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 t
Trang 1Mimicking phosphorylation of the small heat-shock protein
aB-crystallin recruits the F-box protein FBX4 to nuclear SC35 speckles
John den Engelsman1, Erik J Bennink1, Linda Doerwald1, Carla Onnekink1, Lisa Wunderink1,
Usha P Andley2, Kanefusa Kato3, Wilfried W de Jong1and Wilbert C Boelens1
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 two (especially S19D/S45D) or all three (S19D/S45D/
S59D) sites induced the partial translocation of aB-crystallin
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-crystallin
mutants in these speckles were resistant to mild detergent
treatment, and also to DNase I or RNase A digestion,
indicating a stable interaction with one or more speckle 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 speckles also react with an antibody against aB-crystallin endogenously phos-phorylated at Ser45, our 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 protein; 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 many
cell types, but the highest expression is found in eye lens and
muscle cells [4] It occurs in polydisperse hetero-oligomeric
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 serine 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 kinase-activated protein kinase-2, respectively [7,8]
The differential phosphorylation of these serines suggests
specific functional implications for each of them [9,10]
Under stress conditions all three sites become
phosphoryl-ated to some extent, but after proteasomal inhibition and in
disused soleus muscle Ser59 is most prominently
phosphor-ylated [7,11] Biochemical and immunofluorescence analyses
of mitotic cells revealed that phosphorylation at Ser19 and Ser45, but 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 chaperone-like activity, which is 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 filamphosphorylation-independ-ents, 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 different kinds of stress, as well as to apoptosis [18] It inhibits apoptosis by preventing the activation of procaspase 3, in which process 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 to the detergent-insoluble fraction and promote the ubiquitination of an as yet unidentified
Correspondence to W C Boelens, Department of Biochemistry 161,
NCMLS, University of Nijmegen, PO Box 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)
Trang 2protein This suggests that during this process the
aB-crystallin mutants interact with a detergent-insoluble
sub-cellular structure [25] To study this phenomenon in more
detail, we now determined the detergent-insolubility and
cellular localization of a series of aB-crystallin mutants
containing all possible combinations of mimicked
phos-phoserines We 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 recycling of splicing
factors Additionally, we 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 at 37C in Dulbecco’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)1streptomycin, in the presence of 5%
(v/v) CO2
DNA fragments encoding the sequence of human
aB-crystallin and its mutants were cloned in the eukaryotic
expression vector pIRES (Clontech, Palo Alto, CA, USA)
FBX4 was cloned in the pGEX (Amersham Biosciences,
Uppsala, Sweden), pIRES and pEGFP-C1 vector
(Clon-tech) More details about cloning and mutagenesis can be
found in den Engelsman et al [25] Transfections of
plasmids into HeLa cells were performed by lipofection
using the FuGENETM 6 system (Roche Molecular
Bio-chemicals, Basel, Switzerland), as described by the
manu-facturer
To obtain stable cell lines, T-RexTMHeLa cells expressing
the Tet repressor (Invitrogen) were transfected with
pcDNA4/TO (Invitrogen) containing the coding sequences
for wild type aB-crystallin, aB-crystallin S19D/S45D/S59D
or aB-crystallin S19A/S45A/S59A using the FuGENETM
6 system As a vector control, T-RexTMHeLa cells were
transfected with pcDNA4/TO without insert The cells were
grown at 37C in Minimum Essential Medium Eagle (Bio
Whittaker Europe, Verviers, Belgium) supplemented with
10% (v/v) FBS, 100 UÆmL)1penicillin, 200 lgÆmL)1
strep-tomycin, and 5 lgÆmL)1blasticidine (ICN Biomedicals Inc.,
Irvine, CA, USA) in the presence of 5% (v/v) CO2 Stable
transfectants were selected by adding 200 lgÆmL)1zeocin
(Invitrogen) to the culture medium Stable cell lines were
grown with 1 lgÆmL)1 doxycyclin for 3 days to induce
overexpression Overexpression of the different
aB-crystal-lin mutants was assessed by indirect immunofluorescence
and by immunoblotting, as described below
Immunocytochemistry
HeLa cells were seeded on coverslips (18· 18 mm2) one
day prior to transfection Two days after transfection cells
were either fixed in 3% (v/v) paraformaldehyde for 15 min
and permeabilized for 10 min in 0.2% (v/v) Triton in NaCl/
