The Actin Regulatory Protein HS1 Interacts with Arp2/3 and Mediates Efficient Neutrophil Chemotaxis

Background: HS1 is a cortactin homolog, however its role in neutrophil chemotaxis is not known.

Results: HS1 interacts with Arp2/3, regulates Vav1 and Rac signaling, and is necessary for efficient neutrophil chemotaxis.

Conclusion: HS1 tyrosine phosphorylation is critical for its interaction with Arp2/3 and efficient neutrophil chemotaxis.

Significance: This work increases our understanding of how actin regulatory proteins mediate efficient cell migration.

Abstract

HS1 is an actin regulatory protein and cortactin homolog that is expressed in hematopoietic cells. Antigen receptor stimulation induces HS1 phosphorylation, and HS1 is essential for T cell activation. HS1 is also expressed in neutrophils; however, the function of HS1 in neutrophils is not known. Here we show that HS1 localizes to the neutrophil leading edge, and is phosphorylated in response to the chemoattractant formyl-Met-Leu-Phe (fMLP) in adherent cells. Using live imaging in microchannels, we show that depletion of endogenous HS1 in the neutrophil-like PLB-985 cell line impairs chemotaxis. We also find that HS1 is necessary for chemoattractant-induced activation of Rac GTPase signaling and Vav1 phosphorylation, suggesting that HS1-mediated Rac activation is necessary for efficient neutrophil chemotaxis. We identify specific phosphorylation sites that mediate HS1-dependent neutrophil motility. Expression of HS1 Y378F, Y397F is sufficient to rescue migration of HS1-deficient neutrophils, however, a triple phospho-mutant Y222F, Y378F, Y397F did not rescue migration of HS1-deficient neutrophils. Moreover, HS1 phosphorylation on Y222, Y378, and Y397 regulates its interaction with Arp2/3. Collectively, our findings identify a novel role for HS1 and its phosphorylation during neutrophil directed migration.

Introduction

Neutrophils are key mediators of the innate immune response and are the first responders to sites of infection or acute injury. The rapid recruitment of neutrophils requires a highly specialized form of motility that translates chemoattractant signals from the environment into polarized intracellular signals that mediate directed protrusion and efficient migration (1). Furthermore, defects in signaling pathways required for neutrophil chemotaxis are associated with primary immune deficiencies, such as congenital neutropenia and leukocyte adhesion deficiency (2–4).

Chemoattractant-induced cell polarization is characterized by an asymmetric distribution of dynamic F-actin at the leading edge that is regulated by a complex array of actin regulatory proteins and lipid products of PI(3)-kinases, such as phosphatidylinositol (3, 4, 5)-triphosphate (PIP₃) (5, 6). Downstream effectors of PIP₃ include Src and Syk family tyrosine kinases, and Rho GTPases, such as Rac and Cdc42, which regulate the Arp2/3 complex and actin dynamics at the leading edge (7, 7–9). To establish and maintain polarity, these signaling mechanisms become amplified through positive feedback loops. For example, PIP₃ stimulates Rac activation through binding to Rho GTPase guanine exchange factors (GEFs), and active Rac can further activate PI(3)-kinase at the leading edge of chemoattractant-stimulated neutrophils (10). In addition, Hem-1, a scaffold component of the Scar/WAVE complex, can regulate actin polymerization by producing self-organizing waves of actin nucleation that provide a platform for cell signaling to mediate efficient neutrophil motility (11, 12). However, it remains unclear how neutrophils spatiotemporally coordinate these circuits to maximize migration efficiency, and what components are essential to regulate actin dynamics and signaling at the leading edge.

Hematopoietic lineage cell-specific protein 1 (HS1)² (13) is the hematopoietic homolog of the actin binding protein cortactin, that regulates leading edge actin dynamics, invadopodia formation, and cell invasion (14, 15). HS1 is structurally similar to cortactin and contains an N-terminal Arp2/3 binding region, three and one-half 37 amino acid repeating units that bind F-actin, a proline-rich region, and a C-terminal Src homology 3 (SH3) domain important for binding to Vav1 and the WIP and WASp heterodimer (16–18). HS1 is a substrate of Syk and Src family kinases (19, 20), and has been reported to regulate lamellipodial protrusions through stabilization of branched actin filaments (21, 22). Syk phosphorylation of HS1 occurs on tyrosines 378 and 397, and generates high affinity binding sites for SH2 domain-containing proteins. This promotes subsequent association and secondary phosphorylation of HS1 tyrosine 222 by Src family kinases (23, 24). Tyrosine phosphorylation of HS1 is important for regulating lymphocyte trafficking (25), and for mediating antigen-receptor signal transduction, growth arrest, and apoptosis (26, 27). Furthermore, phosphorylation of HS1 can regulate protein-protein interactions, such as HS1 interaction with Vav1 (16).

HS1 has been implicated in T cell signaling and podosome formation (16, 28); however its role in neutrophil chemotaxis is not known. In this study, we used the neutrophil-like PLB-985 cell line to investigate the function of HS1. We demonstrate that HS1 is highly expressed in PLB-985 cells, and HS1 co-localizes with F-actin at the leading edge in neutrophils. We show that HS1 is tyrosine phosphorylated in response to formyl-Met-Leu-Phe (fMLP) in a process that is dependent on Src family kinases. Moreover, HS1 regulates Vav1 and Rac activation in response to fMLP. HS1 and its phosphorylation on tyrosine 222, 378, and 397 are required for efficient neutrophil chemotaxis and binding to Arp2/3 complex. Our findings identify a role for HS1 phosphorylation in neutrophil-directed migration.

