designated C45 (1 : 2500) [38]. Secondary antibodies [fluorescein isothiocy- anate (FITC)-conjugated swine anti-rabbit IgG, FITC- conjugated rabbit a nti-human IgG, FITC-conjugated r abbit anti-mouse IgG, and tetramethylrhodamine isothiocyanate (TRITC)-conjugated rabbit anti-mouse IgG] were used at a 1 : 20 dilution according to the manufacturer (DAKO Corp., Glostrup, Denmark). Nuclei were stained with YOYO-1 iodide (Molecular Probes, Eugene, OR, USA). Images were obtained by confocal laser scanning micro- scopy (Bio-Rad MRC1024, Hercules, CA, USA). Cell fractioning and immunoblotting HeLa cells were transfected with 1 lg of DNA and harvested after 2 days by trypsinization. Cells were washed once with DMEM containing 10% (v/v) FBS, and twice with phosphate buffered saline. Equal numbers of about 10 6 cells were resuspended in 50 lL i ce-cold lysis buffer [10 m M Tris pH 7.5, 100 m M KCl, 1 m M dithiothreitol, 1 m M EDTA, 5 m M MgCl 2 ,1m M phenylmethanesulfonyl fluor- ide, and 0.5% (v/v) Nonidet P-40] and kept on ice for 15 min. The cell extract was centrifuged for 15 min at 1200 g and 4 °C. The supernatant was supplemented with 50 lLof2· SDS sample buffer [ 2% (v/v) SDS, 0 .125 M Tris/HCl pH 6.8, 20% (v/v) glycerol, 0.02% (v/v) 2- mercaptoethanol, 0.05% (w/v) bromophenol blue] heated for 5 min at 95 °C and used as the detergent-soluble fraction. The remaining pellet was washed once with 5 00 lL 4196 J. den Engelsman et al.(Eur. J. Biochem. 271) Ó FEBS 2004 lysis buffer, resuspended in 50 lL lysis buffer supplemented with 50 lLof2· S DS sample buffer, heated for 5 min at 95 °C and used as the detergent-insoluble fraction. The detergent-soluble and detergent-insoluble fractions were separated by S DS/PAGE a nd subs equently blotted onto nitrocellulose membranes (Schleicher & Schu ¨ ll, Dassel, Germany). The membranes were successively incubated with a monoclonal antibody to aB-crystallin (RIKEN) and a horseradish peroxidase conjugated rabbit anti-mouse secondary antibody (DAKO Corp.) to allow visualization by enhanced chemoluminescence (Pierce Chemical Co., Rockford, IL, USA). Images were collected with the BioDoc-It System (UVP Laboratory Products, Cambridge, UK) and quantification was done using the LABWORKS TM software (UVP Laboratory Products). Nuclei were isolated from T-Rex HeLa cells stably transfected with wild type aB-crystallin and induced for expression for 3 days. Cells were harvested by trypsiniza- tion, washed once with Eagle’s minimal essential medium containing 10% (v/v) FBS, and twice with phosphate buffered saline. The pelleted cells were taken up in 100 lL buffer (10 m M Tris/HCl pH 7.8, 10 m M NaCl, 1m M dithiothreitol, 2 m M MgCl 2 ,1m M phenyl- methanesulfonyl fluoride, supplemented with a protease inhibitor c ocktail f rom R oche) a nd incubated on i ce for 20 min. NP-40 was then added to a final concentration of 1% and incubation on ice continued for another 10 min. The cell suspension was passed five tim es t hrough a 21- gauge needle and the nuclei, free of cytop lasmic capping as judged by light microscopy, were pelleted by centrif- ugation for 5 min at 200 g to separate them from the cytoplasmic fraction. The cytoplasmic fraction was col- lected and acetone precipitated. The remaining nuclei were washed twice with 10 m M Tris/HCl pH 7.4, 5 m M MgCl 2 , supplemented with a protease inhibitor cocktail. All fractions were taken up in 2· SDS sample buffer without 2-mercaptoethanol and bromophenol blue, and protein concentrations were determined with the BCA kit (Bio-Rad). Equal a mounts o f p roteins were analyzed by SDS/PAGE and Western blotting with the monoclonal antibody to aB-crystallin (RIKEN) and the polyclonal antibody to phosphorylated aB-crystallin S45p. Results Detergent-insolubility of pseudophosphorylated aB-crystallin Expression constructs containing the cDNAs of wild type and mutated aB-crystallin were transfected into HeLa cells. After 2 days the cells were harvested and separated into a detergent-soluble and