Phosphorylation of the α-chain in the integrin LFA-1 enables β2-chain phosphorylation and α-actinin binding required for cell adhesion

The integrin leukocyte function–associated antigen-1 (LFA-1) plays a pivotal role in leukocyte adhesion and migration, but the mechanism(s) by which this integrin is regulated has remained incompletely understood. LFA-1 integrin activity requires phosphorylation of its β2-chain and interactions of its cytoplasmic tail with various cellular proteins. The α-chain is constitutively phosphorylated and necessary for cellular adhesion, but how the α-chain regulates adhesion has remained enigmatic. We now show that substitution of the α-chain phosphorylation site (S1140A) in T cells inhibits the phosphorylation of the functionally important Thr-758 in the β2-chain, binding of α-actinin and 14-3-3 protein, and expression of an integrin-activating epitope after treatment with the stromal cell–derived factor-1α. The presence of this substitution resulted in a loss of cell adhesion and directional cell migration. Moreover, LFA-1 activation through the T-cell receptor in cells expressing the S1140A LFA-1 variant resulted in less Thr-758 phosphorylation, α-actinin and talin binding, and cell adhesion. The finding that the LFA-1 α-chain regulates adhesion through the β-chain via specific phosphorylation at Ser-1140 in the α-chain has not been previously reported and emphasizes that both chains are involved in the regulation of LFA-1 integrin activity.

Integrins are transmembrane heterodimeric receptors that communicate in two directions across the plasma membrane and mediate interactions with other cells and the extracellular environment (1). Integrins can bind ligands, resulting in outside-in signaling, whereas inside-out signaling is initiated by ligand binding to nonintegrin receptors, such as chemokine receptors or the T-cell receptor (TCR), 4 which activate integrins through intracellular signaling (2,3). The family of leukocyte-specific ␤2-integrins consists of four members that have a common ␤2-chain (CD18) and one of the ␣-chains (␣L, CD11a; ␣M, CD11b; ␣X, CD11c; and ␣D, CD11d). The leukocyte function-associated antigen-1 heterodimer (LFA-1, ␣L␤2, CD11a/ CD18) is primarily expressed on lymphocytes and binds to intercellular adhesion molecules (ICAMs). Mac-1 (macrophage 1 antigen, ␣M␤2, CD11b/CD18) is enriched in the myeloid lineage and is able to bind numerous ligands, among them ICAMs and complement protein iC3b. Complement receptor 4 (CR4, ␣X␤2, CD11c/CD18, p150,95) is expressed in monocytes, macrophages, and dendritic cells, as well as in some subsets of activated T and B cells. It is also capable of binding various ligands, including extracellular matrix molecules, cellular and soluble ligands, and denatured proteins (4,5).
Integrins exist in at least three different conformations: closed, extended, and extended open, each of which possesses different ligand binding affinities and localization in cells. Integrins can modulate their adhesive properties within seconds. The activity of the integrins is for the main part regulated by the binding of different proteins to the cytoplasmic tails, which is mediated by integrin phosphorylation, clustering, and receptor cross-talk (6,7).
The leukocyte integrin ␣-chains are constitutively phosphorylated, whereas the ␤2-chain becomes phosphorylated after activation through chemokines, the TCR, or phorbol esters (8 -12). The integrin cytoplasmic domains are short and devoid of catalytic activity. Only a few cytoplasmic proteins have been found to specifically bind to the integrin ␣-chain cytoplasmic tails (13), whereas signaling, adaptor, and cytoskeletal linker proteins, including talin, kindlins, filamin, ␣-actinin, and 14-3-3 proteins, bind to the ␤2-integrin tails (14). The integrin ␤-chain cytoplasmic domains contain three conserved regions: the two NPX(Y/F) sequences and the serine/threonine sequence between them. Mutation of the ␤2-chain threonines abrogated cell adhesion, but initial experiments did not reveal phosphorylation of these (15). By using the phosphatase inhi-bitor okadaic acid, strong threonine phosphorylation was observed after activation (9 -11). The ␤2-chain is phosphorylated by protein kinase C (PKC) enzymes (16). The phosphorylation of Thr-758 on ␤2 leads to release of bound filamin and promotes binding of 14-3-3 proteins. Talin can bind both to the Thr-758 phosphorylated and unphosphorylated chain (17). The regulation of the association between ␣-actinin and the ␤2-chain is currently poorly understood, but it has been shown that ␣-actinin binding is enhanced by the activation of neutrophils and T cells (18,19).
