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J. Biol. Chem., Vol. 281, Issue 47, 36347-36359, November 24, 2006
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12
3
From the
Regulatory Biology and Functional Genomics Research Group, Siebens-Drake Medical Research Institute, Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario N6A 5C1, Canada, and the Department of Cell Biology and Physiology, Washington University in St. Louis, St. Louis, Missouri 63110
Received for publication, August 9, 2006 , and in revised form, August 30, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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46 kDa with an amino-terminal pleckstrin homology (PH) domain and a carboxyl-terminal leucine-rich region as well as five putative PXXP motifs. The PH domain of CKIP-1 is required for phospholipid binding in vitro and for plasma membrane localization in cells (1, 2). Furthermore, this domain is necessary for interactions with protein kinase CK2, because mutants lacking the PH domain fail to interact with the kinase. Additionally, we have demonstrated that a subpopulation of protein kinase CK2 is targeted to the plasma membrane by CKIP-1 in cells (2). This targeting of CK2 is lost when the PH domain of CKIP-1 is replaced by a myristoylation recognition sequence, even though the CKIP-1 mutant still localizes to the plasma membrane. These results suggest that CKIP-1 may function in an analogous manner to protein kinase A anchoring proteins, which target cAMP-dependent protein kinase A (3-6). In addition to this potential role as a CK2-targeting protein, CKIP-1 appears to have roles independent of CK2. Recent reports have shown that CKIP-1 functions in muscle cell differentiation (7) and AP-1 regulation and apoptosis (8).
To investigate the cellular functions of CKIP-1, we generated cell lines with tetracycline-regulated expression of FLAG-CKIP-1. Induction of FLAG-CKIP-1 in these cells caused changes in cellular morphology, as well as increases in F-actin and total cellular levels of actin (9). To determine the mechanistic basis for these observations, we performed a proteomic screen using Tandem affinity purification (10) and large-scale immunoprecipitations to identify CKIP-1 interaction partners. We identified the heterodimeric actincapping protein as a novel CKIP-1-interacting protein (9). Moreover, we showed that CKIP-1 can partially inhibit the activity of CP at the barbed ends of actin filaments. Collectively, these observations suggested two hypothetical models. First, it is possible that the interaction of CP with CKIP-1 at the plasma membrane inhibits binding of CP to the barbed ends of the actin filament leading to increased actin polymerization and changes in cellular morphology. Alternatively, the effects of CKIP-1 on cell morphology may require interactions with CP to target CKIP-1 to the barbed ends of actin filaments.
To distinguish between these models, we used peptide "walking arrays" and alignments with the CP-binding protein, CARMIL (11), to identify Arg-155 and Arg-157 as residues of CKIP-1 potentially required for its binding with CP. To investigate the importance of these residues for interactions between CKIP-1 and CP, we employed mutants of CKIP-1 harboring substitutions at Arg-155 and Arg-157 as well as a synthetic peptide encompassing the putative CP-binding region of CKIP-1. Finally, to directly test whether changes in cell morphology and the actin cytoskeleton induced by CKIP-1 require its interactions with CP, we generated human osteosarcoma cell lines expressing CP-binding deficient mutants of CKIP-1 under the control of tetracycline.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(mAb 5B12.3) and CP
(mAb 3F2.3 and mAb 1E5.25.4) subunits of actin-capping protein (12) were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD, National Institutes of Health and maintained by the University of Iowa, Department of Biological Sciences (Iowa City, IA). Polyclonal antibodies against CKIP-1 and GST were described in a previous study (1). Monoclonal antibodies against -tubulin were a generous gift from Dr. Lina Dagnino (Department of Pharmacology, University of Western Ontario). Plasmid Constructs—FLAG-CKIP-1 was generated by PCR amplification and subcloned into pRc/CMV and pTRE as previously described (9). CKIP-1 mutations to disrupt interactions with CP were generated for FLAG-CKIP-1, GFP-CKIP-1, GST-CKIP-1, and/or FLAG-CKIP-1/pTRE using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) as described by the manufacturer.
