Characterization of the Ca2+ -regulated ezrin-S100P interaction and its role in tumor cell migration

Abstract

Ezrin is a multidomain protein providing regulated membrane-cytoskeleton contacts that play a role in cell differentiation, adhesion, and migration. Within the cytosol of resting cells ezrin resides in an autoinhibited conformation in which the N- and C-terminal ezrin/radixin/moesin (ERM) association domains (ERMADs) interact with one another. Activation of the ezrin membrane-cytoskeleton linker function requires an opening of this interdomain association that can result from phosphatidylinositol 4,5-bisphosphate binding to the N-ERMAD and threonine 567 phosphorylation in the C-ERMAD. We have shown that ezrin can also be activated by Ca(2+)-dependent binding of the EF-hand protein S100P. We now provide a quantitative analysis of this interaction and map the respective binding sites to the F2 lobe in the ezrin N-ERMAD and a stretch of hydrophobic residues in the C-terminal extension of S100P. Phospholipid binding assays reveal that S100P and phosphatidylinositol 4,5-bisphosphate compete to some extent for at least partially overlapping binding sites in N-ERMAD. Using interaction-competent as well as interaction-incompetent S100P derivatives and permanently active ezrin mutants, we also show that the protein interaction and a resulting activation of ezrin promote the transendothelial migration of tumor cells. Thus, a prometastatic role of ezrin and S100P that had been proposed based on their overexpression in highly metastatic cancers is probably due to a direct interaction between the two proteins and the S100P-mediated activation of ezrin.

FIGURE 1.

Co-localization and co-immunoprecipitation of ezrin and S100P in SK-BR-3 cells. A, ezrin and S100P co-localize to plasma membrane protrusions of stimulated SK-BR-3 cells. Serum-starved SK-BR-3 cells were stimulated with fetal calf serum for 30 min and then fixed with paraformaldehyde, permeabilized, and stained with rabbit anti-ezrin polyclonal and mouse anti-S100P monoclonal antibodies followed by appropriate fluorescently labeled secondary antibodies. The lower panels show a higher magnification of the area indicated highlighting the co-localization of ezrin and S100P to microvillar membrane protrusions. Bars, 10 μm. B, co-immunoprecipitation. A PNS from SK-BR-3 cells expressing GFP-S100P was subjected to immunoprecipitation with an anti-ezrin polyclonal antibody (IP) or with an unspecific polyclonal antibody as control (C). The starting material (PNS) and the precipitated proteins were probed by immunoblotting with either anti-ezrin polyclonal (upper panel) or anti-GFP monoclonal antibodies (lower panel).

FIGURE 2.

Interaction of S100P with immobilized N-ERMAD analyzed by SPR. A, ∼200 response units (RU) of N-ERMAD were immobilized on the CM5 sensor chip using the amine coupling method as described under “Experimental Procedures.” S100P at concentrations of 0.006–3.5 μm was injected and allowed to react with the sensor chip surface at a flow rate of 40 μl/min and an injection time of 3 min. The average binding level at the end of injection (equilibrium) was used for calculation of the steady state affinity. B, steady state affinity determined from the level of binding at equilibrium (R_eq) as a function of the sample concentration. Calculation was carried out using the BIAevaluation software version 4.1, as described under “Experimental Procedures.” The Scatchard plot of the binding experiment is shown in inset C. An approximate dissociation constant of 3e-7 M was calculated for the binding of WT S100P to N-ERMAD.

FIGURE 3.

N-ERMAD and S100P mutant derivatives. A, schematic representation of the different C-terminal deletion constructs. Amino acids predicted to play an important role in dimerization or target protein binding of S100P are highlighted in bold in the sequence given. B, Western blot analysis of the mutant proteins purified after recombinant expression. Lane 1, ezrin 82aa; lane 2, ezrin 173aa; lane 3, ezrin 197aa; lane 4, ezrin 233aa; lane 5, S100P 87aa; lane 6, S100P 91aa. The ezrin derivatives were detected with an anti-GST and S100P proteins with an anti-Penta-His antibody.

FIGURE 4.

Direct interaction between WT S100P and different N-ERMAD derivatives. A, affinity chromatography analysis. The purified GST-tagged ezrin deletion mutants ezrin 233aa, ezrin 197aa, ezrin 173aa, and ezrin 82aa were bound to glutathione-Sepharose columns, and purified WT S100P was added in the fluid phase. Binding reactions were carried out in the presence of Ca²⁺ (see “Experimental Procedures”), and flow-through fractions were collected (FT). After extensive washing with a Ca²⁺-containing buffer, the columns were developed with an EGTA-containing buffer to elute Ca²⁺ dependently bound proteins (E). Finally, all bound proteins were stripped with a glutathione-containing buffer (S). Equivalent amounts of all fractions were subjected to SDS-PAGE and analyzed by Western blotting. S100P was detected using an anti-Penta-His antibody and the N-ERMAD derivates using an anti-GST antibody. Note the binding of WT S100P to all ezrin derivatives except ezrin 82aa. Minor variations in the EGTA elution profiles, e.g. between the experiments using ezrin 197aa and ezrin 173aa, are due to the column and fraction sizes differing slightly from experiment to experiment. B, SPR analysis of the interaction of N-ERMAD and ezrin 82aa with immobilized S100P. ∼200 RU of S100P were amine-coupled to the CM5 sensor chip. N-ERMAD (upper sensogram) and ezrin 82aa (lower sensogram) were injected at a flow rate of 30 μl/min and injection times of 3 min. C, interaction of S100P with amine-coupled N-ERMAD (upper curve) and amine-coupled ezrin 82aa mutant (lower curve) analyzed by SPR. ∼200 RU of N-ERMAD and ezrin 82aa were immobilized on the CM5 sensor chip, and 3.5 μm WT S100P were injected at a flow rate of 40 μl/min and injection times of 3 min. All sensograms are corrected using a blank flow cell as reference. RU, response units.