The leukocyte ␤2-integrin ␣-chains are phosphorylated on Ser-1140 (␣L), Ser-1126 (␣M), and Ser-1158 (␣X) (see Fig. 1A), whereas ␣D phosphorylation has not been studied. The ␣-chain phosphorylation is important for leukocyte adhesion and intracellular signaling (20 -22), but the mechanism(s) has remained unknown. We have now focused on the LFA-1 integrin and show that through the phosphorylation of the ␣-chain, it regulates the phosphorylation, conformation, and protein binding to the ␤2-chain in a specific manner. When cells expressed the LFA-1 S1140A mutation, there was no phosphorylation of the ␤2-chain on Thr-758, but there was a loss of both ␣-actinin and 14-3-3 binding and the presence of the integrin activation-reporter epitope L16 upon treatment with the chemokine stromal cell-derived factor-1␣ (SDF-1␣). Binding of filamin increased. These mutant cells displayed abrogated cell adhesion to the LFA-1 ligand ICAM-1 and impaired directional migration. When LFA-1 S1140A mutated cells were activated through the TCR, the ␤2 phosphorylation and the binding of ␣-actinin to ␤2 decreased as compared with WT cells, whereas the presence of the activation epitope L16 was reduced only slightly as compared with WT cells. This decreased cell adhesion to ICAM-1. The results indicate an important role for integrin ␣-chains in the modulation of leukocyte functions by regulating ␤-chain phosphorylation, which results in altered cytoplasmic protein interactions, followed by changes in adhesion and migration.

Phosphorylation of the LFA-1 ␣-chain regulates the phosphorylation of the ␤2-chain on Thr-758
Cytoplasmic protein interactions with the integrin ␤2-chain have been extensively studied, but the regulation of these has remained incompletely understood. We now show how the ␣-chain of LFA-1 regulates integrin functions by affecting the ␤2-chain. We used the J␤2.7 human Jurkat T cell line expressing equal amounts of WT LFA-1 or LFA-1 containing the mutation S1140A at the known ␣-chain phosphorylation site (Fig.  1B). We have previously shown that this is the only phosphorylation site on the ␣L-chain and that a significant subset (ϳ40%) of ␣L Ser-1140 is phosphorylated in both activated and nonactivated cells (9,20). When the phosphorylation site serine was substituted by alanine, phosphorylation could not be recognized by a Ser(P)-1140 -specific antibody (Fig. 1C). Cells expressing WT LFA-1 could bind ICAM-1 after activation by chemokines such as SDF-1␣ or through the TCR. Cells expressing the ␣L S1140A mutant were unable to bind ICAM-1 after SDF-1␣ activation and showed much reduced binding after activation with anti-CD3 (Fig. 1D). WT cells aggregated on an SDF-1␣coated surface and SDF-1␣-activated cells spread on ICAM-1, which was not seen with cells expressing the LFA-1 S1140A mutant (Fig. 1E).
Phosphorylation of the ␤2-chain Thr-758 is seen only after activation, e.g. by SDF-1␣ or through the TCR (9 -11). In LFA-1 WT expressing cells, there was no phosphorylation of Thr-758 on the ␤2-chain without activation, but after treatment with SDF-1␣ or TCR, a strong label was seen. Importantly, no phosphorylation occurred on ␤2 Thr-758 in cells expressing the ␣L S1140A mutant ␣-chain activated with SDF-1␣. In cells activated through the TCR, a significantly weaker labeling of the Thr-758 was obtained in mutant cells as compared with WT cells (Fig. 1, F and G).

Phosphorylation of Ser-1140 on the LFA-1 ␣-chain affects the binding of cytoskeletal proteins to the ␤2-chain
We further tested whether the ␣-chain mutation affects the interaction with proteins known to bind to the ␤2-chain and to be involved in cell adhesion. Fig. 2A shows immunoprecipitations from Jurkat cells under three conditions; nonactivated, SDF-1␣-activated, or anti-CD3-activated cells. Western blots of supernatants showed equal amounts of proteins in WT and S1140A-expressing cells. Proteins coprecipitated with LFA-1 in the WT and mutant cell line are shown in the IP IB4 lane and quantified from three separate experiments as shown below (Fig. 2B). A significantly reduced binding of ␣-actinin and 14-3-3 was seen in SDF-1␣-activated mutant cells compared with WT cells. Talin binding was consistently reduced, but not significantly. Filamin binding was increased. Activation of the TCR by anti-CD3 resulted in a significant reduction in talin binding. ␣-Actinin and 14-3-3 binding was reduced, but not significantly (Fig. 2, A and B).
