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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Institut für Prophylaxe und
Epidemiologie der Kreislaufkrankheiten, München, Germany;
Genzentrum der Universität, München, Germany;
Friedrich-Miescher-Institut, Basel, Switzerland; Toplab GmbH,
Martinsried, Germany; and Max-Planck-Institut für Biochemie,
Martinsried, Germany.
A 38-kd protein that associates with F-actin structures in
activated platelets and endothelial cells was purified, cloned, and
characterized. The protein contains an N-terminal PDZ motif, a large
intervening sequence, and a C-terminal LIM domain and was identified as
the human homolog of rat CLP36. The study showed that CLP36 associates
with actin filaments and stress fibers that are formed during shape
change and spreading of platelets and during migration and contraction
of endothelial cells. CLP36 binds to The actin cytoskeleton is a complex protein network
that not only provides cellular structure but is also fundamental for cellular dynamics, such as shape change, migration, spreading, and
contraction of nonmotile cells.1,2 Endothelial cells exhibit on activation by inflammatory, atherogenic, or hemostatic stimuli a rapid change of their actin cytoskeleton to rounded contracted cells with stress fibers.3-5 Stress fibers have
been recognized as the contractile organelle of nonmuscle cells bearing structural and functional resemblance to the myofibrils of skeletal muscle.6 They are not only found in vitro in cultured
cells but are also an in vivo phenomenon in arterial vascular
endothelial cells,7,8 where they are prominent in regions
of high-shear stress and above early atherosclerotic
lesions.9,10
Blood platelets show within seconds of activation a dramatic
reorganization of the cytoskeleton such as the formation of new actin
filaments and filament bundles, contractile actomyosin rings, and
stress fiber-like structures. This rapid reorganization of the
cytoskeleton underlies and leads to platelet shape change and
spreading.11,12 It is becoming clear that the cytoskeletal remodeling on cell activation is mediated by high-affinity interactions between specific proteins. On stimulation of platelets, proteins that
interact directly with F-actin, such as myosin and actinin, rapidly
translocate to the F-actin-rich cytoskeleton.13 Also the
small GTP-binding proteins Rho, Rac, and CDC42 that regulate cytoskeletal structures and are activated on platelet stimulation associate with the actin cytoskeleton in stimulated
platelets.14-17 We have observed previously that proteins
of 38-kd molecular mass translocated to the cytoskeleton in activated
platelets.18 Here we report the purification and cloning
of a 38-kd protein that was identified as the human homolog of rat
CLP3619 and almost identical to hCLIM1 isolated from a
human adenocarcinoma complementary DNA (cDNA) library.20
CLP36 contains a N-terminal PDZ domain, a large intervening sequence,
and a C-terminal LIM domain. The PDZ and LIM domains are modular
protein interaction motifs21,22 and mediate protein association with the cytoskeleton and with proteins involved in signal
transduction.23-28 We found that CLP36 associates with
actin filaments and stress fibers in activated human platelets and
endothelial cells. This association is mediated through its
constitutive binding to Cell culture
Purification of CLP36 from platelets