a detergent-insoluble frac- tion. Immunoblotting showed that wild type aB-c rystallin as well as the nonphosphorylatable control aB-crystallin S19A/S45A/S59A were partially found in the detergent- insoluble fraction (Fig. 1A) at levels of 19 ± 4% and 14 ± 4%, respectively (Fig. 1B). Replacement of a single serine by aspartic acid at position 19, 45 or 59 gave a slight but not significant increase in detergent insolubility. Replacing two serines by aspartic acids also gave an increase in detergent insolubility, but only in the case of S19D/S45D (43 ± 3%) was the increase significant. An even more pronounced insolubilization (55 ± 2%) was obtained when all three phosphorylatable serines were replaced by aspartic acids. Mimicking phosphorylation of aB-crystallin reveals a distinct nuclear staining To determine the subcellular localization of aB-crystallin mutants we performed indirect immunofluorescence analy- ses on stably transfected T-Rex TM HeLa cells inducible for aB-crystallin expression (Fig. 2A, panels a–c). Cells induced to express wild type aB-crystallin or the unphosphorylatable aB-crystallin S19A/S45A/S59A showed the expected cyto- plasmic localization, while cells expressing the pseudophos- phorylated aB-crystallin S19D/S45D/S59D additionally displayed localization of aB-crystallin in nuclear bodies. A similar result was obtained with transiently transfected mouse C2 cells, suggesting that this nuclear localization is not cell-specific (data not shown). However, aB-crystallin S19D/S45D/S59D tagged N-terminally with green fluores- cent protein (GFP) did not localize in nuclear bodies (data not shown). This suggests that a free N-terminus is important for nuclear entrance, or that the size of the B A 80 60 40 20 0 Fig. 1. Pseudophosphorylated aB-crystallins are enriched in t he deter- gent-insoluble fraction. (A) H eLa cells were transfected with expression constructs coding for wild type aB-crystallin (WT), pseudophosphor- ylated aB-crystallin mutants containing S to D s ubstitutions at the indicated positions or nonphosphorylatable aB-crystallin S19A/S45A/ S59A. A fixed number of the transfected cells were separated into detergent-soluble (S) and detergen t-insolu ble (I) fractions, and ana- lyzed by Western blo tting using the RIKEN anti-(aB-crystallin) monoclonal antibody. (B) The average level of aB-crystallin in the detergent-insoluble fraction is shown a s a p ercentage of the total aB-crystallin. Values are based on four independent experiments and error bars represent the standard e rror of t he mean (SEM). Asterisks indicate the aB-crystallin mutants that are significantly enriched in the detergent-insoluble fraction compared to wild type aB-crystallin (P <0.005). Ó FEBS 2004 aB-crystallin colocalizes with FBX4 in SC35 speckles (Eur. J. Biochem. 271) 4197 fusion protein or complex becomes too large. The patterns shown in Fig. 2A were obtained with the RIKEN mono- clonal antibody directed against aB-crystallin, but similar cytoplasmic and nuclear staining was observed with the monoclonal anti-(aB-crystallin), 2D2B6, and with a poly- clonal antiserum directed against the N-terminal region of aB-c rystallin. To specifically reveal the localization of detergent-insoluble aB-crystallin, the soluble aB-crystallin was r emoved by treating cells with a detergent solution prior to fixation. Panels d–i in Fig. 2A show that in all cells the cytoplasmic s taining was strongly reduced. Only cells expressing aB-crystallin S 19D/S45D/S59D show the nuclear bodies, indicating that at least part of the detergent- insoluble fraction of the pseudophosphorylated aB-crystal- lin is localized in these structu res. Transiently transfected HeLa cells were used to relate the percentage of cells containing aB-crystallin in nuclear bodies to the number and combinations of Ser to Asp replacements (Fig. 2B). In the case of a single replacement, only S19D and S45D gave an appreciable number of cells with aB-c rystallin in nuclear bodies. In the case of a double