The tyrosine phosphatase CD45 dephosphorylates the negative regulatory tyrosine of the Src family kinase Lck and plays a positive role in TCR signaling. In this study we demonstrate a negative regulatory role for CD45 in CD44 signaling leading to actin rearrangement and cell spreading in activated thymocytes and T cells. In BW5147 T cells, CD44 ligation led to CD44 and Lck clustering, which generated a reduced tyrosine phosphorylation signal in CD45+ T cells and a more sustained, robust tyrosine phosphorylation signal in CD45 T cells. This signal resulted in F-actin ring formation and round spreading in the CD45+ cells and polarized, elongated cell spreading in CD45 cells. The enhanced signal in the CD45 cells was consistent with enhanced Lck Y394 phosphorylation compared with the CD45+ cells where CD45 was recruited to the CD44 clusters. This enhanced Src family kinase-dependent activity in the CD45 cells led to PI3K and phospholipase C activation, both of which were required for elongated cell spreading. We conclude that CD45 induces the dephosphorylation of Lck at Y394, thereby preventing sustained Lck activation and propose that the amplitude of the Src family kinase-dependent signal regulates the outcome of CD44-mediated signaling to the actin cytoskeleton and T cell spreading.

Studies show that the leukocyte-specific transmembrane tyrosine phosphatase CD45 has an important role in lymphocyte development and activation, largely via its effect on regulating the threshold of Ag receptor signaling (reviewed in Refs. 1, 2, 3). CD45 regulates the phosphorylation state and activity of specific Src family kinases expressed in leukocytes. The absence of CD45 affects the phosphorylation state of Lck and, to a lesser extent, Fyn in T cells (4, 5, 6, 7), Lyn in B cells (8, 9, 10, 11), and Lyn and Hck in macrophages (12). The Src family kinases are regulated by phosphorylation at two key tyrosines, the negative regulatory site located close to the C terminus (Y505 in Lck) and an opposing, positive regulatory site located in the activation loop (Y394 in Lck). Phosphorylation at the negative regulatory site induces an intramolecular interaction with the Src homology 2 domain resulting in a closed, inactive conformation (13, 14). Dephosphorylation at this site results in an open conformation allowing autophosphorylation of Y394 and full activation of the kinase (reviewed in Refs. 15, 16). CD45 can affect the phosphorylation state of both the negative and positive regulatory sites in Src family kinases (reviewed in1, 3, 17, 18). Lck in thymocytes (19), Lyn in B cells (10) and Hck and Lyn in macrophages (12) from CD45−/− mice are hyperphosphorylated at both the positive and negative regulatory sites and are more active in in vitro kinase assays, indicating that phosphorylation at the positive regulatory site is dominant over phosphorylation at the negative regulatory site (20).

The positive effect of CD45 in dephosphorylating Lck at the negative regulatory site is important for TCR-mediated signaling events. However, as CD45 is required for the initiation of TCR signaling, it has been difficult to determine whether CD45 also down-regulates Lck activity after TCR stimulation. CD45 negatively regulates CD44-mediated adhesion in T cells, which requires Src family kinase activity (21). CD45 can also affect integrin adhesion, but its effect appears to depend on the integrin and the cell type. CD45 is required for sustained adhesion in macrophages (12), yet negatively regulates α5β1 integrin-, but not α4β1 integrin-mediated adhesion in T cells (22). CD45 has also been reported to have a negative regulatory effect on IL-3 and IFN-α signaling by negatively regulating JAK kinases (23), although another study reported a positive role for CD45 and Lck in IFN-α signaling in T cells (24). Thus CD45 is associated with both positive and negative regulation of signaling in leukocytes, which might at least in part be due to its ability to positively and negatively regulate Src family kinases.

CD44 is a widely expressed cell adhesion molecule that can exist as multiple isoforms due to alternative splicing of 10 variably expressed exons. Although CD44 and its isoforms can bind hyaluronan (a component of the extracellular matrix), most leukocytes require activation before they will bind (reviewed in Refs. 25, 26, 27, 28). Although CD44 is a member of the Link-module superfamily that is distinct from other families of cell adhesion molecules such as the cadherins, selectins, and integrins, it shares several similarities with the selectins and the integrins. For example, like the integrins, ligand binding by CD44 is highly regulated and is induced upon leukocyte activation (29). CD44 shares structural and functional similarities with the selectins as it has an N-terminal C-type lectin domain (30) and can mediate leukocyte rolling (31). Further similarities are found between CD44 and the selectins and integrins when one considers their signaling capabilities. For example, cross-linking of L-selectin or CD44 activates Lck (32, 33) and both LFA-1 and CD44 cross-linking can induce protein kinase C-regulated migration of T cells (34, 35). Overall, CD44 exhibits overlapping functions with the selectins and integrins that together contribute to leukocyte extravasation. Signals transmitted to the cell as a consequence of their interactions may facilitate leukocyte adhesion and subsequent migration.

CD44 cross-linking activates Lck (33) and can lead to an increase in intracellular calcium levels (36) and activation of protein kinase C (35). CD44 can associate with Lck and Fyn in the low-density sucrose fraction after solubilization in 1% Brij 58 or Triton X-100, although the amount varies with the cell line and solubilization conditions (21, 37). Localization to this low-density fraction is equated with localization in cholesterol and sphingolipid-rich membrane domains referred to as lipid rafts. The low level of CD45 in this fraction (∼1%) and the hyperphosphorylation of Lck in this fraction (38) led to the suggestion that CD45 is excluded from lipid rafts. However, other data do not support this suggestion and instead suggests that CD45 is dynamically associated with these detergent resistant domains (39, 40). This suggests that localization of CD45 may be one mechanism to control its activity. Presently, little is known about how the tyrosine phosphatase activity of CD45 is regulated. In this study, we investigate how CD45 regulates CD44-mediated T cell signaling events leading to cell adhesion and cell spreading. Previous work indicated that CD44-mediated signaling leading to Pyk2 phosphorylation and elongated T cell spreading was mediated by Src family kinases and negatively regulated by CD45 (21). In this study, we compare CD44-mediated signaling events leading to T cell adhesion in the presence and absence of CD45 and propose a role for CD45 in down-regulating Lck activity and in determining the duration and strength of signals that affect actin rearrangement and T cell spreading. CD44 clustering in CD45+ T cells led to a small tyrosine phoshorylation signal, the formation of an F-actin ring and round spreading, whereas in the absence of CD45, a robust tyrosine phosphorylation signal led to more extensive, elongated cell spreading. This Src family kinase-dependent tyrosine phosphorylation in the CD45 T cells led to activation of phospholipase C (PLC)4 and PI3K, which were both required for elongated T cell spreading.

