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Molecular and Cellular Biology, June 2008, p. 3776-3789, Vol. 28, No. 11
0270-7306/08/$08.00+0     doi:10.1128/MCB.00507-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

PIX Rho GTPase Guanine Nucleotide Exchange Factor Regulates Lymphocyte Functions and Antigen Receptor Signaling

Karine Missy,^1, Bin Hu,^1, Kerstin Schilling,^1,2 Anke Harenberg,¹ Vadim Sakk,¹ Kerstin Kuchenbecker,³ Kerstin Kutsche,³ and Klaus-Dieter Fischer^2*

Institute of Physiological Chemistry, University of Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany,¹ Institute of Biochemistry and Cell Biology, Otto von Guericke University, Leipziger Str. 44, 39120 Magdeburg, Germany,² Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, Butenfeld 42, 22529 Hamburg, Germany³

Received 22 March 2007/ Returned for modification 30 April 2007/ Accepted 17 March 2008

	ABSTRACT

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
PIX is a Rho GTPase guanine nucleotide exchange factor domain-containing signaling protein that associates with other proteins involved in cytoskeletal-membrane complexes. It has been shown that PIX proteins play roles in some immune cells, including neutrophils and T cells. In this study, we report the immune system phenotype of PIX knockout mice. We extended PIX expression experiments and found that whereas PIX was specific to immune cells, its homolog βPIX was expressed in a wider range of cells. Mice lacking PIX had reduced numbers of mature lymphocytes and defective immune responses. Antigen receptor-directed proliferation of PIX^– T and B cells was also reduced, but basal migration was enhanced. Accompanying these defects, formation of T-cell-B-cell conjugates and recruitment of PAK and Lfa-1 integrin to the immune synapse were impaired in the absence of PIX. Proximal antigen receptor signaling was largely unaffected, with the exception of reduced phosphorylation of PAK and expression of GIT2 in both T cells and B cells. These results reveal specific roles for PIX in the immune system and suggest that redundancy with βPIX precludes a more severe immune phenotype.

	INTRODUCTION

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The activation of lymphocytes by antigen is critical to the generation of specific immune responses. An antigen stimulates signaling cascades in T and B cells via the T-cell antigen receptor (TCR) and the B-cell antigen receptor (BCR). These signaling cascades produce multiple measurable outputs, including tyrosine phosphorylation of proteins, mitogen-activated protein kinase activation, calcium fluxing, and protein degradation. On a larger scale, activation of signaling causes remodeling of macromolecular complexes, such as immune synapses or focal adhesions, enabling a cell to differentiate or to migrate (6, 46). One family of proteins that is important for organizing such signaling complexes in immune cells is the Rho GTPase guanine nucleotide exchange factors (RhoGEFs) (21, 55). RhoGEFs are associated with cytoskeletal remodeling, since they are enzymes that activate Rho family GTPases, such as Rho, Rac, or Cdc42, by catalyzing the exchange of GTP for GDP on the GTPase (26). RhoGEFs contain multiple protein interaction domains and bind to a variety of signaling proteins. The PIX (p21-interacting exchange factor) family RhoGEFs were identified through binding to the PAK kinases (serine/threonine p21-activated kinases) (2, 34, 50).

Members of the PIX family of GEFs include PIX/Cool-2 (cloned out of library), βPIX/Cool-1/p85SPR, and an alternate isoform of βPIX called p50^cool-1 (2, 34, 40). PIX proteins have in common an SH3 domain, a paired Dbl homology (DH) domain, and a pleckstrin homology (PH) domain for activation of Rho GTPases (also known as a RhoGEF domain), but they differ in the lengths of their N- and C-terminal regions (15, 30): PIX contains an N-terminal calponin homology (CH) domain (51), while βPIX does not. Also, the PIX gene, but not the βPIX gene, maps to the X chromosome (32). Both PIX proteins share a coiled-coiled domain implicated in dimerization and a domain called the GIT-binding sequence (50). Although PIX GEFs can activate Rac1 and Cdc42 GTPases, they are subject to many levels of control, including requirements for phosphorylation (54), for monomerization or dimerization (16), for relief from an inhibitory domain (15), and for binding to activated GTPases (3).

PIX proteins associate with a wide variety of proteins, from the neuronal synapse protein Shank (41) and the polarity complex protein Scribble (1), to signaling proteins such as PAK or phosphatidylinositol 3-kinase (p85 subunit) (34, 60), to actin-associated proteins such as β-parvin/affixin (49) and Abi-1 (12). PIX proteins also bind to degradation-related proteins, such as E3 ubiquitin ligases c-Cbl (18) and atrophin-interacting protein 4 (28), and calpain regulatory subunit (48). PIX proteins may play roles in lymphocyte disease by facilitating human immunodeficiency virus Nef functions (9) and through binding to X-linked lymphoproliferative disease protein SAP (23). The predominant binding partners for PIX proteins, however, are GIT proteins (G-protein-coupled receptor kinase-interacting proteins 1 and 2), also known as CAT proteins/p95PKL/APP1/2 (25). PIX proteins and GIT proteins associate in large, stable oligomeric complexes that recruit Rac1 and Cdc42 GTPases and PAK kinases (45). These associations enable PIX protein participation in actin-dependent cell functions, such as migration (57), cell spreading (48), neurite extension (53, 61), and focal complexes (50). It is likely that these functions are tightly coordinated with those of GIT proteins, which include membrane recycling and endosomal dynamics (25).