FIGURE 5.

Direct interaction between N-ERMAD and C-terminal-truncated S100P. A, affinity chromatography analysis. Purified GST-tagged N-ERMAD was immobilized on glutathione-Sepharose beads, and purified WT S100P, S100P 91aa, and S100P 87aa were added in the fluid phase. Binding reactions were carried out in the presence of Ca²⁺, and after extensive washing with a Ca²⁺-containing buffer, the columns were developed with an EGTA-containing buffer to elute Ca²⁺ dependently bound proteins (E). Subsequently, all bound proteins were stripped with a glutathione-containing buffer (S). Equivalent amounts of all fractions were analyzed by Western blotting using anti-Penta-His (S100P) and anti-GST antibodies (N-ERMAD). B and C, SPR analysis of the interaction of WT S100P (B) and S100P 87aa (C) with amine-coupled N-ERMAD. WT S100P and S100P 87aa (0.006–3.5 μm) were injected into a flow cell containing the immobilized N-ERMAD at 40 μl/min flow rate and injection time of 3 min. Note the complete loss of binding in the case of S100P 87aa. RU, response units.

FIGURE 6.

Chemical cross-linking of WT and mutant S100P. WT S100P and the S100P truncation mutants S100P 91aa and S100P 87aa were adjusted to 0.5 mm Ca²⁺ and incubated with the homobifunctional cross-linker BS3 (C). In control experiments, samples were incubated without cross-linker (-). Products of the reactions were subjected to 15% SDS-PAGE and visualized by silver staining. For representation of this figure, the original gel was cut between lanes 1 and 2 and lanes 4 and 5 to remove lanes not relevant for the experiment.

FIGURE 7.

S100P interferes with the PtdIns(4,5)P₂ binding of ezrin. A and B, liposome co-sedimentation assays. A, POPC vesicles containing 3% PtdIns(4,5)P₂ were incubated with N-ERMAD in the absence or presence of WT S100P and S100P 87aa, respectively. Liposomes were pelleted by high speed centrifugation and then subjected to two successive washing steps. The resulting supernatants (Sn1, Sn2, and Sn3) and final pellets (P) were analyzed by SDS-PAGE and silver staining. B, statistical evaluation of three independent liposome pelleting assays (SN, Sn1, Sn2 and Sn3 combined; P, pellet). Note that WT S100P interferes to some extent with the binding of N-ERMAD to PtdIns(4,5)P₂-containing liposomes, i.e. the amount of N-ERMAD in the liposome pellet fraction is markedly decreased in the presence of WT S100P. This inhibition is not seen with S100P 87aa. C, lipid plate assay revealing the binding of N-ERMAD to immobilized PtdIns(4,5)P₂ in the absence or presence of S100P derivatives. After PtdIns(4,5)P₂ immobilization, lipid binding of N-ERMAD was assessed as described under “Experimental Procedures” in the presence or absence of WT S100P, S100P 91aa, and S100P 87aa. Statistical significance in all analyses was calculated using unpaired student's t test (N-ERMAD-WT S100P, p = 0.0003, n = 4; N-ERMAD-S100P 91aa, p = 0.0001, n = 4). Note the reduction in PtdIns(4,5)P₂-bound N-ERMAD in the presence of WT S100P and S100P 91aa that is not seen with the S100P 87aa mutant. The inhibitory effect of S100P on the PtdIns(4,5)P₂ binding of N-ERMAD is less pronounced in the lipid plate as compared with the liposome co-sedimentation assay, most likely due the higher density and local concentration of PtdIns(4,5)P₂ present on the lipid plate.

FIGURE 8.

Transendothelial migration of HTB-58 cell lines stably expressing different ezrin and S100P derivatives. A, stable expression of GFP fusion proteins of WT S100P, S100P 91aa, S100P 87aa, and ezrin T567D in HTB-58 cells was verified by Western blotting of total cell lysates with a monoclonal anti-GFP antibody. Probing of the same blot with anti-vimentin antibodies served to control for equal loading. B, statistical evaluation of transendothelial migration assays employing the different cell lines. The migration of GFP-positive HTB-58 cells through a monolayer of HMEC-1 grown on microporous filter support was measured as described under “Experimental Procedures.” Note that cells expressing WT S100P, S100P 91aa, or the permanently active ezrin mutant T567D show increased migration as compared with control cells expressing GFP alone. In contrast, expression of S100P 87aa caused no significant effect on the transendothelial migration of HTB-58 cells.