EXPERIMENTAL PROCEDURES

Reagents

Antibodies and reagents used in this report are mouse anti-HS1 (BD Biosciences), rabbit anti-HS1 (D83A8) XP, rabbit anti-phosphoHS1 (Y397), and rabbit anti-Rac1/2/3 (Cell Signaling Technology). Mouse anti-vinculin clone VIN-11–5 and mouse anti-β-actin clone AC-15, mouse anti-HA clone HA-7 (Sigma-Aldrich). Mouse anti-cortactin clone 4F11 and mouse anti-phosphotyrosine clone 4G10 (Millipore). Mouse anti-Rac2 (Proteintech Group), mouse anti-Vav1 clone B-6 (Santa Cruz Biotechnology), rabbit anti-phospho-Vav1(Y174) (Epitomics), goat anti-rabbit Alexa Fluor 680, goat anti-mouse Alexa Fluor 800, mouse anti-GFP (Invitrogen). FITC donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories) and Rhodamine phalloidin (Invitrogen). Glutathione-agarose (Clontech) and GammaBind G Sepharose (GE Healthcare). Human fibrinogen, f-Met-Leu-Phe (fMLP), PP2, BSA, and phosphatase inhibitor mixture 2 (Sigma-Aldrich). ICAM-1 was purified from transfected CHO cells by affinity purification as previously described (29), fibronectin was purified from human plasma by affinity chromatography as previously described (30), and human serum albumin (American Red Cross Blood Services).

Cell Culture and Primary Neutrophil Isolation

Primary human neutrophils were obtained from healthy donors with informed consent and purified using polymorphprep solution (Nycomed, Oslo, Norway) according to the manufacturer's instructions. PLB-985 cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin at a concentration of 0.1–1 × 10⁶ cells/ml. To differentiate, 1.25% DMSO was added to 2 × 10⁵ cells/ml for 6 days. HEK293T cells (American Type Culture Collection) and Platinum-GP cells (Cell Biolabs, Inc.) were maintained in Dulbecco's minimal essential medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin.

DNA Constructs, Transfections, and Viral Infections

HS1 was PCR amplified using the following primers: forward, 5′-ATGTGGAAGTCTGTAGTGGGCCATGATGTGTCTG-3′ and reverse, 5′-CTCCAGAAGCTTGACATAATTTGCAGGGAAGAGTCC-3′ and cloned into pEGFP-N1 (Takara Bio Inc.). HS1-GFPN1 was subcloned into the retroviral vector pMX-IRES-GFP (Cell Biolabs, Inc) from which the IRES-GFP sequence was excised using the following primers: forward, 5′-ATGTGGAAGTCTGTAGTGGGCCATGATGTGTCTG-3′ and reverse, 5′-CTTTTATTTTATCGTCGACTTACTTGTACAGCTCGTCCATGCCGAG-3′. HS1-ΔPS-GFP was generated by cloning HS1 amino acids 1–280 into pEGFP-N1 and pMX using the following primers: forward, 5′-ATGTGGAAGTCTGTAGTGGGCCATGATGTGTCTG-3′, and reverse, 5′-CGGTGGATCCCCCTGTGGAGCCTCAGGGCTCCTCTTTGTCACAGCCTTTCGC-3′.HS1-PS-GFP was generated by cloning HS1 amino acids 280–486 into pEGFP-N1 and pMX using the following primers: forward, 5′-TCGAATTCGCCACCATGCCAGTGATAGCTATGGAAGAG-3′, and reverse 5′-CTCCAGAAGCTTGACATAATTTGCAGGGAAGAGTCC-3′. An shRNA resistant full length HS1-GFP was generated by Quikchange site-directed mutagenesis (Agilent Technologies) of pMX-HS1-GFP using the following primers: sense, 5′-GATGAGCTTTCCTTTGATCCGGATGATGTGATTACGGATATCGAGATGGTGGACGAGGGC-3′ and antisense, 5′-GCCCTCGTCCACCATCTCGATATCCGTAATCACATCATCCGGATCAAAGGAAAGCTCATC-3′. HS1 phospho-mutants were subsequently generated by Quikchange site-directed mutagenesis of the shRNA-resistant pMX-HS1-GFP using the following primers: pMX-HS1(Y397F)-GFP sense 5′-GGATGAACCAGAGGGGGACTTTGAGGAGGTGCTCGAGCCTGAAGATT-3′ and antisense, 5′-AATCTTCAGGCTCGAGCACCTCCTCAAAGTCCCCCTCTGGTTCATCC-3′.pMX-HS1(Y2F)-GFP was generated by using site-directed mutagenesis of pMX-HS1(Y397F)-GFP using the following primers: sense, 5′-GCCCGAGCCTGAGAATGACTTTGAGGACGTTGAGGAGATG-3′ and antisense, 5′-CATCTCCTCAACGTCCTCAAAGTCATTCTCAGGCTCGGGC-3′. pMX-HS1(Y3F)-GFP was generated by site-directed mutagenesis of pMX-HS1(Y2F)-GFP using the following primers: sense, 5′-GAGGCCCCGACCACAGCTTTTAAGAAGACGACGCCCATAG-3′ and antisense 5′-CTATGGGCGTCGTCTTCTTAAAAGCTGTGGTCGGGGCCTC-3′.

Lentiviral HS1 shRNA targets were purchased (Thermo Fisher Scientific). Targets used in this study were control shRNA 5′-TGTCTCCGAACGTGTCACGTT-3′, HS1 shRNA 1 5′-CCTGCAAATTATGTCAAGCTT-3′, and HS1 shRNA 2 5′-GACGACGTAATCACTGACATT-3′. HEK293T cells were grown to 70% confluency in a 10-cm tissue culture dish for each lentiviral target and transfected using 6 μg of HS1 or control shRNA, 0.6 μg VSV-G, and 5.4 μg CMV 8.9.1. The 72-h viral supernatant was collected and concentrated using a lentivirus concentrator (Lenti-X; Takara Bio Inc.) following the manufacturer's instructions. 1 × 10⁶ PLB-985 cells were infected with viral supernatant for 3 days in the presence of 15 μg/ml polybrene. Stable cell lines were generated with 1 μg/ml puromycin selection.