FLAG-CKIP-1 R133,K135E was generated using the forward primer: 5'-CTC AAC TCT GCC ATC ACC GAG GCC GAG AAC CGT ATC TTG GAT GAG-3' and the reverse primer: 5'-CTC ATC CAA GAT ACG GTT CTC GGC CTC GGT GAT GGC AGA GTT GAG-3'. FLAG-CKIP-1 R133A,K135A,R137A was similarly generated using the forward primer: 5'-CTC AAC TCT GCC ATC ACC GCT GCC GCT AAC GCT ATC TTG GAT GAG GTC ACC-3' and the reverse primer: 5'-GGT GAC CTC ATC CAA GAT AGC GTT AGC GGC AGC GGT GAT GGC AGA GTT GAG-3'. CKIP-1 R155A,R157A constructs were generated using the forward primer: 5'-TCT TGC CCA TCC CAC TGC AGA CGC AGC AAA AAT CCA GCA CTC CCG C-3' and the reverse primer: 5'-GCG GGA GTG CTG GAT TTT TGC TGC GTC TGC AGT GGG ATG GGC AAG A-3'. CKIP-1 R155E,R157E constructs were generated using the forward primer: 5'-TCT TGC CCA TCC CAC TGA GGA CGA GGC AAA AAT CCA GCA CTC CCG C-3' and the reverse primer: 5'-GCG GGA GTG CTG GAT TTT TGC CTC GTC CTC AGT GGG ATG GGC AAG A-3'. Similarly, CKIP-1 R155A,R157A,K159A constructs were generated using the forward primer: 5'-CCC ACT GCA GAC GCA GCA GCA ATC CAG CAC TCC CGC CGC-3' and the reverse primer: 5'-GCG GCG GGA GTG CTG GAT TGC TGC TGC GTC TGC AGT GGG-3' using CKIP-1 R155A,R157A as a template. All mutations were confirmed by DNA sequencing.
Immunoprecipitations—For endogenous CKIP-1 immunoprecipitations, U2-OS cells (9 x 15-cm plates) or C2C12 cells (3 x 10-cm plate) were harvested in radioimmune precipitation assay lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.l% SDS, 1% aprotinin and 0.1 mM phenylmethylsulfonyl fluoride). The lysates were sonicated on ice with four 4-s bursts and then centrifuged at 55,000 rpm in a Beckman TL120.2 rotor for 15 min at 4 °C. The cleared lysates were divided into three aliquots, and precleared with pansorbin and protein G beads. Immunoprecipitations were then performed with anti-CKIP-1 or as a control with anti-GST antibodies bound to protein A-Sepharose for 1 h at 4 °C. An additional control employed protein A-Sepharose in the absence of antibody. For FLAG and GFP immunoprecipitations, U2-OS cells were lysed on ice in 0.5 ml of Nonidet P-40 lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 1% aprotinin, and 0.1 mM phenylmethylsulfonyl fluoride) or radioimmune precipitation assay lysis buffer. The lysates were sonicated and cleared as described above. The cleared lysates were subjected to immunoprecipitation using anti-FLAG M2 antibodies bound to protein G-Sepharose or anti-GFP antibodies bound to protein A-Sepharose for 1 h at 4 °C. In all cases, the lysates were centrifuged briefly, and the protein A/G-Sepharose beads were washed four times with the appropriate lysis buffer. Following the last wash, bound proteins were eluted from the beads by the addition of sample buffer.
Immunoblot Analysis—Samples were separated by 12% SDS-PAGE using the method of Laemmli (13). Proteins were transferred to polyvinylidene difluoride membranes for 1 h at 15 V, 0.3 A using a semidry transfer apparatus (Bio-Rad, Hercules, CA). Anti-FLAG blots were performed according to the manufacturer's instructions using anti-FLAG M2 at 1:2,500 and goat anti-mouse secondary antibody (1:20,000) conjugated to horseradish peroxidase. Anti-actin blots were performed according to the manufacturer's instructions using anti-actin at 1:2,000 and goat anti-rabbit secondary antibody (1:20,000) conjugated to horseradish peroxidase. Anti-GFP immunoblots were performed by blocking for 1 h in 3% gelatin in TBST (Tris-buffered saline with 0.05% Tween-20), followed by incubation with primary antibody at 1:3,000. Immune complexes were detected with goat anti-rabbit secondary antibodies (1:20,000) conjugated to horseradish peroxidase. Immunoblotting with anti--tubulin antibodies was performed by blocking for 1 h in 5% skim milk powder in TBST, followed by incubation with primary antibody (1:100). Immune complexes were detected using goat anti-mouse secondary antibodies (1:5,000) conjugated to horseradish peroxidase. Anti-CP and -CP blots were performed by blocking membranes for 1 h in 3% bovine serum albumin in TBST followed by incubation with primary antibodies at dilutions of 1:200 and 1:1,000, respectively. In both cases, secondary goat anti-mouse antibodies were used at a dilution of 1:5,000. Where indicated, membranes were stripped with 0.1 M NaOH for 30 min.