The most significant difference between ␣L WT and S1140A-expressing cells after SDF-1␣ was seen for ␣-actinin binding to ␤2. The ␣-actininbinding site on the LFA-1 integrin has been mapped to the ␤2-chain amino acids 736 -745, partly overlapping with the talin membrane-proximal binding site (23) (Fig. 1A). To verify that there is no ␣-actinin-binding site on ␣L, synthetic peptides of the cytoplasmic chains of ␣L, ␣L/Ser(P)-1140, ␤2, or ␤2/Thr(P)-758 were linked to Sepharose beads, and Jurkat cell lysates were added. Bound proteins were separated by SDS-PAGE and analyzed by immunoblotting for ␣-actinin, 14-3-3, and talin (Fig. 2C). In another experiment, peptides were spotted on a nitrocellulose membrane, and purified ␣-actinin was added. After extensive washing, protein binding was determined with specific antibodies. Experiments verified that ␣-actinin binds to the ␤2 or ␤2/Thr(P)-758 peptides but not to ␣L or ␣L/Ser(P)-1140 (Fig. 2D).
To study the differences in LFA-1-mediated adhesion complex formation in cells adhering to the ligand, the complexes from Jurkat cells binding to coated ICAM-1 or poly-L-lysine were purified, and their protein content was analyzed. Proteins found in the adhesion sites of LFA-1 WT-expressing cells and bound to ICAM-1 included the integrin ␤2 and ␣L chains, ␣-actinin, 14-3-3, and talin, after both SDF-1␣ and anti-CD3 activation. In contrast, ICAM-1 adhesion complexes from LFA-1 S1140A-expressing cells contained none of the proteins Integrin ␣-chain regulates the ␤-chain found in WT LFA-1-expressing cells activated with SDF-1␣, indicating that stable adhesion complexes were not formed (Fig. 2E). Instead, these proteins were detected in the Western blotting of unbound cells (Fig. 2F). After anti-CD3 activation of S1140A-expressing cells, the same proteins were present in the ICAM-1 adhesion complexes, although less than in WT LFA-1-expressing cells. The proteins in the adhesion complexes were quantified from three separate experiments (Fig. 2G). This result indicates that J␤2.7 LFA-1 S1140A-expressing cells can be at least partly activated to bind ligand by anti-CD3, which is not the case for SDF-1␣ activation (Figs. 1, D and E, and 2E). J␤2.7 Jurkat cells lacking LFA-1 expression did not bind to ICAM-1, which indicates that binding takes place solely through LFA-1 (not shown).
A negative charge on both the ␣and ␤2-chain phosphorylation sites increases binding of ␣-actinin to LFA- 1 We were not able to transfect J␤2.7 cells with the ␣L S1140D mutant and therefore used COS7 cells, which have been shown to express functional heterodimeric integrins on their cell surface after transfection with ␤2-integrins (24). The ␣L-chain is phosphorylated in COS7 cells on Ser-1140 (20). COS7 cells do not contain the SDF-1␣ receptor or TCR, but integrins can be activated by phorbol esters. PMA activation of COS7 cells transfected with WT LFA-1 led to increased Thr-758 phosphorylation on ␤2 (Fig. 3A) and increased ␣-actinin and 14-3-3 binding (Fig. 3B). COS7 cells were then transfected with WT ␣L, the nonphosphorylatable S1140A or the S1140D mutant that mimics phosphorylated Ser-1140. ␤2 Thr-758 was phosphorylated in cells expressing the S1140D mutant but not in cells expressing S1140A (Fig. 3A). Lysates from PMA-activated cells were immunoprecipitated with the LFA-1 antibody IB4, and the precipitates were immunoblotted for ␣-actinin, ␤2, and ␣L. Binding of ␣-actinin to LFA-1 was reduced in LFA-1 S1140A-expressing cells in accordance with the results from J␤2.7 cells. The LFA-1 S1140D mutation resembled that of WT LFA-1 phosphorylated on Ser-1140, supporting the model that the negative charge on the ␣-chain is important for ␤2 Thr-758 phosphorylation, enabling stronger ␣-actinin binding (Fig. 3C). Mutation of Thr-758 to alanine resulted in decreased binding of ␣-actinin (Fig. 3D). Cells expressing ␤2 T758D and the S1140A ␣L-chain were able to bind ␣-actinin; however, binding was further increased when a negative charge was present on both chains, represented by cells expressing ␣L WT Ser-1140 and ␤2 T758D. ␣L S1140D-expressing cells did not bind ␣-actinin without PMA activation (Fig. 3E). This verifies that the negative charge on the ␣L-chain Ser-1140 is important for ␤2 Thr-758 phosphorylation and for increased ␣-actinin binding, which requires the phosphorylated ␤2 Thr-758.