Retroviral transduction of all pMX-GFP constructs was performed as follows: Platinum-GP cells were grown to 70% confluency in a 10-cm tissue culture dish and transfected using 10 μg of retroviral DNA and 3 μg VSV-G. The 72-h viral supernatant was collected and added to 1 × 10⁶ PLB-985 cells for 3 days in the presence of 15 μg/ml polybrene. Cells were sorted for equal GFP expression levels using the cell sorter (FACSAria; BD) at the University of Wisconsin flow cytometry facility.

Transwell Chemotaxis Assay

Transwell filters (Corning) with 3-μm pores were coated with 10 μg/ml fibrinogen for 1 h at 37 °C, blocked in 2% BSA for 30 min at 37 °C, and allowed to dry. 2 × 10⁵ differentiated PLB-985 cells were plated in the top chamber and were allowed to migrate for 90 min toward mHBSS (HBSS, 20 mm HEPES, 0.1% HSA) containing 10 nm fMLP at 37 °C, 5% CO₂. Cells in the lower chamber were detached by treatment with 45 mm EDTA for 15 min at 4 °C. Cells were quantified by flow cytometry. Percentage of cells transmigrated relative to loading control was determined and graphed either as percent migrated or percent migrated relative to control shRNA -fMLP from at least three independent experiments.

Rho GTPase Pull-down Assay

Rho GTPase pull-down assays were performed as described (31). In brief, differentiated PLB-985 cells were serum starved for 24 h in RPMI supplemented with 0.1% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin, and plated on 10 μg/ml fibrinogen for 10 min. Cells were stimulated with 1 μm fMLP for 1 min, immediately placed on ice, and lysed in lysis buffer (25 mm HEPES, pH 7.5, 150 mm NaCl₂, 1% Nonidet P-40, 10 mm MgCl₂, 1 mm EDTA, 10% glycerol, 1 μg/ml pepstatin A, 2 μg/ml aprotinin, 1 μg/ml leupeptin, and 200 nm phenylmethanesulfonyl fluoride, 1 mm sodium orthovanadate, 1% protease inhibitor mixture 2). 30 μg of bacterially expressed GST-PAK-Rac/Cdc42 (p21)-binding domain (PBD) was incubated with the lysates for 1 h at 4 °C. The affinity-precipitated products were run on SDS-PAGE gel, transferred to nitrocellulose, and immunoblotted for Rac. Quantification of total Rac for all samples was normalized to control shRNA -fMLP. The mean ± S.E. ratio of Rac-GTP/Rac was graphed relative to control shRNA-fMLP from three independent experiments.

Immunoblot Analysis and Immunoprecipitation

Differentiated PLB-985 or HEK293 cells were placed in lysis buffer (25 mm HEPES, pH 7.5, 150 mm NaCl₂, 1% Nonidet P-40, 10 mm MgCl₂, 1 mm EDTA, 10% glycerol, 1 μg/ml pepstatin A, 2 μg/ml aprotinin, 1 μg/ml leupeptin, and 200 nm phenylmethanesulfonyl fluoride, 1 mm sodium orthovanadate, 1% protease inhibitor mixture 2) on ice for 10 min and clarified by centrifugation. Protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Immunoblotting of cell lysates was performed and blots were imaged with an infrared imaging system (Odyssey; LI-COR Biosciences). For immunoprecipitations, differentiated PLB-985 cells were plated on 10 μg/ml fibrinogen or 2% BSA for 10 min at 37 °C, 5% CO₂. In some cases cells were pre-treated for 30 min with 10 μm PP2 or pervanadate (100 μm sodium orthovanadate, 3 mm H₂O₂). Cells were stimulated with 1 μm fMLP for the indicated time and were lysed by addition of ice-cold lysis buffer and clarified by centrifugation. Protein concentrations were determined using a BCA assay and equal protein amounts were incubated for 1 h at 4 °C with 3 μg of mouse anti-HS1 (BD Biosciences), rabbit anti-HS1 (Cell Signaling), mouse anti-Vav1 (Santa Cruz Biotechnology), or mouse anti-GFP (Invitrogen). Immune complexes were captured on GammaBind G-Sepharose beads (GE Healthcare), washed three times in lysis buffer, and analyzed by immunoblotting. For HS1-GFP and Vav1-HA coimmunoprecipitations, HEK293 cells were transfected with GFP or HS1-GFP and Vav1-HA (32). GFP was immunoprecipitated with mouse anti-GFP (Invitrogen), captured on GammaBind G-Sepharose beads, and analyzed by immunoblotting for HA.

Adhesion Assays

100,000 control shRNA and HS1 shRNA 2 cells were labeled with calcein AM (Invitrogen) and plated in the presence or absence of 100 nm fMLP for 30 min in quadruplicate in a 96-well black microplate (Greiner Bio-one) coated with fibrinogen, fibronectin, or ICAM-1 followed by blocking with 2% BSA at 37 °C and 5% CO₂. Fluorescence was measured using a plate reader (Victor3 V; PerkinElmer). Plates were gently washed with mHBSS (HBSS, 20 mm HEPES, 0.1% HSA) in between readings until the remaining control shRNA -fMLP cells were <10% of the original input.

