CD44-initiated cell spreading induces Pyk2 phosphorylation, is mediated by Src family kinases, and is negatively regulated by CD45.

CD44 is a cell adhesion molecule implicated in leukocyte adhesion and migration, co-stimulation of T cells, and tumor metastasis. CD45 is a leukocyte-specific protein tyrosine phosphatase that dephosphorylates the Src family kinases, Lck and Fyn, in T cells. Positive regulation of Lck by CD45 is required for its effective participation in T cell receptor signaling events. Here, immobilized CD44 antibody induced a distinctive cell spreading in CD45(-), but not CD45(+), T cells, and this correlated with the induction of tyrosine-phosphorylated proteins. Two focal adhesion family kinases, Pyk2 and, to a lesser extent, FAK were inducibly phosphorylated, as was a potential substrate, Cas. CD44-mediated cell spreading and induced tyrosine phosphorylation were prevented by the Src family kinase inhibitor, PP2. Furthermore, 2-fold more Lck associated with CD44 in the low density sucrose fraction from CD45(-) T cells compared with CD45(+) T cells, suggesting that CD45 may regulate the association of Lck with CD44 in this fraction. Therefore, in CD45(-) T cells, CD44 signaling is mediated by Src family kinases, and this leads to Pyk2 phosphorylation, cytoskeletal changes, and cell spreading. This implicates CD45 in the negative regulation of Src family kinase-mediated CD44 signaling leading to T cell spreading.

CD44 is a widely expressed cell adhesion molecule that binds hyaluronan (HA) 1 , a component of the extracellular matrix. HA is present in most tissues and is found at high levels in cartilage and synovial fluids. CD44 and HA have been implicated in several biological processes, including wound healing, tissue regeneration, embryogenesis, leukocyte extravasation, and tumor metastasis (reviewed in Refs. [1][2][3][4]. CD44 can also bind other ligands in addition to HA, and these include serglycin, a proteoglycan secreted by cytotoxic T lymphocytes; osteopontin, a multifunctional secreted glycoprotein with adhesive and migratory properties; and certain types of collagen and fibronec-tin (reviewed in Refs. 1 and 3). CD44 antibodies can block the migration of pro-T cells to the thymus (5,6) and can prevent leukocyte extravasation at inflammatory sites (7,8), implicating a role for CD44 in cell adhesion and migration. CD44 antibodies can also stimulate leukocyte adhesion and augment T cell activation, indicating that CD44 is capable of signaling to the cell (9 -11). Adhesion of activated lymphocytes or T cell lines to immobilized CD44 antibody can induce cell spreading, indicating that CD44 can also induce cytoskeletal changes (12,13). Cross-linking CD44 on T cells activates the tyrosine kinase, p56 Lck (Lck), which associates with CD44 (14). Lck and Fyn are both associated with CD44 isolated from the low density fraction of a sucrose gradient after cell lysis in 0.5% Brij-58 (15). This low density fraction is thought to contain specialized plasma membrane microdomains or lipid rafts enriched in glycosphingolipids and glycophosphoinositol-linked proteins (reviewed in Refs. 16 and 17). In T cells, various signaling molecules, such as LAT, have been localized to this fraction, and the T cell receptor (TCR) and other signaling molecules are thought to be recruited into this fraction upon antibody ligation or antigen recognition (reviewed in Refs. 18 and 19). These lipid domains are thought to be crucial domains in the plasma membrane where T cell signaling proteins reside and where TCR signaling occurs. Whether CD44 interacts directly or indirectly with Lck and Fyn in this fraction is not known, and the significance of CD44 in this fraction is not understood.
CD45 is a tyrosine phosphatase known to dephosphorylate Lck at Tyr 505 , its negative regulatory site, and in T cells this is essential for its effective participation in TCR signaling events (reviewed in Refs. 20 and 21). To a lesser extent, CD45 can also dephosphorylate the analogous site in Fyn. However the effects of this are less noticeable because TCR signaling and T cell activation are less affected by the absence of Fyn (22,23). Csk is a tyrosine kinase that phosphorylates Src family kinases at the negative regulatory site (24) and thus phosphorylation at this site depends on the balance of CD45 and Csk activities. Under some circumstances, CD45 can dephosphorylate Lck at Tyr 394 , a positive regulatory site located in the activation loop of the kinase domain (reviewed in Refs. 20 and 25). This indicates that CD45 has the potential to down regulate, as well as up-regulate, Lck activity.