Pior first permeabilized in 0.2% (v/v) Triton in NaCl/Pifor
1 min and then fixed in 3% (v/v) paraformaldehyde for
10 min For DNase I (Roche) and RNase A (Roche) treatment, T-RexTM HeLa cells expressing aB-crystallin S19D/S45D/S59D were fixed in methanol for 2 min at
20C and treated with DNase I (400 UÆmL)1) or RNase A (1 mgÆmL)1) for 1 h at 37C
A monoclonal 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 peptide antibody to the N-terminal 10 residues of aB-crystallin (NCL-ABCrys, Novocastra, Newcastle upon Tyne, UK) were also used (undiluted and at 1 : 50 dilution, respectively), and gave the same results as the RIKEN antibody We further tested a polyclonal antiserum (K79) to the C-terminal 13 residues of aB-crystallin, as has been widely used in other studies Because this antiserum was earlier suggested to give nonspecific staining of nuclear bodies [36], we used primary cultures of lens epithelial cells derived from 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 pronounced 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 antibody further in our 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 at 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 anti-human IgG, FITC-FITC-conjugated rabbit 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 106 cells were resuspended in 50 lL ice-cold lysis buffer [10 mM Tris pH 7.5, 100 mM KCl, 1 mM dithiothreitol, 1 mM EDTA, 5 mMMgCl2, 1 mMphenylmethanesulfonyl 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 4C The supernatant was supplemented with
50 lL of 2· SDS sample buffer [2% (v/v) SDS, 0.125M 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 95C and used as the detergent-soluble fraction The remaining pellet was washed once with 500 lL
Trang 3lysis buffer, resuspended in 50 lL lysis buffer supplemented
with 50 lL of 2· SDS sample buffer, heated for 5 min at
95C and used as the detergent-insoluble fraction The
detergent-soluble and detergent-insoluble fractions were
separated by SDS/PAGE and subsequently 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 theLABWORKSTM
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 mM Tris/HCl pH 7.8, 10 mM NaCl,
1 mM dithiothreitol, 2 mM MgCl2, 1 mM
phenyl-methanesulfonyl fluoride, supplemented with a protease
inhibitor cocktail from Roche) and incubated on ice 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 times through a
21-gauge needle and the nuclei, free of cytoplasmic 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 mM Tris/HCl pH 7.4, 5 mM
MgCl2, 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 amounts of proteins 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-crystallin
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-RexTMHeLa 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 the deter-gent-insoluble fraction (A) HeLa cells were transfected with expression constructs coding for wild type aB-crystallin (WT), pseudophosphor-ylated aB-crystallin mutants containing S to D substitutions 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 detergent-insoluble (I) fractions, and ana-lyzed by Western blotting using the RIKEN anti-(aB-crystallin) monoclonal antibody (B) The average level of aB-crystallin in the detergent-insoluble fraction is shown as a percentage of the total aB-crystallin Values are based on four independent experiments and error bars represent the standard error of the 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).
Trang 4fusion 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-crystallin To specifically reveal the localization of
detergent-insoluble aB-crystallin, the soluble aB-crystallin
was removed by treating cells with a detergent solution prior
to fixation Panels d–i in Fig 2A show that in all cells
the cytoplasmic staining was strongly reduced Only
cells expressing aB-crystallin S19D/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 structures
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-crystallin in nuclear bodies In the case of a double replacement all three possible aB-crystallin mutants could
B
A
e
h
g
b
c
Fig 2 Detergent-insoluble pseudophosphorylated aB-crystallin localizes in nuclear bodies (a) T-RexTMHeLa cell lines stably transfected with aB-crystallin wild type (WT), S19D/S45D/S59D (STD) or S19A/S45A/S59A (STA) were induced for expression Part of the cells were fixed and permeabilized (No detergent) while other cells were permeabilized prior to fixation (Detergent) Localization of aB-crystallin was visualized by indirect immunofluorescence with the RIKEN anti-(aB-crystallin) mAb and TRITC-conjugated secondary 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 judged by fluorescence microscopy Per slide 200 transfected cells were counted at a magnification of 400· The average of two independent experiments is shown.