Peptide Array Generation—The SPOT method (14, 15) was employed in the synthesis of peptide arrays for use in determining the residues of CKIP-1 required for interaction with CP. Peptides were synthesized on Amino-PEG500-UC540 membranes derivatized with a polyethylene glycol spacer (Intavis AG). Stock solutions of amino acid derivatives (Peptides International) were prepared at a concentration of 0.33 M in a solution of 0.5 M 1-hydroxybenzotriazole/N-methyl-2-pyrrolidone. Immediately prior to each cycle of synthesis, amino acid solutions were activated with the addition of one part diisopropyl-carbodiimide (DIC) solution (1.1 M DIC in N-methyl-2-pyrrolidone) to three parts of the amino acid stock solution. Each cycle of peptide synthesis consisted of (i) coupled spotting of amino acids on the membrane, (ii) capping of the membrane with a solution of 2% acetic anhydride in DMF (1 x 30 s, 1 x 5 min), (iii) wash with DMF (3 x 2 min), (iv) deprotection with 20% piperidine in DMF (10 min), (v) wash with DMF (4 x 2 min), (vi) wash with ethanol (2 x 2 min), and (vii) drying of membranes in a stream of cold air and preparation for the next cycle of synthesis. After the final cycle in the peptide synthesis, the membrane was thoroughly dried overnight in a fume hood to prepare the peptides for side-chain deprotection. Side-chain deprotection was carried out on the membrane in the following steps: (i) deprotection in 95% trifluoroacetic acid, 3% triisopropylsilane, 2% water for 2 h, (ii) wash with dichloromethane (4 x 2 min), (iii) wash with DMF (4 x 2 min), (iv) wash with ethanol (2 x 2 min), and (v) dry with a stream of cold air.
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1- and 1-subunits or mouse 1- and 2-subunits from a single plasmid (16). CP was expressed and purified to homogeneity from BL21(DE3) Escherichia coli, as described (17). Chicken CP was used for GST pulldown assays and peptide array assays. Mouse CP was used for in vitro actin polymerization assays.
Peptide Overlay Assay—Prior to the overlay assay, the membrane was wetted with 95% ethanol and water. The peptide filter was then blocked for 1 h in 2% bovine serum albumin in Tris-buffered saline followed by incubation with CP (1 µM) overnight at 4 °C with rocking. Following washing, the filter was incubated with antibodies against native CP (anti-CP 1E5.25.4) at a dilution of 1:200. Bound CP was detected with goat anti-mouse secondary antibodies (1:5000) conjugated to horseradish peroxidase.
Isothermal Titration Calorimetry—ITC experiments were carried out on a MicroCal VP-ITC isothermal titration calorimeter (18). The heterodimeric actin-CP was purified as described (17). Purified CP and CKIP-1 peptide (SYLAHPTRDRAKIQHSRRPPTR) were dialyzed into 10 mM potassium phosphate, pH 7.2, 100 mM KCl, 1 mM Tris (2-carboxyethyl)phosphine hydrochloride and 5% glycerol. For titration, purified CP and CKIP-1 peptide were diluted to 18 and 180 µM, respectively, using the equilibrating solution obtained after dialysis was complete, and each was degassed prior to titration. CP protein was loaded into the cell (1.4 ml), and CKIP-1 peptide was loaded into the syringe. Titration of the CKIP-1 peptide was performed at 25 °C starting with an initial 1-µl injection, followed by 65 5-µl injections, with a spacing of 300 s. The sample cell was stirred at 300 rpm throughout the experiment. The buffer blank performed under the same conditions showed negligible heats of binding (<0.015 µcal/s). The dissociation constant (KD) and stoichiometry of binding (n) were obtained by non-linear least-squares fitting of the CP versus CKIP-1 pep-tide data to a one-site model provided with the data analysis software (Origin). Baseline was subtracted automatically using Origin software. The results of two independent experiments are shown.
