Haartman Institute and Helsinki University Central Hospital, Biomedicum Helsinki, P.O. Box 63, 00014 University of Helsinki, Finland
* Author for correspondence (e-mail: tomi.makela{at}helsinki.fi )
Accepted 6 March 2002
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Summary |
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Key words: PDZ, LIM, Stress fibers, Kinase
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Introduction |
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Interactions between kinases and PDZ-LIM proteins are mediated via their
LIM domains. Thus the third LIM domain of Enigma has been reported to
associate with the cytoplasmic tail of the insulin receptor, and this
interaction is apparently important for the endocytosis of the receptor
(Wu and Gill, 1994).
Association of the second LIM domain of Enigma with Ret/ptc2 is required for
the plasma membrane localization and mitogenic activity of Ret/ptc2
(Durick et al., 1998
;
Durick et al., 1996
). The LIM
domains of ENH bind to PKC (Kuroda et al.,
1996
), and subsequently Cypher1 was also found to associate with
PKC via its LIM domain (Zhou et al.,
1999
).
PDZ domains of this family were first found associated with the Z lines of
striated muscle (Xia et al.,
1997). Thus the PDZ domains of Enigma, ALP and ZASP/Cypher1
proteins mediate localization to the Z line of striated muscle, where Enigma
interacts with ß-tropomyosin (Guy et
al., 1999
) and ALP (Xia et
al., 1997
) as well as ZASP/Cypher1
(Faulkner et al., 1999
;
Zhou et al., 1999
) with
-actinin-2. Accordingly, both ALP and Cypher1 appear to be muscle
specific on the basis of their expression patterns as well as of the
phenotypes of mice with targeted mutations of these genes.
Alp-/- mice gradually develop a cardiomyopathy
(Pashmforoush et al., 2001
),
and Cypher1-/- mice die postnatally with a severe form of
congenital myopathy (Zhou et al.,
2001
). On the basis of the histology, it was suggested that
Cypher1 is not required for formation but rather maintenance of sarcomeres
(Zhou et al., 2001
).
The association of PDZ-LIM proteins with the cytoskeleton is not limited to
striated muscle. The CLP-36, also called CLIM1,
(Kotaka et al., 1999;
Wang et al., 1995
) PDZ-LIM
protein is expressed in several epithelial tissues
(Vallenius et al., 2000
).
Furthermore, the PDZ domain of CLP-36 mediates its stable association with
nonmuscle forms of
-actinin (
-actinin-1 and
-actinin-4)
(Vallenius et al., 2000
), and
CLP-36 is accordingly localized to actin stress fibers in nonmuscle cells.
Actin stress fibers form a dynamic organelle that is important in processes
involving cell shape, such as cell migration, cell polarity and cytokinesis.
These functions are thought to be mediated by actin filaments in response to
several only partially characterized signal transduction pathways. One of
these involves the LIM-kinase that acts downstream of Rac and Rho, and
phosphorylates cofilin, thereby inactivating its F-actin depolymerization
activity (Arber et al., 1998;
Yang et al., 1998b
).
Biochemical purification of stress fibers has revealed that it is composed of
at least 20 polypeptides (Katoh et al.,
1998
), one of which is likely to be CLP-36. However, a number of
other potential novel regulators of stress fibers remain to be identified. In
this report we have characterized a novel kinase targeted to actin stress
fibers by the CLP-36 PDZ-LIM protein.
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Materials and Methods |
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Cell culture and antibodies
The U2OS osteosarcoma cell line was propagated in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10% fetal calf serum, penicillin and
streptomycin and glutamine at 37°C in 5% CO2. The mouse
monoclonal anti-Myc antibody (9E10) was from Babco Inc., Berkeley, CA. The
rabbit polyclonal anti-CLP-36 antibody was raised against purified full-length
GST-CLP-36. The ALP antibody is described by Pomies et al.
(Pomies et al., 1999).
Mammalian expression vectors and transfections
The open reading frame of Clik1 was PCR amplified from (C1/pDR2) with the
primers 5'-GGA ATT CCG TCG ACT GGC AGC CAT GGA AAC GGG GA-3' and
5'-TCT AGA TTA AGC GGC ACA TGT GAC CTG GTC-3', then subcloned
further to pAMC (Tiainen et al.,
1999) as an EcoR1-XbaI insert to generate
Myc-Clik1. The mutagenesis kit (Promega) was used to generate the ATP-binding
site mutant of Clik1 (Clik1-K98M/pAMC) by mutating lysine 98 to methionine
using primer 5'-GGC GGT CAT GAA GAT CCG-3'. The Myc-tagged or
HA-tagged CLP-36 plasmids were generated by subcloning an
EcoRI-XhoI fragment from 38/JG4-5
(Vallenius et al., 2000
) into
EcoRI-SalI sites of pAMC or pAHC
(Tiainen et al., 1999
).