The positive role of CD45 in regulating Lck function in TCR signaling and T cell activation is well established (reviewed in Refs. 26 and 21). More recently, CD45 has been implicated in the regulation of integrin-induced leukocyte adhesion, which has suggested a new role for CD45 in the negative regulation of cell adhesion (reviewed in Ref. 20). CD45 was shown to negatively regulate ␣ 5 ␤ 1 integrin, but not ␣ 4 ␤ 1 , mediated adhesion to fibronectin in T cells (27). Also, in CD45 Ϫ bone marrowderived macrophages, ␤ 2 integrin-mediated cell adhesion to plastic occurred more rapidly than in CD45 ϩ macrophages, but this adhesion was not sustainable (28). Integrin-mediated adhesion occurs at points of focal contact and signaling via integrins involves the activation of Src and focal adhesion kinases (reviewed in Refs. 29 and 30). In the CD45 Ϫ macrophages, the Src family kinases, Hck and Lyn were found to be hyperphosphorylated at their negative regulatory site, yet were more active in an in vitro kinase assay than the dephosphorylated form present in the CD45 ϩ cells (28). This suggests that CD45 may contribute to the down-regulation of these kinases in macrophages, possibly by dephosphorylation of the positive regulatory site during integrin-mediated adhesion. These hyperphosphorylated, hyperactivatable kinases may contribute to the aberrant integrin-mediated adhesion in these cells.
Previous work in CD45 ϩ and CD45 Ϫ BW5147 T cells indicated that Lck, and to a lesser extent Fyn, were hyperphosphorylated at their negative regulatory site in the CD45 Ϫ cells (31,32). Despite this, Lck was just as active in an in vitro kinase assay as Lck isolated from CD45 ϩ cells (33). TCR signaling was also greatly attenuated in the CD45 Ϫ BW5147 T cells expressing the TCR/CD3 complex (33), indicating a positive role for CD45 in TCR signaling. Here we provide evidence implicating a negative role for CD45 in regulating CD44 mediated cell spreading in TCR ϩ BW5147 T cells by preventing CD44 mediated signaling events. In the absence of CD45, CD44 mediated adhesion to immobilized antibodies resulted in the signaling via Src family kinases and the induction of tyrosine-phosphorylated proteins, including two focal adhesion family kinases, Pyk2 and to a lesser extent, FAK. In the CD45 ϩ cells, no tyrosine phosphorylation was induced implying that the CD44 associated Src family kinases are not activated. This suggests a negative regulatory role for CD45 in Src family kinase activation and in CD44 mediated signaling events leading to cell adhesion and spreading. This work supports an emerging role for CD45 in negatively regulating adhesion molecule signaling leading to cell spreading in T cells.

EXPERIMENTAL PROCEDURES
Cells and Antibodies-Murine BW5147 T lymphoma cells (CD45 ϩ and CD45 Ϫ ; ATCC) and these cells transfected with CD3 and ␦ to express surface T cell receptor (TCR)/CD3 were used (34). These TCR/ CD3 ϩ -transfected BW5147 T cells, hereafter referred to as CD45 ϩ BW and CD45 Ϫ BW cells, were maintained at 37°C equilibrated with 5% CO 2 in DMEM with 10% v/v horse serum and 3 mM of Histidinol (Sigma-Aldrich) to maintain plasmid expression. CD45 ϩ and CD45 Ϫ SAKR T cell lines were from R. Hyman (35).
Antibody Immobilization-50 l of purified anti-CD44 antibody KM201 (40 g/ml) or 2% w/v bovine serum albumin (BSA, Life Technologies, Inc.) in phosphate-buffered saline (PBS), were coated on 96well tissue culture plates at 37°C for 3 h. Plates were washed 2ϫ with PBS then blocked with 2% w/v BSA in PBS for another 2 h at 37°C and washed 3ϫ with PBS prior to use.
Cell Adhesion and Cell Spreading Assay-10 5 BW cells were added to the antibody or BSA-coated 96-well tissue culture plates at a concentration of 2 ϫ 10 6 cells/ml in DMEM and 0.1% v/v fetal calf serum for various time periods. 30 l of 3ϫ reducing SDS sample buffer was added directly to the cells after each time point and treated at 100°C for 5 min before electrophoresis. In some experiments, cells were preincubated with 10 M PP2 (Calbiochem) or 25 M cytochalasin D (Calbiochem) in DMEM, 0.1% fetal calf serum for 30 min at 37°C, before the cells and media were transferred to the antibody-immobilized plates.