Rat anti-mouse CD44 mAbs, IM7, KM81, and KM201 from American Type Culture Collection (ATCC) were used as well as the following rabbit antisera: J1WBB against the cytoplasmic domain of mouse CD44 (21), R54-3B against residues 34–150 of Lck (41), R02.2 against the cytoplasmic domain of mouse CD45 (42), and pY394 specific for phosphorylated Y394 of Lck (43). Soluble ICAM-1 was a gift from Dr. F. Takei (Terry Fox Laboratory, Vancouver, British Columbia, Canada). I3/2 is a rat anti-mouse CD45 mAb (44) and was conjugated to Alexa Fluor 488, whereas IM7 was conjugated to Alexa Fluor 647 (Invitrogen). Anti-Fyn (sc-16), anti-Lck (sc-13), anti-PLCγ1 (sc-1249), and anti-Csk (sc-286) were from Santa Cruz Biotechnology; the anti-phosphotyrosine mAb 4G10 was from Upstate Biotechnology. Anti-phospho-Akt (S473) Ab and anti-phospho-Lck (Y505) Ab were from Cell Signaling Technology. Anti-β-actin was from Sigma-Aldrich. Anti-mouse IgG-HRP and anti-rabbit IgG-HRP were from Southern Biotechnology Associates. Protein A-HRP was from Bio-Rad. Alexa Fluor 488 phalloidin, anti-mouse IgG Alexa Fluor 488, anti-rabbit IgG Alexa Fluor 568, and anti-rabbit IgG Alexa Fluor 488 were from Molecular Probes and used at 1/100 dilution for confocal microscopy. Biotinylated anti-CD19 (MB19-1), NK1.1 (PK136), Ter119, and CD11b (M1/70) were from eBioscience or BD Pharmingen. Biotinylation of anti-CD4 (GK1.5) and anti-CD8 (53-6.7) mAbs (ATCC) were performed with EZ-Link Sulfo-NHS-Biotin Reagents (Pierce) as per the manufacturer’s instructions. Anti-biotin MicroBeads were from Miltenyi Biotec. The pharmacological reagents PP2, LY294002, U73122, and U73343 were from Calbiochem.

CD45+ and CD45 BW5147 T cells transfected with CD3 ζ-chain and δ-chain for TCR expression (45) were grown in DMEM, 10% heat-inactivated horse serum with 3 mM l-histidinol (Sigma-Aldrich).

CD45+/+ thymocytes were harvested from C57BL/6 mice (The Jackson Laboratory) and CD45−/− thymocytes from CD45−/− mice (46) backcrossed nine times onto a C57BL/6 background. A total of 2 × 108 thymocytes from three mice were labeled with 19 μg of biotinylated anti-CD4 and anti-CD8 Abs, then anti-biotin MicroBeads were added before passing through LS columns (Miltenyi Biotec) according to the manufacturer’s instructions, to enrich for double negative (DN) thymocytes. Thymocytes were then cultured in stimulation medium (RPMI 1640, 10% heat-inactivated FBS, 0.055 mM 2-ME, 10 mM HEPES) at 2 × 106/ml containing 12.5 ng/ml PMA (Sigma-Aldrich) and 250 ng/ml ionomycin (Sigma-Aldrich) in the presence of 20 U/ml recombinant mouse IL-2 (R&D Systems). On the fourth day, the medium containing PMA and ionomycin was removed and replaced with fresh medium containing IL-2 only. Activated DN thymocytes were used for cell spreading assays on the seventh day.

Lymph nodes (LN) harvested from C57BL/6 and CD45−/− mice were passed through a strainer to generate a single cell suspension. Cells (1.5–2 × 107) were treated with RBC lysis buffer (0.83% w/v NH4Cl, 10 mM Tris-HCl (pH 7)) before being labeled with biotinylated anti-CD19 (15 μg), CD11b (7.5 μg), NK1.1 (15 μg), and Ter119 (7.5 μg) Abs and anti-biotin MicroBeads were added before passing through LS columns to enrich for T cells. Typically, more than 90% of the cells were T cells and we obtained ∼4 × 105 T cells from the LN of three CD45−/− mice and ∼8 × 106 from three C57BL/6 mice. CD19 was used as the B cell marker because the conventional B cell marker, B220 is an isoform of CD45 and is not expressed in CD45−/− mice. T cells were activated with 2.5 ng/ml PMA and 500 ng/ml ionomycin for 48 h in stimulation medium at 1 × 106/ml, then used in the spreading assay. All animal protocols were approved and performed according to the University Animal Care Committee and Canadian Council of Animal Care Guidelines.

The anti-CD44 mAb, KM81, was immobilized on 96-well plates (tissue culture-treated plates, Falcon or Nunc) at 40 μg/ml in PBS overnight at 4°C, essentially as described (21). Cells (5 × 104) in 50 μl of spreading medium (DMEM with 0.1% heat-inactivated FBS) were added for various times at 37°C before fixation with a final concentration of 4% paraformaldehyde for 20 min at room temperature. Light images of cells were taken with a Nikon Coolpix 950 mounted on a Nikon inverted microscope (Eclipse TS100) with a 20X or 40X objective and imported into Adobe Photoshop. Measurement of cell length for BW5147 T cells was calibrated with a microscope stage ruler. Cell length for activated primary thymocytes and T cells was measured from F-actin-labeled confocal images imported into NIH ImageJ. Polarized cells were identified as cells showing a significant asymmetrical, elongated phenotype and were distinguished from nonpolarized cells, which were predominantly round. Three independent experiments were performed (two independent experiments for LN T cells), and at least 85 cells were measured. Significance was determined with the unpaired Student’s t test.