The mutation of PIX in mice results in neutrophils defective in orienting and migrating toward a chemoattractant (33). This phenotype resembles that of GIT2 knockout mice, which also have neutrophils with direction-sensing defects (36). In humans, PIX mutations are associated with X-linked mental retardation (32). PIX knockout lymphocytes have not been described in detail; however, results of studies on PIX in Jurkat T cells point to multiple roles for PIX proteins in T cells. Using overexpression of a mutant βPIX to block PIX signaling in Jurkat T cells, it was shown that PIX proteins are complexed with GIT and PAK and are required for PAK activation by TCR (31). A subsequent study revealed that PIX proteins target PAK to the immune synapse and are required for TCR signaling to phospholipase C-1 and transcription (43). As the overexpression of a βPIX mutant would likely block PIX signaling as well, the functions that can be specifically ascribed to PIX in T cells have not yet been determined.

We aimed to investigate PIX alone, and not βPIX, in lymphocytes. To this end, we generated PIX-deficient mice by gene targeting. We describe here several cellular and molecular defects that can be specifically attributed to PIX. On a systemic level, immune responses were defective and mature lymphocyte populations were reduced. At the cellular level, antigen receptor-induced proliferation was decreased and spontaneous migration was increased in the absence of PIX. Molecular events that were altered in PIX^– T and B cells included decreased GIT2 expression, PAK phosphorylation, and recruitment of PAK and Lfa-1 to the immune synapse. Thus, this study of PIX^– mice identifies nonredundant roles for PIX that cannot be compensated for by βPIX and reveals new functions for RhoGEFs in the immune system.

	MATERIALS AND METHODS

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PIX gene targeting. A genomic mouse 129/Ola library (RZPD Center, Berlin, Germany) was screened with an PIX cDNA probe amplified by reverse transcription-PCR. Cosmids were isolated, and a portion of the genomic DNA of one of these cosmids (clone MPMGc121F09630) was subcloned into pBluescript and used to generate the targeting vector. Genomic DNA encoding amino acids 400 to 465 was exchanged by a neomycin cassette flanked by loxP sites. The targeting construct was linearized and electroporated into E14 (129/Ola) embryonic stem (ES) cells. Homologous recombinants were analyzed by Southern blotting and PCR. Targeted ES clones were aggregated with eight-cell-stage embryos (39), which were subsequently transferred to pseudopregnant mice to generate chimeric offspring. Chimeric males were then bred with C57BL/6 females to obtain PIX knockout mice. OT-II mice were kindly provided by F. R. Carbone. Mice used in this study were backcrossed to C57BL6 mice for four to five generations, and 6- to 12-week-old littermates or age-matched mice were analyzed. Animals were housed under specific-pathogen-free conditions under institutional guidelines.

Flow cytometry, cell purification, proliferation, and migration. Thymuses, spleens, and lymph nodes were dissected and crushed in RPMI medium, and samples were depleted of red blood cells. Cell surface marker expression was analyzed using a four-color flow cytometer (FACScalibur; Becton Dickinson) and CellQuest software. The following antibodies were used: CD21-fluorescein isothiocyanate (FITC), CD23-phycoerythrin (PE), B220-peridinin-chlorophyll-protein complex (PerCP), Thy1.2-allophycocyanin (APC), CD4-PE, CD8-FITC, CD8-PE, B220-FITC, immunoglobulin M (IgM)-PE, B220-Cy5, IgD-FITC, and IgM-FITC (Pharmingen). Cell analysis was performed by complement lysis purification and fluorescence-activated cell sorting (FACS) as previously described (21, 55). In brief, B cells were purified from mouse spleens by complement lysis of cells precoated with anti-CD4 (no. 172) and anti-CD8 (31 M) followed by gradient purification over Lympholyte M (Cedarlane). T cells were purified in the same manner, except splenocytes were precoated with anti-B220 (RA3-3A1) and anti-major histocompatibility complex II (MHC-II; anti-IAb, IAd hybridoma; ATCC HB-35). Cell purity was assessed by flow cytometry. Purified (>90%) B cells (1 x 10⁵) were seeded into round-bottom 96-well plates (Costar) in freshly prepared Iscove's modified Dulbecco's medium, 10% fetal calf serum, and 10^–5 M β-mercaptoethanol or RPMI and activated with B7.6 monoclonal antibody (MAb) anti-IgM, F(ab')2 anti-IgM (Jackson Laboratories), anti-CD40 [FGK45(47)], interleukin-4 (IL-4), or lipopolysaccharide (LPS). B cells were harvested at 3 days after a 12-h pulse with 0.5 to 1 µCi [³H]thymidine/well. Purified lymph node T cells (1 x 10⁵/well) were stimulated with anti-CD3 alone (2.5 µg/ml; MAb 145-2C11) or together with anti-CD28 (0.5 µg/ml; MAb 37.51; Pharmingen) and concanavalin A (ConA; 5 µg/ml), or with a combination of phorbol myristate acetate (PMA; 5 ng/ml) plus ionomycin (0.5 µg/ml; Sigma). Supernatants were collected for cytokine enzyme-linked immunosorbent assays (ELISAs) before [³H]thymidine for proliferation assays was added. To assay migration, Lympholyte M-purified splenocytes or lymph node cells (1 x 10⁶) were incubated at 37°C for 0.5 h in RPMI containing 0.25% fatty acid-free bovine serum albumin (Sigma) and 10 mM HEPES. Transmigration to SDF/1 (R&D) was assayed in 5-µm-pore-size Costar transwell plates. Cells were stained with anti-CD4-FITC, anti-CD8-PE, and anti-B220-PerCP and counted by FACS. Migration was calculated as the percentage of the "input" samples.