Microfluidic Chemotaxis Assays and Immunofluorescence

Microfluidic devices were fabricated as previously described (33). In brief, the devices enable the generation of a static gradient of chemoattractant between a source chamber containing fMLP and a sink chamber containing the PLB-985 cells, which were connected by a 1-mm-long, 400-μm-wide channel in which the fMLP gradient is generated. Microfluidic chambers were coated with 20 μg/ml fibrinogen for 1 h at 37 °C in PBS. 3 μl of neutrophil suspension (4 × 10⁶ cells/ml) was added to the device, and 1 μm fMLP was added to the source port. The gradient was allowed to set up and equilibrate in a 37 °C humidified chamber for 15 min before imaging. Time-lapse imaging was performed using a 10×, NA of 0.45, or 60×, NA of 1.4 objective and motorized stage (Ludl Electronic Products) on an inverted microscope (Eclipse TE300) using a charge-coupled device camera (CoolSNAP ES2) and captured into MetaVue imaging software v6.2. Images were taken every 30 s for 30–45 min with up to eight devices being imaged simultaneously. Tracking and velocity measurements were performed using ImageJ analysis software (National Institutes of Health). For immunofluorescence, cells were allowed to chemotax in the microfluidic devices for 20 min, then fixed in 3.7% paraformaldehyde and stained with antibodies following standard protocols.

Statistical Analysis

For statistical comparisons, unpaired t test or one-way analysis of variance (ANOVA) were used with Tukey post-tests with p < 0.05 considered significant.

RESULTS

HS1 Localizes to the Leading Edge during Neutrophil Chemotaxis

To determine how HS1 regulates amoeboid cell chemotaxis, we used the myeloid leukemia cell line, PLB-985, which can be terminally differentiated into neutrophil-like cells with DMSO (34). Using immunoblotting to detect cortactin and HS1 in PLB-985 cells, we found that HS1, but not cortactin, is expressed in both differentiated and undifferentiated cells (Fig. 1B). HS1 is up-regulated in terminally differentiated PLB-985 cells, similar to actin and vinculin as reported previously (2), suggesting that HS1 expression is important for neutrophil function (Fig. 1B).

FIGURE 1.

HS1 is expressed in neutrophils and localizes to the leading edge during chemotaxis. A, schematic diagram of HS1 illustrating the Arp2/3 and F-actin binding regions, the proline-rich and SH3 domain, and tyrosines 222, 378, and 397 that are phosphorylated by Syk and Src family kinases. B, HS1 and cortactin expression analysis by Western blot. Equal total protein loaded from HEK293, undifferentiated, and differentiated PLB-985 cells were immunoblotted for cortactin, HS1, and actin. Cortactin expression is limited to HEK293 cells while HS1 expression is only in PLB-985 cells. Furthermore, HS1 and actin protein expression are up-regulated with terminal differentiation induced by DMSO for 6 days. Western immunoblot is representative of three independent experiments. C, immunofluorescent localization of HS1 (green) and F-actin (red) in differentiated PLB-985 cells (dPLB-985) and primary neutrophils in the presence and absence of a linear gradient of fMLP (1 μm). During chemotaxis in response to fMLP, HS1 colocalizes with F-actin in both cell types. Scale bar, 10 μm.

To characterize the intracellular localization of HS1 during chemotaxis, we detected endogenous HS1 using immunofluorescent staining in both differentiated PLB-985 cells and human peripheral blood neutrophils (Fig. 1C). In unstimulated neutrophils, HS1 colocalized with F-actin at the cell periphery; upon stimulation with fMLP, HS1 colocalized with F-actin at the leading edge in polarized neutrophils during chemotaxis. To determine if Arp2/3 and F-actin binding mediates this localization, we ectopically expressed the N-terminal Arp2/3 and F-actin binding repeats of HS1 (HS1-ΔPS-GFP) in PLB-985 cells (Fig. 2). We found that the F-actin and Arp2/3 binding region of HS1 was sufficient for leading edge localization. In contrast, a HS1-GFP construct containing only the C-terminal proline-rich region and SH3 domain (HS1-PS-GFP) displayed a more diffuse localization, similar to GFP expression alone, suggesting that the F-actin binding region is critical for HS1 localization (Fig. 2 and supplemental Movies S1 and S2).

FIGURE 2.

The N-terminal Arp2/3 and F-actin binding regions mediate HS1 localization. A, schematic of HS1 and two truncation mutants. HS1-ΔPS-GFP contains amino acids 1 through 280 including the N-terminal acidic domain that binds Arp2/3, and the F-actin binding region. HS1-PS-GFP contains amino acids 280 through 486 including the proline-rich region and SH3 domain. B, representative images from time-lapse movies of HS1-deficient PLB-985 cells stably expressing GFP, HS1-PS-GFP (supplemental Movie S1), or HS1-ΔPS-GFP (supplemental Movie S2) during chemotaxis to fMLP (1 μm) in microfluidic gradient generators. GFP- and HS1-PS-GFP-expressing PLB-985 cells showed diffuse cytoplasmic localization. HS1-ΔPS-GFP localized to the leading edge. Scale bar, 10 μm.

Chemoattractant-induced Phosphorylation of HS1 Is Dependent on Adhesion and Src Family Kinases

To determine if HS1 is phosphorylated in chemoattractant stimulated neutrophils, we immunoprecipitated HS1 from fMLP-stimulated PLB-985 cells and immunoblotted for total phosphotyrosine (Fig. 3, A and B). We found that fMLP induced HS1 phosphorylation with maximal phosphorylation after 3 min of stimulation. Treatment of cells with the Src inhibitor PP2 abolished fMLP-induced HS1 phosphorylation, indicating that Src family kinases are necessary for HS1 phosphorylation. In addition, tyrosine phosphorylation of HS1 was dependent on adhesion to fibrinogen because fMLP stimulation of cells plated on BSA did not significantly induce phosphorylation of HS1 (Fig. 3). To further characterize HS1 phosphorylation, an antibody to phospho-tyrosine 397 was used in fMLP-treated cells (Fig. 3, C and D). Accordingly, phosphorylation on Y397 induced by fMLP required both Src signaling and adhesion to fibrinogen (Fig. 3, C and D). Collectively, the data show that fMLP induces tyrosine phosphorylation of HS1 in adherent neutrophils.