cDNA cloning Total RNA was isolated from HEL cells, using RNeasy (Qiagen, Hilden, Germany) and purified through oligo(dT)-cellulose (Sigma, Deisenhofen, Germany). First-strand cDNAs were synthesized with oligo(dT)-priming and Moloney murine leukemia virus (M-MLV) reverse transcriptase (GIBCO BRL, Karlsruhe, Germany). To amplify the clp36 cDNA, polymerase chain reaction (PCR) was performed with specific primers, based on the open reading frame (ORF) in THC 94 235 (sense 5'-ACCACCCAGCAGATAGAC-3' and antisense 5'-AACCAAAGTAAGCAGAGAAC-3'). The PCR product of 1.1 kilobase (kb; expected length 1.093) was electro-eluted from the gel at a constant voltage of 2 V/cm for 3 hours, purified by phenol/chloroform extraction, reamplified by PCR, and extracted from low-melt agarose (QIAquick Gel Extraction Kit, Qiagen). The clp36 cDNA was blunt-end inserted into the HincII site of the pCR-Script Amp SK+ vector (Stratagene, La Jolla, CA) and cloned in Escherichia coli XL1-blue (Stratagene). With the use of the Sac I and Hind III site, the clp36 cDNA was subcloned into the pQE-32 vector. For synthesis of glutathione-S-transferase (GST)-tagged LIM motif of CLP36 (amino acid residues (aa) 257-329; GST-LIM), the lim cDNA was subcloned into the pGEX-5X-2 (Pharmacia, Freiburg, Germany) vector by digestion with Xho I and Eco RI.clp36 cDNA was also obtained from human umbilical venous endothelial cells (HUVECs). RNA was isolated from HUVECs using PureScript (Biozym, Oldendorf, Germany), and cDNA was synthesized using the CapFinder PCR cDNA Synthesis Kit (Clontech). Endothelial clp36 cDNA was obtained by PCR using the clp36 specific primers. DNA sequencing The clp36 cDNA from endothelial cells and HEL cells, the various His-tag and enhanced green fluorescent protein (EGFP)-fusion constructs from clp36, and the various clp36 constructs for the yeast 2-hybrid were sequenced on both strands by MediGenomix (München, Germany) and MWG (Ebersberg, Germany).Expression of recombinant CLP36 The His-tagged CLP36 and His-tagged LIM of CLP36 were expressed in E coli XL1-blue and purified nondenaturating by Ni-NTA affinity chromatography according to instructions of the manufacturer (Quiagen). GST-LIM of CLP36 was expressed in E coli NM 522 and purified with glutathione agarose beads (Sigma).Antibody production Two potentially immunogenic peptides of CLP36 were chosen according to their hydrophilicity and sequence difference to homologous proteins. Peptide 14 (EDQIYCEKHARER; CLP36 [302-314]) and Peptide 17 (ESEEKGDPNKPSGFRS, CLP36 [224-239]) were synthesized and coupled through an N-terminal cysteine to KLH (Pierce, Rockford, IL). Antibodies to recombinant His-CLP36 and the 2 peptides of CLP36 were raised in rabbits (pab production, Herbertshausen). Antisera were purified using protein A columns (Protein A-Sepharose CL-4B, Sigma).Immunoblots Proteins were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, blotted, and detected by enhanced chemoluminescence as described.30 The dilutions of the rabbit anti-CLP36 immunoglobulin and the monoclonal mouse anti- -actinin immunoglobulin M (IgM) (BM-75.2; Sigma) were
1:20 000 and 1:5000, respectively. The dilutions of the
horseradish-peroxidase-linked secondary antimouse and antirabbit
antibodies (Amersham) were both 1:10 000.
Expression of CLP36 in platelets was estimated by loading a SDS gel
with increasing amounts of GST-LIM and platelet proteins of a defined
cell number. The amount of loaded GST-LIM molecules was calculated by
measuring absorption at 280 nm, using the extinction coefficient of GST
( Immunoprecipitation Suspensions of washed platelets were adjusted to a concentration of 1 × 109 cells/mL. Unstimulated platelets, platelets preincubated with cytochalasin D (2 µmol/L) for 2 minutes, or BAPTA-AM (60 µmol/L) and EGTA (5 mmol/L) for 30 minutes, or platelets activated by 1 U/mL thrombin (Boehringer Mannheim) for 1 minute were lysed in an equal volume of 2 × lysis buffer ("RIPA"; pH 7.4, 2% Triton X-100, 2 mmol/L EGTA, 100 mmol/L HEPES, 150 mmol/L NaCl, 2 mmol/L sodium orthovanadate, 1 tablet protease inhibitor cocktail/5 mL [No. 1 836 153, Boehringer Mannheim]) for 20 minutes on ice. The lysates were clarified by centrifugation at 15 600g for 10 minutes at 4°C. The supernatant was precleared by