We then extended the study to another ␣-chain, ␣X␤2. This integrin is expressed in myeloid cells. We therefore stably expressed ␣X␤2 in the myeloid/erythroleukemic cell line K562. Activation of these cells with SDF-1␣ resulted in phosphorylation of ␤2 Thr-758 in WT ␣X␤2-expressing cells, whereas no phosphorylation of ␤2 Thr-758 was detected in cells expressing the ␣-chain phosphorylation mutant ␣X S1158A (Fig. 3F). This result further emphasizes the role of ␣-chain phosphorylation for ␤2-chain phosphorylation. The double band could mean that some ␤2-chains are phosphorylated on additional sites (16).

Phosphorylation of ␣L affects the cellular localization of ␣-actinin and the activation epitope of LFA-1
We next studied whether there is a difference in the localization of ␣-actinin in cells expressing LFA-1 WT or S1140A. The cells were activated with SDF-1␣ or anti-CD3 and allowed to adhere to ICAM-1, fixed, and stained for ␤2, for ␣-actinin, or by phalloidin ( Fig. 4, A, and B). In cells expressing WT LFA-1, ␣-actinin localized to the membrane and ruffling edges and colocalized with ␤2 and phalloidin both in SDF-1␣ and anti-CD3 activated cells (Pearson's correlation coefficient for ␤2 and ␣-actinin at the membrane was 0.78 for SDF-1␣ and 0.70 for anti-CD3 activation). Colocalization was also seen in anti-CD3-activated cells expressing the S1140A mutant, but less in mutant cells activated with SDF-1␣ where ␣-actinin staining was more diffused (Pearson's correlation coefficient for ␤2 and ␣-actinin at the membrane was 0.47 for SDF-1␣ and 0.69 for anti-CD3 activation).
The effect of ␣L S1140A phosphorylation on LFA-1 activation was next identified by labeling cells with the NKI-L16 antibody recognizing the fully active epitope L16 of LFA-1 (25). Cells expressing the WT LFA-1 activated with SDF-1␣ were strongly positive for the L16 epitope, which was not detected in the S1140A-expressing cells. In contrast, L16 positivity was detected after anti-CD3 activation of S1140A-expressing cells (Fig. 4, C and D).

Phosphorylation of the LFA-1 ␣and ␤-chains and binding of ␣-actinin are needed for chemotaxis
The linking of integrins to the actin cytoskeleton by ␣-actinin is important for cell adhesion and migration (26). Cell adhesion was studied by transfecting Jurkat cells expressing LFA-1 with a ␤2-peptide containing the ␣-actininbinding site of ␤2, which blocks the interaction between ␣-actinin and the ␤2-chain but does not affect talin binding (19). Static adhesion to ICAM-1 was inhibited by ␤2-peptide transfection into cells activated by SDF-1␣, but not by the control peptide (Fig. 5A). To analyze the role of the ␣-actinin/integrin interaction in migration, Jurkat or expressing LFA-1 WT or S1140A were stained with ␤2 (CD18-PE) or ␣L (CD11a-APC) and analyzed by flow cytometry. Mean fluorescence intensity (MFI) is shown for each graph. C, J␤2.7 cells expressing ␣L WT or S1140A were analyzed by Western blotting and immunoblotted with ␣L/pS1140 or ␣L antibodies. D, J␤2.7 cells expressing ␣L WT or S1140A were activated with SDF-1␣ or anti-CD3 and allowed to adhere to ICAM-1, and bound cells were quantified. E, in the upper panels, J␤2.7 cells expressing ␣L WT or ␣L S1140A were allowed to adhere to a SDF-1␣-coated surface. In the lower panels, SDF-1␣-activated cells were allowed to adhere to ICAM-1 and spread for 1 h. F, lysates of J␤2.7 cells expressing ␣L WT or S1140A were activated with SDF-1␣ or anti-CD3, analyzed by SDS-PAGE, and immunoblotted with ␤2/Thr(P)-758, ␤2, ␣L, or actin as a loading control. G, the amount of ␤2/Thr(P)-758 per ␤2 was quantified from five separate experiments. Molecular mass markers (kDa) are shown to the left of the blots. *, p Ͻ 0.05; ***, p Ͻ 0.005.