replacement all three possible aB-crystallin mutants could B A ad e fi h g b c Fig. 2. Deterge nt-insol uble pseudophosphorylated aB-crystallin localizes in nuclear bodies. (a) T -Rex TM HeLa cell lines stably transfected with aB-crystallin wild type (WT), S19D/S45D/S59D (S TD) or S19A/S45A/S59A (STA) were induced for expression. Part of the cells were fixed and permeabilized (No detergent) while other ce lls were permeabilized prior to fixation (Dete rgent). Localization of aB-crystallin was visualized by indirect immunofluorescence with the RIKEN anti-(aB-crystallin) mAb and TRITC-conjugated se condary antibody ( a–f), and nuclei were stained with YOYO-1 (g–i). (B) Percentage of HeLa cells, transiently transfected with wild type (WT) or mutated aB-crystallin, which exhibit nuclear bodies as ju dged by fluorescence microscopy. Per slide 200 transfected cells were counted at a magnification of 400·. The average of two independent experimentsisshown. 4198 J. den Engelsman et al.(Eur. J. Biochem. 271) Ó FEBS 2004 be detected in nuclear bodies, but the c ombination S19D/ S45D had the strongest effect. The largest number of positive cells was obtained with the S19D/S45D/S59D mutant. These results confirm the correlation between detergent-insolubility and nuclear localization of the pseu- dophosphorylated aB-crystallins (compare Figs 1B and 2B). It may be noted that even in the case of S19D/S45D/ S59D not all nuclei detectably displayed such bodies, as is also the case for this same mutant in the stably transfected cells (Fig. 2 A, panel e). aB-crystallin S19D/S45D colocalizes with SC35 speckles The nucleus contains various types of subnuclear struc- tures, such as nucleoli, SC35 speckles, Cajal bodies and polymorphonuclear leukocyte bodies, each having different nuclear activities [39,40]. Based on the morphological appearance we speculated t hat the nuclear aB-crystallin bodies might b e localized at the SC35 speckles [41]. A double immunofluorescence analysis was therefore per- formed on de tergent-treated HeLa cells transiently trans- fected with aB-crystallin S19D/S45D, using a human autoimmune anti-Sm serum suitable for staining SC35 speckles [41,42] in combination with monoclonal a nti-(aB- crystallin). aB-crystallin S19D/S 45D indeed perfectly colo- calized with the most intensely stained Sm speckles (Fig. 3A, a–c). A s imilar result was obtained with aB- crystallin S19S/S45D/S59D (data not shown). To confirm that the a nti-Sm serum indeed stains S C35 speckles, the colocalization o f t he Sm epitope with the splicing f actor SC35, which i s the antigen b y which these speckles w ere originally characterized [41], is shown using a monoclonal antibody, anti-SC35 (Fig. 3A, d–f). These findings establish that mimicking phosphorylation of aB-crystallin results in its association with S C35 speckles. Localization of aB-crystallin S19D/S45D/S59D in SC35 speckles is resistant to DNase I and RNase A treatment To find out if the association of pseudophosphorylated aB-crystallin with SC35 speckles is dependent on intact DNA or RNA, we subjected T-Rex TM HeLa cells expres- sing the m utant S19D/S 45D/S59D to DNase I or RNase A treatment [41]. The localization of aB-crystallin S19D/ S45D/S59D was visualized by indirect immunofluorescence (Fig. 3B, a and d). The DNase treated cells were costained with YOYO-1 (Fig. 3B, panel b). Hardly any DNA staining was observed after DNase treatment; only the staining of the nucleoli r emained, indicating that most of the DNA was digested. However aB-crystallin could s till be de tected in SC35 speckles (Fig. 3B, a and c). The RNase-treated cells were costained w ith anti-Sm serum, because the localization of Sm proteins at SC35 speckles is more RNA-dependent than the Sm proteins that are diffusely distributed through- out the nucleoplasm. No Sm protein could be detected in theSC35specklesafterRNasetreatment(Fig.3B,eandf), as shown before [41], indicating that most of the RNA was digested, but aB-crystallin was still present