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In Vitro Translation Assays and GST Pull-down Assays—Radiolabeled (35S-lableled) FLAG-CKIP-1 and FLAG-CKIP-1 R155E,R157E were produced by in vitro transcription and translation using a TNT kit (Promega, Madison, WI) with T7 polymerase according to the manufacturer's recommendations. GST fusion proteins encoding CK2 or GST alone were expressed in bacteria and purified using glutathioneagarose as described previously (20). Following in vitro transcription and translation, GST pull-down assays were performed by diluting 5 µl of the TNT reactions with 50 µl of Nonidet P-40 buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet P-40) and adding 11 µl of GST or GST-CK2 beads. Incubation was at 4 °C for 1 h with rocking. The beads were collected by centrifugation, the supernatant was removed, and the beads were washed three times with 400 µl of Nonidet P-40 buffer. Bound proteins were eluted by the addition of sample buffer and were subjected to SDS-PAGE on a 12% gel. After drying, the radiolabeled proteins were visualized using a PhosphorImager (Amersham Biosciences) and quantitated for two independent experiments using ImageQuaNT software.
Generation of Cell Lines—UTA6 cells were derived from the human osteosarcoma cell line U2-OS and express the tetracycline-regulated transcriptional activator protein (generous gift from Dr. Christoph Englert, Forschungszentrum, Germany (21)). Generation of a cell line with tetracycline-regulated expression of FLAG-CKIP-1 has been previously described (9). Cell lines expressing FLAG-CKIP R155E,R157E were generated by transiently transfecting UTA6 cells with mutant FLAG-CKIP-1/pTRE and pTK-Hyg in the presence of tetracycline (1.5 µg/ml). Drug selection with hygromycin (500 µg/ml) and G418 (460 µg/ml) began 4 days after transfection. Once stably transfected colonies were formed, they were picked and transferred to 96-well plates. Individual colonies of FLAG-CKIP-1 R155E,R157E-expressing cells were grown up and screened for highly regulated inducible expression by immunoblot analysis.
Immunofluorescence—Cells (200,000) grown on sterile coverslips were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Invitrogen) at 37 °C in an atmosphere of 5% CO2. Coverslips were washed three times with PBS and fixed for 20 min in 3.7% paraformaldehyde at 37 °C. Cells were permeabilized for 5 min with 0.1% Triton X-100 in PBS and then treated with for 5 min with 0.1 M glycine in PBS. Coverslips were washed with PBS and then incubated with anti-FLAG M2 at 1:250 for 1 h at 37 °C. Following washing with PBS, secondary antibodies conjugated to TRITC (1:1,000) were added for 1 h in the dark. Coverslips were washed, mounted with Gelvatol (Celvol 205, Celanese Chemicals, Dallas, TX), and examined with a Zeiss LSM 510 confocal microscope.