A total of 5 × 104 T cells or 1.5 × 105 activated DN thymocytes were incubated on Ab or BSA-coated chamber slides (Lab-Tek) in 150 μl of spreading medium (DMEM or RPMI 1640 with 0.1% heat-inactivated FBS) for 30 min or 2 h at 37°C then fixed as mentioned. Alternatively, BW5147 T cells were pretreated with 20 μM PP2, 20 μM LY294002, 0.5 μM U73343, or 0.5 μM U73122 for 30 min at 37°C before being incubated and fixed on the coated slides. The cells were washed and permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature, then incubated with 1% BSA at room temperature for 30 min to prevent nonspecific binding. BW5147 T cells were incubated for 1 h with primary Abs in 1% BSA/PBS or overnight in 150 μl of 10 U/ml Alexa Fluor 488-conjugated phalloidin in PBS. Cells were washed three times, incubated with corresponding Alexa Fluor-conjugated Abs for 1 h. Activated primary cells were incubated for 1.5 h with 100 μl of 10 U/ml Alexa Fluor 488-conjugated phalloidin in PBS. Cells were washed three times with PBS before being mounted in 90% v/v glycerol/2.5% w/v DABCO (1,4-diazabicyclo-(2, 2, 2)-octane) in PBS (Sigma-Aldrich). For double labeling, the staining was conducted in a sequential manner with four Ab-labeling steps. Control labeling without the primary Ab was included to ensure no cross-reactivity occurred between the secondary Abs.

Images of labeled BW5147 T cells were captured with Bio-Rad Radiance 2000 or Plus on a Nikon Eclipse 300 or Zeiss Axiovert, respectively, using a 60X oil immersion objective. The images were collected with Kalman collection filter (2X) with a step size of 0.3 μm. Image size was 512 × 512 pixels covering the dimension of 162 × 162 μm. Images of labeled BW5147 T cells, activated T cells or thymocytes were captured with Olympus FluoView FV1000 using a 60X or 100X objective, with a step size of 0.19 μm covering dimensions of 212 × 212 μm or 127 × 127 μm. For double labeling experiments, images were collected with the same settings in a sequential manner. For any given experiment, the same laser power and gain controls settings were used to ensure consistent signal intensity. Cells from random fields were collected and analyzed from at least three independent experiments.

Fluorescent images were processed in either NIH Image or NIH ImageJ. One of the images close to the interface between the cells and the slide was selected from the stack of confocal images and reopened in Adobe Photoshop. Alternatively, a projection of five images close to the interface was made to enhance signal-to-noise ratio. The image/adjustment/level command was used to adjust image contrast.

Cells were pretreated with or without 20 μM PP2 or LY294002 at 37°C for 30 min before being added to a 6-well plate (Nunc) containing immobilized CD44 Ab, and incubated at 37°C for various times. Then, 5 × 106 cells/ml were lysed with 250 μl of ice-cold 5X lysis buffer (5% Triton X-100, 50 mM Tris-HCl (pH 7.2), 700 mM KCl, 10 mM EDTA (pH 8.0), 2.5 mM sodium orthovanadate, 1 mM sodium molybdate, 5 μg/ml aprotinin, 5 μg/ml leupeptin, 5 μg/ml pepstatin, and 1 mM PMSF). Lysates were centrifuged and 1 μg of anti-PLCγ1 Ab, or 2.5 μl of R54-3B was added at 4°C for 1 h. Then 20 μl of protein A beads (Repligen) was added for 1 h, then beads were washed with lysis buffer and samples resolved in a 7.5% polyacrylamide gel and transferred to polyvinylidene fluoride membrane (Immobilon P; Millipore).

For anti-phosphotyrosine detection, blots were incubated with 1/5000 4G10; phosphoY505 of Lck was detected by incubation with 1/1000 anti-phospho Lck (Y505) Ab in 0.5% BSA in TBS-T (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% v/v Tween 20) for 1.5 h at room temperature. After several brief washes with TBS-T, the blot was incubated with 1/5000 anti-mouse IgG-HRP or protein A-HRP for 1 h. The blot was then washed several times with TBS-T and bands visualized with ECL or ECLplus (Amersham Biosciences). Membranes were stripped in 50 mM glycine (pH 2.5), 150 mM NaCl, 0.1% v/v Nonidet P-40 for 30 min at room temperature, then washed with TBS-T and reprobed with 1/200 anti-PLCγ1 or 1/5000 anti-Lck in 5% skim milk/TBS-T and 1/5000 anti-rabbit IgG-HRP or protein A-HRP.

For whole cell lysate blots, polyvinylidene fluoride membranes of cell lysates from 2.5 × 104 cells were probed with 1/1000 phospho-Akt (S473) and 1/5000 anti-rabbit IgG-HRP in 5% skim milk/TBS-T before being stripped and reprobed with 1/1000 Akt Ab and 1/5000 anti-rabbit IgG-HRP. Alternatively, membranes were probed with 1/200 anti-Csk Ab and 1/5000 protein A-HRP before stripped and reprobed with 1/5000 anti-β-actin Ab and 1/5000 anti-mouse IgG-HRP.

Comparison of the extent of cell spreading between CD45+ and CD45 BW5147 T cells on immobilized CD44 mAb, KM81, revealed that the CD45+ cells underwent a small, but significant increase of 11% in cell diameter and the cells exhibited round spreading (Fig. 1,A). No spreading was observed with cells on immobilized BSA. In the absence of CD45, the cells adopted an elongated phenotype resulting in a much larger increase in cell length of 69% (Fig. 1 A). Cells started to spread at 5 min and the majority of cells had spread by 30 min. A similar pattern of spreading was observed with immobilized LFA-1 mAb (TIB213) and immobilized recombinant soluble ICAM (data not shown).