in SC35 speckles (Fig. 3B, d and f). I t thus appears that the localization of pseudophosphorylated aB-crystallin in nuclear speckles is not dependent on intact DNA or RNA. aB-crystallin S19D/S45D recruits FBX4 to the SC35 speckles We have shown previously that the aB-crystallin mutants S19D/S45D and S19D/S45D/S59D interact with the F-box protein FBX4 [25]. These same mutants also associate most strongly with SC35 sp eckles (Fig. 2B). FBX4 normally is a detergent-soluble p rotein, but upon coexpression with aB-crystallin S19D/S45D a fraction of FBX4 becomes detergent-insoluble [25]. This suggests that FBX4 may well colocalize with aB-crystallin S19D/S45D at the SC35 speckles. We investigated this possibility using a C-termin- ally GFP-tagged FBX4 expression construct. When this construct alone was overexpressed in H eLa cells, fluores- cence was found in cytoplasm and nucleus, but excluding the nucleoli (data not shown, and [43]). Upon pretreatment with detergent before fi xation, any cells transfected with FBX4–GFP could no longer be detected, although we obtained a transfection efficiency of 40–45%. This indicates that most of the FBX4–GFP, similar to untagged FBX4, is detergent-soluble (data not shown and [25]). However, when FBX4–GFP was coexpressed with aB-crystallin S19D/S45D, a colocalization of detergent-insoluble FBX4–GFP with aB-crystallin S19D/S45D at SC35 speck- les could be observed (Fig. 3C, a–c). FBX4–GFP was not observed in s peckles when coexpressed with aB-crystallin wild type or S19A/S45A/S59A (data not shown). These results indicate that aB-crystallin S19D/S45D is able to recruit FBX4–GFP to SC35 speckles. SC35 speckles contain aB-crystallin endogenously phosphorylated at Ser45 To be physiologically relevant, our results obtained w ith the phosphomimicking aB-crystallin mutants would suggest that endogenously phosphorylated aB-crystallin should also be present in SC35 speckles. However, antibodies against aB-crystallin did not stain any speckles in cells expressing wild type aB-crystallin (Fig. 2A, a and d). In contrast, an antibody that specifically recognizes aB-crystallin phos- phorylated at Ser45 [8] clearly revealed speckles in the diffusely stained nucleoplasm (Fig. 4A, panel a), colocaliz- ing with the Sm staining of SC35 speckles (panel b). This phosphospecific antibody, S45p, thus is clearly much more sensitive in detecting its antigen than the anti-(aB-crystallin) sera. While nuclear speckles staining for aB-crystallin were not observed in every cell expressing the phosphomimicking mutants (Fig. 2A, panel e; Fig. 2B), the phosphospecific antibody stained speckles in all cells, i ndicating that the presence of phosphorylated aB-crystallin in nuclear speckles is a constitutive feature. To confirm that the speckle staining is indeed due to the presence of phosphorylated aB-crystallin, we performed Western blotting with the anti- (aB-crystallin) and anti-S45p IgGs on the isolated nuclei of these cells. It appears that only a t iny p roportion of the total aB-crystallin is present in t he nuclear fraction (Fig. 4B, panel a), while aB-crystallin phosphorylated at Ser45 is exclusively found in this fraction (panel b). With respect to their localization in SC35 speckles, the phosphomimicking aB-crystallin mutants t hus resemble the endogenously Ser45-phosphorylated aB-crystallin. Ó FEBS 2004 aB-crystallin colocalizes with FBX4 in SC35 speckles (Eur. J. Biochem. 