FIGURE 3.

Tyrosine phosphorylation of HS1 is induced by fMLP and is mediated by adhesion and Src family kinases. A and C, immunoblot of HS1 immunoprecipitation from PLB-985 cells treated with fMLP (1 μm) and blotted for total phospho-tyrosine (A), or immunoblot of phospho-HS1(Y397) from cells treated with fMLP (1 μm) (C). Note maximal tyrosine phosphorylation of HS1 after 3 min with fMLP. Adhesion to fibrinogen (10 μg/ml) was necessary for significant HS1 phosphorylation. Treatment of cells plated on fibrinogen with PP2 (10 μm) impaired tyrosine phosphorylation in response to fMLP. B and D, quantification of HS1 tyrosine phosphorylation (B), or phospho-HS1 (Y397) (D). The ratio of phospho-HS1 to total HS1 was quantified and graphed relative to unstimulated cells in suspension. Graph represents mean ratio ± S.E. from at least three independent experiments. *, p < 0.05 by one-way ANOVA with Tukey post-test compared with unstimulated cells. Immunoblots are representative of three independent experiments.

HS1 Is Required for Efficient Neutrophil Chemotaxis

To characterize the function of HS1 during neutrophil motility, HS1-deficient PLB-985 cells were generated using lentiviral delivered shRNA (Fig. 4A). Two unique shRNA targets were found to significantly deplete endogenous HS1 levels. Using transwell assays to investigate chemotaxis to fMLP, we found that HS1-deficient cells had impaired migration compared with control cells (Fig. 4B). The effects on migration were dependent on the efficiency of the knockdown, with more significant effects observed with HS1 shRNA 2. Neutrophil random motility was not affected by HS1 depletion, suggesting that the defect in motility was downstream of chemoattractant-induced signaling. To determine if the defects in migration were due to altered adhesion, adhesion assays to fibrinogen, fibronectin, and ICAM-1 were performed (Fig. 4C). We observed no significant defects in fMLP-induced neutrophil adhesion in HS1-deficient cells.

FIGURE 4.

Neutrophil chemotaxis is impaired in HS1-deficient PLB-985 cells. A, lentiviral shRNAs were generated against two unique HS1 targets, both of which displayed a significant knockdown compared with scrambled control shRNA-expressing cells. Western immunoblot shows representative data from three independent experiments using differentiated PLB-985 cells. Actin is used as a loading control. B, Transwell migration assays showed reduced migration to fMLP (10 nm) in HS1 shRNA 1 and 2 cells compared with control shRNA-expressing cells; HS1 shRNA 2 displayed the most significant impairment of migration. Cell migration was quantified by flow cytometry and graphed as a percentage of the initial total cell input. The mean ± S.E. from three independent experiments are shown. p values were obtained by ANOVA with Tukey post-test. C, adhesion assay of differentiated control shRNA and HS1 shRNA 2 cells plated on fibrinogen (10 μg/ml), fibronectin (10 μg/ml), and ICAM-1 (2.5 μg/ml) showed no significant differences in adhesion between HS1-deficient cells relative to control. Cells were graphed as a percentage of input from three independent experiments in quadruplicate. Statistical significance was determined by ANOVA with Tukey post-test. D, schematic diagram of the microfluidic device illustrating the simple loading protocol used to analyze neutrophil chemotaxis. E, images taken from time-lapse movie over the course of 30 min of control (supplemental Movie S3) and HS1 knockdown cells (supplemental Movie S4) during chemotaxis on fibrinogen (20 μg/ml) showing automated cell tracking (yellow lines), scale bar, 100 μm. F and G, quantification of velocity (F) and chemotactic index (G) of control shRNA and HS1 shRNA 2-expressing cells during chemotaxis to fMLP. HS1 shRNA 2 cells exhibit decreased velocity and directionality compared with control. Graph represents mean ± S.D. of greater than 1000 cells from three independent experiments. p values were obtained by t test.

To further characterize the chemotaxis of HS1-deficient PLB-985 cells, we used microfluidic gradient generation in custom-designed chemotaxis chambers that allow the precise control of chemoattractant gradients over extended durations under flow free conditions (Fig. 4D) (35). Using live imaging, HS1-deficient cells had impaired chemotaxis to fMLP compared with control cells (Fig. 4E and supplemental Movies S3 and S4). Automated cell tracking revealed a significant decrease in cell speed and directionality in HS1-deficient cells compared with control cells in response to fMLP (Fig. 4, F and G). Taken together, our findings suggest that HS1 is essential for efficient neutrophil chemotaxis to fMLP.

HS1 Is Necessary for Rac Activation and Vav1 Phosphorylation

HS1 phosphorylation regulates Rac and Cdc42 activation in Natural Killer cells (36). To determine if Rac activation is altered in HS1-deficient neutrophils, we performed GST-PAK-PBD pulldown assays for Rac-GTP. We found a significant reduction in total Rac-GTP levels in HS1-deficient cells compared with control cells treated with fMLP (Fig. 5, A and B). Moreover, there was a reduction of Rac2-GTP, the predominant Rac isoform in neutrophils (37) (Fig. 5, A and C). Vav1, a GEF for Rac, binds to the SH3 domain of HS1 (16). To determine if HS1 is necessary for Vav1 phosphorylation induced by fMLP, we tested the effects of HS1 depletion on fMLP-induced phosphorylation of Vav1 using phospho-Vav1(Y174) antibodies (Fig. 6A). Control cells exhibited maximal phosphorylation of Vav1 after 3 min of fMLP stimulation, however depletion of HS1 significantly impaired chemoattractant induced Vav1 phosphorylation (Fig. 6). Together, these data demonstrate that HS1 is required for activation of Rac2 and Vav1 phosphorylation in response to fMLP.