FIGURE 1.

CD44-mediated cell spreading and actin rearrangement in CD45+ and CD45 T cells. A, CD45+ and CD45 BW5147 T cells were incubated on immobilized BSA or CD44 mAb for 2 h. Cells were then fixed with 4% paraformaldehyde and photographed. The longest length of the cells was measured. Graph shows the average cell length with the SD, and data represent the average length of over 250 cells taken from three separate experiments. ∗∗, p < 0.01 for a significant difference between the length of the two different samples. B, CD45+ and CD45 BW5147 T cells were untreated or treated with 20 μM PP2 and incubated on CD44 mAb or BSA for 2 h and fixed with 4% paraformaldehyde. F-actin was labeled with Alexa Fluor 488-conjugated phalloidin after the cells were permeabilized with 0.1% Triton X-100. The confocal images are a single 0.3-μm horizontal slice close to the interface between the cells and the immobilized CD44 mAb. Images are representative of 150 cells examined over three experiments. Actin rings were observed in 61% of the CD45+ cells (n = 79) and this percentage was observed in only 19% of the CD45+ cells (n = 77) in the presence of PP2. Scale bar represents 10 μm.

FIGURE 1.

CD44-mediated cell spreading and actin rearrangement in CD45+ and CD45 T cells. A, CD45+ and CD45 BW5147 T cells were incubated on immobilized BSA or CD44 mAb for 2 h. Cells were then fixed with 4% paraformaldehyde and photographed. The longest length of the cells was measured. Graph shows the average cell length with the SD, and data represent the average length of over 250 cells taken from three separate experiments. ∗∗, p < 0.01 for a significant difference between the length of the two different samples. B, CD45+ and CD45 BW5147 T cells were untreated or treated with 20 μM PP2 and incubated on CD44 mAb or BSA for 2 h and fixed with 4% paraformaldehyde. F-actin was labeled with Alexa Fluor 488-conjugated phalloidin after the cells were permeabilized with 0.1% Triton X-100. The confocal images are a single 0.3-μm horizontal slice close to the interface between the cells and the immobilized CD44 mAb. Images are representative of 150 cells examined over three experiments. Actin rings were observed in 61% of the CD45+ cells (n = 79) and this percentage was observed in only 19% of the CD45+ cells (n = 77) in the presence of PP2. Scale bar represents 10 μm.

Close modal

Confocal microscopy of the spread cells labeled with Alexa Fluor 488-conjugated phalloidin revealed that the CD45+ T cells formed an F-actin ring close to the interface between the cell and the slide (Fig. 1,B, top panel). A time course indicated that the rings started to form by 5 min and were present in the majority of cells (70%, n = 92) by 30 min. The actin rings were still present at 2 h (63%, n = 136). Often the F-actin ring would be located within the perimeter of the cell, which may represent the edge of the contact zone between the cell and the slide. In CD45 T cells, the F-actin ring was not observed. Instead, actin filaments were observed at the sides of the elongated cell (Fig. 1 B, top panel). The F-actin cables were always aligned with the longitudinal axis of the cell, suggesting a directional axis. None of these F-actin structures were observed when the T cells were incubated on immobilized BSA. This suggests that the presence of CD45 can influence F-actin formation and cell shape induced by CD44-mediated signaling.

We had previously shown that Src family kinase activity is required for elongated spreading in CD45 T cells (21). To determine whether Src family kinase activity was also required for F-actin rearrangement and cell spreading, BW5147 T cells were pretreated for 30 min with 20 μM of the Src family kinase inhibitor, PP2, then immobilized on CD44 mAb for 2 h and the formation of F-actin examined after staining with Alexa Fluor 488-conjugated phalloidin. Treatment of the CD45+ cells with PP2 resulted in the loss of F-actin rings, with the F-actin staining resembling that of cells on immobilized BSA (Fig. 1 B, bottom panels). CD45 cells also lost the characteristic F-actin staining, which became more punctate as the cells lost their elongated shape and rounded up. This indicates that Src family kinase activity is required for CD44-induced actin rearrangement in both the CD45+ and CD45 T cells. However, the difference in F-actin formation and cell shape between the CD45+ and CD45 cells suggests that CD45 can influence the outcome of the Src family kinase-mediated signal.

To determine whether CD45 also affected CD44-mediated cell spreading in primary cells, we compared the spreading of CD44+, CD4 and CD8 DN thymocytes, and LN T cells isolated from CD45−/− (46) and CD45+/+ mice. Because it had previously been demonstrated that CD44-mediated spreading only occurs with activated T cells (47), DN thymocytes and T cells were activated with PMA and ionomycin before spreading on the immobilized CD44 mAb (Fig. 2). Analysis of DN1 and DN2 populations ex vivo indicated that CD44 was expressed at slightly higher levels on the CD45−/− cells (data not shown), suggesting that CD45 may also influence the expression of CD44. However, there was no significant difference in CD44 expression after stimulation and at day 7, the majority of activated DN thymocytes were CD44+ and levels were comparable between CD45+/+ and CD45−/− cells (Fig. 2,A). Both the CD45+/+ and CD45−/− activated DN thymocytes spread on the CD44 mAb and induced CD44 clustering (data not shown). As with the CD45 BW5147 T cells, the CD45−/− activated DN thymocytes spread more extensively on the CD44 mAb than the CD45+/+ cells (Fig. 2,B) and this was significant after quantitation (Fig. 2,C, left). The CD45−/− activated DN thymocytes not only spread significantly longer than the CD45+/+ cells, but were also significantly more polarized (Fig. 2,C). The CD45+/+ activated DN thymocytes spread round or round with a slight extension or polarization. To examine the effect of CD45 on F-actin formation in the spread thymocytes, cells were immobilized on CD44 mAb for 30 min and then labeled with Alexa Fluor 488-conjugated phalloidin. In keeping with the round spreading, F-actin was primarily localized at the periphery of the cell in the majority of CD45+/+ activated DN thymocytes (Fig. 2 D). This contrasts with the CD45−/− cells where the majority of cells had localized F-actin accumulation at one or both ends of the cell. No significant F-actin organization was observed when the cells were plated on BSA (data not shown).