Integrin ␣-chain regulates the ␤-chain cells expressing WT LFA-1 or LFA-1 S1140A were allowed to migrate over an ICAM-1-coated surface toward SDF-1␣. In the mutant cells, with reduced interaction between the integrin and ␣-actinin, directional migration was impaired ( Fig. 5B and videos S1 and S2). Cells transfected with the ␤2 peptide, but not the control peptide, were also unable to migrate (Fig. 5B). Mutant cells were not able to strongly adhere to ICAM-1 and could be washed off by pipetting, which was not the case for WT cells. The mutant cells formed extensions but did not spread or become polarized to the same extent as WT cells and were not able to move toward the chemokine gradient. Instead they ruffled in different directions, but the cell body remained stationary (Fig. 5, C and D). Both the accumulated and euclidean distances, as well as directionality, were reduced in cells with impaired ␣-actinin-integrin binding (Fig. 5E).

Discussion
Integrin activity has to be tightly regulated to allow rapid and precise changes between different activation states. The roles of the ␤-chain cytoplasmic tails have been extensively studied and shown to be essential for integrin function. Chemokines, such as SDF-1␣, bind to their receptors resulting in phospholipase C activation followed by signaling through CalDag, Rap1, and RapL (27). The proximal signaling events from the TCR are different and include the Lck tyrosine kinase (28), which then phosphorylates the ZAP-70 kinase, and phospholipase C␥ (27). PKCenzymesthenbecomeactivatedinbothpathwaysandphosphorylate Thr-758 on ␤2. Phosphorylation of the ␤2-chain is known to regulate the affinity for different binding proteins (7). Filamin binds primarily to the unphosphorylated ␤2-chain, whereas ␤2 phosphorylated on Thr-758 binds 14-3-3 proteins and initiates intracellular signaling through Tiam1-Rac1 and inhibitory signaling to ␣4␤1 (12,17,29,30).
Previous work indicates a regulatory role for the membraneproximal part of ␣-chain cytoplasmic domains in integrin regulation. The proximal portions of both ␣ and ␤ cytoplasmic domains are ␣-helical and associate with each other (31). The GFFKR motif in the ␣-chain is well conserved and is important in keeping the integrins in a nonadhesive state by interacting by ionic bonds with the ␤-chain. When this motif is deleted or mutated, integrins become active (32)(33)(34)(35)(36)(37). Binding of talin can perturb the membrane-proximal association, disrupting the intersubunit interactions, which results in activation (38,39). In fact, most of the known negative regulators of integrins bind to the conserved membrane-proximal site (40 -42).
The ␣-chain membrane distal regions vary in lengths and sequences, and their roles in integrin activation have been less defined. Deletion of the membrane distal regions of some ␣-chains affects cell adhesion and spreading, but similar deletions in other ␣-chains have no effect on adhesion (15,(43)(44)(45)(46)(47)(48)(49). This shows that there are different regulatory roles of the membrane distal regions among different integrins. Interestingly, deletion of the ␣L membrane distal part diminishes talin-and kindlin-induced integrin conformational change and ligand binding (50). We have previously shown that ␤2-integrin ␣-chain phosphorylation is needed for adhesion after activation induced by chemokine, ligand, or active Rap1 (20 -22). The regulatory mechanisms have not been known. We now show that phosphorylation of the ␣-chain of LFA-1 is needed for ␤2-chain phosphorylation and its protein interactions.
A significant part of the LFA-1 ␣-chain is phosphorylated on the single site Ser-1140 (9,20), and mutation of the serine residue to alanine inhibited or reduced phosphorylation of Thr-758 in the ␤2-chain. Treatment with the phosphatase inhibitor okadaic acid increased ␣-chain phosphorylation (9), indicating that there is a cycle of phosphorylation and dephosphorylation events of the ␣-chain. This could, in turn, regulate the phosphorylation of Thr-758 of the ␤2-chain, enabling rapid cell adhesion and deadhesion for example during movement. We extended the study to include another ␣-chain, ␣X, which is known to be phosphorylated on Ser-1158 (22). Phosphorylation of ␤2 Thr-758 was impaired also in myeloid/erythroid K562 cells expressing ␣X S1158A.