Cell signaling and PIX expression. For analysis of PIX expression, tissues and hematopoietic cells (purity, >90%) from wild-type mice were isolated and purified as previously described (21). Protein content was estimated by a Bradford assay (Bio-Rad, Richmond, CA), and equal amounts of protein (70 µg) from each lysate were used to determine PIX expression. Proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed with an anti-PIX antibody recognizing both - and βPIX as described previously (34) or commercially obtained (Chemicon International, Temecula, CA) with similar results. To analyze lymphocyte signaling, purified T cells were preincubated for 1 h at 37°C in RPMI and then incubated with 15 µg/ml anti-CD3 alone or in combination with 5 µg/ml anti-CD28 for 10 min on ice. Cells were warmed at 37°C for 1 min, and stimulations were started by the addition of 30 µg/ml anti-hamster antibody (Serotec). B-cell stimulation was performed using anti-CD40 (FGK 45) (47) as described elsewhere (55). Immunoblot assays were performed with anti-phospho-PAK1/2 (Ser199/204 of PAK1 and Ser192/197 of PAK2; Cell Signaling), anti-phospho-Erk1/2 (E4; Santa Cruz), anti-phospho-c-Cbl (Y774; Cell Signaling), and anti-PAK (N20; Santa Cruz), anti-GIT1 (H-170; Santa Cruz sc-1396), anti-GIT1/2 (p95PKL/GIT; BD 611388), anti-SAP (Chemicon AB4069), and anti-Cbl (Santa Cruz sc-170). Equal loading of membranes was confirmed by Erk1/2 (K-23; Santa Cruz) staining. GTPase activity assays using glutathione S-transferase-PAK as activation probe and anti-Rac1 (Santa Cruz) and anti-CDC42 antibodies (Transduction Laboratories) were performed as described elsewhere (24). To measure calcium fluxing, lymph node or splenic cells (5 x 10⁶) were incubated with 4 µg/ml of Fluo-3-AM per ml and 10 µg/ml of FuraRed (Molecular Probes) in RPMI for 45 min at 37°C. T cells were stained with anti-Thy1-APC (Pharmingen) on ice and stimulated with anti-CD3 (5 µg/ml) cross-linked with anti-hamster IgG (10 µg/ml) to start stimulation. B cells were stained with anti-B220 (Pharmingen) on ice and stimulated with F(ab')2 anti-IgM (10 µg/ml). Calcium influx was monitored by flow cytometry, and data were analyzed with FlowJo software. TCR-induced actin polymerization using anti-CD3 at 5 µg/ml, followed by anti-hamster IgG at 5 µg/ml, was performed essentially as described previously (17).

GIT2 expression analysis. Quantitative reverse transcription-PCR was performed as described elsewhere (10). In brief, total RNA isolated from mouse lymph node T cells using the High Pure RNA isolation kit (Roche Diagnostics) was used to prepare random-primed cDNA. Quantitative PCR was conducted using the QuantiTect Sybr green PCR kit (Qiagen). The real-time PCR was performed in a Light Cycler (Roche) using Light Cycler software (Roche) for fluorescence detection and data evaluation. The housekeeping gene PBGD was used to standardize the cDNA content. The following GIT2 oligonucleotide primers were used: 5'-AACACTCTCTGCTGGACCCT-3' and 5'-GGACGAACGCTAACATCTGA-3' (N-terminal primer) and 5'-CAGGAGACTCCAGCTTACCG-3' and 5'-CATAGGCACACTGGATGACC-3' (C-terminal primer). For Northern blot analysis, total RNA from lymph nodes was separated by gel electrophoresis, blotted, and probed with an N-terminal GIT2 probe generated by reverse transcription-PCR using the following oligonucleotide primers: 5'-CCTGCTCCAGATGGTTGAGA-3' and 5'-CGCCTGTCAACTTCGTCGTA-3'. Specific protein expression was analyzed by Western blotting using an antibody recognizing GIT2 long and GIT2 short (Becton-Dickinson).

T-cell-APC conjugate formation and synapse microscopy. For peptide-induced cell-cell conjugation, LB27.4 cells (H-2^d/b-restricted B-cell hybrid; ATCC) were pulsed with 1 µg/ml OVA II peptide (OVA_329-339) at 37°C for 4 h and stained with 5 µM CellTracker Orange 5 (and 6)-{[(4-chloromethyl)benzoyl]amino}tetramethylrhodamine (CMTMR). Purified OT-II TCR-transgenic CD4⁺ T cells were stained with 0.1 µM 5 (and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), mixed with B cells at a 1:1 ratio, and centrifuged at 40 x g for 5 min to initiate conjugate formation and then incubated at 37°C for 30 min. Resulting conjugates were analyzed by FACS. To assess immune synapses, purified C57BL/6 wild-type splenic B cells were pulsed overnight at 37°C with OVA II peptide (10 µg/ml) in the presence of LPS (30 µg/ml). Cells were collected, stained with CellTracker Blue 7-amino-4-chloromethylcoumarin (CMAC; (Molecular Probes), and mixed at a 1:1 ratio with purified CD4⁺ OT-II CD4⁺ T cells from lymph nodes. The mixed cell suspension was briefly centrifuged (30 seconds at 4,500 x g) to initiate conjugation. After incubation at 37°C for 5 min, cells were carefully resuspended, settled on an eight-well mask slide, and fixed with 4% paraformaldehyde. Fixed cells were permeabilized with 0.1% Triton X-100 and stained with Alexa 488-conjugated anti-CD3 (145-2C11; Pharmingen) and either anti-PIX (Chemicon), anti-PAK2 (Cell Signaling), or anti-phospho-PAK1/2 (PAK1 S199/S204, PAK2 S192/S197; Cell Signaling) followed by Alexa 594-conjugated goat anti-rabbit IgG (Molecular Probes) as a secondary antibody. To observe integrin clustering in synapses, the mixed cells were incubated at 37°C for 30 min and then allowed to settle on the slide, fixed, permeabilized, and stained with biotinylated anti-mouse CD11a (L, LFA-1 chain; BD) and Cy3-conjugated streptavidin. Slides were observed with both a conventional Leica DM IRB/E microscope (Leica Microsystems Wetzlar GmbH, Germany) and Openlab software (Improvision) for quantification experiments and a Zeiss Axiovert 200 M microscope equipped with an Apotome device (Carl Zeiss, Germany) to produce images with a similar quality as those produced by confocal microscopy.