Sucrose Gradient Density Equilibrium Centrifugation-5 ϫ 10 7 cells were lysed in 1 ml of ice-cold TK buffer (10 mM Tris-HCl, pH 7.2, 140 mM KCl, 0.5 mM sodium orthovanadate, 0.2 mM sodium molybdate, 1 g/ml aprotinin, 1 g/ml leupeptin, 1 g/ml pepstatin, 0.2 mM phenylmethylsulfonyl fluoride) containing 1% v/v Brij-58 (Pierce) or 1% v/v Triton X-100 (Fisher Scientific Ltd.). The lysate was mechanically disrupted with a Dounce homogenizer for 15 strokes, then incubated on ice for 20 min. The lysate was transferred to an ultracentrifuge tube diluted with 1 ml of 80% w/v sucrose in TK buffer, overlaid with 6 ml of 30% w/v sucrose and 3.5 ml of 5% w/v sucrose in TK buffer, and then Ultracentrifuged at 230,000 ϫ g for 16 h at 4°C. The gradients were collected from the top into 8-ϫ 1.5-ml fractions. The pellet was washed twice with TK buffer and dissolved in 150 l of 2% w/v SDS, 10 mM Tris-HCl, pH 7.2. 20 l from each fraction and 2 l from dissolved pellet were boiled in the presence of 3ϫ reducing SDS sample buffer prior to electrophoresis. Immunoprecipitation and Immunoblotting-10 7 cells were lysed in 0.5 ml of ice-cold TK buffer with 1% Brij-58 or 1% Triton X-100, incubated on ice for 10 min, then centrifuged at 16,000 ϫ g for 10 min at 4°C. Detergent-soluble material was precleared with 50 l of 50% slurry of Sepharose CL-4B beads (Sigma-Aldrich) for 1 h at 4°C, then 50 l of 25% slurry of IM7 conjugated CNBr-Sepharose beads (4 mg/ml) or 50 l of Sepharose beads alone was added as a control and incubated for 2 h at 4°C.
For immunoprecipitation from sucrose gradient fractions, fractions 2 to 4 (low density), and fractions 7 and 8 (high density), were pooled separately. The high density pool was diluted two times with TK buffer to reduce the sucrose concentration. 60 l of 50% slurry of Sepharose beads was added to preclear the mixture. Then 60 l of 25% slurry of IM7-conjugated CNBr-Sepharose beads or 30 l of Sepharose beads alone (control) was added and incubated for 2 h at 4°C.
For Pyk2, FAK, and Cas immunoprecipitation, 5 ϫ 10 6 cells were added to 1 well of a 6-well tissue culture plate (Life Technologies, Inc.) after the wells were coated with 0.5 ml of 40 g/ml KM201. The cells were allowed to flatten at 37°C for various times before they were lysed on ice on the plate in the presence of 1 ml of media with 250 l of 5ϫ lysis buffer containing 5% Triton X-100, 50 mM Tris-HCl, pH 7.2, 140 mM KCl, 10 mM EDTA, 2.5 mM sodium orthovanadate, 1 mM sodium molybdate, 5 g/ml aprotinin, 5 g/ml leupeptin, 5 g/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride. Cell lysate was either pipetted or scraped off the plate, collected into Eppendorf tubes, and centrifuged at 16,000 ϫ g for 10 min at 4°C. Soluble cell lysate was incubated with 1 g of anti-Pyk2 or anti-FAK or 0.5 g of anti-Cas antibody at 4°C, rotating for 1 h. Then 20 l of 50% slurry protein A-agarose beads (Repligen Corp.) or protein G-Sepharose 4 Fast Flow (Amersham Pharmacia Biotech) was added, and the mixture was further rotated at 4°C for 45 min.