Phalloidin Staining, Extraction, and Quantitation—For TRITC-phalloidin staining of cells, coverslips were washed three times with PBS and fixed for 20 min in 3.7% paraformaldehyde at 37 °C. Following permeabilization with 0.1% Triton X-100, coverslips were stained with TRITC-phalloidin (1 µg/ml) for 40 min in the dark, washed with PBS, and mounted with Gelvatol. Slides were visualized using a Zeiss Axiovert inverted fluorescence microscope and Northern Elite software. F-actin quantitation experiments were performed by the method of Machesky et al. (22). Briefly, triplicate plates of cells were washed 2x with PBS and scraped into 400 µl of F-actin stabilizing extraction buffer (10 mM Pipes, pH 7.0, 20 mM potassium phosphate, 5 mM EGTA, 2 mM MgCl2, 0.1% Triton X-100) with 3.7% paraformaldehyde and 2 µM TRITC-phalloidin and rocked for 1 h at room temperature. The cells were pelleted by brief centrifugation and washed 2x with 200 µl of 0.1% saponin, 20 mM potassium phosphate, 10 mM Pipes, pH 7.0, 5 mM EGTA, and 2 mM MgCl2. The bound phalloidin was extracted in 1 ml of methanol by rocking1hat room temperature, and TRITC levels were determined by reading the emission at 563 nm following excitation at 542 nm. Values were normalized to the overall protein concentration and represent the average of three 10-cm plates ± S.D. from two independent experiments.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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is detected in anti-CKIP-1 immunoprecipitates from extracts of U2-OS and C2C12 cells, CP is not detected following immunoprecipitation with control anti-GST (IgG) antibodies or with protein A beads alone. These observations reinforce the physiological relevance of CP-CKIP-1 interactions.
Identification of CP-binding Sites on CKIP-1—We have previously demonstrated using deletion analysis that interaction between CKIP-1 and CP is dependent on amino acids 133-193 of CKIP-1 (9) (highlighted in Fig. 1B). To identify residues required for this interaction more precisely, we generated a CKIP-1 peptide filter composed of 16-mers "walking" from the amino to carboxyl termini of this region, with frame shifts of three amino acids between each peptide (Fig. 1C). The resulting filter was incubated with recombinant CP (see inset) followed by detection with antibodies against native CP. As shown in Fig. 1C, two peptides exhibited interactions with CP. A multiple sequence alignment of the CP-binding domain of CKIP-1 reveals that both peptides, designated site 1 and site 2, appear to be very well conserved between species (Fig. 1D).
Recently, a study by Yang et al. (11) demonstrated that the interaction of CARMIL with CP requires two conserved basic residues, Lys-991 and Arg-993. It was therefore intriguing that both site 1 and site 2 (Fig. 1D) contain basic-X-basic motifs. Consequently, we performed an alignment of CARMIL (residues 969-1008) with the region of CKIP-1 encompassing sites 1 and 2. As illustrated in Fig. 1E, the sequence encompassing site 2 could be aligned with the CARMIL sequence suggesting that the residues necessary for interactions between CARMIL and CP might be present in CKIP-1. Moreover, the equivalent residues in CKIP-1 (Arg-155 and Arg-157) are highly conserved between species (Fig. 1D).
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To distinguish between these two binding sites, we tested if mutations within these regions affected interactions with CP by immunoprecipitation assays. U2-OS cells were transfected with FLAG-CKIP-1 constructs as indicated, and the derived lysates were immunoblotted with anti-FLAG M2 antibodies to demonstrate equal loading (Fig. 2B, left panel). Immunoprecipitations were performed on these lysates using anti-FLAG M2 antibodies followed by immunoblotting with anti-CP antibodies (Fig. 2B, right panel). As shown, mutation of R155E,R157E in putative site 2 resulted in greatly diminished binding to CP as did R155A,R157A or R155A,R157A,K156A (data not shown). By comparison, CKIP-1 harboring R133A,K135A,R137A substitutions (Fig. 2B) or R133,K135E substitutions (data not shown) in site 1 had no effect on binding to CP. These results indicate that disruption of Arg-155 and Arg-157 within site 2 on CKIP-1 is sufficient to prevent interaction with CP, whereas similar substitutions within site 1 do not affect CKIP-1-CP interactions.
To further characterize CKIP-1 with substitutions within site 2, we next examined the effects of various mutations on binding to CP using immunoprecipitation and GST pull-down assays (Fig. 3). For immunoprecipitations, U2-OS cells were transfected with FLAG-CKIP-1 constructs, and the lysates derived from these cells were immunoblotted with anti-FLAG M2 antibodies to demonstrate equal loading (Fig. 3A, left panel). The lysates were immunoprecipitated with anti-FLAG M2 antibodies and immunoblotted with anti-CP antibodies (Fig. 3A, right panel). Compared with wild-type CKIP-1, all three mutants exhibit greatly reduced binding to CP. Similar experiments were performed with GFP-tagged constructs (Fig. 3B). Again, the three CKIP-1 mutants exhibit greatly diminished binding to CP.
