Báo cáo khoa học: Mimicking phosphorylation of the small heat-shock protein aB-crystallin recruits the F-box protein FBX4 to nuclear SC35 speckles docx

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:
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. The additional recruitment of FBX4 may
stimulate the ubiquitination of the speckle protein.
Acknowledgements
WethankDrG.Eguchiforhisgenerousgiftoftheanti-(aB-crystallin)
monoclonal antibody 2D2B6, and Dr N. H . Lubsen for useful
discussions. T his work w as supporte d b y a grant from the Netherlands
Organization for Scientific Research (NWO-MW 902-27-227).
References
1. Van Montfort, R., Slingsby, C. & Vierling, E. (2001) Structure and
function of the small heat shock protein/a-crystallin family of
molecular chapero nes. Adv. Protein Chem. 59, 105–156.
2. Arrigo, A.P. & Muller, W.E.G. (2002) Small Stress Proteins.

Springer-Verlag, Berlin/Hei delberg/Ne w York.
3. Horwitz, J. (2003) a-Crystallin. Exp. Eye Res. 76, 145–153.
4. Inaguma, Y., Hasegawa, K., Goto, S., Ito, H. & Kato, K. (1995)
Induction of the synthesis of hsp27 and aB-crystallin in tissues of
heat-stressed rats and its suppression by ethanol or an a1-adren-
ergic antagonist. J. Biochem. (Tokyo) 117, 1238–1243.
5. Kato, K., G oto, S ., Inaguma , Y., Hasegawa, K ., M orishita, R . &
Asano, T. (1994) Purification and characterization of a 20-kDa
protein that i s h ighly h om ologous t o aB-crystallin. J. Biol. C h em.
269, 15302–15309.
6. Sugiyama, Y., Suzuki, A., Kishikawa, M ., Akutsu, R., Hirose, T.,
Waye, M.M., Tsui, S.K., Yoshida, S. & Ohno, S. (2000) Muscle
develops a specific form of small heat shock protein complex
composed o f MK BP/HSPB2 and HSPB3 during myogenic dif-
ferentiation. J. Biol. C hem. 275, 1095–1104.
7. Ito,H.,Okamoto,K.,Nakayama,H.,Isobe,T.&Kato,K.(1997)
Phosphorylation of aB-crystallin in response to variou s types of
stress. J. Biol. Chem. 27 2 , 29934–29941.
8. Kato, K., Ito, H., Kamei, K., Inaguma, Y., Iwamoto, I. & Saga, S.
(1998) Phosphorylation of aB-crystallin in mitot ic cells and iden-
tification of enz ymatic activities responsible f or phosphorylation.
J. Biol. Chem. 273, 28346–28354.
9. Kato, K., Ito, H. & Inaguma, Y. (2002) Expression an d phos-
phorylation of mammalian small heat shock proteins. Prog. Mol.
Subcell. Biol. 28, 129–150.
10. Gaestel, M. (2002) sHsp-phosphorylation: en zymes, signaling
pathways and functional implications. Prog. Mol. Subcell. Biol. 28,
151–169.
11. Kato, K., Ito, H., K amei, K., Iwamoto, I. & Inaguma, Y. (2002)
Innervation-depend ent phosphorylation and accumulation of

aB-crystallin and H sp27 as insoluble complexes in disused muscle.
FASEB J. 16, 1432–1434.
12.Ito,H.,Kamei,K.,Iwamoto,I.,Inaguma,Y.,Nohara,D.&
Kato, K. (2001) Phosphorylation-induced change of the oligo-
merization state o f aB-crystallin. J. Biol. Chem. 276, 5346–5352.
13. Lutsch,G.,Vetter,R.,Offhauss,U.,Wieske,M.,Grone,H.J.,
Klemenz, R., Schimke, I., Stahl, J. & Benndorf, R. (1997)
Abundance and location of the small heat shock proteins HSP25
and aB-crys tallin in ra t and hum an heart. Circulation 96, 3466–
3476.
14. Quinlan, R. (2002) Cytoskeletal competence requires protein
chaperones. Prog. Mol. Subcell. Biol. 28, 219–233.
15. Djabali, K., Piron, G., de Nechaud, B. & Portier, M.M. (1999)
aB-crystallin interacts with cyt oplasmic intermediate filament
bundles during mitosis. Exp. Cell Res. 253, 6 49–662.
16. Djabali, K., de Nechaud, B., Landon, F. & Portier, M.M. (1997)
aB-crystallin interacts with intermediate filaments in response to
stress. J. Cell Sci. 110 , 2759–2769.
17. Verschuure, P., Croes, Y., van den Ijssel, P.R.L.A., Quinlan, R.A.,
de Jong, W.W. & Boelens, W.C. (2002) Translocation of small
heat shock proteins to the actin c ytoskeleton upon proteasomal
inhibition. J. Mol. Cell Cardiol. 34, 117–128.
18. Mehlen, P., Schulze-Osthoff, K. & Arrigo, A .P. (1996) Small stress
proteins as novel regulators o f apoptosis. Heat sh ock protein 27
blocks Fas/APO-1- and staurosporine-induced cell death. J. Biol.
Chem. 271, 16510–16514.
19. Kamradt, M.C., Chen, F. & Cryns, V.L. (2001) The s mall heat
shock protein aB-crystallin negatively regulates cytochrome c-and
caspase-8-dependent activation of caspase-3 b y inhibiting its
autoproteolytic maturation. J. Biol. Chem. 276, 16059–16063.