incubation with 5 µg/mL preimmune serum and Protein A Sepharose CL-4B for 1 hour, followed by centrifugation at 15 600g for 10 minutes. The supernatant was incubated for 1 hour at 4°C with 15 µg anti-CLP36 antibody and, subsequently, with Protein A Sepharose (0.75 mg in 10 µL RIPA) for 1 hour. All steps were performed at 4°C. After centrifugation, the immunoprecipitates were washed 3 times with 1.5 mL of ice-cold lysis buffer, and resuspended in 2 × sample buffer (125 mmol/L Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 3.1% dithiothreitol, 0.01 mg/mL bromphenol blue) and treated for 5 minutes at 95°C before gel separation.To determine the binding domains of CLP36 to Blot overlay assay Platelet proteins resolved by SDS-polyacrylamide gel electrophoresis (PAGE) were blotted onto nitrocellulose membranes (Amersham). Membranes were then blocked overnight at room temperature in triethanolamine-buffered saline (TBS), 0.05% Tween-20, 3% nonfat dry milk (Bio-Rad). Subsequent incubations with biotinylated His-tagged CLP36 (100 ng/mL; biotinylation with N-+-Biotinyl-6-amino-capronsäure-N-succinimidylester) were performed for 4 hours at room temperature in incubation buffer (TBS, 0.05% Tween-20, 0.5% nonfat dry milk [Biorad]). The blots were washed 3 times for 10 minutes with incubation buffer, incubated with Extravidin-peroxidase (Sigma) for 1 hour at room temperature, and washed 3 times with TBS, 0.05% Tween-20. Bands were detected by enhanced chemiluminescence (Amersham).Isolation of cytoskeletal and membrane skeletal fractions of platelets Cytoskeletal fractions were prepared similarly to the method described previously,17,31 using a 2 × Triton lysis buffer (pH 7.3) containing 2% Triton X-100, 0.1 mol/L Tris-HCl, 0.2 mol/L NaCl, 4 mmol/L MgCl2, 20 mmol/L EGTA, 200 µmol/L Pefablock-SC (Boehringer Mannheim), 20 µg/mL leupeptin, and 200 µg/mL aprotinin.Platelet spreading Platelet spreading was carried out as described32 with some modifications. Round glass coverslips (Eppendorf) were coated for 1 hour with 100 µL poly-L-lysine (0.1 mg/mL) (Sigma), washed twice with phosphate buffered saline (PBS), air dried, and put into 24-well culture dishes. Aliquots (0.2 mL) of washed platelet suspensions or, in some experiments, platelet-rich plasma were adjusted to 3 × 107 cells/mL and pipetted on the coverslips, which were centrifuged for 5 minutes at 250g at room temperature. Platelets were allowed to spread by incubation at 37°C for 5 to 10 minutes. Platelets not attached were removed by rinsing the coverslips with PBS.Immunofluorescence microscopy Spread platelets or endothelial cells (subconfluent and confluent), grown on coverslips, were fixed with fixation buffer (60 mmol/L Pipes, pH 6.1, 25 mmol/L Hepes, 10 mmol/L EGTA, 3 mmol/L MgCl2) containing 3.7% formaldehyde (Sigma) for 10 minutes at room temperature or overnight at 4°C. Coverslips were rinsed briefly with PBS containing 1 mmol/L MgCl2 and 1 mmol/L CaCl2, bathed in permeabilization buffer (0.2% Triton X-100 in PBS) for 5 minutes at room temperature and washed 3 times with PBS. Cells were incubated with the primary antibodies or rhodamine-phalloidin (Molecular Probes, Leiden, The Netherlands) in a moist chamber at room temperature for 1 hour. F-actin was stained with rhodamine-phalloidin (50-fold dilution). For primary antibodies we used: The polyclonal anti-CLP36 immunoglobulin (25 µL of 30 µg/mL corresponding to 0.75 µg) or anti-17-peptide immunoglobulin, the monoclonal anti--actinin antibody (clone BM-75.2; Sigma) and the
monoclonal antivinculin antibody (ICN, Aurora, OH), diluted as
recommended. Preimmune serum immunoglobulin (25 µL of 30 µg/mL) or
incubation of permeabilized cells with His-CLP36 (16 µg) for 1 hour
before or 12 hours after addition of anti-CLP36 antibody (0.75 µg)
served as control for the specific staining of CLP36.