Myc-CDK7 was expressed from MO15-3M
(Makela et al., 1994
).
HA-tagged ALP was PCR amplified from smALP/pBS (a kind gift from Pascal
Pomies) with primers 5'-CG ACG CGT ACC ACC ATG CCA CAG AAC GTT
ATT-3' and 5'-GC GTC GAC CTA GCA TTT TGG GTA AAC AG-3',
followed by digestion with MluI and SalI and subcloned into
MluI-SalI sites of pAHC. EGFP-CLP-36 plasmids were generated
by subcloning an EcoRI-XhoI fragment from 38/JG4-5
(Vallenius et al., 2000
) into
EcoRI-SalI sites of EGFP-C2 (Clontech). EGFP-CLP1-231 was
generated from EGFP-CLP-36 by BamH1 digestion to delete the
C-terminal amino acids 232-329. U2OS osteosarcoma cells were transfected by
using the calcium phosphate transfection method as described previously
(Sambrook, 1989).
Cell extracts, immunoprecipitation and western blotting
Adult mouse testes were frozen in liquid nitrogen, homogenized, lysed in
ELB (150 mM Nacl, 50 mM HEPES pH 7.4, 5 mM EDTA, 0.1% NP-40, 1 mM
dithiothreitol (DTT), 2.5 µg/ml aprotinin, 0.5 mM phenylmethylsulfonyl
fluoride, 10 mM ß-glycerol-phosphate and 1 µg/ml leupeptin) and
subsequently treated the same as the U2OS cell extracts (see below). 48 hours
after transfection, U2OS cells were washed in PBS and lysed in ELB buffer 40
minutes on ice. The lysates were centrifuged at 14 000 g for 20
minutes at +4°C. Equal amounts (200-400 µg) of cleared supernatant was
incubated with 3 µl monoclonal anti-Myc antibody or 2µl anti-CLP-36
antibody for 2 hours at +4°C prior to adding 25% protein A-sepharose beads
for 1 hour. Subsequently beads were washed four times with ELB. Western
blotting analysis was performed according to standard procedures (Sambrook,
1989).
Immunofluorescence
Transfected cells on coverslips were fixed with 3.5% (w/v) paraformaldehyde
(PFA), permeabilized with 0.1% Triton X-100 for 5 minutes, blocked with 5%
goat serum in PBS for 30 minutes, labeled with primary antibody for 30
minutes, washed three times with PBS, labeled with secondary antibody for 30
minutes, washed, labeled with Hoechst, mounted with Elvanol and analyzed with
Zeiss Axiovert microscope. The 0.5% Triton X-100 treatment was performed as
described previously (Sainio et al.,
1997). The expression of the fusion proteins used in
immunofluorescence studies was confirmed using western blotting analysis.
Frozen sections were sliced from adult mouse testis, fixed with 94% ethanol
overnight at +4°C and labeled as transfected coverslips (see above).
In vitro kinase assay
Anti-Myc immunoprecipitates from Myc-Clik1 overexpressing lysates were
washed twice in kinase buffer (20 mM Tris pH 7.5, 50 mM KCl, 5 mM
MgCl2, 2.5 mM MnCl2, 1 mM DTT) and resuspended in 15
µl kinase buffer with 10 µCi [32P]ATP without any
added substrates (Fig. 1B) or
with added substrates including HistoneH1, MBP, CTD, GST-CDK2(K33R),
-actinin and GST-CLP-36 for 30 minutes at 30°C. Samples were
resolved on SDS-PAGE, transferred to nitrocellulose membranes and stained with
Ponceau to detect substrates. Subsequently radiolabeled bands were detected
with autoradiography.
|
Yeast two-hybrid screening
A LexA-Clik1 fusion protein encoding amino acids 40-406 of Clik1 was used
to screen a human fetal liver cDNA library in pJG4-5 (Clontech Laboratories,
cat. HL4507AK), essentially as described
(Gyuris et al., 1993). Positive
clones were subjected to sequence analysis or analyzed by digestion, and
representative clones (38/pJG4-5, amino acids 1-329; 51/pJG4-5, amino acids
24-329; and 47/pJG4-5, amino acids 158-329) were used to subclone various
forms of CLP-36 into other vectors.