Immunizations. Mice were immunized by intraperitoneal injection of 100 µg TNP-keyhole limpet hemocyanin (KLH) or 10 µg trinitrophenyl (TNP)-Ficoll, respectively. TNP-KLH mice were boosted 14 days after the first injection. Sera were collected before immunization and on days 7 and 14 after TNP-Ficoll immunization or on days 14 and 21 after TNP-KLH immunization. Immunoglobulins were detected by alkaline phosphatase-conjugated antibodies to mouse IgG1, IgG2a, and IgG3 and biotinylated antibody to mouse IgM (all from Pharmingen) by ELISA. The amount of each antigen-specific isotype was determined by comparing test samples to a standard serum pooled from immunized wild-type and PIX-deficient mice (21).

	RESULTS

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Generation of PIX knockout mice. To investigate PIX functions in the immune system in vivo, we generated a null mutation of the mouse apix locus (Fig. 1A). Correct integration of the targeting vector was identified by Southern blotting using the indicated 5' and 3' probes (Fig. 1B). As the PIX locus is located on the X chromosome and the ES cells used for homologous recombination were obtained from a male (XY), targeted clones did not contain the wild-type allele. Male PIX mutant mice were null for PIX, while female mice could be either heterozygous or homozygous for the mutation. Mice generated from ES cells were genotyped by PCR (Fig. 1C). Mice carrying the targeted PIX allele did not express PIX protein (Fig. 1D). In the analyses presented here, we did not use heterozygous PIX mice as experimental controls because X chromosome inactivation would lead to PIX being inactivated in some cells and tissues, possibly resulting in PIX^– cells as controls. Rather, strain- and age-matched mice that were wild type for PIX were used as controls. The PIX mutant mice referred to here are described as PIX^– regardless of gender and were born at Mendelian frequencies, were fertile, and did not exhibit any obvious physical abnormalities.

View larger version (30K): [in this window] [in a new window]   FIG. 1. Generation of PIX– mice. (A) Schematic representation of the domains of PIX, the apix genomic locus, the targeting vector, and the targeted allele. Domains are indicated by open boxes, exons XI to XIV are represented by solid boxes, and locations of primers used for PCR are shown as arrowheads. A segment of the PH domain was replaced with the neomycin resistance cassette (neo) flanked with BamHI sites, with the direction of transcription indicated by an arrowhead. Predicted sizes of fragments generated by digestion with BamHI are shown. Probes A and B were used for Southern blot detection of short and long arms, respectively. B, BamHI; S, SacI; X, XbaI. (B) Southern blot analysis of genomic DNA from wild-type (Y/+) and hemizygous (Y/–) PIX-deficient ES cell clones. Genomic DNA was digested with BamHI and hybridized with probe A or probe B. (C) PCR was performed to analyze littermates derived from crosses between PIX heterozygous females (+/–) and wild-type males (Y/+). DNA fragments derived from wild-type (500-bp) and targeted (650-bp) alleles are indicated. (D) Immunoblot analysis of lymph node (LN) and spleen whole-cell lysates prepared from wild-type (Wt) and PIX– mice using an anti-PIX antibody. - and βPIX isoforms are indicated. (E) Expression of PIX in hematopoietic cells. Cell homogenates from the indicated tissues were resolved by SDS-PAGE and immunoblotted with a polyclonal anti-PIX antibody. (F) Hematopoietic cells were purified and analyzed as for panel E.

PIX expression primarily in immune cells.

Reduced numbers of mature lymphocytes in PIX^– mice. The high levels of PIX expression in lymphoid organs pointed to a role for PIX in lymphocyte development. Flow cytometry analysis of lymphocyte development in PIX^– mice revealed that immature lymphocytes, including thymocyte subsets (CD4⁺ CD8⁺ double-positive immature thymocytes and CD4⁺ or CD8⁺ single-positive thymocytes) (Fig. 2A) were normal. Immature bone marrow B-cell subsets (B220⁺ IgM^lo-hi) were also normal (Fig. 2B). However, absolute numbers of mature T and B cells were significantly reduced in peripheral lymphoid organs (Table 1). T cells (CD4⁺ or CD8⁺ single positive) in spleen and lymph nodes were present in normal relative numbers but were reduced in overall number by approximately half (Table 1). Similarly, mature B-cell numbers in lymph nodes were reduced by about half (Table 1). B-cell numbers in spleens were reduced, although not significantly, but some subsets were present in abnormal ratios: marginal zone B cells (CD21^hi CD23^lo) in the spleen were increased approximately 1.5- to 2-fold and immature B cells (IgM^hi IgD^lo), which include marginal zone B cells, and transitional B cells (IgM^hi IgD^hi) in spleen were increased, while mature B cells (IgM^lo IgD^hi) were decreased in PIX^– mice (Fig. 2B). Thus, PIX is dispensable for development of most subsets of lymphocytes but is required for limiting marginal zone B-cell numbers and for promoting numbers of mature T cells and B cells.