All immunoprecipitates were washed three times with 1 ml of lysis buffer, and the proteins were eluted from beads by boiling in reducing SDS sample buffer, analyzed on SDS-PAGE using 7.5% polyacrylamide gel, and then transferred to a PVDF membrane (Immobilon P, Millipore). The membrane was air-dried and incubated with primary antibodies in TTBS buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% v/v Tween 20) with 0.5% w/v BSA for 1 h. For anti-phosphotyrosine immunoblotting, the PVDF membrane was air-dried or incubated directly with 5% w/v BSA in TBS or TTBS buffer for 1 h prior to incubation with the primary antibody. After incubation with the primary antibody, the membrane was washed three times with TTBS and further incubated with horseradish peroxidase-conjugated secondary reagents in 0.5% w/v BSA TTBS for another hour, then washed again in TTBS three times. Antibody-reactive bands were visualized using ECL or ECL plus (Amersham Pharmacia Biotech). Primary antibodies used included JIWBB at 1/1000 dilution, R54-3B and anti-actin at 1/2500 dilution, R02.2 at 1/5000 dilution, 4G10 at 0.4 g/ml, anti-Csk at 0.5 g/ml, anti-Pyk2 and anti-FAK at 1 g/ml, and anti-Cas at 0.25 g/ml. Horseradish peroxidase-conjugated secondary reagents included protein A (Bio-Rad Laboratories) and goat anti-rat or mouse IgG (Jackson ImmunoResearch Laboratories, Inc.), both were used at 1/5000 dilution. If reprobing of the same membrane was required, the membrane was stripped by incubating with stripping buffer (50 mM glycine, pH 2.5, 150 mM NaCl, 0.1% v/v Nonidet P-40) for 30 min at room temperature.
Data Analysis-Blots were scanned and spot densitometry was carried out using IMAGE software (National Institutes of Health).

CD45 Regulates CD44-induced Signaling and Adhesion
and CD45 Ϫ BW, both adhered to HA and anti-CD44-coated plates, but only CD45 Ϫ BW T cells underwent a distinct morphological change and flattened and spread lengthwise on the immobilized anti-CD44 mAb, KM201 (Fig. 1A). This elongated flattening was observed with an immobilized antibody concentration ranging from 40 to 250 g/ml, with a higher percentage of cells flattened at the higher concentrations. Differences in cell spreading were also observed between the CD45 ϩ and CD45 Ϫ BW cells lacking TCR expression and between the SAKR CD45 ϩ and CD45 Ϫ T cell lines, although the differences were less pronounced (data not shown). The binding of CD45 Ϫ BW cells to other immobilized CD44 mAbs, IM7 and IRAW, also resulted in their elongation and spreading in a distinct manner from CD45 ϩ BW cells, although the CD45 ϩ BW cells exhibited some circular spreading with IM7 and high concentrations of KM201 (data not shown). The anti-CD44 antibody, KM201, binds to the HA binding site of CD44, yet adhesion of either CD45 ϩ or CD45 Ϫ BW cells to immobilized HA did not cause cell spreading under any condition tested. Thus, in addition to CD45, the epitope or affinity of the CD44 antibody may influence the signaling ability of CD44. The cell flattening and spreading was observed in the CD45 Ϫ cells after 30 min but increased over a period of 2 h and was still observed after 16 h (Fig. 1B). Cell spreading was abolished by pretreatment of cells with 25 M cytochalasin D for 30 min, indicating that cell spreading required an intact actin cytoskeleton (data not shown). Overall, these data indicate that the presence or absence of CD45 can affect CD44-induced cell spreading in T cell lines, indicating that CD45 may negatively regulate CD44mediated signaling events.
One of the main functions of CD45 in T cells is to regulate the function of the Src family tyrosine kinase, Lck. To determine if Src family kinases played a role in CD44 triggered cell spreading, the CD45 Ϫ BW T cells were pretreated for 30 min with 10 M PP2, a Src family kinase inhibitor (40). PP2 did not prevent the binding of either CD45 ϩ or CD45 Ϫ BW T cells to the immobilized CD44 mAb, KM201, but it did prevent the cell spreading observed in the CD45 Ϫ BW T cells (Fig. 1A). This indicates that CD44-mediated spreading in CD45 Ϫ BW T cells is mediated by Src family kinases.