The coverslips were washed 3 times and incubated in a solution containing the secondary antibodies fluorescein (FITC)-conjugated goat antirabbit IgG (Dianova, Hamburg, Germany) or Alexa 568 goat-anti mouse IgG dye (sulfonated rhodamine derivative, Molecular Probes), all diluted 1:200. After 3 washing steps, the cells were mounted in Immuno Floure Mounting Medium (ICN). An inverted Leica microscope equipped with the TCS confocal system and an Ar/Kr laser was used to obtain images through × 63 and × 100 objectives (type UV 1,32NA oil PL APO). Stacks of images were analyzed using the LeicaNT software. Cells in Figure 3Ai-Ciii, Figure 4, and Figure 6 were viewed on a Leica fluorescence microscope RBM 3. Cloning of EGFP-clp36 constructs Sequences of clp36 (nt 1-987), clp36 LIM (nt
1-771), and clp36LIM (nt 772-987) were cloned in
p actin-EGFP developed in the laboratory of Dr A. Matus (Friedrich
Miescher-Institute, Basel). pactin-EGFP is identical to the vector
described by Ludin et al,33 except that EGFP (Clontech)
was used instead of GFP. The HindIII site and the ClaI site in the
polylinker at the 5'-end of the EGFP sequence were used in cloning. The
amino terminal primers contained the Kozak consensus sequence GCC GCC
AGC CAT GA after the HindIII site for efficient expression. cDNA
sequences for the construction of gene fusion were obtained by PCR for
clp36 on a pCR-Script Amp SK+ vector containing clp36 cDNA
from HEL cells (see above). The EGFP fusion plasmids were transfected
in HUVECs by electroporation.
Electroporation of HUVECs Cells (2 × 106) were treated with trypsin/EDTA, spun down, washed with PBS, and resuspended in 1 mL cold PBS. Cells were incubated with 20 µg EGFP fusion plasmid for 5 minutes at 0°C and electroporated at 240 V and 960 µF, using the BioRad gene pulser II. Transfected cells were incubated at 0°C for 7 minutes, resuspended in 10 mL pre-warmed RPMI 1640, and plated onto collagen-coated coverslips (see below). After an overnight period of gene expression, cells were fixed and mounted as described (see below) and viewed on a Leica fluorescence microscope RBM 3.Yeast 2-hybrid interactions To define the interacting domains between CLP36 and-actinin-1, the Gal4-based Matchmaker 2-Hybrid System 2 (Clontech)
was used. Nonmuscle -actinin-1 cDNA was obtained from endothelial cell cDNA, using the primers (based on the -actinin-1 sequence) 5'-ATGGACCATTATGATTCTCAGC-3' (forward) and 5'-TTAGAGGTCACTCTCGCCG-3' (backward). The PCR-product was used to reamplify the spectrinlike repeat 1, spectrinlike repeats 1-2, spectrinlike repeats 2-3, spectrinlike repeats 1-3 of -actinin-1, and full-length
-actinin-1. These constructs were subcloned into pACT-2
("prey"), using the EcoRI and XhoI cloning sites. CLP36, CLP36 (aa
1-82), CLP36 (aa 1-104), CLP36 (aa 1-127), CLP36 (aa 81-256), CLP36 (aa
1-256), and CLP36 (aa 257-329) were amplified from the CLP36 cDNA of
HEL cells and subcloned into pAS2-1 ("bait"), using the NdeI and
PstI cloning sites. Bait and prey plasmids were cotransformed into electrocompetent Saccharomyces cerevisiae Y190
(Clontech) (pulse 1, 5 kV, 25 µF, 200  ) and plated onto synthetic
dropout agar (SD medium) containing histidine but no tryptophan
and leucin to select for clones carrying both plasmids. Protein
expression of CLP36 constructs was confirmed by Western blot analysis.
To screen for 2-hybrid interactions, the -galactosidase activity was
determined by qualitative blue/white screening using the Colony-Lift Filter Assay as recommended by the manufacturer (Clontech). The LacZ reporter gene in yeast strain Y190 is under the control of the
GAL1 upstream activating sequence and, therefore, inducible by a
positive 2-hybrid interaction. To assay yeast transformants for LacZ
reporter gene expression, colonies were plated on SD medium lacking
tryptophan and leucine.