View larger version (57K): [in this window] [in a new window]   FIG. 2. Analysis of lymphocyte populations in PIX– mice. (A) T-cell development in normal (Wt) and PIX– mice. Thymocytes and splenic and lymph node T cells from 6- to 8-week-old wild-type and PIX– littermates were stained with anti-CD4-PE and anti-CD8-FITC and analyzed by FACS. Percentages of cells within the lymphocyte populations are indicated in the relevant quadrants. (B) B-cell development alterations in PIX– mice. Bone marrow B-cell precursors (left panel), splenic B cells (middle two panels), and lymph node B cells (right panel) from 8- to 10-week-old wild-type and PIX– littermates were analyzed by FACS following staining with anti-B220-FITC and anti-IgM-PE (bone marrow), anti-B220-PerCP, anti-IgM-PE and anti-IgD-FITC (spleen and lymph nodes), and anti-B220-PerCP, CD21-FITC, and CD23-PE (spleen). Percentages of cells within the lymphocyte population are indicated in the relevant quadrants.

View this table: [in this window] [in a new window]   TABLE 1. Lymphocyte populations in PIX– mice

Reduced immune responses in PIX^– mice.

View larger version (15K): [in this window] [in a new window]   FIG. 3. Decreased humoral immune responses in PIX– mice. Wild-type (Wt) and PIX– mice (12 to 14 weeks old) were immunized with the thymus-dependent antigen TNP-KLH (10 Wt and 12 PIX– mice) or the thymus-independent type 2 antigen TNP-Ficoll (10 Wt and 10 PIX– mice). Primary TNP-KLH immune responses were measured after 14 days. Mice were challenged on day 14, and secondary immune responses were assessed on day 21. TNP-Ficoll immune responses were analyzed on day 7 and day 14. Significance was verified by Student's t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Defective TCR-induced proliferation and signaling in the absence of PIX.

View larger version (15K): [in this window] [in a new window]   FIG. 4. TCR-induced cellular responses. (A) Decreased TCR-induced proliferation of PIX– T cells. Proliferation of wild-type (Wt) or PIX– CD4+ T cells was assessed after stimulation with coated anti-CD3 alone or with anti-CD28, ConA, or PMA plus ionomycin and measured by [3H]thymidine incorporation. (B) IL-2 production by PIX– T cells. CD4+ T cells were stimulated as indicated for panel A, and IL-2 collected 16 h later from cell supernatants was assessed by ELISA. Data are expressed as means ± standard deviations of triplicate values and are representative of four independent experiments.

View larger version (20K): [in this window] [in a new window]   FIG. 5. PIX in TCR signaling. (A) Normal calcium fluxing. Lymph node T cells from wild-type (Wt) and PIX– mice were stained with calcium-sensitive dyes and, for Thy1, stimulated on TCR and analyzed by FACS. (B) Normal actin polymerization. Lymph node T cells were stimulated on TCR, fixed, stained with FITC-phalloidin and anti-CD4, and analyzed by FACS. (C) Normal tyrosine phosphorylation in PIX– T cells. Purified lymph node T cells from wild-type (Wt) or PIX– mice were stimulated on TCR (CD3) for the indicated times (minutes), and lysates were resolved by SDS-PAGE and immunoblotted for proteins phosphorylated on tyrosine residues. (D) Normal GTPase activation. Purified lymph node T cells from wild-type and PIX– mice were stimulated on TCR. Cell extracts were incubated with glutathione S-transferase-PAK1 RBD to precipitate GTP-bound Rac1 (Rac1-GTP). Anti-Rac1 antibodies were used to detect GTP-bound GTPases. To confirm equal loading, 10% of cell lysates was resolved separately and immunoblotted using anti-Rac. (E) Normal ERK1/2 and c-Cbl phosphorylation. Purified lymph node T cells were stimulated on TCR and CD28 and analyzed for ERK1/2, Cbl, and PAK phosphorylation using phospho-specific antibodies. (F) Defective PAK phosphorylation in PIX-deficient T cells. Purified lymph node T cells were stimulated on TCR and CD28 and analyzed for PAK phosphorylation using phospho-specific antibodies. Antibodies against the nonphosphorylated proteins served as a control. All data are representative of at least three independent experiments.

PIX is required for GIT2 expression.

View larger version (28K): [in this window] [in a new window]   FIG. 6. PIX is required for GIT2 expression. (A) Decreased GIT2 protein levels. Purified lymph node T cells were analyzed for the expression of PIX-associated proteins PAK2, Cbl, SAP, Rac1, CDC42, GIT1, and GIT1/2 by Western blotting. The identities of the bands corresponding to GIT1 and GIT2 in the lane probed with anti-GIT1/2 are indicated. The blot was reprobed with anti-ERK1/2 to demonstrate equal loading. (B) Normal GIT2 mRNA expression. GIT2 expression in splenocytes was analyzed by Northern blotting using an N-terminal probe recognizing GIT2-long and GIT2-short. The positions of RNA molecular markers are indicated. (C) Quantitative PCR. Expression analysis of GIT2 in purified T cells by real-time PCR using primers recognizing either GIT2-long and GIT2-short (N-terminal primers) or primers specific for GIT2-long (C-terminal primers). (D) GIT2-short protein expression. Expression of GIT2 proteins was determined in T cells using an antibody recognizing GIT2-long and GIT2-short only and not GIT1. (E) In vitro GIT2 turnover in T cells. Purified wild-type T cells were cultured on plates coated with anti-CD3 (5 µg) and soluble anti-CD28 antibody (1 µg) with or without 50 µg/ml of cycloheximide (CHX) for the indicated times and analyzed for GIT2 expression by Western blotting.