Induction of Tyrosine Phosphorylation by CD44-triggered Cell Spreading Occurs in CD45 Ϫ BW T Cells and Is Inhibited by a Src Family Kinase
Inhibitor-To further examine the role of Src family kinases in CD44-mediated signaling events, we examined whether adhesion of CD45 ϩ or CD45 Ϫ BW T cells to immobilized CD44 antibody involved the induction of tyrosine phosphorylation. The induction of tyrosine-phosphorylated proteins was observed in the CD45 Ϫ BW cells that had undergone cell spreading but was not observed to any significant degree in CD45 ϩ BW cells that had not spread on the immobilized CD44 mAb (Fig. 2). The tyrosine-phosphorylated doublet (ϳ55 kDa) observed in lysates from CD45 Ϫ BW cells co-migrated with Lck and Fyn (data not shown). There were at least two prominent tyrosine-phosphorylated proteins of 120 -130 kDa that were induced in the CD45 Ϫ BW cells upon binding immobilized CD44 antibody. There was also a less defined band at ϳ80 kDa; however, this was not consistently observed. The induction of tyrosine phosphorylation correlated with the initiation of the morphological changes, becoming noticeable after 30 min and being sustained up to 16 h (data not shown). Therefore, the induction of tyrosine phosphorylation correlates with the induction of cell spreading. It was noted that, after 4 h, induction of some tyrosine phosphorylation was occasionally observed in CD45 Ϫ BW T cells in wells coated with BSA (Fig.  2B). However, these tyrosine-phosphorylated bands were distinct from those observed upon incubation with immobilized CD44 antibody, and the phosphorylation of these proteins did not result in cell spreading (Fig. 1A). The addition of PP2 inhibited both the spreading and the induction of tyrosine phosphorylation induced by immobilized anti-CD44 antibody (Figs. 1A and 2B). Therefore, CD44 signaling resulted in cytoskeletal changes and cell spreading in CD45 Ϫ BW T cells that required Src family kinase activity and resulted in the sustained tyrosine phosphorylation of cellular proteins.
Immobilized CD44 Antibody-triggered Tyrosine Phosphorylation of Pyk2-Several proteins of ϳ120 -130 kDa are known to become tyrosine-phosphorylated after stimulation and activation of Lck and Fyn, two Src family kinases expressed in T cells. These include p130 Cas, p130 SLAP or fyb, FAK, and Pyk2. Both FAK and Pyk2 have been implicated in mediating integrin-triggered cell adhesion and cell spreading, by linking the integrin-mediated signal to the cytoskeleton (reviewed in Refs. 29 and 41). FAK and Pyk2 are also inducibly tyrosine-phosphorylated in response to TCR ligation and can associate with Lck (42). It was therefore determined whether these kinases became phosphorylated upon immobilization of the BW T cells by anti-CD44 antibody. Fig. 3 demonstrates that Pyk2 becomes strongly tyrosine-phosphorylated after 30 min in the CD45 Ϫ BW cells. This timing correlated with the induction of cell spreading and was sustained over the measurement period of 2 h. Much lower levels of tyrosine phosphorylation of Pyk2 were seen in the CD45 ϩ BW T cells, suggesting that, in the presence of CD45, the induction of Pyk2 phosphorylation could occur but was greatly attenuated. Low levels of FAK phosphorylation were observed only in the CD45 Ϫ BW T cells. Thus, strong Pyk2 tyrosine phosphorylation and weak FAK phosphorylation correlated with the induction of cell spreading in the CD45 Ϫ cells. This suggests that CD44 signaling leading to cell spreading in CD45 Ϫ BW cells is mediated by Src family kinases, Pyk2, and, to a lesser extent, FAK. It is not known whether the low levels of observed FAK tyrosine phosphorylation represents a preference for Pyk2 in the CD44 signaling pathway or whether FAK is expressed at low levels in these cells.
Paxillin is a cytoskeletal protein that can recruit both FAK and Src kinases to the plasma membrane, and activation of these kinases by integrin engagement leads to paxillin phosphorylation. Paxillin also co-localizes with Pyk2, FAK, and Src at the microtubule-organizing center (reviewed in Ref. 43). However, in this study, paxillin was not reproducibly tyrosinephosphorylated in CD45 Ϫ BW cells after immobilization on anti-CD44 antibody (data not shown). Thus, cell spreading initiated by immobilized CD44 antibody in the CD45 Ϫ BW cells requires Src family kinase activity for induction of the tyrosine phosphorylation of Pyk2 and, to a lesser extent, FAK but does not require the tyrosine phosphorylation of paxillin. Cas family members (p130 Cas, HEF-1, and Efs) are docking proteins implicated in integrin receptor signaling to the actin cytoskeleton. They can associate with FAK and Pyk2 via SH3 domain interactions and can be tyrosine-phosphorylated by either FAK or Pyk2 or by cooperative interaction between Src family kinases and FAK or Pyk2 (reviewed in Ref. 44). Fig. 3C demonstrates that the p130 Cas mAb detects two molecular mass species in BW cells and that the lower molecular mass species becomes tyrosine-phosphorylated in the CD45 Ϫ cells 30 min after immobilization on anti-CD44 mAb. Unlike the Pyk2 phosphorylation, this tyrosine phosphorylation is not sustained and is reduced at the 2-h time point.