Although activation of cells with both SDF-1␣ and through the TCR results in phosphorylation of Thr-758 on ␤2, differences were seen between the LFA-1 WT and S1140A-expressing cells. LFA-1 ␤2 could not be phosphorylated on Thr-758, nor bind to ICAM-1 in mutant cells activated with the chemokine, but showed only a reduced phosphorylation compared with WT after anti-CD3 activation. This reflects the differences in signaling downstream of the TCR and chemokine receptor and indicates that additional factors play a role in the regulation of ␤2 phosphorylation. In WT expressing cells, Thr-758 on ␤2 is readily phosphorylated upon SDF-1␣ treatment, and S1140D is functionally similar evidently because of the negative charge at position Ser-1140.
The S1140A ␣-chain phosphorylation mutation markedly decreased ␣-actinin binding to the ␤2-chain and impaired cell migration toward the SDF-1␣ chemokine. Decreased binding was also seen with 14-3-3 and an increase in binding of filamin, which is most likely a cause of decreased Thr-758 phosphorylation in the mutant cell line. There was also a decrease in talin binding in the mutant cells activated with anti-CD3. Because the most prominent difference in binding was seen with ␣-actinin, we investigated this further. ␣-Actinin is essential for cell migration (26). Phosphorylation of both ␣L and ␤2 regulated the binding between ␣-actinin and LFA-1. The strongest binding occurred when both ␣L Ser-1140 and ␤2 Thr-758 were phosphorylated. Figure 2. ␣-Chain phosphorylation regulates binding of cytoplasmic interaction partners to ␤2 differently. A, lysates of J␤2.7 cells expressing ␣L WT or S1140A were not treated or activated with SDF-1␣ or anti-CD3, immunoprecipitated with the LFA-1 antibody IB4, and immunoblotted for ␣-actinin, 14-3-3, talin, filamin, ␤2, or ␣L. B, the amount of coprecipitated proteins per immunoprecipitated ␤2 was quantified from three separate experiments. C, full-length cytoplasmic peptides of ␣L, ␣L/Ser(P)-1140, ␤2, or ␤2/Thr(P)-758 were bound to beads and Jurkat cell lysate added. Bound proteins were analyzed by immunoblotting with indicated antibodies. D, peptides were spotted on nitrocellulose, and purified ␣-actinin was added. After washing, binding was assessed with the ␣-actinin antibody. E and F, J␤2.7 cells expressing ␣L or ␣L S1140A were allowed to adhere on poly-L-lysine or ICAM-1 and adhesion complexes (E) or unbound cells (F) collected, and lysates were analyzed by immunoblotting using indicated antibodies. G, the amount of protein found in the adhesion complexes was quantified from three separate experiments. SN, supernatant. Molecular mass markers (kDa) are shown to the left of the blots. Lines depict borders between two separate gels. *, p Ͻ 0.05; **, p Ͻ 0.01.

Integrin ␣-chain regulates the ␤-chain
A negative charge on the ␣L-chain, either by Ser-1140 phosphorylation or by expressing the S1140D mutant, was needed to enable phosphorylation of the ␤2 Thr-758. ␣-Actinin was able to bind the ␤2-chain also without ␣-chain phosphorylation only if there was a negative charge on Thr-758, as in the ␤2 T758D mutant, which could override the S1140A mutation and bind ␣-actinin. Binding was, however, stronger with a negative charge on both chains, indicating that the ␣-chain also directly regulates ␣-actinin binding. The ␣L cytoplasmic tail forms a triple-helical structure. Helix 3 makes contacts with both helices 1 and 2, and helices 1 and 3 are in contact with the ␤2-tail. Interestingly, Ser-1140 is located in helix 3 at the negatively charged surface of the ␣L tail, and its phosphorylation can enhance the negative charge of this surface (51). We conclude that ␣L Ser-1140 phosphorylation is required for ␤2 Thr-758 phosphorylation, possibly by making the site accessible to PKC enzymes, and as the chains move apart allowing new cytoskeletal interactions, as shown by increased ␣-actinin binding. This, in turn, would have an effect on cytoskeleton-mediated processes in cells.