To complement the results of the immunoprecipitation assays, we employed GST pull-down assays. GST and GST-CKIP-1 were purified on glutathione beads and stained with GelCode Blue to demonstrate equivalent protein expression (Fig. 3C, left panel). The GST fusions were used to perform pull-down assays with purified recombinant CP (Fig. 3C, right panel). In good agreement with the results described above, both mutants exhibit greatly reduced binding to CP. Collectively, these data suggest that Arg-155, Arg-157, and possibly Lys-159 of CKIP-1 are required for interaction with CP and imply that CKIP-1 and CARMIL may share similar CP-binding residues.
Dissociation Constant for CP Interactions with CKIP-1 Peptide—To further characterize interactions between CKIP-1 and CP, we employed a synthetic 22-residue peptide corresponding to the area of CKIP-1 responsible for interactions with CP as determined in Fig. 1. Based on two independent measurements, the average dissociation constant for this interaction was determined to be 9.5 x 10-8 M, fitting the data to a one-site binding model (Fig. 4). Notably, this value compares favorably with measurements recently made for the interactions between CP and V-1 (23) as well as interactions between CP and CD2AP, another CP-binding protein with a similar basic-X-basic motif to that found in CKIP-1 and CARMIL (24). It is also notable that in the study by Bruck et al. (24), the CKIP-1 peptide performed at least as well as analogous peptides derived from CD2AP and CARMIL-1 in inhibiting interactions between CD2AP and CP, an observation consistent with the conclusion that CKIP-1 shares a CP-binding motif with these other proteins. Collectively, these data extend the biochemical characterization of interactions between CKIP-1 and CP and further reinforce the physiological relevance of these interactions.
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50% inhibition, consistent with our previous results (9). In contrast, the R155E,R157E and R155A,R157A,K159A CKIP-1 mutants had no effect on the capping activity of CP (Fig. 5B). Thus, the functional assays agree that these CKIP-1 mutants do not interact with CP. We extended the functional analysis of the CP-binding region of CKIP-1 in similar assays with the 22-amino acid CKIP-1 peptide spanning site 2 (Fig. 5C). First, we found that addition of the peptide had a small inhibitory effect on the capping activity of CP. We also attempted to use a wider range of peptide concentrations, but we were only successful at inhibiting CP activity at concentrations up to 500 nM. We do not know the precise reason for the inability to use higher peptide concentrations; however, we anticipate that this may be a limitation of the polymerization assay and/or the solubility of the peptide. In the presence of full-length CKIP-1, addition of the peptide suppressed the inhibitory effect of CKIP-1 on the activity of CP. Both full-length CKIP-1 and the peptide were present at concentrations near saturation in this assay, so the results are consistent with the peptide competing with CKIP-1 for binding CP. If the peptide and CKIP-1 interacted with different regions of CP, one would have expected an additive effect from the combination. These results are consistent with the proposed CP-binding region of CKIP-1 being sufficient for binding CP and having a direct role in the interaction of CKIP-1 with CP.
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CKIP-1 R155E,R157E Retains the Ability to Interact with Protein Kinase CK2 and to Self-associate—To further characterize the properties of this CKIP-1 mutant, we designed experiments to examine interactions between CKIP-1 and CK2 as well as self-association of CKIP-1. Because the PH domain of CKIP-1 is responsible for interactions with protein kinase CK2 (2), mutation of Arg-155 and Arg-157, which lie outside of this domain, would not be expected to influence interactions with CK2. Nevertheless, to test that the R155E,R157E mutations have not disrupted interactions between CKIP-1 and protein kinase CK2, plasmids encoding wild-type FLAG-CKIP-1 and FLAG-CKIP-1 R155E,R157E were subjected to in vitro transcription and translation in the presence of [35S]methionine (Fig. 7A). Translated products were utilized in pull-down assays with GST-CK2 or GST alone, and bound proteins were separated by SDS-PAGE and visualized using a PhosphorImager (Fig. 7A, top panel). As shown, both wild-type and mutant CKIP-1 retain the ability to interact with protein kinase CK2. To quantitate these results, the data for two independent experiments were analyzed using ImageQuaNT software and expressed as percentage of bound radiolabeled protein (Fig. 7A, lower panel). These data suggest that the two CKIP-1 constructs bind protein kinase CK2 to the same degree.