271) 4199 A B C a d a def bc abc ef bc Fig. 3. Pseudophosphorylated aB-crystallin localizes in SC35 speckles, independent of intact DNA and RNA, and recruits FBX4 to these s peckles. (A) HeLa cells, transiently transfected w ith aB-crystallin S19D/S45D (a–c) or nontransfected (d–f), were first permeabilized and subsequently fixed. Cells were stained with t he RIKEN mAb anti-(aB-crystallin) (a) or the monoclonal antibody to S C35 (d) and costained with anti-Sm (b and e). The yellow pseudocolour shows the extent of colocalization between the two antigens (c and f). Primary a ntibodies to aB-crystallin and S C35 were detected with TRITC-conjugated secondary antibo dies, whereas Sm was detected by FITC-conjugated secondary antibodies. (B) T-Rex TM HeLa cells expressing aB-crystallin S19D/S45D/S59D were fixed in methanol, without prior permeabilization, and treated with DNase I (a–c) or RNase A (d–f). Cells were costained with the RIKE N mAb anti-(aB-crystallin) ( a and d) and YOYO-1 ( b) or anti-Sm (e). P anels c and f show the o verlays. (C) HeLa cells were cotransfected with expression constructs encoding aB-crystallin S19D/S45D and C-terminally GFP-tagged FBX4. Before fixation cells were permeabilized to remove detergent-soluble proteins. aB-crystallin was detected by indirect immunofluorescence using the RIKEN mAb anti-(aB-crystallin) (a), and FBX4 was d etected by GFP fluorescence (b). The merge picture (c) shows their colocalization. 4200 J. den Engelsman et al.(Eur. J. Biochem. 271) Ó FEBS 2004 Discussion We report here that mimicking th e phosphorylation of aB-crystallin at two of its three phosphorylatable serines, especially at Ser19 and Ser45, or at all three serines, results in colocalization with SC35 speckles. The pseudophosphor- ylated aB-crystallin that localizes with these speckles is detergent-insoluble, and i ts localization i s resistant to DNase I and RNase A, indicating that these mutants form a stable interaction with one or more speckle-associated proteins. SC35 speckles are interchromatin granule clusters that contain snRNPs and other splicing components, and may f unction as sites for storage or recycling o f splicing factors [ 41]. D uring mitosis SC35 speckles d issociate, resulting mainly in a diffuse distribution of SC35 compo- nents throughout the cell. Using an antibody that specific- ally recognizes aB-crystallin phosphorylated at Ser45, Kato et al. [8] observed a similar diffuse staining pattern in mitotic glioma cells. Based on our finding that transfected pseudophosphorylated aB-crystallin localizes in nuclear speckles i n interphase cells, one would expect that this phospho-specific antibody S45p should also stain nuclear speckles containing endogenously phosphorylated aB-crys- tallin. As shown in Fig. 4 A, this is indeed the case. The next question is whether the observed recruitment of FBX4 to SC35 s peckles by p seudophosphorylated aB-crystallin (Fig. 3C) reflects a genuine property of endogenously phosphorylated aB-crystallin, too. We could not observe colocalization of endogenous FBX4 or trans- fected FBX4–GFP with nuclear speckles in any cells other than t hose coexpressing FBX4–GFP and the phosphomimicking aB-crystallins. A plausible explanation for this difference between transfected pseudophosphoryl- ated and endogenously phosphorylated aB-crystallin is that the overexpressed Ser-Asp mutants are likely to be trapped together with FBX 4–GFP in sta ble interactions within the speckles, while the same interactions are transient for endogenously and reversibly phosphorylated aB-crystallin. The transient presence of FBX4 in SC35 speckles might be too low for detection. The actual function of endogenously phosphorylated aB-crystallin in relation to FBX4 and speckle proteins need not be lo calized in the SC35 speckles themselves. aB-crystallin is a chaperone-like protein, and it is possible that the function of the putative i nteraction with one or more speckle-specific proteins simply is to stabilize them during mitosis, when SC35 speckles are dissociated. Such a function might be related to the observation that in heat- stressed H9C2 cells Hsp25 colocalizes with heat labile proteins in nuclear granules [44]. However, this does not explain the involvement of FBX4. Because pseudophos- phorylation of aB-crystallin also recruits FBX4 to t he SC35 speckles (Fig. 3C), it might be more likely that the combined association of phosphorylated aB-crystallin and FBX4 with a speckle p rotein results in ubiquitination of the latter during mitosis, targeting it for degradation. We have