Defective migration of PIX^– lymphocytes. PIX proteins have been implicated in cytoskeletal functions, such as cell spreading and focal complexes (48, 50). GIT2 is an inhibitor of epithelial cell migration (19). Therefore, we tested spontaneous migration of PIX^– T and B cells and chemokine-induced migration to SDF-1 (CXCL12) using flow cytometry analysis of the output of a transwell chamber. We found that the number of PIX^– T cells migrating to SDF-1 was consistently higher than that of the wild type (Fig. 7A). However, the migration of the uninduced sample was also increased, suggesting that the basal migration rate of PIX^– T cells was higher than the wild type (Fig. 7A). Additional testing of PIX^– T-cell migration in the absence of stimuli confirmed that the PIX^– basal migration was higher than in the wild type (Fig. 7B). Similar results were observed in PIX^– B cells (Fig. 7A and B, lower panels). Lymphocytes squeezing through a restrictive extracellular matrix radically deform their shape in an amoeboid movement (20). To discover if PIX regulates this type of migration, we tested migration through membranes of smaller pore sizes, 3 µm instead of 5 µm, as was previously described for βPIX (57). Overall migration rates were reduced 5- to 10-fold, but we found similar results as in Fig. 7A and B: PIX^– cells migrated consistently more than wild-type cells (data not shown). Together, these data show that PIX is an inhibitor of lymphocyte migration.

View larger version (15K): [in this window] [in a new window]   FIG. 7. Elevated T- and B-cell migration. CD4+ T cells (upper panels) and B220+ B cells (lower panels) from wild-type (Wt) or PIX– mice were tested for chemokine-induced (A) or spontaneous (B) migration through the filter of a transwell plate (5 µm). Results are shown as the percentage of input cells that migrated to the lower chamber containing either increasing concentrations of SDF-1 (CXCL12) (A) or medium alone (B). Data are expressed as the means ± standard deviations and represent at least three independent experiments.

PIX is required for immune synapses.

View larger version (35K): [in this window] [in a new window]   FIG. 8. Reduced recruitment of PAK to the T-cell-APC contact site in PIX– T cells. T-cell-B-cell conjugates were formed by mixing LPS-activated B cells pulsed with Ova II peptide and stained with CMAC (blue) with OT-II-transgenic CD4+ lymph node T cells from wild-type and PIX– mice. T cells were stained with anti-CD3-FITC (TCR; green). Cell conjugates are shown in differential interference contrast images. PIX, PAK2, and pPAK were stained with anti-PIX, anti-PAK2, and anti-pPAK antibodies and an Alexa 594 (red) secondary antibody (upper panels). The histogram represents the mean fluorescent intensity of TCR, PIX, PAK, or pPAK at the immune synapse (± the standard deviation), defined here as the interface between a T cell and a B cell featuring concentrated TCR fluorescence. Fluorescent intensity was assessed within a region that was redrawn for each protein at each synapse, with the background subtracted. Over 50 T-cell-B-cell conjugates were analyzed in each of two independent experiments.

View larger version (32K): [in this window] [in a new window]   FIG. 9. Defective immune synapse formation in PIX– T cells. (A) Reduced numbers of PIX– T-cell-B-cell conjugates. Purified OT-II CD4+ lymph node T cells from wild-type or PIX– transgenic mice and B cells either loaded with OVA II peptide or left without peptide were stained with CFSE (T cells) and CMTMR (B cells). Equal numbers of CD4+ T cells and APCs were mixed and analyzed for conjugate formation by FACS. APCs were gated and analyzed for the presence of CD4+ cells. The dashed line divides numbers of conjugates that were specifically induced by peptide from background numbers of uninduced conjugates. Results are representative of three independent experiments performed in duplicate. (B) Normal integrin expression. CD4+ cells from OT-II TCR transgenic wild-type or PIX– mice were stained for 4, L, β1, and β2 integrin chains and analyzed by FACS. (C) Reduced L recruitment. T-cell-B-cell conjugates formed with OT-II CD4+ T cells and peptide-loaded APCs (blue) were stained with anti-CD3-FITC (TCR; green) and anti-CD11a-biotin (L; a component of Lfa-1 integrin), followed by streptavidin-Cy3 (light green). The histogram (right) shows the percentage of 200 T-cell-B-cell conjugates of wild-type and PIX– synapses with polarized L. Data are representative of three independent experiments.

Defective B-cell proliferation in the absence of PIX. Since B-cell numbers were also reduced in PIX^– mice, we next investigated cellular functions of PIX^– B cells. In proliferation assays, PIX^– B cells showed a severe reduction in proliferation after stimulation by either B7.6 or F(ab')₂ anti-IgM antibodies against the BCR (approximately 70% and 80% inhibition, respectively). However, proliferation induced by LPS was normal, demonstrating that there were no intrinsic cell cycle defects in PIX-deficient B cells and that PIX is not downstream of LPS receptors (Fig. 10A). These defects were lessened when costimulation with anti-CD40 or IL-4 was introduced (Fig. 10A), suggesting that additional stimuli can compensate for the BCR defect in PIX^– B cells. To further explore the activation defects of PIX^– B cells, we quantified cell surface levels of B-cell activation markers. Levels of MHC class II were about 1.5- to 2-fold lower on resting PIX^– B cells. After 9 h of BCR stimulation, MHC class II levels were increased on PIX^– B cells but did not rise to normal levels (Fig. 10B). Expression of CD69 on PIX^– B cells was also decreased relative to the wild type after 6 h of anti-IgM stimulation but rose to normal levels after 16 h of activation (data not shown), indicating that PIX is required for early BCR signaling to activation.