CD45 Negatively Regulates the Association of CD44 with Lck-To further investigate the link between CD44 signaling and Src family kinases, CD44 was immunoprecipitated from both CD45 ϩ or CD45 Ϫ BW T cells and its association with the Src family kinases was assessed. Lck co-precipitated with CD44 under a variety of conditions, including 0 -150 mM KCl, with or without 2 mM EDTA and in both 1% Brij-58 or 1% Triton X-100 (data not shown). Solubilization of cells in 1% Brij-58 compared with 1% Triton X-100 resulted in a lower amount of CD44 immunoprecipitated but with a higher percentage of associated Lck. This suggested that 1% Brij-58 selectively solubilizes the CD44 pool associated with Lck. A comparison of the amount of Lck co-immunoprecipitating with equal amounts of CD44 indicated that ϳ2-fold more Lck coprecipitated with CD44 isolated from the CD45 Ϫ BW T cells than the CD45 ϩ BW T cells after cell lysis in either 1% Brij-58 or 1% Triton X-100 (Fig. 4). Similar results were also found for the other T cell Src family kinase, Fyn, which also co-precipitated with CD44 (data not shown). This suggested that CD45 was negatively regulating the association of Lck and Fyn with CD44. The fact that more Lck and Fyn associates with CD44 in the CD45 Ϫ BW cells than the CD45 ϩ BW cells may be a contributing factor toward the enhanced CD44-mediated signaling observed in the CD45 Ϫ BW cells.

Distribution of Lck to the Low Density Sucrose Fraction Is Increased in CD45 Ϫ BW T Cells-A subset of both CD44 and
Lck have been shown to migrate to the low density fraction after cell lysis and sucrose density gradient centrifugation (15,45). This has been equated with the localization of these proteins to a low density lipid domain or raft in the plasma membrane of cells. This lipid fraction, typically enriched in glycolipids, sphingomyelin, and cholesterol, is not well solubilized by detergents such as Brij and Triton and is thought to exist on the membrane of unstimulated cells as microdomains or lipid rafts (reviewed in Refs. 16,17). In the BW cells, sucrose gradients were performed on both 1% Brij-58-and 1% Triton X-100-treated cells, and the distribution of CD44, Lck, Csk, and CD45 was determined (Fig. 5 and Table I). In CD45 ϩ and CD45 Ϫ BW cells, low levels of CD44 (ϳ4%) were present in the low density fraction in Triton-treated cells, whereas considerably more CD44 was present (ϳ50%) after cell lysis in 1% Brij-58. Also, slightly more Lck (40 -60%) resided in the low density fraction after cell lysis in 1% Brij-58 compared with 25-40% after lysis in 1% Triton X-100. In contrast, very low levels of CD45 or Csk (ϳ1%) were detected in the low density fraction, regardless of which detergent was used. In addition, it was observed that ϳ1.5-fold more Lck was present in the low density fraction when lysates from CD45 Ϫ cells were compared with lysates from CD45 ϩ cells, and this was true for either detergent used. This raises the possibility that CD45 may negatively regulate the localization of Lck to this low density fraction in T cells.
CD44 and Lck Association Occurs in the Low Density Fraction-To determine if the association of Lck with CD44 occurred in the low density fraction in both the CD45 ϩ and CD45 Ϫ BW cells, CD44 was immunoprecipitated from both the low (fractions 2-4) and high (fractions 7 and 8, Fig. 5) density regions of the sucrose gradient. Fig. 6 demonstrates that Lck associates with CD44 in the low density fraction and that ϳ2-fold more Lck co-precipitates with CD44 isolated from this fraction in the CD45 Ϫ cells than in the CD45 ϩ cells. Thus, in the CD45 Ϫ BW cells, more Lck is present in the low density fraction and more Lck associates with CD44 isolated from this fraction.

CD44-mediated Spreading in CD45
Ϫ BW T Cells-Engagement of CD44 with immobilized antibody resulted in the polarized cell spreading of CD45 Ϫ but not CD45 ϩ BW T cells. At higher CD44 mAb concentrations and particularly with the CD44 mAb, IM7, the CD45 ϩ BW cells did begin to spread but exhibited a non-polarized, circular spreading (data not shown). This type of spreading of CD45 ϩ T cells on high concentrations of CD44 mAbs has also been observed previously (12,13), and dendrite formation has been reported in activated B cells (46). Here we demonstrate that BW T cells lacking CD45 have an enhanced ability to flatten and spread in a polarized manner in response to binding immobilized anti-CD44 antibody. As noted by others (13), cell spreading did not occur in response to binding immobilized HA, a physiological ligand for CD44. However, HA fragments have been shown to induce cytokine and chemokine secretion, to up-regulate integrin function, and to induce nitric-oxide synthase via an NF-B-dependent mechanism in activated macrophages, indicating that CD44 can signal to the cell in response to binding a physiological ligand (47)(48)(49). Both CD44 cross-linking and fragmented HA can induce integrin-mediated adhesion in colon carcinoma cells (50), suggesting that, in these cells, CD44 ligation by mAbs may mimic the interaction of CD44 with fragmented HA. Although HA is the best characterized ligand for CD44, CD44 can also bind to osteopontin, serglycin, itself, fibronectin, and some types of collagen. Therefore it is possible that CD44 crosslinking may mimic the binding of one of these ligands.