To verify positive and negative interactions from the
Protein purification and cloning of CLP36 We purified a 38-kd protein from human platelets and obtained 7 peptides. Their sequences mapped completely with a tentative human consensus sequence present in the EST database. On the basis of this sequence information, specific primers were designed, and the cDNA of the 38-kd protein was obtained by PCR using cDNA from HEL cells. The cDNA contains a 987-bp ORF encoding a protein of 329 amino acids with a calculated molecular mass of 36 072 Da, and a pI of 6.56. Within the deduced amino acid sequence of the ORF, all 7 peptides, which had been obtained by microsequencing of purified P38, were identified (underlined in Figure 1). The protein shows 88% sequence identities with the C-terminal LIM-domain protein of 36 kd from both rat and mouse (Genbank/EMBL accession no. U 23769 and AF 053367). The protein was, therefore, denoted human CLP36. Rat CLP36 was previously cloned from rat hepatocytes by differential screening of a substractive (normoxic minus hypoxic) cDNA library.19 Clp36 was also sequenced after PCR amplification of HUVEC cDNA and found to be identical with that from HEL cells. Furthermore, alignment of the clp36 nucleotide sequence with the sequence data in EST databases (dbEST and preblast_est), which were obtained from many human tissues and cells, showed complete identity to more than a hundred different EST sequences. This is important, because an almost identical human clp36 sequence has been described recently and has been designated as hCLIM1.20 This gene has 3 different nucleotides in positions 60, 61, and 63 that could not be found in the available EST-sequence databases. The latter 2 nucleotide exchanges lead to a glycine-to-arginine exchange in position 21 of hCLIM1.20 Glycine in position 21 is within a conserved domain of the PDZ motif and also present in the other proteins of the PDZ/LIM-domain proteins (see below). One possible reason for the sequence difference between clp36 and hCLIM1 is that hCLIM1, which had been isolated from a human adenocarcinoma cDNA library,20 is a mutated form of clp36.
Human CLP36 contains a C-terminal LIM domain of 51 amino acid residues
displaying the known LIM-consensus motif and an N-terminal PDZ domain
of 81 amino acid residues (shaded in Figure 1). CLP36 belongs to a new
group of LIM proteins that contain an N-terminal PDZ domain and 1 or 3 C-terminal LIM domains. Human CLP36 shows a 47% overall identity to
the human actinin-associated LIM protein (hALP) in striated muscle that
binds to Immunoprecipitation and immunoblotting of CLP36 Polyclonal antibodies directed against the recombinant His-CLP36 and 2 specific peptides of CLP36 (aa 302-314, designated as "14-peptide," and aa 224-239, designated as "17-peptide,"; bold in Figure 1) were used for immunoblot analysis. All 3 antibodies recognized specifically one band of about 38 kd in human platelets. The anti-CLP36 and anti-peptide-17 antibodies were able to specifically immunoprecipitate CLP36 from platelet lysate. Preimmune serum or anti-17-peptide antiserum preadsorbed with the 17-peptide did not immunoprecipitate platelet CLP36. Also, recombinant His-CLP36 added to the platelet lysate effectively competed with endogenous CLP36 for immunoprecipitation (Figure 2A). We found an expression of 105 molecules CLP36 per platelet, corresponding to about 0.3% of the total protein content in platelets (for the measurement and calculation see "Materials and methods").