indeed previously demonstrated that pseudophosphorylated aB-crystallin together with FBX4 promotes the ubiquitina- tion of one or a few specific proteins [25]. Unfortunately, the identity of this ubiquitinated protein remains to be estab- lished. However, a role for phosphorylated aB-crystallin in degradation of a speckle protein would be i n a greement with the increasing evidence for an important function of aB-crystallin in the ubiquitin proteasome system [17,22–25]. Such a function is also apparent from the desmin-related myopathy mutant aB-crystallin R120G [32]. Characteristic for this myopathy i s the presence of cytoplasmic bodies containing desmin and aB-crystallin [33,34]. Two o ther papers have recently reported the localization of endogenous aB-crystallin in SC35 speckles [45,46]. In contrast to our findings, this localization was found to be phosphorylation-independent. M oreover, speckles were only observed with antisera raised against the C-terminal residues of aB-crystallin [45,46], and with the monoclonal antiserum 2D2B6 [45]. With an antiserum against the C-terminal sequence o f aB-crystallin (K79, see Materials and methods) we also found nuclear speckles in all cell lines studied, transfected or not, but the 2D2B6 monoclonal o nly stained speckles in cells transfected with pseudophosphory- lation mutants of aB-crystallin (data not shown). To t he best of our knowledge, aB-crystallin in nuclear speckles has previously only been reported when using antisera against the C-terminal sequence [36,47,48]. It has been claimed that this speckle staining is nonspecific [36], as has been confirmed by t he prominent staining of nuclear speckles by K79 in lens epithelial c ells of aB-crystallin knock-out mice (see Materials and methods). Because of this apparent cross-reactivity, nuclear speckles visualized with antibodies A ab B ab Fig. 4. aB-crystallin endogenously phosphorylated at Ser45 colocalizes with SC35 speckles. (A) T-Rex TM HeLa cells s tably t ransfected with aB-crystallin wild type (WT) were induced for expression, and after 3 days fixed and permeabilized. Cells wer e sta ined with the polyclonal anti-(aB-crystallin) S45p (a) and cost ained with anti-Sm (b). The S45p antibody was used because phosphorylation at Ser45 is the most rep- resentative for the thre e possible p seudoph osphorylation sites in aB-crystallin (Figs 1 B and 2B). The S45p antibody was detected with TRITC-conjugated se condary antibodies, whereas Sm was detected by FITC-conjugated secondary ant ibod ies. Arrows indicate some of the speckles that contain both aB-crystallin S45p and Sm. (B) T-Rex TM HeLa cells stably transfected with aB-crystallin wild type (WT) were induced f or expression and harvested af ter 3 days. Part of the cells was usedastotalcelllysate(T),whilethe other part was fractionated into a soluble fraction (S) and a nuclear fraction (N). Fractions were analyzed by Western blotting using the RIKEN mAb anti-(aB-crystallin) (a) and the polyclonal anti-(aB-crystallin) S45p (b). Ó FEBS 2004 aB-crystallin colocalizes with FBX4 in SC35 speckles (Eur. J. Biochem. 271) 4201 against the C-terminal sequence of aB-crystallin should be interpreted with caution. This means t hat localization of aB-c rystallin in SC35 speckles has only been demonstrated unambiguously in the c ase of t he pseudophosphorylated mutants, stained with the anti-(aB-crystallin) mAbs, and in the case of endogenously phosphorylated aB-crystallin, stained with the antiserum against phosphorylated Ser45. In summary, these results indicate that phosphorylation of aB-crystallin induces its association with a SC35 speckle- specific protein. 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Mimicking phosphorylation of the small heat-shock protein aB-crystallin recruits the F-box protein FBX4 to nuclear SC35 speckles John den. interact with the F-box protein FBX4 [25]. FBX4 is an adaptor molecule of the ubiquitin -protein isopeptide ligase SKP1/CUL1 /F-box (SCF). The mutant aB-crystal- lins