We show that LFA-1 S1140A-expressing cells with impaired ␣-actinin binding exhibited impaired directional migration toward the SDF-1␣ chemokine. Cells expressing the LFA-1 S1140A mutant had a rounded and less polarized morphology compared with WT expressing cells. The same could be seen with cells where the ␤2-␣-actinin interaction was disrupted by a peptide corresponding to the ␣-actininbinding site on ␤2. This peptide has shown selectivity in binding ␣-actinin and not talin, although it contains part of the talin membrane-proximal site (19). Impaired migration in S1140A-expressing cells appears to be a result of the lack of ␣-actinin linking ␤2 to the cytoskeleton, which is known to be important for cell migration. A conserved region (residues 733-742) in the ␤2 cytoplasmic domain is critical for its cytoskeletal association (34). This region overlaps with the mapped ␣-actinin site (residues 736 -746). Furthermore, a regulatory domain in ␤2 between residues 748 -762 inhibits the constitutive association of the ␤2 tail with ␣-actinin (23). This sequence contains both the Thr-758 phosphorylation site and binding sites for talin and 14-3-3. Phosphorylation of Ser-1140 on ␣L and Thr-758 on ␤2 may release the inhibitory structure of the ␤2-chain, resulting in ␣-actinin binding, activation of the integrin binding site as shown with the NKI-L16 antibody, and migration of cells. We cannot, however, rule out the possibility that the reduced migration of LFA-1 S1140A-expressing cells is due to impaired binding of other proteins to the ␣-chain. Cells lacking ␣-actinin have a decreased ability to translocate but adhere more strongly to ICAM-1 (19). In our migration experiment, the LFA-1 S1140A-expressing cells were able to weakly adhere and make membrane projections but did not migrate toward the chemokine. This indicates that ␣-actinin may link the active integrin to the cytoskeleton to mediate directional migration.
At the leading edge, adhesions are constantly turned over, and the cell membrane attaches and then detaches from ICAM-1 when making new ligand contacts. Phosphorylationdephosphorylation of ␣L Ser-1140 and ␣X Ser-1158 could provide a fast way of regulating integrin-␣-actinin cytoskeleton linkage, dynamic adhesion, and migration. Our data establish an essential role for the ␣-chain cytoplasmic domain phosphorylation in the regulation of the ␤-chain and thus integrin activity.
Integrin ␣-chain regulates the ␤-chain units/ml penicillin-streptomycin. The cells were treated with SDF-1␣ (50 ng/ml) for 20 min, anti-CD3 (10 g/ml) for 30 min or left untreated. For immunoblotting with the Thr(P)-758/␤2 antibody, cells were treated with 1 g/ml of okadaic acid for 5 min. Cells were washed once with cold PBS, lysed on ice in 2% immunoprecipitation assay buffer (50 mM Tris-HCl, pH 7.8, 150 mM NaCl, 1% Triton X-100, 1% Nonidet P-40, 15 mM MgCl 2 , and 5 mM EDTA) with protease and phosphatase inhib-  Integrin ␣-chain regulates the ␤-chain itors (Roche Applied Science) for 30 min. Lysates were centrifuged at 20,000 ϫ g for 1 h at 4°C. Prewashed protein G-Sepharose beads were added for 2 h at 4°C. The unbound fraction was mixed with IB4 or ␣-actinin antibody overnight at 4°C, and protein G-Sepharose beads for another 2 h at 4°C. The beads were washed three times with 1% immunoprecipitation assay buffer (50 mM Tris-HCl, pH 7.8, 150 mM NaCl, 0.5% Triton X-100, 0.5% Nonidet P-40, 15 mM MgCl 2 , and 5 mM EDTA), and bound proteins were eluted with Laemmli sample buffer, separated on SDS-PAGE, immunoblotted, and detected using ECL (Pierce). The figures show one representative picture of at least three experiments. The amount of coprecipitated proteins per immunoprecipitated ␤2 was quantified from three separate experiments by ImageJ. For the calculation of p values, one-way analysis of variance (Bonferroni post hoc) or unpaired Student's t test was used. The mean standard deviations are given in the figures.

Cell transfections
␣L was subcloned into the pcDNA3.1 vector (Thermo Scientific). ␤2 in the pcDNA3.1 vector was from Addgene (Cambridge, MA) (plasmid 8640 (55); the ␣X WT and ␣X S1158A mutant in the pcDM8 vector have been described (22). The mutations S1140A and S1140D in ␣L were introduced by sitedirected mutagenesis (56). All constructs were checked by sequencing. COS7 cells were transiently cotransfected using FuGENE HD transfection reagent according to the manufacturer's instructions (Promega, Madison, WI) and harvested 48 h after the transfection.
The ␣-actininbinding site-containing the ␤2 peptide HLS-DLREYRRFEKEKLKSC and the ␤2 cytoplasmic chain peptides with and without phosphate on Thr-758 C-NNDNPLFK-SA(pT)-TTVMNPK and C-NNDNPLFKSATTTVMNPK were obtained from GenicBio Ltd. (Shanghai, China). As a control, the P621 peptide VDVDSDGSTDLVIGA was used. The ␤2 peptide containing the ␣-actininbinding site or the control peptide P621 was transfected into J␤2.7 expressing LFA-1 WT using Pro-Ject protein transfection reagent kit according to the manufacturer's instructions (Thermo Scientific). Briefly, 5 g of peptide in 50 l of PBS was added to 10 l of the dried Pierce reagent tube, incubated for 5 min at room temperature, and added to 500,000 cells/ml in RPMI 1640 without serum in a six-well plate. After 4 h, the cells were washed twice with PBS and stored in RPMI 1640.