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Inducible Expression of FLAG-CKIP-1 R155E,R157E—Our results in Figs. 2, 3, 4, 5, 6 and 7 indicate that the CKIP-1 R155E,R157E mutant is an ideal candidate to distinguish between our working models for the effects of CKIP-1 on cell morphology, because it fails to interact with CP yet retains other known characteristics of wild-type CKIP-1. Therefore, we generated U2-OS cell lines with tetracycline-regulated expression of FLAG-CKIP-1 R155E,R157E. For these experiments, FLAG-CKIP-1 R155E,R157E was ligated into the pTRE vector immediately downstream of the tet-responsive promoter. Following transfection and drug selection, stably transfected colonies were picked and screened for highly regulated inducible expression of mutant FLAG-CKIP-1 in the absence of tetracycline. U2-OS cells expressing wild-type CKIP-1 (DC1.4) or CKIP-1 R155E,R157E (DC3.20) were grown in the presence (+) or absence (-) of tetracycline, fixed, and labeled with anti-FLAG M2 antibodies and visualized by confocal microscopy (Fig. 8A). Both cell lines exhibit strong induction and tight regulation of CKIP-1 expression. To examine protein expression, cells grown in the presence (+) of tetracycline or induced (-) for 24 h were harvested. These lysates were subjected to immunoblotting with anti-FLAG M2 antibody (Fig. 8B, upper panel). Again, induction is tightly regulated, and both cell lines appear to express similar levels of FLAG-CKIP-1. To ensure equal protein loading, the membrane was stripped and reprobed with anti--tubulin antibodies (Fig. 8B, lower panel).
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100 cells from numerous fields of view (Fig. 8D). The results from this experiment clearly show that cells induced to express wild-type CKIP-1 have a significantly greater perimeter than noninduced cells, whereas induction of CKIP-1 R155E,R157E expression has no effect on cell shape. FLAG-CKIP-1 R155E,R157E Does Not Increase Phalloidin Binding or Cellular Actin Levels—Previously, we documented a 1.5-fold increase in phalloidin staining and modest increases in cellular actin levels associated with CKIP-1 expression (9). Based on these results, we asked whether induction of CKIP-1 R155E,R157E would elicit the same response by examining the F-actin profile of these cells using fluorescently-labeled phalloidin. The wild-type and mutant CKIP-1 cell lines were grown in the presence or absence of tetracycline and then stained with TRITC-phalloidin (Fig. 9A). Induction of FLAG-CKIP-1 caused an increase in overall phalloidin staining, along with increased numbers of transverse actin stress fibers and actin aggregates, consistent with our previous report (9). In contrast, induction of FLAG-CKIP-1 R155E,R157E failed to cause any apparent change in the actin cytoskeleton. To extend these results, we performed quantitative measurements of TRITC-phalloidin binding (Fig. 9B). In accordance with our previous study, the data for wild-type CKIP-1 expression, normalized to extract protein, clearly shows a 1.5-fold increase in phalloidin binding. On the other hand, increased phalloidin staining was not observed following induction of FLAG-CKIP-1 R155E,R157E. These data demonstrate that induction of wild-type FLAG-CKIP-1 results in an increase in the overall amount of cellular F-actin, whereas induction of a CKIP-1 mutant that fails to interact with CP does not.
Finally, we examined the total cellular levels of actin in these cell lines, grown in the presence of tetracycline or induced for 18 h. As reported, induced expression of FLAG-CKIP-1 results in a modest increase in the cellular levels of actin (Fig. 9C, left panel) (9). However, induction of FLAG-CKIP-1 R155E,R157E expression failed to cause a similar increase. The membrane was stripped and reprobed with anti--tubulin antibodies to ensure equal protein loading on the gel (Fig. 9C, right panel). Quantification of actin levels by densitometry and normalization to -tubulin levels from two independent experiments illustrated that actin levels are increased by 30% in cells expressing FLAG-CKIP-1, whereas there was no corresponding increase in FLAG-CKIP-1 R155E,R157E cells. Taken together, these data suggest that interaction with CP is necessary for induced CKIP-1 to increase levels of total and F-actin in cells.