FIGURE 5.

HS1-deficient PLB-985 cells have impaired Rac activation in response to fMLP. A, differentiated control shRNA and HS1 shRNA 2-expressing cells were plated on fibrinogen (10 μg/ml) in mHBSS for 5 min, and then stimulated with a uniform concentration of 1 μm fMLP for 1 min. GST-PAK-PBD pulldown assays were performed and showed decreased total Rac-GTP and Rac2-GTP in HS1-deficient cells compared with control shRNA. B and C, mean ratio of Rac-GTP/Rac ± S.E. (B), or Rac2-GTP/Rac2 ± S.E. C was graphed relative to control from three independent experiments. *, p < 0.05; one-way ANOVA with Tukey post-test.

FIGURE 6.

HS1-deficient PLB-985 cells have impaired Vav1 phosphorylation in response to fMLP. A, immunoprecipitation of Vav1 and immunoblot of phospho-Vav1(Y174) from control shRNA and HS1 shRNA 2 cells in suspension, or plated on fibrinogen (10 μg/ml), in the absence or presence of fMLP (1 μm) for 1, 3, and 5 min. B, quantification of the ratio of phospho-Vav1(Y174) to total Vav1 relative to non-plated and unstimulated control shRNA cells. In control shRNA cells Vav1 was maximally phosphorylated after 3 min of stimulation with fMLP, and this was significantly reduced upon HS1 depletion. Graph represents mean ± S.E. from three independent experiments. *, p < 0.01; one-way ANOVA with Tukey post-test.

Phosphorylation of Tyrosines 222, 378, and 397 Is Necessary for Efficient Neutrophil Chemotaxis

Syk phosphorylation of HS1 tyrosines 378 and 397 is required for Src family kinase interaction with HS1 and subsequent phosphorylation of tyrosine 222 (24). To investigate the role of HS1 tyrosine phosphorylation during neutrophil chemotaxis, we generated control shRNA cells that stably express GFP, and HS1-deficient cells that stably express either GFP, HS1-GFP, or one of two phospho-mutant HS1 constructs: HS1(Y2F)-GFP with Y378,397F mutations, and HS1(Y3F)-GFP with Y222,378,397F mutations (Fig. 7A). To detect phosphorylation of these constructs, GFP- and HS1-GFP-expressing cells were treated with pervanadate and analyzed for phosphorylation at tyrosine 397 (Fig. 7A). HS1-deficient cells expressing wild-type HS1-GFP showed specific phosphorylation, however, HS1(Y2F)-GFP and HS1(Y3F)-GFP were not phosphorylated at tyrosine 397. Differences in phosphorylation at tyrosine 397 and total tyrosine phosphorylation were also observed in response to fMLP (supplemental Fig. S1). To determine if phosphorylation of HS1 regulates localization to the leading edge of cells during chemotaxis, live-cell imaging in microfluidic chemotaxis chambers was performed (Fig. 7B). GFP-expressing cells showed diffuse localization throughout the cytoplasm (supplemental Movies S5 and S6), however wild-type HS1-GFP, HS1(Y2F)-GFP, and HS1(Y3F)-GFP localized to the leading edge of cells during chemotaxis, similar to Fig. 1C (supplemental Movies S7–S9). These results demonstrate that phosphorylation of HS1 tyrosines 222, 378, and 397 is not necessary for leading edge localization of HS1 in neutrophils.

FIGURE 7.

Phosphorylation of HS1 tyrosines 222, 378, and 397 mediates neutrophil chemotaxis. A, immunoblot of control shRNA PLB-985 cells expressing GFP, or HS1-deficient cells expressing GFP, HS1-GFP, HS1(Y2F)-GFP, or HS1(Y3F)-GFP. HS1 immunoprecipitation and immunoblot of phospho-HS1(Y397) in pervanadate-treated cells showed increased phosphorylation of endogenous and wild-type HS1-GFP at tyrosine 397, while the HS1(Y2F)-GFP and HS1(Y3F)-GFP phospho-mutants had little to no phosphorylation. Lower bands in the HS1 immunoblot are endogenous HS1, and upper bands are exogenous HS1-GFP fusion proteins. B, representative image from time-lapse movies of HS1 shRNA 2 cells expressing HS1-GFP, HS1(Y2F)-GFP, or HS1(Y3F)-GFP (supplemental Movies S7–S9) during chemotaxis to fMLP (1 μm) in microfluidic gradient generators. HS1-GFP, HS1(Y2F)-GFP, and HS1(Y3F)-GFP all showed restoration of leading edge localization similar to endogenous HS1 in Fig. 1C. Scale bar, 10 μm. C, Transwell migration assays to fMLP (10 nm) of control shRNA cells expressing GFP or HS1 shRNA 2 cells expressing GFP, HS1-GFP, HS1(Y2F)-GFP, or HS1(Y3F)-GFP. HS1 shRNA 2 cells expressing GFP exhibited significantly reduced migration compared with control shRNA GFP cells, similar to Fig. 4B. Ectopic expression of wild-type HS1-GFP or HS1(Y2F)-GFP was able to fully rescue the migration defect to control shRNA levels. However, expression of HS1(Y3F)-GFP showed no change in chemotaxis in HS1-deficient cells. Graph represents mean ± S.E. from three independent experiments performed in duplicate. Means with common letters indicate p > 0.05 by ANOVA with Tukey post-test. D, immunoblot of control shRNA PLB-985 cells expressing GFP, or HS1-deficient cells expressing GFP, HS1(Y222F)-GFP, HS1(Y378F)-GFP, or HS1(Y397F)-GFP. Immunoblot of phospho-HS1(Y397) in fMLP-treated cells showed increased phosphorylation of endogenous HS1 and HS1(Y222F)-GFP at tyrosine 397, while the HS1(Y378F)-GFP and HS1(Y397F)-GFP phospho-mutants had little to no phosphorylation. Lower bands in the HS1 immunoblot are endogenous HS1, and upper bands are exogenous HS1-GFP fusion proteins. E, Transwell migration assays to fMLP (10 nm) of control shRNA cells expressing GFP or HS1 shRNA 2 cells expressing GFP, HS1(Y222F)-GFP, HS1(Y378F)-GFP, or HS1(Y397F)-GFP. HS1 shRNA 2 cells expressing GFP exhibited significantly reduced migration compared with control shRNA GFP cells, similar to Fig. 4B. Ectopic expression of the three single phospho-mutants of HS1 was able to fully rescue the migration defect to control shRNA levels. Graph represents mean ± S.E. from three independent experiments performed in duplicate. *, p < 0.05 by ANOVA with Tukey post-test.