Northern blot analysis
Clontech Multiple Tissue Northern Blot filters (7757-1, 7760-1, 7766-1)
were probed with 630 bp (nucleotides 404-1034) probe of Clik1 cDNA according
to standard protocols (Sambrook, 1989). The Raji (Burkitt's lymphoma) lane
that was present in the Northern filter was excluded from the analysis because
of low levels of mRNA.
Recombinant proteins and solution binding assays
GST and GST-CLP-36 proteins were purified as described previously
(Vallenius et al., 2000). For
detection of association between cellular proteins and GST-CLP-36, 300 µg
of U2OS cell extracts in ELB were incubated with 5 µg of purified GST or
GST-CLP-36 for 2 hours at +4°C prior to adding 7.5 µl (packed volume)
glutathione sepharose beads for 1 hour. Subsequently the beads were washed
four times with ELB and subjected to SDS-PAGE, Ponceau staining and western
blott analysis.
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Results |
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The predicted 44.6 kDa Clik1 protein contained a serine-threonine kinase
domain between amino acids 69 and 390 (underlined in
Fig. 1A), with the
serine-threonine active site signature between amino acids 223-235. Other
functional domains in Clik1 included four potential NLS signals (dotted boxes
in Fig. 1A), yielding an NLS
score of 1.14 by PSORT (Nakai and
Kanehisa, 1992), and a proline-rich region in its N-terminal end.
Despite our original observation that EST H85389 was similar to Csk1,
full-length Clik1 shares only 20-30% identity to any known serine/threonine
kinases, including Csk1, and thus does not clearly belong to characterized
kinase families. Similar homologies were observed with a group of
uncharacterized CaM-kinase related kinases (e.g. GenBank CAC29064), with
several CDKs, and with the PAK family of kinases.
Autophosphorylation of Clik1
To determine whether the predicted protein encoded a kinase, a plasmid
expressing a Myc-epitope tagged Clik1 (Myc-Clik1) was transiently transfected
into COS-7 or U2OS osteosarcoma cells. At the same time, we also transfected a
Clik1 mutant (Myc-K98M) with a methionine substitution of the conserved
ATP-binding lysine (asterisk in Fig.
1A) to disrupt kinase activity, but this mutant behaved the same
as the wild-type Clik1 in other regards
(Fig. 5D). Anti-Myc
immunoprecipitates from Myc-Clik1- or Myc-K98M-transfected cells were
subsequently subjected to an in vitro kinase reaction. Autoradiography of the
samples following SDS-PAGE and transfer to nitrocellulose membranes indicated
the presence of a broad radiolabeled band at 46-48 kDa in the wild-type
Clik1-transfected but not in the Clik1-K98M lane
(Fig. 1B, IP-kinase). This band
co-migrated with the wild-type Myc-Clik1 protein detected as a tight doublet
in western blot analysis (Fig.
1B, IP-Western). A band co-migrating with the faster migrating
band was noted in the Clik1-K98M lane, indicating similar expression of the
mutant and suggesting that the slower migrating band in wild-type Clik1
represented a phosphorylated form of Clik1. These results indicate that Clik1
undergoes autophosphorylation in vitro. Moreover, analysis of wild-type Clik1
and Clik1-K98M by western blot analysis from cell lysates showed the same
difference in the bands, strongly suggesting that Clik1 undergoes partial
autophosphorylation in cells (data not shown). The immunopurified Clik1 kinase
did not phosphorylate any tested substrates including CDK2 (as GST-CDK2),
Histone H1, myelin basic protein (MBP), the YSPTSPS heptapeptide C-terminal
repeat of RNA Pol II, -actinin, actin or CLP-36 (data not shown).
|
Clik1 associates with the PDZ-LIM protein CLP-36
To identify cellular proteins interacting with the Clik1 kinase, a yeast
two-hybrid screen with LexA-Clik1 (amino acids 40-406) was performed. 36 out
of 37 identified clones represented CLP-36, of which the longest (full length,
clone 38) and the shortest (amino acids 158-329; clone 47) clones are shown in
Fig. 2A. All clones contained
the C-terminal LIM domain, suggesting that the association between CLP-36 and
the Clik1 kinase is mediated by this protein-protein interaction motif. To
investigate whether CLP-36 also interacts with cellular Clik1, a U2OS cell
extract overexpressing Myc-Clik1 was incubated with baculovirus-produced
GST-CLP-36 or GST alone. Subsequent purification of the GST proteins and
analysis of associated proteins by western blot analysis indicated that
Myc-Clik1 specifically interacted with GST-CLP-36
(Fig. 2B, anti-Myc, lane 2) and
with GST-CLP1-158 generated from clone 47 (data not shown). GST-CLP-36
did not associate with cellular Myc-CLP-36 under the same conditions (data not
shown), suggesting that the PDZ and LIM domains of CLP-36 do not dimerize
unlike the related PDZ-LIM protein Ril
(Cuppen et al., 1998
).