Immunofluorescence
Jurkat J␤2.7 cells were activated with SDF-1␣ (50 ng/ml) for 20 min or anti-CD3 (10 g/ml) for 30 min and allowed to adhere on ICAM-1 (6 g/ml) coated 8-well Ibidi chambers (-Slide 8-well, ibiTreat) for 30 min and fixed in 4% formaldehyde for 20 min at 37°C. The plates were washed twice with PBS. The cells were stained with primary antibodies against ␤2 (R2E7B) and ␣-actinin (EP2527Y) and Alexa Fluor 488 or 633 secondary antibodies or TRITC-phalloidin (Invitrogen), washed, and mounted with Prolong Gold antifade reagent (Thermo Scientific). The Images were acquired using a Leica TCS SP8 STED 3X CW 3D confocal microscope (Wetzlar, Germany) with the HC PL Apo 20ϫ/0.75 IMM CORR CS 2 (glycerol) objective at room temperature and analyzed with Leica Application Suite and Fiji (National Institutes of Health). For colocalization studies, confocal images were automatically analyzed, and individual cells were manually selected as region of interest.

Flow cytometry
Jurkat J␤2.7 cells were activated with SDF-1␣ (100 ng/ml) or anti-CD3 (10 g/ml) or left untreated. 1 ϫ 10 6 cells per treatment were incubated with NKI-L16 (1:100) or CD18-PE in Hepes buffer on ice for an hour. APC-conjugated goat antimouse was used as the secondary antibody. Flow data were acquired using an LSR II flow cytometer (Becton Dickinson) and analyzed using BD FACSDiva software.

Cell adhesion and migration assays
For static adhesion assays, soluble ICAM-1 (6 g/ml) was coated on flat-bottomed 96-well microtiter plates (NUNC MaxiSorp, Thermo Scientific) overnight at 4°C. The plates were then washed twice with PBS and blocked with 1% heatdenatured BSA for 1 h. 200,000 J␤2.7 cells expressing LFA-1 WT or ␣L S1140A/␤2 with or without activation with SDF-1␣ (50 ng/ml) for 20 min or anti-CD3 (10 g/ml) for 30 min were allowed to adhere for 30 min. Unbound cells were removed by washing three times in PBS, and the bound cells were lysed and detected using p-nitrophenyl phosphate substrate tablets (Sigma-Aldrich, S0942) dissolved in buffer (50 mM NaCH 3 COO, pH 5, 1% Triton X-100) at a concentration of 3 mg/ml. 100 l of substrate solution was added to each well containing lysate and incubated for 45 min at 37°C. The reaction was stopped with 1 M NaOH, and the reactivity was detected by measuring absorbance at 405 nm.
For the chemotactic cell migration assay, -slide chemotaxis chambers (Ibidi, Germany) were used. The assays were performed according to the manufacturer's instructions. Briefly, the -Slide chemotaxis slides were coated with 6 g/ml ICAM-1 at 37°C for 1 h and washed with PBS. 6 l of cells at a concentration of 3 ϫ 10 6 cells/ml were seeded in the center chamber of a -slide. Two reservoirs were filled with 60 l of serum-free RPMI 1640. One of the filling ports was filled with 10 l of chemoattractant SDF-1␣ (1 g/ml) solution by removing 10 l of medium from the other port on the same side of the device. Cell migration was recorded by mounting the -Slide on the stage of an inverted microscope in a cell incubator. For the trajectory analysis, pictures were taken every 4 min for 4 h using the Cell-IQ high content screening microscope (ϫ10 lens) (Chip-Man Technologies Ltd., Tampere, Finland). The cells were tracked with the Fiji ImageJ manual tracking program (National Institutes of Health), tracks of 15 cells from one representative experiment are shown in the figure, and tracks of 50 cells each from three separate experiments were analyzed with the Chemotaxis and Migration Tools software (Ibidi for ImageJ). Statistical analyses were performed using one-way analysis of variance (Bonferroni post hoc) or unpaired Student's t test. Mean standard deviations are included in figures.