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DISCUSSION |
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Given that expression of CKIP-1 affects cell morphology and the cytoskeleton, it is interesting that expression of CARMIL in human glioblastoma cells was shown to increase the number and size of lamellipodia (11). Moreover, small interference RNA-induced depletion of CARMIL caused a decrease in F-actin and in lamellipodia that could be rescued by expression of wild-type CARMIL but not deletions lacking a region implicated in binding CP. These data suggest that CARMIL, through interactions with CP, can alter both cell morphology and the actin cytoskeleton. Our results also reinforce the conclusion that CKIP-1 and CARMIL contain conserved CP-interaction residues, or a possible CP-binding motif as recently suggested by Bruck et al. (24). This observation suggests that CKIP-1 is a member of a family of proteins that can modulate the activity of capping protein, and therefore actin polymerization and cell morphology.
We proposed two working models for the role of CKIP-1 in cell morphology. Our first model suggested that the interaction of CP with CKIP-1 at the plasma membrane inhibits binding of CP to the barbed ends of the actin filament leading to the generation of a "capping-free" zone at the plasma membrane and to increased actin polymerization and changes in cellular morphology. However, the inhibition of CP by CKIP-1 is only partial (9) raising the alternative model in which the effects of CKIP-1 on cell morphology involve targeting of CKIP-1 to the barbed ends of actin filaments by CP. Our results indicate that disruption of binding to CP did not affect the localization of CKIP-1 in cells, arguing against the second model. In addition, the disruption of CP-binding did cause CKIP-1 to lose the ability to change the actin cytoskeleton and the shape of cells, favoring the first model. This hypothetical model is illustrated on the left hand side of Fig. 10. Inhibition of CP activity would lead to an increase in free barbed ends and, therefore, an increase in F-actin. This model is also consistent with reports in the literature. For example, expression of CP mutants that fail to interact with actin in Saccharomyces cerevisiae led to an increase in F-actin (42). In a similar vein, as noted earlier, depletion of CP from D. discoideum or L. monocytogenes resulted in affects on the actin cytoskeleton as did short hairpin RNA-induced depletion of the CP-inhibitory protein CARMIL in human glioblastoma cells (11). Taken together, these studies suggest that inhibition of CP leads to polymerization of actin filaments in vivo. As illustrated in Fig. 10, our previous studies demonstrated that CK2 phosphorylates CP at S9 and that CK2 increases the inhibitory activity of CKIP-1 on CP, although this inhibitory activity appears to be phosphorylation-independent (9). In the future, it will clearly be of interest to define the precise role of CK2 in the regulation of CP and the role of CKIP-1 in the control of this event.
In addition to providing new insights regarding the role of CKIP-1 and its interactions with CP in the regulation of cell morphology and the cytoskeleton, this study establishes a foundation for further investigation. We have shown that both the morphology change and the increase in F-actin are dependent on interactions between CKIP-1 and CP, because these changes are prevented by disrupting the interactions CKIP-1 and CP. In this respect, these studies have led us closer to an understanding of how CKIP-1 regulates the actin cytoskeleton through interactions with CP.
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FOOTNOTES |
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1 Supported by a Canadian Graduate Scholarship from the CIHR.
2 Supported by a CIHR Doctoral Award.
3 To whom correspondence should be addressed: Dept. of Biochemistry, University of Western Ontario, Medical Sciences Bldg., London, Ontario N6A 5C1, Canada. Tel.: 519-661-4186; Fax: 519-661-3175; E-mail: litchfi{at}uwo.ca.
4 The abbreviations used are: CKIP-1, CK2-interacting protein 1; CK2, protein kinase CK2 or casein kinase II; CP, actin-capping protein; GFP, green fluorescent protein; mAb, monoclonal antibody; GST, glutathione S-transferase; DMF, N,N-dimethylformamide; ITC, isothermal titration calorimetry; PBS, phosphate-buffered saline; TRITC, tetramethylrhodamine isothiocyanate.
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