View larger version (18K): [in this window] [in a new window]   FIG. 10. Reduced BCR-induced proliferation and MHC-II expression in PIX– mice. (A) Purified wild-type and PIX– splenic B cells were stimulated with medium alone or with anti-IgM MAb B7.6 or anti-IgM F(ab')2 plus, where indicated, anti-CD40 or IL-4, or with LPS, and proliferation was measured by [3H]thymidine incorporation. Data are means ± standard deviations of triplicate values and are representative of four experiments. (B) Reduced MHC-II expression on PIX-deficient B cells. Purified splenic B220+ cells from wild-type and PIX mutant mice were stained for MHC-II directly after purification or after a 9-hour stimulation with anti-IgM. Similar results were obtained in two different experiments.

PIX is required for BCR signaling to PAK.

View larger version (15K): [in this window] [in a new window]   FIG. 11. Altered BCR signaling in PIX– B cells. (A) Increased tyrosine phosphorylation. Lysates from purified wild-type (Wt) or PIX– splenic B cells stimulated with F(ab')2 anti-IgM (10 µg/ml) for the indicated times were analyzed for phosphotyrosine. (B) Normal BCR-induced calcium flux. B cells from wild-type and PIX– mice were stained with calcium-sensitive dyes and for B220 surface expression, stimulated on BCR by F(ab')2 anti-IgM, and analyzed by FACS. (C) Increased ERK activation. Cell lysates prepared as for panel A were immunoblotted for phospho-ERK. Blots were stripped and reprobed for ERK1/2 as loading controls. (D) Defective PAK phosphorylation in PIX– B cells. Purified splenic B cells were stimulated on BCR by F(ab')2 anti-IgM and analyzed for PAK phosphorylation using phospho-specific antibodies. Blots were stripped and reprobed for PAK2 as a loading control.

	DISCUSSION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

βPIX is a close homolog of PIX. We wanted to determine the phenotypes of mice lacking either PIX or βPIX alone before analyzing mice with combined mutations in both PIX proteins. In ongoing work, we found that mutation of βPIX alone is embryonic lethal at an early stage (data not shown). Unlike PIX, βPIX is widely expressed, albeit also highly in immune cells. The broad expression of βPIX is consistent with the lethal phenotype, while the restricted expression of PIX is in line with the more moderate phenotype we describe here. Additionally, the expression pattern of PIX proteins suggests that PIX has evolved for some specific immune function. For example, the fact that PIX has a CH domain while βPIX does not may influence the composition of protein complexes that form around PIX, determining the specific immune function of PIX. CH domains are often found in proteins that have a strong relationship to the cytoskeleton and may modify actin filaments (51).

We found that PIX was required to stabilize GIT2-long protein, consistent with a previous report showing that βPIX regulates GIT2 protein levels in HeLa cells (19). A similar function has been reported for adhesion and degranulation promoting adaptor protein (ADAP) and its binding partner, SKAP55 (27). ADAP and SKAP55 relay TCR signals to integrin adhesion (37). In the absence of ADAP, SKAP55 and its close homolog, SKAP-HOM, are constitutively degraded (27). Thus, T cells from ADAP knockout mice are actually triple knockouts for ADAP, SKAP55, and SKAP-HOM. Similarly, the PIX^– lymphocytes are akin to a double knockout of PIX and GIT2-long. PIX and GIT proteins associate tightly in a large, stable oligomeric complex that could contain as many as 10 to 20 proteins of the approximate size of PIX and GIT proteins (45). PIX and GIT proteins do not disassociate from each other freely nor can they be assembled into the complex in a preparation of purified proteins, and it was suggested that this is because in vivo they are cotranslationally assembled into a complex (45). Therefore, PIX may stabilize GIT2-long by binding tightly to it in the PIX-GIT complex and preventing it from being accessible to protease cleavage or ubiquitin tagging. Since PIX also binds to a common calpain protease subunit and to two ubiquitin ligases (18, 28, 48), it may act as a protective barrier between these and GIT2-long. This would be important in cellular microenvironments regulated by high protein turnover, such as focal complexes at the membrane.

We have shown that PIX knockout lymphocytes lack GIT2-long protein; however, GIT2-short was still expressed. GIT2-short lacks the paxillin-binding site and the coiled-coil domain involved in dimerization of GIT1 and GIT2-long and is differently localized at the perinuclear regions (35). It has been suggested that GIT2-short regulates Golgi complex organization rather than focal complexes (35). Little is known about GIT2 in lymphocytes, as the knockout phenotype, like that of PIX, has only been characterized in neutrophils (36). It will be important for the understanding of the PIX-GIT complex to establish which defects in the PIX lymphocytes are due to loss of PIX and which are due to loss of GIT2-long.

Although PIX is a RhoGEF, we did not observe any defects in TCR activation of PIX targets Rac1 and Cdc42 in PIX^– T cells. This result was surprising, since it was reported that in PIX^– neutrophils Cdc42 activation by C5a is impaired, and we expected a similar result for lymphocytes (33). One possible explanation for the difference is that the GTPases in the neutrophil study were activated by C5a, a G-protein-coupled receptor which may differ in signaling requirements than the TCR that we assessed. Alternatively, it may be the case that PIX^– neutrophils have a severe signaling defect in the PIX-GIT complex, since neutrophils do not express GIT1 (36). Thus, PIX^– neutrophils lack PIX, GIT1, and likely GIT2-long, which could impair signaling to GTPases more than in PIX^– lymphocytes, as they still express GIT1. Another possible explanation for the normal GTPase activation in PIX^– lymphocytes may be that βPIX can compensate for PIX in GTPase activation. There is evidence from studies on Jurkat cells that mice with double mutations in PIX and βPIX could potentially have defective GTPase activation. It was shown that Jurkat T cells overexpressing a mutant SH3 domain from βPIX have defective calcium fluxing and phospholipase C-1 activation (43). The mutant SH3 domain from βPIX probably out-competes endogenous PIX and βPIX in binding to signaling components, effectively creating cells with a double inactivation of PIX and βPIX that manifest stronger signaling defects than those of the PIX knockout T cells described here. Thus, the normal calcium fluxing, actin polymerization, and GTPase activation we observed may be due to compensation by βPIX.