20. Kamradt, M.C., Chen, F., Sam, S. & Cryns, V.L. (2002) The small
heat shock protein aB-crystallin negatively regulates apoptosis
during myogenic differentiation by inhibiting caspase-3 activation.
J. Biol. Chem. 277, 38731–38736.
21. Morrison, L.E., Hoover, H.E., T hue rauf, D.J. & Glembotski,
C.C. (2003) Mimicking p hosphorylation of aB-crystallin on ser-
ine-59 i s necessary and sufficient to provide maximal protection o f
cardiac myocytes from apoptosis. Circ. Res. 92, 203–211.
22. Conconi, M., Djavadi-Ohaniance, L., Uerkvitz, W., Hendil, K.B.
& Friguet, B . ( 1999) Conformational ch anges i n the 20S protea-
some upon macromolecular ligand binding analyzed with mono-
clonal antibodies. Arch. B iochem. Biophys. 362, 325–328.
23. Boelens, W.C., Croes, Y. & de Jong, W.W. (2001) Interaction
between aB-crystallin and the human 20S proteasomal subunit
C8/alpha7. Biochim. Biophys. Acta 1544, 311–319.
24. Ito, H., Kamei, K., Iwamoto, I., I naguma, Y., Garcia-Mata, R.,
Sztul, E. & K ato, K. (2002) Inhibition of proteasomes induc es
accumulation, p hosp horylation, and recruitment of HSP27 and
aB-crystallin to aggresomes. J. Biochem. (Tokyo) 131, 593–603.
25. den Engelsman, J., Keijsers, V., de Jong, W.W. & Bo elens, W.C.
(2003) The small heat-shock protein aB-crystallin promotes
FBX4-dependent ubiquitination. J. Biol. Chem. 278, 4699–4704.
26. Head, M .W. & Goldman, J.E. (2000) Small h eat shock proteins,
the cytoskeleton, an d in clusion bo dy fo rmation. Ne uropathol.
Appl. Neurobiol. 26, 304–312.
27. Schultz, C., Dick, E .J., Cox, A.B., Hu bbard, G.B., Braak , E. &
Braak, H. (2001) Expression of stress proteins aB-crystallin,
ubiquitin, and hsp27 in pallido-nigral spheroids of aged rhesus
monkeys. Neurobiol. Aging 22, 677–682.
28. Lowe, J., Mayer, J., Landon, M. & Layfield, R. (2001) Ubiquitin

and the molecular pathology of neurodegenerative diseases. Adv.
Exp. Med. Biol. 487, 169–186.
29.Fischer,D.,Matten,J.,Reimann,J.,Bonnemann,C.&
Schroder, R. (2002) Expression, localization and functional
divergence of aB-crystallin and heat shock protein 27 i n core
myopathies and ne urogenic atrop hy. Acta Neuropathol. (Berlin)
104, 297–304.
30. Selcen, D., Krueger, B.R. & Engel, A.G. (2002) Familial cardio-
neuromyopathy w ith h yaline ma sses a nd ne maline r ods: a novel
phenotype. Ann. Neurol. 51, 224–234.
31. Ryan, M .C., Shooter, E.M. & Notterpek, L. (2002) Aggresome
formation in neuropathy models based on peripheral myelin
protein 22 mutations. Neurobiol. Dis. 10, 109–118.
32. Ito,H.,Kamei,K.,Iwamoto,I.,Inaguma,Y.,Tsuzuki,M.,Kis-
hikawa,M.,Shimada,A.,Hosokawa,M.&Kato,K.(2003)
Hsp27 suppresses the formation o f inclusion bod ies induced by
4202 J. den Engelsman et al.(Eur. J. Biochem. 271) Ó FEBS 2004
expression of R120G aB-crystallin, a caus e o f desmin-related
myopathy. Cell Mol. Life Sci. 60, 1217–1223.
33. Chavez Z obel, A.T., Loranger, A., Marceau, N., Theriault, J .R.,
Lambert, H. & Landry, J. (2003) Distinct chaperone mechanisms
can delay the formation of aggresomes by the myopathy-causing
R120G aB-crystallin mutant. Hum. Mol. Gen et. 12, 1609–1620.
34. Vicart, P., Ca ron, A., Guicheney, P., Li, Z., Prevost, M.C., Faure,
A.,Chateau,D.,Chapon,F.,Tome,F.,Dupret,J.M.,Paulin,D.
& Fardeau, M . (1998) A missense mutation in the aB-crystallin
chaperone gene causes a desmin-related myopathy. Nat. Genet. 20,
92–95.
35. Sawada, K., Agata, K., Yoshiki, A. & Eguchi, G. (1993) A set of
anti-crystallin monoclonal antibodies for detecting lens specifi-