FIGURE 2.

CD44-mediated cell spreading and F-actin rearrangement in activated primary CD45+/+ and CD45−/− thymocytes and T cells. A, CD44 expression on activated CD45+/+ and CD45−/− DN thymocytes determined by flow cytometry and shown as the average percentage of CD44-expressing cells and mean fluorescence intensity (MFI) ± SEM from six independent experiments. B, Phase-contrast image of activated DN thymocytes incubated on immobilized BSA or CD44 mAb for 30 min. C, Average length ± SD of activated CD45+/+ and CD45−/− DN thymocytes immobilized on CD44 mAb for 30 min. At least 85 cells were counted over three independent experiments. ∗∗∗, p < 0.001. Percentage of polarized cells ± SEM from four independent experiments. ∗, p < 0.05. D, F-actin labeled activated CD45+/+ and CD45−/− DN thymocytes after immobilization on CD44 mAb for 30 min. The confocal images are a stack of five 0.19-μm horizontal slices close to the interface between the cells and the immobilized CD44 mAb. Images are representative of cells examined over three experiments. Scale bar represents 10 μm. E, Average cell length ± SD of at least 221 cells for CD45+/+ and CD45−/− activated LN T cells immobilized on CD44 mAb (left). ∗∗∗, p < 0.001. The percentage of polarized cells ± SEM obtained from six mice over two independent experiments is also shown (right). ∗∗, p < 0.01. F, F-actin labeled, activated CD45+/+ and CD45−/− LN T cells after immobilization on CD44 mAb for 30 min. The confocal images are a stack of five 0.19-μm horizontal slices close to the interface between the cells. The image is one representative of two experiments.

FIGURE 2.

CD44-mediated cell spreading and F-actin rearrangement in activated primary CD45+/+ and CD45−/− thymocytes and T cells. A, CD44 expression on activated CD45+/+ and CD45−/− DN thymocytes determined by flow cytometry and shown as the average percentage of CD44-expressing cells and mean fluorescence intensity (MFI) ± SEM from six independent experiments. B, Phase-contrast image of activated DN thymocytes incubated on immobilized BSA or CD44 mAb for 30 min. C, Average length ± SD of activated CD45+/+ and CD45−/− DN thymocytes immobilized on CD44 mAb for 30 min. At least 85 cells were counted over three independent experiments. ∗∗∗, p < 0.001. Percentage of polarized cells ± SEM from four independent experiments. ∗, p < 0.05. D, F-actin labeled activated CD45+/+ and CD45−/− DN thymocytes after immobilization on CD44 mAb for 30 min. The confocal images are a stack of five 0.19-μm horizontal slices close to the interface between the cells and the immobilized CD44 mAb. Images are representative of cells examined over three experiments. Scale bar represents 10 μm. E, Average cell length ± SD of at least 221 cells for CD45+/+ and CD45−/− activated LN T cells immobilized on CD44 mAb (left). ∗∗∗, p < 0.001. The percentage of polarized cells ± SEM obtained from six mice over two independent experiments is also shown (right). ∗∗, p < 0.01. F, F-actin labeled, activated CD45+/+ and CD45−/− LN T cells after immobilization on CD44 mAb for 30 min. The confocal images are a stack of five 0.19-μm horizontal slices close to the interface between the cells. The image is one representative of two experiments.

Close modal

Although there are greatly reduced numbers of peripheral T cells in the CD45−/− mice, we sought to determine whether this observation also occurred in activated T cells. LN were isolated from multiple CD45+/+ and CD45−/− mice and T cells were enriched for by negative selection (see Materials and Methods). All the T cells expressed CD44 and the levels were enhanced upon activation with PMA and ionomycin. Activated CD45+/+ and CD45−/− T cells expressed comparable levels of CD44, yet, as with the T cell line and DN thymocytes, showed distinct spreading and polarization ability in response to the ligation of CD44. Activated CD45−/− T cells spread significantly longer and were significantly more polarized than the CD45+/+ T cells (Fig. 2,E). F-actin labeling in the activated T cells also revealed distinct differences between the CD45+/+ and CD45−/− T cells (Fig. 2 F). Similar to the CD45+ BW5147 T cells and activated CD45+/+ DN thymocytes, CD45+/+ T cells showed a cortical distribution of F-actin, forming a ring around the periphery of the cell. In contrast, F-actin was more heterogeneously distributed in the CD45−/− cell and was often enriched in cell protrusions at one end of the cell. Small filamentous spikes of F-actin were also often observed protruding from one end of the cell. Together, these data establish a role for CD45 in regulating CD44-mediated cell spreading and F-actin rearrangement in activated primary CD44+ DN thymocytes and peripheral T cells.

To investigate how CD45 can affect the outcome of Src family kinase mediated F-actin rearrangement and cell spreading, we investigated the temporal and spatial organization of Src family kinases in CD45+ and CD45 BW5147 T cells spread on immobilized CD44 mAb. Antiserum against the cytoplasmic domain of CD44 was used to label CD44. Confocal microscopy revealed that at 5 min, CD44 had redistributed into clusters close to the interface between the cell and immobilized mAb (Fig. 3,A). These CD44 clusters were observed in both the CD45+ and CD45 T cells and were still present at 2 h (Fig. 3 A and data not shown). Approximately 80% of CD45+ T cells (n = 152) and 89% of CD45 T cells (n = 175) had clusters of CD44 at 30 min, indicating that their formation was independent of the presence of CD45. Incubation of the cells on BSA did not induce cluster formation. Treatment of the T cells with latrunculin A, which prevents actin polymerization, significantly reduced the number of cells showing CD44 clusters and also reduced cluster size, suggesting that F-actin polymerization promoted cluster formation (data not shown).