The analysis of expression of CLP36 in other human cell types by Western blot analysis revealed the presence of CLP36 in venous and arterial human umbilical endothelial cells and HeLa cells, but not in the monocytic cell line U937 (Figure 2B). The presence of CLP36 messenger RNA (mRNA) in endothelial cells was confirmed by performing reverse transcriptase-polymerase chain reaction (RT-PCR) and cDNA sequencing. Immunocytochemical localization of CLP36 in resting and activated platelets Immunofluorescence studies using either anti-CLP36 or anti-17-peptide antiserum were performed on spread platelets that allow, because of their larger size, a higher resolution of cytoskeletal structures. Adherent platelets show various stages of activation from shape change characterized by an irregular cell surface and extrusion of pseudopodia to spreading with the extension of veils between the radially outgrowing filopodia.32,39 These processes are associated with the formation of new actin structures: microfilament bundles in the pseudopodia (Figure 3Ai), lamellipodia at the edge of the extending veil (Figure 3Bi), microfilaments radiating outward from the platelet center, and a thin filament ring at the rim in the fully spread platelet (Figure 3Ci). After completion of the spreading phase, platelets exhibit further distinct actin patterns: Parallel cables of actin filaments resembling stress fibers traversing the platelet or microfilament bundles arranged in a triangular pattern or concentric rings with a center free of filamentous actin are seen (Figure 3Di). In this late stage, platelets can also exhibit a vortexlike actin filament arrangement (Figure 3Eii).40 Double labeling of CLP36 and F-actin in spread human platelets revealed a colocalization (Figure 3). CLP36 was associated with microfilaments in long pseudopodia (Figure 3Ai-ii) and short filopods and lamellipods (Figure 3Bi-ii). In the fully spread platelet, CLP36 was highly concentrated along the radially outgrowing actin filaments without being present in the F-actin-rich center the region where the secretory granules are concentrated. CLP36
could also be found at the rim of the spread platelet, on the inner
side of the cortical actin belt (Figure 3Ci-iii). In late stages of
spreading, CLP36 is seen along actin stress fibers in a dotted regular
pattern along parallel actin bundles (Figure 3Di-ii), although the
overlay (Figure 3Diii) showed only in part a colocalization because of
the different and inhomogeneous intensities of the F-actin and
CLP36 signals.
CLP36 was absent in focal adhesions that localize to the tips of actin stress fibers in these late forms of spread platelets.40 This was visualized by costaining of CLP36 with the focal adhesion protein vinculin (Figure 3Ei,iii). Immunofluorescence microscopy in resting and activated endothelial cells In subconfluent endothelial cells, stress fibers are abundant. In contrast to the controls (Figure 4Ai-ii), specific staining of the cells with the anti-CLP36 immunoglobulin revealed that CLP36 was localized on stress fibers showing a dotted staining pattern (Figure 4Bi-ii). Like in platelets, CLP36 was absent from focal adhesions (visualized by staining with antivinculin antibodies) that are prominent in subconfluent endothelial cells (Figure 5Ci-ii).
In resting confluent endothelial cells, CLP36 colocalized with the peripheral actin filaments beneath the plasma membrane (Figure 4Ci-ii). Confluent endothelial cells activated with thrombin for various times exhibit a dramatic reorganization of their actin cytoskeleton. CLP36 translocated to the newly formed actin structures after thrombin activation (Figure 4Di-ii). The periodic dotted staining pattern of CLP36 on actin stress fibers in
subconfluent and thrombin-stimulated endothelial cells resembled the
decoration of stress fibers with anti- Targeting of heterologously expressed CLP36 to actin stress fibers To examine more directly the localization of CLP36 in vivo and to identify the domain of CLP36 responsible for its targeting to the actin cytoskeleton, we transfected endothelial cells with CLP36 gene constructs that result in a C-terminal fusion protein with EGFP. Transfection of the vector, which only contained EGFP, served as control. Examination of fixed migrating endothelial cells showed that CLP36 and CLP36 containing the PDZ domain and the large intervening sequence but lacking the LIM domain were able to target EGFP to actin stress fibers in a dotted patternsimilar to that seen with indirect
immunofluorescence (Figure 6A-B). The EGFP-CLP36 fusion protein did not target to focal adhesion, confirming our indirect immunofluorescence studies. These proteins also localized to cortical actin filaments. EGFP fused to the LIM domain did not bind
to the actin cytoskeleton (Figure 6Ci-ii). It was found in the
cytoplasm and concentrated in the nucleus. This nuclear trapping may be
due to the small size of the fusion protein (35 kd), letting it pass
the nuclear pores. It was observed also with EGFP alone and is,
therefore, not specific.
