A, endogenous CKIP-1 and CP interact in U2-OS and C2C12 cells. Lysates derived from U2-OS and C2C12 cells were immunoprecipitated with anti-CKIP-1 antibodies and immunoblotted with anti-CPβ antibodies. Control immunoprecipitations were performed with anti-GST antibodies (IgG) or protein A-Sepharose beads alone. B, schematic illustrating the 60-amino acid domain of CKIP-1 required for interaction with CP. C, peptide walking array analysis of the region of CKIP-1 required for CP binding. CKIP-1 peptides (16-mers, frame shifted by three amino acids) were synthesized using the SPOT method as described under “Materials and Methods.” An overlay assay was performed using recombinant CP (1 μm, see inset) followed by detection with antibodies against native CPβ. Two peptides found to mediate interactions with CP are indicated. D, the region spanning these two peptides (101–199 amino acids) was aligned with sequences of CKIP-1 from various species using ClustalW. Completely conserved residues (*), highly conserved residues (:), and conserved residues (.) are indicated. Both CP-binding sites are well conserved between species. E, alignment of the putative CP-binding region on CKIP-1 with the CP-binding site on CARMIL. The residues highlighted in red in the CARMIL sequence were shown to be necessary for its interactions with CP.

A, schematic representation of the CKIP-1 constructs generated to disrupt interaction with CP. B, U2-OS cells were transfected with FLAG-CKIP-1 constructs or empty vector as indicated. Lysates were immunoblotted with anti-FLAG M2 antibodies to demonstrate equivalent expression (left panel). Immunoprecipitations were performed with anti-FLAG M2 antibodies, followed by immunoblotting with anti-CPβ antibodies (right panel). The positions of FLAG-CKIP-1, CPβ, and the heavy and light chains (HC and LC) of IgG are indicated.

A, U2-OS cells were transfected with FLAG-CKIP-1 constructs or empty vector as indicated. Lysates were immunoblotted with anti-FLAG M2 antibodies to demonstrate equivalent expression (left panel). Anti-FLAG M2 immunoprecipitations were performed followed by immunoblotting with anti-CPβ antibodies (right panel). B, U2-OS cells were transfected with GFP-CKIP-1 constructs or GFP alone as indicated. Cell extracts were immunoblotted with anti-GFP antibodies to demonstrate equivalent expression (left panel). Immunoprecipitations were performed with anti-GFP antibodies, followed by immunoblotting with anti-CPβ antibodies (right panel). C, GST and GST-CKIP-1 were purified as described under “Materials and Methods” and visualized using GelCode Blue to demonstrate equivalent protein expression (left panel). Recombinant CP (0.4 μg) was incubated with GST or GST-CKIP-1 constructs bound to glutathione beads. Pull-down assays were immunoblotted with anti-CPα antibodies (right panel). The positions of CPα, CPβ, and the heavy and light chains (HC and LC) of IgG are indicated. The designation of CKIP-1 mutants is as in Fig. 2.

ITC was performed at 25 °C using purified CP (18 μm) and CKIP-1 peptide (180 μm). Data were fitted to a one-site binding model using Origin software. The results of two independent experiments are shown, with the estimated number of binding sites (n) and the dissociation constant (K_D) indicated.

Actin polymerization from seeds is measured over time. The addition of 4 nm CP blocks actin polymerization by capping the growing barbed ends, and CKIP-1 inhibits the capping activity of CP. A, CKIP-1 (80 nm) inhibits CP activity by ~50%. B, point mutations of the critical conserved residues of CKIP-1 abolish its ability to inhibit CP. Two different CKIP-1 point mutants have no effect at 200 nm, under conditions where 20 nm and 80 nm of wild-type CKIP-1 inhibit CP. C, peptides corresponding to the critical conserved region of CKIP-1 inhibit CKIP-1 interaction with CP. Addition of the CKIP-1 peptide lessens the inhibitory effect of whole CKIP-1 on CP. The peptide alone has a small effect on CP. The concentration of the peptide was 500 nm, and the CKIP-1 concentration was 80 nm. In each panel, a control without seeds is shown. The designation of CKIP-1 mutants is as in Fig. 2.