FIGURE 3.

Distribution of CD44, Lck, and Fyn upon CD44-mediated cell spreading. CD45+ and CD45 T cells were fixed with 4% paraformaldehyde after incubation on immobilized BSA for 30 min or CD44 mAb for 5 and 30 min. Confocal images are one horizontal 0.3-μm slice close to the interface between the cells and the immobilized mAb, as described in Materials and Methods. A, CD44 was then labeled with J1WBB, which recognizes the cytoplasmic domain of CD44. B, Lck was labeled with R54-3B and Alexa Fluor 488-conjugated anti-rabbit IgG Ab. C, Shows the labeling of Fyn. D, Double labeling of cells with CD44 (green) and Lck (red) mAbs after 30 min of incubation on immobilized CD44 mAb. Confocal image of five 0.19-μm slices is shown. Scale bar represents 10 μm in each image. These are representative images observed in over 65 cells in each observation, from at least three independent experiments.

FIGURE 3.

Distribution of CD44, Lck, and Fyn upon CD44-mediated cell spreading. CD45+ and CD45 T cells were fixed with 4% paraformaldehyde after incubation on immobilized BSA for 30 min or CD44 mAb for 5 and 30 min. Confocal images are one horizontal 0.3-μm slice close to the interface between the cells and the immobilized mAb, as described in Materials and Methods. A, CD44 was then labeled with J1WBB, which recognizes the cytoplasmic domain of CD44. B, Lck was labeled with R54-3B and Alexa Fluor 488-conjugated anti-rabbit IgG Ab. C, Shows the labeling of Fyn. D, Double labeling of cells with CD44 (green) and Lck (red) mAbs after 30 min of incubation on immobilized CD44 mAb. Confocal image of five 0.19-μm slices is shown. Scale bar represents 10 μm in each image. These are representative images observed in over 65 cells in each observation, from at least three independent experiments.

Close modal

Lck was also recruited into clusters in a similar time frame as CD44, suggesting an intimate association between CD44 and Lck. Over 80% of both CD45+ and CD45 T cells (n > 100 in both cases) had recruited Lck into clusters by 5 min (Fig. 3,B). Clustering is known to activate Lck and this response was consistent with Src family kinase-dependent actin rearrangement occurring in both the CD45+ and CD45 cells. However, it also suggests that even though Lck is hyperphosphorylated at the negative regulatory tyrosine (Y505) in the CD45 T cells (4), it is still active upon clustering of CD44. Co-labeling of CD44 and Lck in both CD45+ and CD45 T cells demonstrated their colocalization in the clusters (Fig. 3 D).

Because the Src family kinase Fyn resides in the low-density sucrose fraction along with CD44 and Lck, and can coprecipitate with CD44 (21), its distribution was also examined. Interestingly, by 5 min only 25% of CD45+ cells (n = 68) had recruited Fyn into clusters, whereas 74% of CD45 cells (n = 84) had recruited Fyn (Fig. 3 C). By 30 min, 48% of the CD45+ T cells (n = 93) and 91% of the CD45 T cells (n = 67) had clusters of Fyn. This result indicated that the presence of Lck in CD44 clusters was independent of CD45 expression, whereas Fyn recruitment was delayed and less efficient in the CD45+ T cells.

To determine the outcome of Lck and Fyn clustering with CD44 in the CD45+ and CD45 cells, the cells were colabeled with an anti-CD44 cytoplasmic domain and anti-phosphotyrosine Ab (Fig. 4). At 5 min, clusters of phosphotyrosine were observed in CD45 T cells, whereas virtually no staining was observed in the CD45+ cells. By 30 min, more phosphotyrosine was present in the CD45 T cells and the majority of the CD44 clusters (97%, n = 175) were positive for phosphotyrosine. In contrast, only the occasional spot of phosphotyrosine was seen in the CD45+ T cells. This observation indicates that CD44 clustering leads to robust, sustained tyrosine phosphorylation in the CD45 T cells. Induction of tyrosine phosphorylation in the CD45+ T cells occured at a much lower intensity and to a much lesser extent than in the CD45 T cells. This finding indicates that CD45 negatively regulates the CD44-induced tyrosine phosphorylation signal.

FIGURE 4.

Induction of tyrosine phosphorylation in CD45+ and CD45 T cells upon CD44-mediated cell spreading. CD45+ and CD45 T cells were labeled with CD44 and phosphotyrosine mAbs after immobilization on CD44 mAb for 5 and 30 min. Each image is a 0.3-μm slice of a confocal image taken close to the interface between the cell and the slide. No cross-reactivity between the secondary Abs was observed. Scale bar represents 10 μm. Data are from three independent experiments from over 120 cells analyzed. After 30 min, clusters of CD44 were observed in 80% of the CD45+ T cells (n = 152) and 89% of the CD45 T cells (n = 175), and 97% of CD45 T cells (n = 175) showed anti-phosphotyrosine labeling.

FIGURE 4.

Induction of tyrosine phosphorylation in CD45+ and CD45 T cells upon CD44-mediated cell spreading. CD45+ and CD45 T cells were labeled with CD44 and phosphotyrosine mAbs after immobilization on CD44 mAb for 5 and 30 min. Each image is a 0.3-μm slice of a confocal image taken close to the interface between the cell and the slide. No cross-reactivity between the secondary Abs was observed. Scale bar represents 10 μm. Data are from three independent experiments from over 120 cells analyzed. After 30 min, clusters of CD44 were observed in 80% of the CD45+ T cells (n = 152) and 89% of the CD45 T cells (n = 175), and 97% of CD45 T cells (n = 175) showed anti-phosphotyrosine labeling.