Transwell migration assays were used to determine if phosphorylation of HS1 affects neutrophil chemotaxis. Similar to Fig. 4B, HS1-deficient cells expressing GFP had a significant reduction in chemotaxis compared with control shRNA cells (Fig. 7C). This defect could be fully rescued by expression of wild-type HS1-GFP. Surprisingly, expression of HS1(Y2F)-GFP was also able to rescue the chemotaxis defect, suggesting that phosphorylation of tyrosines 378 and 397 is not necessary for fMLP-mediated chemotaxis. However, HS1(Y3F)-GFP-expressing cells displayed a significant reduction in neutrophil chemotaxis compared with control, suggesting that phosphorylation of tyrosines 222, 378, and 397, is required for efficient neutrophil chemotaxis.

To determine if phosphorylation of tyrosine residues 222, 378, or 397 alone is required for neutrophil chemotaxis we generated HS1 shRNA cells that stably express HS1(Y222F)-GFP, HS1(Y378F)-GFP, or HS1(Y397F)-GFP (Fig. 7D). In response to fMLP, cells expressing HS1(Y222F)-GFP showed increased phosphorylation on tyrosine 397 similar to control. However, phosphorylation of tyrosine 397 in both HS1(Y378F)- and HS1(Y397F)-expressing cells was reduced (Fig. 7D), raising the possibility that the commercially available polyclonal antibody against phospho-HS1(Y397) cross-reacts with phospho-HS1(Y378). Using transwell migration assays we found that expression of HS1(Y222F), HS1(Y378F), or HS1(Y397F) were sufficient to rescue migration of HS1-deficient cells (Fig. 7E). Collectively, these findings indicate that phosphorylation of tyrosines 222, 378, and 397 of HS1 is important for efficient neutrophil chemotaxis.

HS1 Phosphorylation Regulates Interactions with Vav1 and Arp2/3

To determine how phosphorylation on tyrosines 222, 378, and 397 regulates neutrophil chemotaxis, we characterized the role of tyrosine phosphorylation on interactions with Vav1 and Arp2/3. HEK293 cells were co-transfected with HA-tagged Vav1, and either GFP, HS1-GFP, or the two phospho-mutant constructs, HS1(Y2F)-GFP and HS1(Y3F)-GFP, and treated with pervanadate. HS1(Y2F) and HS1(Y3F) phospho-mutants had significantly impaired interactions with Vav1 compared with wild-type HS1 (Fig. 8, A and B). In accordance with previous publications, this suggests that Y378 and Y397 are necessary for Vav1 binding (17, 36), but this does not explain the specific defect in chemotaxis in HS1-deficient cells that express HS1(Y3F).

FIGURE 8.

Phosphorylation of HS1 Y222, Y378, and Y397 mediate interactions with Vav1 and Arp2/3. A, immunoprecipitation of GFP and immunoblot for HA-tagged Vav1 from pervanadate treated HEK293 cells co-expressing Vav1-HA and GFP, HS1-GFP, HS1(Y2F)-GFP, or HS1(Y3F)-GFP. Full-length HS1-GFP was able to interact with Vav1, however HS1(Y2F)-GFP and HS1(Y3F)-GFP had a significant decrease in Vav1 interaction. B, quantification of the average ratio of Vav1-HA to immunoprecipitated HS1-GFP constructs relative to wild-type HS1-GFP-expressing cells. *, p < 0.05; one-way ANOVA with Tukey post-test from three independent experiments. C, immunoprecipitation of GFP and immunoblot for Arp2/3 complex subunit 2 (ARPC2) in control PLB-985 cells expressing GFP, or HS1-deficient cells expressing HS1-GFP, HS1(Y2F)-GFP, or HS1(Y3F)-GFP in response to fMLP (1 μm) stimulation for 3 min. HS1(Y3F)-GFP had impaired interactions with Arp2/3 compared with wild-type HS1-GFP and HS1(Y2F)-GFP. D, quantification of the average ratio of ARPC2 to immunoprecipitated HS1-GFP constructs relative to wild-type HS1-GFP-expressing cells. *, p < 0.05; one-way ANOVA with Tukey post-test from three independent experiments.