CLP36 binds to -actinin
in endothelial cells was due to a direct interaction of the 2 proteins,
CLP36 was immunoprecipitated from platelet lysates (Figure
7A). Western blot analysis showed that
-actinin specifically coimmunoprecipitated with CLP36. Vinculin,
which binds also to -actinin42 but is not expected to
bind to CLP36 (see above), was not coimmunoprecipitated with CLP36
(Figure 7B). The interaction between CLP36 and -actinin was also
detected by the blot overlay assay. Biotinylated CLP36 bound to a
single platelet protein that co-migrated with platelet -actinin
(Figure 7C).
To establish a direct interaction of CLP36 with
To further test whether the LIM domain may be involved in Coordinate and reversible translocation of CLP36 and -actinin (Figure 9). CLP36
and -actinin were present in the membrane skeleton, but no
significant changes of these proteins on stimulation could be observed.
Given the finding that CLP36 binds to -actinin-1 in resting
platelets, these results indicate that both proteins might translocate
as a complex from the cytosol to the F-actin-rich cytoskeleton.
Human CLP36 was purified from platelets and cloned from HEL cells. We found by PCR amplification/cDNA sequencing and Western blot analysis that CLP36 was also expressed in endothelial cells and HeLa cells. CLP36 could not be detected in various human monocytic cell lines. Analysis of the cDNA database revealed that the EST tags, which matched completely the CLP36 cDNA, were derived from many tissues (eg, lung, liver, pancreas, spleen, colon, thyroid, placenta, and retina) and cell types (endothelial cells, senescent fibroblasts, Jurkat T cells, and colon carcinoma cells). Similarly, Northern blot analysis, using hCLIM1 as probe, has revealed a wide human tissue distribution with strong expression in heart and skeletal muscle; moderate expression in spleen, small intestine, colon, placenta, and lung; and weak expression levels in liver, thymus, kidney, prostate, and pancreas.20 Immunofluorescence studies showed that CLP36 translocated to newly
formed actin filaments and stress fibers, but not to focal adhesions,
in both activated platelets and endothelial cells. CLP36 did not
interact directly with F-actin in F-actin cosedimentation assays (data
not shown). Instead we found by coimmunoprecipitation, yeast 2-hybrid
analysis and blot overlay assay that CLP36 bound to The nonmuscle Yeast 2-hybrid analysis indicated that the intervening region of CLP36,
but not its PDZ or LIM domain, bound to the spectrinlike repeats 2 and
3 within the rod domain of CLP36 belongs to a new structurally and functionally distinct group of
proteins containing a C-terminal PDZ and one or more N-terminal LIM
domains. These proteins might link cell signaling to actin dynamics,
because, as far as it has been elucidated, they bind to the actin
cytoskeleton as well as to signaling enzymes: ALP and Cypher expressed
in striated muscle bind through their PDZ domain to
The binding of CLP36 to
A very recent study reports that CLP36 binds via its PDZ-domain to actinin-1 and actinin-4 in colon epithelial cells.54
We greatly appreciate the excellent technical assistance of U. Wielert and C. Meister. Some of the experiments are part of the thesis of K.B. at the University of Munich.
Submitted May 11, 2000; accepted August 23, 2000.
Supported by the Deutsche Forschungsgemeinschaft (Si 274; GRK 438) and the August-Lenz-Stiftung.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Wolfgang Siess, Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Universität München, Pettenkoferstr. 9, D 80336 München, Germany; e-mail: wolfgang.siess{at}klp.med.uni-muenchen.de.
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© 2000 by The American Society of Hematology.
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| Copyright © 2000 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
-actinin-1 and associates with actin filaments and stress fibers
in activated platelets and endothelial cells
Top
Abstract
Introduction
= 40 920 M
1 cm
LIM (aa 1-257) or His-LIM (aa 258-329) (1 hour, 10 µg
each). The his-tag fusion proteins and associated proteins were
subsequently pulled down with anti-RGS-[His]4 antibody (5 µg; Qiagen). The precipitates were analyzed on silver-stained protein SDS gels for the presence of 
indicates colonies that are white in the
X-Gal screening assay; nt, interaction not tested.