Trang 5be detected in nuclear bodies, but the combination 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 2A, 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 that the nuclear aB-crystallin
bodies might be localized at the SC35 speckles [41] A
double immunofluorescence analysis was therefore
per-formed on detergent-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
anti-(aB-crystallin) aB-crystallin S19D/S45D indeed perfectly
colo-calized with the most intensely stained Sm speckles
(Fig 3A, a–c) A similar result was obtained with
aB-crystallin S19S/S45D/S59D (data not shown) To confirm
that the anti-Sm serum indeed stains SC35 speckles, the
colocalization of the Sm epitope with the splicing factor
SC35, which is the antigen by which these speckles were
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 SC35 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-RexTMHeLa cells
expres-sing the mutant S19D/S45D/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 remained, indicating that most of the DNA was
digested However aB-crystallin could still be detected in
SC35 speckles (Fig 3B, a and c) The RNase-treated cells
were costained with 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
the SC35 speckles after RNase treatment (Fig 3B, e and f),
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) It 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 speckles (Fig 2B) FBX4 normally is a detergent-soluble protein, 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 HeLa cells, fluores-cence was found in cytoplasm and nucleus, but excluding the nucleoli (data not shown, and [43]) Upon pretreatment with detergent before fixation, 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 speckles 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 with 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 staincolocaliz-ing 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, indicating 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 tiny proportion of the total aB-crystallin is present in the 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 thus resemble the endogenously Ser45-phosphorylated aB-crystallin
Trang 6B
C
a
d a
Fig 3 Pseudophosphorylated aB-crystallin localizes in SC35 speckles, independent of intact DNA and RNA, and recruits FBX4 to these speckles (A) HeLa cells, transiently transfected with aB-crystallin S19D/S45D (a–c) or nontransfected (d–f), were first permeabilized and subsequently fixed Cells were stained with the RIKEN mAb anti-(aB-crystallin) (a) or the monoclonal antibody to SC35 (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 antibodies to aB-crystallin and SC35 were detected with TRITC-conjugated secondary antibodies, whereas Sm was detected by FITC-conjugated secondary antibodies (B) T-RexTMHeLa 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 RIKEN mAb anti-(aB-crystallin) (a and d) and YOYO-1 (b) or anti-Sm (e) Panels c and f show the overlays (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 detected by GFP fluorescence (b) The merge picture (c) shows their colocalization.
Trang 7We report here that mimicking the 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 its localization is 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 function as sites for storage or recycling of splicing
factors [41] During mitosis SC35 speckles dissociate,
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 in 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 4A, this is indeed the case
The next question is whether the observed recruitment
of FBX4 to SC35 speckles by pseudophosphorylated
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 those coexpressing FBX4–GFP and the phosphomimicking aB-crystallins A plausible explanation for this difference between transfected pseudophosphoryl-ated and endogenously phosphorylpseudophosphoryl-ated aB-crystallin is that the overexpressed Ser-Asp mutants are likely to be trapped together with FBX4–GFP in stable 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 localized in the SC35 speckles themselves aB-crystallin is a chaperone-like protein, and it is possible that the function of the putative interaction 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 the SC35 speckles (Fig 3C), it might be more likely that the combined association of phosphorylated aB-crystallin and FBX4 with
a speckle protein 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 in agreement 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 is the presence of cytoplasmic bodies containing desmin and aB-crystallin [33,34]
Two other 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 Moreover, 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 of aB-crystallin (K79, see Materials and methods) we also found nuclear speckles in all cell lines studied, transfected or not, but the 2D2B6 monoclonal only stained speckles in cells transfected with pseudophosphory-lation mutants of aB-crystallin (data not shown) To the 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 the prominent staining of nuclear speckles
by K79 in lens epithelial cells of aB-crystallin knock-out mice (see Materials and methods) Because of this apparent cross-reactivity, nuclear speckles visualized with antibodies
A
Fig 4 aB-crystallin endogenously phosphorylated at Ser45 colocalizes
with SC35 speckles (A) T-RexTMHeLa cells stably transfected with
aB-crystallin wild type (WT) were induced for expression, and after
3 days fixed and permeabilized Cells were stained with the polyclonal
anti-(aB-crystallin) S45p (a) and costained with anti-Sm (b) The S45p
antibody was used because phosphorylation at Ser45 is the most
rep-resentative for the three possible pseudophosphorylation sites in
aB-crystallin (Figs 1B and 2B) The S45p antibody was detected with
TRITC-conjugated secondary antibodies, whereas Sm was detected by
FITC-conjugated secondary antibodies Arrows indicate some of the
speckles that contain both aB-crystallin S45p and Sm (B) T-RexTM
HeLa cells stably transfected with aB-crystallin wild type (WT) were
induced for expression and harvested after 3 days Part of the cells was
used as total cell lysate (T), while the 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).
Trang 8against the C-terminal sequence of aB-crystallin should be
interpreted with caution This means that localization of
aB-crystallin in SC35 speckles has only been demonstrated
unambiguously in the case of the 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 The additional recruitment of FBX4 may
stimulate the ubiquitination of the speckle protein
Acknowledgements
We thank Dr G Eguchi for his generous gift of the anti-(aB-crystallin)
monoclonal antibody 2D2B6, and Dr N H Lubsen for useful
discussions This work was supported by a grant from the Netherlands
Organization for Scientific Research (NWO-MW 902-27-227).
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