It has also been reported that HS1 can interact with Arp2/3, a key regulator of cell motility. We therefore investigated how tyrosine phosphorylation of HS1 regulates binding to the Arp2/3 complex. HS1-deficient PLB-985 cells stably expressing wild-type HS1 or HS1(Y2F)-GFP were able to interact with endogenous Arp2/3 complex, subunit 2 (ARPC2). However, cells expressing the triple-mutant HS1(Y3F)-GFP had a significant reduction in ARPC2 interactions (Fig. 8, C and D). Collectively, these data demonstrate that tyrosine phosphorylation of HS1 residues 378 and 397 regulates its interaction with Vav1, and that tyrosine phosphorylation of 222, 378, and 397 is required for interactions with the Arp2/3 complex and for efficient neutrophil chemotaxis.

DISCUSSION

There is substantial interest in understanding how the leading edge is regulated during amoeboid motility, including how directional signals are amplified to spatially coordinate cytoskeletal rearrangements during directed motility. We have now identified HS1 as a central component of the leading edge that provides a key link between chemoattractant stimulation and downstream signaling to Vav1 and Rac. Rac is important for regulating dynamic F-actin at the leading edge and is part of a positive feedback loop to PI(3,4,5)P₃, which requires actin polymerization (38). Furthermore, HS1 is important in PI(3,4,5)P₃ signaling through its direct interaction with PI(3)K and Vav1 (16, 39). Therefore, it is intriguing to speculate that HS1 is a central adaptor protein that regulates spatiotemporal localization of PI(3)K, Vav1, and Rac to promote actin polymerization and amplification of signaling at the leading edge during chemotaxis.

Using indirect immunofluorescence we found that HS1 colocalizes with F-actin at the leading edge of neutrophils during chemotaxis and that chemoattractant stimulation induces HS1 phosphorylation through Src kinase signaling. Previous reports have shown that Syk phosphorylation of HS1 tyrosines 378 and 397 promotes Src family kinase binding and subsequent phosphorylation of tyrosine 222 (23, 24). However, activation of Syk by integrin signaling is dependent on Src family kinases (40), therefore inhibition of Src kinases using PP2 may disrupt Syk activation and HS1 tyrosine phosphorylation. In any case, our findings suggest that both chemoattractant and integrin-mediated adhesion is critical for phosphorylation of HS1 through a Src-dependent pathway. However, the identity of the key Src kinase family member in neutrophils that mediates HS1 phosphorylation is not known.

Few studies have addressed how HS1, the hematopoietic specific homolog of cortactin, regulates leukocyte motility. It is known that HS1 can regulate the actin cytoskeleton in leukocytes through its ability to bind and activate Arp2/3, and by interacting with Vav1 (16, 22). More specifically, a recent report demonstrated that HS1 mediates dendritic cell podosome formation and motility through its interactions with the WIP and WASp heterodimer (28). We now report for the first time, that HS1 is a key regulator of neutrophil chemotaxis. Using time-lapse microscopy in microchannels we show that HS1-deficient cells have impaired velocity and directional persistence in response to a gradient of fMLP. We had previously reported that the HS1 binding partner Hax1 regulates neutrophil chemotaxis through its effects on integrin-mediated adhesion and Rho GTPase activation (2). However, HS1-deficient neutrophils displayed normal integrin-mediated adhesion. This is in contrast to HS1-deficient natural killer cells, which have impaired adhesion to ICAM-1 (36).

T-cell receptor ligation induces HS1 phosphorylation that mediates Vav1 recruitment and activation at the immune synapse (16). Moreover, phosphorylation of tyrosines 378 and 397 is critical for HS1 function in natural killer cells (36). In contrast, we found that phosphorylation on tyrosines 378 and 397 is not necessary for efficient neutrophil chemotaxis since ectopic expression of HS1(Y2F)-GFP was sufficient to rescue chemotaxis of HS1-deficient cells. However, tyrosine 222 is critical for efficient neutrophil chemotaxis since the triple mutant (Y222F, Y378F, and Y397F) was not sufficient to rescue migration of HS1-deficient cells. Our findings also show that HS1-deficient neutrophils have impaired activation of Rac and Vav1 signaling. Based on our findings we hypothesize that phosphorylation of HS1 regulates its interaction with Vav1 and the spatiotemporal activation of Rac. Accordingly, we showed that phosphorylation of tyrosines 378 and 397 is required for HS1 interaction with Vav1. However, our findings demonstrate the surprising finding that HS1-(Y3F) but not HS1-(Y2F) had impaired interactions with Arp2/3, suggesting that HS1 interaction with Arp2/3 is critical for its effects on neutrophil directed migration. Our findings are consistent with a previous report that the HS1 coiled-coil region is required for F-actin binding and synergistic Arp2/3 activation (22), however our report is the first to show that phosphorylation of HS1 regulates its interaction with Arp2/3. Taken together these findings support a model in which fMLP induced phosphorylation of HS1 mediates Vav1 phosphorylation and activation of Rac and Arp2/3 that amplify leading edge signaling to mediate efficient neutrophil chemotaxis.

In summary, we identified a novel role for HS1 in neutrophil chemotaxis through chemoattractant-induced phosphorylation of tyrosines 222, 378, and 397. An important remaining question is how phosphorylation of tyrosine 222 of HS1 specifically modulates the interaction with Arp2/3 in the N-terminal acidic domain, and what key effector proteins bind to HS1 through the regulated phosphorylation of tyrosine 222. In addition it will be important to address how HS1 orchestrates cross talk between Syk and Src kinases to mediate efficient chemotaxis. These types of questions are challenges for future investigation. Collectively, our findings suggest that HS1 is an important adaptor molecule that links Rac and Vav1 signaling to actin dynamics and represents a central point of regulation to spatiotemporally coordinate feedback mechanisms during chemotaxis.