|
Coexpression of CLP-36 leads to relocalization of the nuclear Clik1
kinase
When transfected alone, Myc-Clik1 was localized mainly in the nucleus of
U2OS osteosarcoma cells by immunofluorescence analysis
(Fig. 3A). Although these cells
express no detectable endogenous CLP-36
(Fig. 3B and data not shown),
transfected CLP-36 is localized to the cytoplasm and to actin stress fibers
(Fig. 3E). Strikingly, in cells
expressing both Clik1 and CLP-36, the majority of Clik1 was relocalized to the
cytoplasm and colocalized with CLP-36 (Fig.
3G). This redistribution of nuclear Clik1 by CLP-36 was specific
to Clik1, as coexpression of CLP-36 did not lead to relocalization of another
nuclear kinase, CDK7 (Fig. 3J).
The redistribution of Clik1 was not kinase dependent, as it was also observed
with the Clik1-K98M mutant (Fig.
5D). Taken together with the physical association of Clik1 and
CLP-36, these results indicate that when in association with CLP-36, Clik1 is
mostly cytoplasmic and suggest that Clik1 may have distinct functions in the
nucleus and the cytoplasm.
|
CLP-36 targets Clik1 kinase to actin stress fibers
The weak actin filament-like staining pattern of Clik1 in cells expressing
CLP-36 (Fig. 3G) prompted us to
study whether CLP-36 is able to target the Clik1 kinase to actin stress
fibers. To this end, coverslips were subjected to detergent extraction prior
to fixation to remove soluble material and to visualize actin stress fibers.
As demonstrated in Fig. 4, the
Clik1 kinase is colocalized with CLP-36 in actin stress fibers
(Fig. 4A). In these cells,
Clik1 staining was also detected in the nucleus where it accumulated into
bright clusters. The significance of this observation is still unclear, but we
could not detect these clusters with other tested kinases. The more diffuse
nuclear staining was caused by background after detergent extraction as it was
detected also with other constructs (data not shown) To get more insight into
the specificity of the ability of CLP-36 to relocalize Clik1 from the nucleus
to actin stress fibers, a similar experiment was carried out with ALP
(Pomies et al., 1999;
Xia et al., 1997
), a close
relative of CLP-36. Interestingly, whereas ALP did localize to actin stress
fibers, as does CLP-36 (Fig.
4E), ALP failed to relocalize Clik1 from its nuclear localization
(Fig. 4D), indicating that
these proteins do not interact. These results indicate that CLP-36
specifically targets Clik1 to actin stress fibers.
|
C-terminal part of CLP-36 is essential to relocalize Clik1
The yeast two-hybrid analysis as well as the GST-binding assay suggested
that the C-terminal part of CLP-36 is needed for the association between
CLP-36 and Clik1. To test whether this was also the case in living cells, U2OS
cells were transfected either with GFP-tagged CLP-36 (EGFP-CLP-36) or a mutant
CLP-36 lacking the C-terminal LIM domain (EGFP-CLP1-231) together with Clik1.
The results demonstrate that in cells expressing the full-length CLP-36, Clik1
was relocalized to the cytoplasm (Fig.
5B), whereas the C-terminal truncated CLP-36 that also localized
to actin stress fibers was unable to relocalize nuclear Clik1
(Fig. 5F). Owing to the low
background of GFP-constructs, we were able to study the localization of CLP-36
in actin stress fibers in more detail. Interestingly, in cells expressing both
Clik1 and CLP-36, the continuous periodic staining pattern of CLP-36 was
partly disrupted (Fig. 5A,I).
Previous studies demonstrate that CLP-36 localizes to actin stress fibers
(Vallenius et al., 2000), and
therefore it was of interest to study the actin filaments in cells expressing
both CLP-36 and Clik1. In cells expressing GFP-tagged CLP-36 alone the actin
filaments were intact when stained with phalloidin
(Fig. 5H), and CLP-36 localized
along actin filaments as expected (Fig.
5G). However, in cells expressing both CLP-36 and Clik1, the actin
filament bundles were partially disorganized, suggesting that in those cells
actin stress fibers is also altered (Fig.
5J) (data not shown). These results suggest a role for CLP-36 in
maintaining normal stress fibers. The exact role of the Clik1 kinase in this
regard is difficult to assess as a similar disorganization was noted even when
a kinase-deficient Clik1 was used (Fig.
5C).