Close modal

To investigate how CD45 might be down-regulating CD44 signaling, its localization was monitored upon CD44-mediated signaling. Surprisingly, like CD44 and Lck, CD45 was also recruited to the clusters within 5 min and was still associated at 30 min (Fig. 5,A). In addition to localizing in the clusters, CD45 also formed a ring around the immobilized cell, similar to that observed for F-actin. Double labeling confirmed that CD45 colocalized to the same clusters as CD44 and Lck (Fig. 5, B and C). This colocalization of the tyrosine phosphatase CD45 with the CD44/Lck clusters is consistent with the reduced phosphotyrosine levels observed in the CD45+ T cells (Fig. 4).

FIGURE 5.

CD45 distribution upon CD44-mediated cell spreading. A, CD45+ T cells were immobilized on CD44 mAb for 5 min (left) and 30 min (right). Cells were fixed and CD45 was labeled with R02.2. One representative 0.3-μm horizontal slice from a confocal image taken from close to the interface of the cell and slide is shown. The experiment was repeated at least three times, and CD45 was located in clusters in 49% of T cells (n = 138) and 52% of T cells (n = 149) at 5 and 30 min, respectively. B and C, Double labeling of cells with CD45 (green) and CD44 (red) or Lck (red) Abs after 5 min of incubation on immobilized CD44 mAb. Confocal image of five 0.19-μm slices. Scale bar represents 10 μm in each case.

FIGURE 5.

CD45 distribution upon CD44-mediated cell spreading. A, CD45+ T cells were immobilized on CD44 mAb for 5 min (left) and 30 min (right). Cells were fixed and CD45 was labeled with R02.2. One representative 0.3-μm horizontal slice from a confocal image taken from close to the interface of the cell and slide is shown. The experiment was repeated at least three times, and CD45 was located in clusters in 49% of T cells (n = 138) and 52% of T cells (n = 149) at 5 and 30 min, respectively. B and C, Double labeling of cells with CD45 (green) and CD44 (red) or Lck (red) Abs after 5 min of incubation on immobilized CD44 mAb. Confocal image of five 0.19-μm slices. Scale bar represents 10 μm in each case.

Close modal

Localization of CD45 to CD44 clusters could decrease phosphotyrosine levels directly or indirectly by dephosphorylating and inactivating Lck. To investigate the latter, CD45+ and CD45 T cells were labeled with the phosphospecific Lck Y394 antiserum, which recognizes Lck phosphorylated at the positive regulatory site. At 30 min, Y394 phosphorylation was clearly observed in the CD45 T cells and was detectable in the clusters of ∼38% (n = 276) of the cells (Fig. 6 A). In contrast, very little phosphorylated Y394 was observed in the CD45+ T cells and no signal was observed in the clusters (0%, n = 147). Given the colocalization of CD45 and Lck with the CD44-induced clusters and the absence of phosphospecific Lck Y394 staining in these clusters, we conclude that CD45 is down-regulating CD44-induced signaling events by inducing the dephosphorylation of Lck Y394 and thereby limiting Lck activation.

FIGURE 6.

Phosphorylation of Lck upon CD44-mediated spreading. A, Phosphorylation of Lck at Y394 in CD44-immobilized CD45+ and CD45 T cells. CD45+ and CD45 T cells were incubated on immobilized CD44 mAb for 30 min. The cells were then fixed, permeabilized and labeled with anti-Lck phospho-Y394-specific antiserum, as described in Materials and Methods. The image from the projection of five 0.3-μm slices taken from close to the interface between the cells and immobilized mAb is shown. This one image is representative of at least three independent experiments. Phospho-Y394 labeling in clusters was detectable in 38% of the CD45 T cells (n = 276), but was not present in any of CD45+ cells (n = 147) examined. Scale bar represents 10 μm. B, Lck was immunoprecipitated from 5 × 106 CD45+ and CD45 T cells, and samples were probed with a phosphospecific Y505 Ab (pY505, top), stripped, and reprobed with an anti-Lck Ab (bottom). C, Whole cell lysates from CD45+ and CD45 T cells were probed with a Csk Ab (top) before being stripped and reprobed with anti-β-actin Ab (bottom). Bands were detected with ECL or ECLplus.

FIGURE 6.

Phosphorylation of Lck upon CD44-mediated spreading. A, Phosphorylation of Lck at Y394 in CD44-immobilized CD45+ and CD45 T cells. CD45+ and CD45 T cells were incubated on immobilized CD44 mAb for 30 min. The cells were then fixed, permeabilized and labeled with anti-Lck phospho-Y394-specific antiserum, as described in Materials and Methods. The image from the projection of five 0.3-μm slices taken from close to the interface between the cells and immobilized mAb is shown. This one image is representative of at least three independent experiments. Phospho-Y394 labeling in clusters was detectable in 38% of the CD45 T cells (n = 276), but was not present in any of CD45+ cells (n = 147) examined. Scale bar represents 10 μm. B, Lck was immunoprecipitated from 5 × 106 CD45+ and CD45 T cells, and samples were probed with a phosphospecific Y505 Ab (pY505, top), stripped, and reprobed with an anti-Lck Ab (bottom). C, Whole cell lysates from CD45+ and CD45 T cells were probed with a Csk Ab (top) before being stripped and reprobed with anti-β-actin Ab (bottom). Bands were detected with ECL or ECLplus.

Close modal

To further determine that this robust Lck activation, evidenced by Y394 phosphorylation, was due to the absence of CD45 and not to changes in Y505 phosphorylation by another phosphatase, the phosphorylation state of Y505 was monitored upon CD44 mediated cell spreading. Fig. 6,B shows that as expected, Lck was hyperphosphorylated at Y505 before CD44 signaling and that the level did not change significantly upon CD44-mediated cell spreading. This is in agreement with other work that shows that Lck can be phosphorylated at both Y394 and Y505 in the absence of CD45 (17) and that despite Y505 phosphorylation, Y394 phosphorylation is a necessary prerequisite and accurate predictor of Lck kinase activity (48, 49). Finally, we confirmed that the differences in Lck Y505 phosphorylation were consistent with the loss of CD45 and were not due to any differences in expression of Csk in the CD45+ and CD45 T cells (Fig. 6 C).