FLAG- and GFP-tagged CKIP-1 mutants, designated as in Fig. 1, exhibit comparable membrane localization to wild-type CKIP-1. A, U2-OS cells transfected with FLAG-CKIP-1 constructs were subjected to anti-FLAG M2 immunofluorescence and visualized by confocal microscopy. B, U2-OS cells transfected with GFP-CKIP-1 constructs were fixed to visualize GFP fluorescence by confocal microscopy.

A, constructs expressing FLAG-CKIP-1, FLAG-CKIP-1 R155E,R157E, or empty vector were subjected to in vitro transcription and translation and GST pull-down assays as described under “Materials and Methods.” Radiolabeled proteins bound to the GST fusion proteins were visualized using a PhosphorImager (top panel). The positions of FLAG-CKIP-1 and CKIP-1 are indicated. Data were analyzed using ImageQuaNT software and expressed as the percentage of bound radiolabeled product for two independent experiments (lower panel). B, U2-OS cells were co-transfected with GFP-CKIP-1 along with FLAG-CKIP-1, FLAG-CKIP-1 R155E,R157E, or empty vector as indicated. Cell extracts were immunoblotted with anti-GFP antibodies to demonstrate equivalent expression (left panel). Lysates were then subjected to immunoprecipitations with anti-FLAG M2 antibodies and immunoblotting with anti-GFP antibodies.

A, anti-FLAG M2 immunofluorescence of DC1.4 and DC3.20 cells grown in the presence or absence of tetracycline. B, DC1.4 and DC3.20 cells were grown in the presence or absence of tetracycline for 24 h to induce expression of wild-type FLAG-CKIP-1 or FLAG-CKIP-1 R155E,R157E. Total cell lysate (40 μg) was immunoblotted with anti-FLAG M2 antibodies (top panel). To ensure equal loading, the membrane was stripped and reprobed with anti-β-tubulin (lower panel). C, DC1.4 and DC3.20 cells were grown in the presence or absence of tetracycline and microscopic images were captured after 24-h induction. D, perimeters of DC1.4 and DC3.20 cells were measured and quantitated using Northern Elite software. Results shown represent the average of ~100 cells in 6 fields ± S.D.

A, DC1.4 and DC3.20 cells grown in the presence of tetracycline or induced for 24 h were stained with TRITC-phalloidin. B, F-actin quantitation of DC1.4 and DC3.20 cells grown in the presence or absence of tetracycline for 24 h was performed as described under “Materials and Methods.” Data represent the average of two independent experiments each using three 10-cm plates of cells normalized for protein concentration (±S.D.). C, DC1.4 and DC3.20 cells were grown in the presence or absence of tetracycline for 18 h. Lysates prepared with radioimmune precipitation assay buffer were immunoblotted with anti-actin antibodies (left panel). To ensure equal loading, the membrane was stripped and reprobed with anti-β-tubulin (right panel).

Interactions between CKIP-1 and CP at the plasma membrane inhibit binding of CP to the barbed ends of actin filaments leading to increased actin polymerization and changes in cellular morphology (left side). CK2 has been shown to phosphorylate CPα at serine 9. The presence of CK2 increases the inhibitory effect of CKIP-1 on CP; however, this inhibition is phosphorylation-independent. In cells, CP is also inhibited by other cellular components, including PIP2 and CARMIL. Disruption of the interactions between CKIP-1 and CP rescues the ability of CP to bind to the barbed ends of actin filaments (right side). This results in basal actin polymerization and parental U2-OS cell morphology. The inset illustrates microscopic images of cells expressing wild-type CKIP-1 or CKIP-1 R155E,R157E. Actin monomers are shown as the “v” symbols. CP is shown as a monomer for simplicity. CKIP-1 is shown as a dimer, but its exact oligomeric structure is unknown.