Another observation that emerged from this study is that PIX^– lymphocytes migrated more than wild-type cells, indicating that PIX is an inhibitor of lymphocyte migration. A previous report showed that GIT2 also represses cell motility by inhibiting lamellipodia (19). PIX and GIT proteins bind to many proteins involved in focal complexes, such as paxillin, a scaffold-type protein that is central to focal adhesions (25, 50). It is therefore a strong possibility that PIX and GIT2 inhibit cell migration by participating in focal complexes such as those required during interaction between a T cell and an antigen-presenting cell. A defect in focal adhesions could explain both the increased migration of PIX^– T cells and the defective immune synapses of PIX^– T cells. We found that PAK was not efficiently recruited to or activated at immune synapses on PIX^– T cells, consistent with a previous report showing impaired recruitment and activation of PAK in Jurkat cells (43). We also found that Lfa-1 clusters at PIX^– immune synapses were reduced, as were overall numbers of PIX^– immune synapses. Since Lfa-1 is an integrin that regulates adhesion of T cells to APCs, our results are consistent with a model of PIX as a regulator of adhesion complexes formed upon TCR activation that restrain a T cell from migrating and allow it to form a contact zone with another cell.

Activation of TCR results in inside-out signaling to integrins, including Lfa-1 (11, 29). Some of the known signaling proteins in this pathway include ADAP and SKAP55. SKAP-HOM regulates BCR activation of adhesion in B cells (56). Many parallels between the phenotypes of these mice with that of PIX^– mice suggest that PIX may function in the same pathway. First, Lfa-1 and ADAP knockout mice both have decreased lymphocyte cellularity in the periphery, presumably due to a role for Lfa-1 in adhesion during lymphocyte recirculation (8, 22, 42, 52). Similarly, PIX^– lymphocytes migrated excessively and may migrate out of their developmental niches in the spleen and lymph nodes due a failure to adhere to the extracellular matrix, resulting in decreased cell numbers. In addition, Lfa-1 and ADAP are both required for normal T-cell proliferation (8, 22, 42, 52), consistent with our finding that PIX is required for both T- and B-cell proliferation. Moreover, ADAP, SKAP55, and Lfa-1 are all implicated in T-cell-APC conjugation or synapse formation (38, 58, 59). We also found that efficiency of conjugation and the recruitment of Lfa-1 and PAK to the synapse were all decreased in the absence of PIX. Finally, proximal TCR signaling events in ADAP knockout T cells, such as tyrosine phosphorylation, calcium fluxing, actin polymerization, and ERK activation, were normal (22, 42); proximal BCR signaling events in SKAP-HOM B cells, such as tyrosine phosphorylation, calcium fluxing, and ERK activation, were also normal (56). We found that these events were normal in PIX^– T cells, too, and virtually normal in PIX^– B cells. The similarities in these phenotypes suggest that like ADAP and SKAP proteins, PIX plays a role in TCR activation of focal complexes that is independent of immediate TCR signaling events. Further studies are required to investigate these propositions, and these experiments are under way in our laboratory.

In conclusion, we have identified an essential role for PIX in both T cells and B cells in lymphocyte development and immune functions. The disruption of PIX resulted in wide-ranging defects in lymphocytes. It has been shown that in neuronal cells, PIX proteins regulate critical functions: βPIX binds to neuronal proteins and regulates neurite extension (53, 61), and PIX mutation is linked to mental retardation (32). Our results now highlight the importance of the PIX-GIT complex to lymphocytes and suggest that investigating the parallels between neurons and lymphocytes will provide insights that may contribute to understanding pathological disorders in both systems. Future issues to be resolved are the role of the PIX-GIT complex in membrane dynamics at lymphocyte adhesions: perhaps PIX proteins control actin extensions such as lamellipodia or filopodia, while GIT proteins transport membrane vesicles to the site of cellular extension. It also will be interesting to assess the roles of PIX in other immune cell types, such as macrophages or dendritic cells. For example, mutation of PIX may also affect the functioning of the B-cell synapse (7). Finally, it is conceivable that the phenotype of mice with an PIX mutation combined with a conditional βPIX mutation will be more severe than that of PIX^– mice, and the analysis of the double knockout mice will reveal mechanistic insights into synapse formation and lymphocyte migration.

	ACKNOWLEDGMENTS

 
We thank N. Ushmorova for excellent technical assistance, I. Girkontaite for scientific support, and K. Tedford for scientific editing. We also thank Ed Manser for the gift of PIX polyclonal antibody.

This work was supported by Deutsche Forschungsgemeinschaft grants SFB 497 and DFG Fi-639/2-2 to K.D.F. K.M. is supported by a Marie Curie Fellowship (contract HPMF-CT-1999-00128).

	FOOTNOTES

 
* Corresponding author. Mailing address: Institute of Biochemistry, Otto von Guericke University, Leipziger Str. 44, 39120 Magdeburg, Germany. Phone: 49-391-671-4288. Fax: 49-391-671-5898. E-mail: klaus.fischer{at}med.ovgu.de

Published ahead of print on 31 March 2008.

K.M. and B.H. contributed equally to this work.

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