cities: b-crystallin as a specific marker for detecting lentoidogenesis
in cultures of chicken lens epithelial cells. Jpn J. Ophthalmol. 37,
355–368.
36. van de Klundert, F.A., Gijsen, M.L., van d en IJssel, P.R.L.A.,
Snoeckx, L.H. & de Jong, W.W. (1998) aB-crystallin and hsp25 in
neonatal cardiac c ells differences in cellular l ocalization under
stress conditions. Eur. J. Cell Biol. 75, 38–45.
37. Andley, U.P., Song, Z., Wawrousek, E.F., Brady, J.P., Bassnett, S.
& Fleming, T.P. (2001) Lens epithelial cells derived from
aB-crystallin knockout mice demonstrate hyperproliferation and
genomic instability. FASEB J. 15, 221–229.
38. Pruijn, G .J., Schoute, F., Thijsse n, J.P., S meenk, R.J. & van
Venrooij, W.J. (1997) M apping of SLE-spec ific Sm B cell epitopes
using murine m on oclonal a ntibodies. J. Autoimmun. 10, 1 27–136.
39. Spector, D.L. (2001) Nuclear domains. J. Cell Sci. 114, 2891–2893.
40. Lamond, A.I. & Earnshaw, W .C. (1998) Structure and function in
the nucleus. Science 280, 547–553.
41. Spector, D.L., Fu, X.D. & Maniatis, T. (1991) Associations
between distinct p re-mRNA splicing components and the cell
nucleus. EMBO J. 10 , 3467–3481.
42. Nyman,U.,Hallman,H.,Hadlaczky,G.,Pettersson,I.,Sharp,G.
& Ringertz, N.R. (1986) Intranuclear localization of snRNP
antigens. J. Cell Biol. 102, 137–144.
43. Cenciarelli, C., Chiaur, D.S., Guardavaccaro, D., Parks, W.,
Vidal, M. & Pagano, M. (1999) Identification o f a family of hu-
man F-box proteins. Curr. Biol. 9, 1177–1179.
44. Bryantsev, A.L., Loktionova, S .A., Ilyinskaya, O .P., Tararak,
E.M., Kampinga, H.H. & Kabakov, A.E. (2002) Distribution,
phosphorylation, and activities of Hsp25 in heat-stressed H9c2
myoblasts: a functional link to cytoprotection. Cell Stress

Chaperones 7, 146–155.
45. van d en Ijssel, P.R.L.A., W heelock, R., Prescott, A., Russell, P. &
Quinlan, R.A. (2003) Nuclear speckle localisation of the small heat
shock p rotein aB-crystallin and its inhibition by the R120G car-
diomyopathy-linke d mutation. Exp. Cell Res. 287, 2 49–261.
46. van Rijk, A.F., Stege, G.J., Bennink, E .J., May, A. & B loemendal,
H. (2003) Nuclear staining for the small heat shock protein
aB-crystallin co-localizes with splicin g factor SC35. Eur. J. Cell
Biol. 82, 361–368.
47. Brzyska, M., Stege, G.J., Renkawek, K. & B osman, G.J. (1998)
Heat shock, but not the reactive state per se, induces increased
expression of the small stress proteins hsp25 and aB-crystallin in
glial cells in vitro. Neuroreport 9, 1549–1552.
48. Bajramovic, J.J., Bsibsi, M., Geutskens, S.B., Hassankhan, R.,
Verhulst, K.C., Stege, G.J., de Groot , C.J. & van Noort, J.M.
(2000) Differential expression of stress proteins in human adult
astrocytes in respon se to cyto kines. J. Ne uroimmunol. 106, 14–22.
Ó FEBS 2004 aB-crystallin colocalizes with FBX4 in SC35 speckles (Eur. J. Biochem. 271) 4203