Role of Src Family Kinases in CD44 Signaling and Cell Spreading and Comparison with Integrin-mediated Signaling
and Adhesion-Here we show that CD44 signaling occurs in CD45 Ϫ BW T cells where the Src family kinases are predominantly phosphorylated at the negative regulatory site (31)(32)(33). We demonstrate that Src family kinases play an important role in CD44-mediated signaling leading to T cell spreading in CD45 Ϫ T cells. This suggests that CD45 has a negative regulatory effect on CD44-associated Src family kinases and provides additional data supporting an emerging role for CD45 in negatively regulating leukocyte adhesion and spreading. In CD45 Ϫ macrophages, the Src family kinases, Hck and Lyn, are also hyperphosphorylated at the negative regulatory site (28). This suggests that both integrin and CD44 signaling to the cytoskeleton is enhanced by the hyperphosphorylated form of Src family kinases, which are more prevalent in CD45 Ϫ cells.
In CD45 Ϫ BW T cells, CD44-induced Src family kinase activity results in the phosphorylation of Pyk2, a member of the focal adhesion family of tyrosine kinases that is normally expressed in the brain and hematopoietic system (41). Although FAK is considered the primary kinase mediating integrin ad- hesion events (29), FAK was phosphorylated to a lesser extent in the CD44 signaling pathway, suggesting a possible divergence between CD44 and integrin signaling pathways. Paxillin, a common downstream target of FAK and Pyk2, was not consistently tyrosine-phosphorylated upon CD44 signaling, suggesting that paxillin phosphorylation is not essential for CD44-mediated cell spreading to occur. Paxillin phosphorylation is thought to be a key event in integrin-mediated signaling leading to cell spreading (43), indicating another possible divergence between the CD44 and integrin signaling pathways. Despite these potential differences, many similarities exist in the signaling pathways induced by the integrins and CD44, including the involvement of FAK and Src family kinases and the negative regulatory effect of CD45 on leukocyte adhesion and spreading. In addition, Cas family members are phosphorylated upon integrin signaling, and FAK and Pyk2 together with Src family kinases have been implicated in this process (reviewed in Ref. 44). Here we find that the lower molecular mass species detected by the p130 Cas antibody is inducibly phosphorylated in the CD45 Ϫ BW cells, but notably, this phosphorylation is not sustained.
CD44-Lck Association in Lipid Domains-Localization of proteins to the low density fraction of sucrose gradients has been associated with protein localization in the cell to specific lipid domains or rafts in the cell membrane (17). Here we have shown that in CD45 Ϫ T cells, CD44-mediated cell spreading requires Src family kinase activity. Because CD44 associates with Src family kinases in the low density sucrose fraction this implies that Src family kinases present in this fraction can be activated in the absence of CD45. Because CD44-mediated Src family kinase activation is severely attenuated in CD45 ϩ BW cells, CD45 may exert a negative regulatory effect on this pool of Lck. However, to do this directly, CD45 would have gain access to Lck in this fraction, and we and others have shown that very little CD45 is present in the low density fraction (51,52). CD45 may therefore transiently access these domains, may access microdomains from the periphery, may be solubilized from these lipid microdomains upon detergent lysis, or may act indirectly to down-regulate Lck activity. In this report, lysis in 1% Brij-58 or 1% Triton X-100 resulted in different amounts of CD44 and Lck being present in the low density fraction, indicating that different detergents can differentially solubilize CD44 and Lck, suggesting that caution should be exercised when extrapolating the presence or absence of a protein in the low density fraction to its presence or absence in lipid microdomains or rafts in the cell membrane. As suggested by others (53), the low density fraction may contain more than one type of lipid vesicle or membrane domain. However, despite the differences in CD44 distribution between the two detergents, the CD44-Lck association was consistently found to occur only in the low density fraction and to occur to a greater extent in CD45 Ϫ T cells.