Both Clik1 and CLP-36 are expressed in adult testis
To investigate the possible physiological role of the association of Clik1
and CLP-36, the expression patterns of these proteins were compared. CLP-36
mRNA has been detected at variable levels in a wide variety of tissues
(Kotaka et al., 2001;
Kotaka et al., 1999
;
Vallenius et al., 2000
). To
determine the tissue distribution of the Clik1 mRNA, northern blotting
analysis using various adult tissues was performed
(Fig. 6A). A 7.0 kb band was
present at low levels in all tissues and cell lines examined except for the
testis, where strong expression of Clik1 mRNA was noted.
|
Subsequently, analysis of expression levels of the corresponding proteins was attempted. Unfortunately, we were not been able to observe any endogenous Clik1 protein with the polyclonal peptide antibodies that were generated against Clik1, although they did recognize Clik1 overexpressed following transfections. By contrast, a rabbit polyclonal CLP-36 antiserum raised against GST-CLP-36 detected a 38 kDa band in total cell extracts from a variety of tissues, including adult testes (Fig. 6B, lane 1). This antiserum was also capable of immunoprecipitating a 38 kDa band from non-denaturing testes lysates (Fig. 6B, lane 2), and this band was not observed with pre-immune serum or after blocking of the immune serum with antigen (Fig. 6B, lane 3). The specificity of the CLP-36 antibody was further demonstrated by its inability to detect overexpressed ALP or RIL, whereas a strong band was seen with overexpressed CLP-36 (data not shown). On the basis of these results, we conclude that our antiserum is capable of recognizing endogenous CLP-36. Also the results indicate that the testis represents a tissue where both Clik1 and CLP-36 are expressed.
The availability of the CLP-36 antiserum further allowed characterization of the cell types expressing CLP-36 in the testis. Immunofluorescence analysis indicated strong CLP-36 staining in the peritubular contractile cells surrounding the seminiferous tubuli and in elongating spermatids (Fig. 6C). In addition, a weaker but specific staining was noted throughout the seminiferous epithelium when compared with the block control.
![]() |
Discussion |
---|
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Transfected Clik1 was found to localize predominantly to the nucleus, suggesting a nuclear function that will require further studies. However, a dramatic redistribution of Clik1 was noted following coexpression with CLP-36, where Clik1 shifted to the cytoplasm and into actin stress fibers, suggesting that CLP-36 levels determine the subcellular localization of Clik1. This was supported by a slightly less nuclear localization of Clik1 in COS-7 cells expressing higher levels of endogenous CLP-36 than U2OS cells used for the studies in Fig. 3 (data not shown).
Regarding the mechanism of this relocalization, it is interesting to note
that some wild-type CLP-36 was detected in the nucleus
(Fig. 3), and this localization
was more pronounced in the CLP-36 mutant with a disrupted PDZ domain
(Vallenius et al., 2000). This
suggests that CLP-36 may shuttle between the cytoplasm and the nucleus as has
been reported for LIMK-1 containing both PDZ and LIM domains
(Yang et al., 1998a
). In this
case, the observed relocalization of Clik1 could be caused by nuclear export
following association with CLP-36.
Our results demonstrate the ability of the CLP-36 PDZ-LIM protein to
recruit the Clik1 kinase to actin cytoskeleton. Thus, we provide direct
evidence for an adapter role for the PDZ-LIM proteins. Several previous
studies have suggested similar roles, especially for the more distantly
related PDZ-LIM proteins (Enigma/LMP-1, ENH and Zasp/Cypher1 with 28-36%
identity in LIM domains with CLP-36), whose LIM domains have all been found to
associate with various isoforms of PKC
(Kuroda et al., 1996;
Zhou et al., 1999
). However,
the PDZ-LIM subfamily with a higher overall similarity and domain structure to
CLP-36 (ALP and Ril with 60-67% identity in LIM domains with CLP-36) have not
been reported to associate with kinases. Our results definitely indicate that
within this subfamily the observed kinase interaction demonstrates a high
specificity, as no interaction was observed between Clik1 and either ALP
(Fig. 4) or Ril (data not
shown).
Taken together with our previous data
(Vallenius et al., 2000), the
results presented in this report demonstrate an association of the Clik1
kinase with actin stress fibers. This association is mediated through a bridge
formed by the PDZ-LIM protein CLP-36 and
-actinin
(Vallenius et al., 2000
). The
observed changes in CLP-36 localization in actin stress fibers in cells
expressing Clik1 kinase suggest that Clik1 may represent a novel regulator of
the actomyosin cytoskeleton in nonmuscle cells.
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Acknowledgments |
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