Opposing Roles for CD45 and Src Family Kinases in TCR Signaling and CD44-mediated Cell Spreading-Phosphorylation of FAK and Pyk2 has been reported after immobilization of T cell lines by anti-CD3 antibody (42). However, in contrast to TCR⅐CD3 stimulation, immobilized anti-CD44-induced cell spreading did not induce the same tyrosine-phosphorylated proteins. For example, no significant tyrosine phosphorylation was observed between 20 and 40 kDa where both LAT (36 kDa) and CD3 (21 kDa) migrate. Thus, CD44 signaling may induce the tyrosine phosphorylation of only a subset of proteins that become tyrosine-phosphorylated upon TCR⅐CD3 ligation. One key difference between TCR⅐CD3 signaling and CD44-mediated signaling in BW T cells is that efficient TCR⅐CD3 signaling only occurs in CD45 ϩ cells (33), whereas CD44-mediated signaling leading to polarized cell spreading only occurs in CD45 Ϫ cells. Enigmatically, the T cell Src family kinases are required for the initiation of both signals. This leads us to propose that there are at least two pools or states of Lck, and possibly Fyn, in the T cell that have distinct functions and that the distribution between these two pools is regulated by CD45. One pool is dephosphorylated by CD45 at the negative regulatory site and primed for participation in TCR signaling events. The other pool is phosphorylated at Tyr 505 , associated with CD44, and can be activated in the absence of CD45. We suggest that this pool of Lck is rapidly inactivated by CD45 as CD44-mediated signaling is severely attenuated in the CD45 ϩ BW cells. In CD45 Ϫ T cells, TCR signaling is severely attenuated, indicating a positive role for CD45 in regulating Lck activity in TCR TABLE I Distribution of Lck, CD44, CD45, and Csk on sucrose density gradients from CD45 ϩ and CD45 Ϫ BW T cells Low and high density represents sucrose density fractions 2 to 4, and 7 and 8 respectively, after cell lysis in either 1% Brij58 or 1% Triton-X-100, as indicated (see "Experimental Procedures"). Numbers represent the percentage of the molecule in that fraction Ϯ the S.D. n ϭ the number of times the experiment was performed. ϩ 50 Ϯ 9% (n ϭ 4) 49 Ϯ 8% (n ϭ 4) Ͻ1 Ϯ 2% (n ϭ 3) 100 Ϯ 3% (n ϭ 3) Ϫ 46 Ϯ 3% (n ϭ 4) 48 Ϯ 6% (n ϭ 4) 7 Ϯ 6% (n ϭ 3) 93 Ϯ 6% (n ϭ 3) CD45 ϩ Ͻ 1 Ϯ 2% (n ϭ 4) 99 Ϯ 4% (n ϭ 4) 1 Ϯ 1% (n ϭ 4) 100 Ϯ 3% (n ϭ 4) Csk ϩ 1 Ϯ Ͻ1% (n ϭ 2) 99 Ϯ Ͻ1% (n ϭ 2) 2 Ϯ 2% (n ϭ 2) 97 Ϯ 3% (n ϭ 2) Ϫ 1 Ϯ 1% (n ϭ 2) 98 Ϯ Ͻ1% (n ϭ 2) 1 Ϯ 1% (n ϭ 2) 96 Ϯ 5% (n ϭ 2) FIG. 6. CD44-Lck interaction occurs in the low density fraction after sucrose density gradient centrifugation. CD45 ϩ and CD45 Ϫ BW cells (CD45 ϩ and CD45 Ϫ cells) were lysed in 1% Brij-58 and then centrifuged on a sucrose gradient for 16 h (see "Experimental Procedures"). Low density fractions (fractions 2-4, see signaling events. In contrast, CD44 signaling leading to cell spreading is enhanced in CD45 Ϫ cells, suggesting a negative role for CD45 in regulating Src family kinase activity involved in cell adhesion signaling and cell spreading events. Thus CD45 and Src family kinases appear to have a dual role in T cells, one in promoting antigen-induced T cell activation and another in preventing cell spreading in response to signaling through the cell adhesion molecule, CD44. We propose that one function of CD45 is as an anti-adhesion molecule in leukocytes that acts to prevent unwanted firm cell adhesion and cell spreading in response to the binding of cell adhesion molecules such as CD44. CD45 may achieve this by down-regulating the activity of the pool of Src family kinases associated with CD44.