To further understand why Lck activation and sustained tyrosine phosphorylation led to enhanced, elongated cell spreading in the CD45 T cells, we examined downstream signaling events. Upon TCR ligation, Src family kinase activation can result in the activation of several downstream signaling molecules including PI3K, PLCγ, and MAPK (50, 51, 52). To determine whether these pathways were activated upon CD44 signaling, we first examined activation of the PI3K pathway by monitoring AKT phosphorylation. Phospho-AKT (S473) was induced upon CD44 ligation in the CD45 T cells (Fig. 7,A) to a much greater extent than observed in the CD45+ T cells. This induction was significantly reduced by 20 μM PP2 (Fig. 7 A), indicating that activation of the PI3K pathway was Src family kinase dependent.

FIGURE 7.

Induction of phosphorylation of AKT and PLCγ1 upon immobilization on CD44 mAb. CD45+ and CD45 T cells were treated with or without 20 μM PP2, incubated on immobilized CD44 mAb for the indicated times, and cell lysates were prepared and resolved by SDS-PAGE, as described in Materials and Methods. A, Whole cell lysates (the equivalent of 2.5 × 104 cells) from CD45+ and CD45 T cells were loaded in each lane and probed with phospho-S473-specific AKT Ab (pS473, top) before being stripped and reprobed with AKT Ab (bottom). Bands were detected with ECL or ECLplus. B and C, PLCγ1 was immunoprecipitated from 5 × 106 CD45+ and CD45 T cells, and samples were probed for phosphotyrosine with 4G10 (pTyr, top), stripped and reprobed with anti-PLCγ1 (bottom). L indicates lysate. In C, CD45 T cells were immobilized on CD44 mAb for the time indicated after being treated or not with 20 μM PP2. Cells incubated on BSA for 30 min before PLCγ1 immunoprecipitation (B) or lysate only (L) are indicated. CD45 T cells immobilized on CD44 mAb for the time indicated are shown with medium alone (M), DMSO (D), or 20 μM LY294002 (LY). A 10-min incubation on BSA (B) is also indicated.

FIGURE 7.

Induction of phosphorylation of AKT and PLCγ1 upon immobilization on CD44 mAb. CD45+ and CD45 T cells were treated with or without 20 μM PP2, incubated on immobilized CD44 mAb for the indicated times, and cell lysates were prepared and resolved by SDS-PAGE, as described in Materials and Methods. A, Whole cell lysates (the equivalent of 2.5 × 104 cells) from CD45+ and CD45 T cells were loaded in each lane and probed with phospho-S473-specific AKT Ab (pS473, top) before being stripped and reprobed with AKT Ab (bottom). Bands were detected with ECL or ECLplus. B and C, PLCγ1 was immunoprecipitated from 5 × 106 CD45+ and CD45 T cells, and samples were probed for phosphotyrosine with 4G10 (pTyr, top), stripped and reprobed with anti-PLCγ1 (bottom). L indicates lysate. In C, CD45 T cells were immobilized on CD44 mAb for the time indicated after being treated or not with 20 μM PP2. Cells incubated on BSA for 30 min before PLCγ1 immunoprecipitation (B) or lysate only (L) are indicated. CD45 T cells immobilized on CD44 mAb for the time indicated are shown with medium alone (M), DMSO (D), or 20 μM LY294002 (LY). A 10-min incubation on BSA (B) is also indicated.

Close modal

Although linker for activation of T cells (LAT) and MAPK (ERK1/2) phosphorylation are key events in TCR signaling, these proteins were not phosphorylated upon CD44 ligation in either the CD45+ or CD45 cells (data not shown). In TCR signaling, LAT phosphorylation facilitates the recruitment and activation of PLCγ1, which initiates the release of intracellular calcium (53). As CD44 signaling has been shown to involve calcium (36), it was of interest to determine whether PLCγ1 was phosphorylated in the absence of LAT phosphorylation. CD44 ligation induced the transient tyrosine phosphorylation of PLCγ1 in the CD45, but not the CD45+ T cells (Fig. 7,B). This phosphorylation was inhibited by 20 μM PP2 but not 20 μM LY294002 (Fig. 6 C), indicating a dependence on Src family kinase activity, but not PI3K activity. Thus both PI3K and PLCγ1 were activated to a much greater extent in the CD45 T cells, in a Src family kinase dependent manner.

To determine whether either of these two proteins accounted for the difference in F-actin organization and cell spreading observed between the CD45+ and CD45 T cells, the cells were treated with PI3K or PLC inhibitors. Interestingly, treatment with the PI3K inhibitor (20 μM LY294002) resulted in the formation of F-actin rings and round spreading in the CD45 T cells, similar to that observed in the CD45+ cells (Fig. 8). The inhibitor did not affect actin ring formation in the CD45+ T cells, although in both cases the actin rings were smaller in the presence of the PI3K inhibitor. Thus the PI3K inhibitor prevented elongated cell spreading and induced round spreading in the CD45 T cells. Addition of U73122, an inhibitor of PLC, but not the inactive analog U73343, also prevented the elongated cell spreading and formation of F-actin cables in the CD45 T cells and led to the formation of F-actin rings and round spreading (Fig. 8, bottom panels). The inhibitor had no observable effect on F-actin ring formation and round spreading in the CD45+ T cells. This experiment indicates that PLC and PI3K activities are both necessary, but alone not sufficient, for F-actin rearrangement and elongated cell spreading in the CD45 T cells. Without this activity, the cells revert to the F-actin ring formation and round spreading that is observed in the CD45+ T cells, indicating that their activation is important for polarized F-actin reorganization and directed, elongated cell spreading.

FIGURE 8.

CD44-mediated F-actin rearrangement in the presence of PI3K and PLC inhibitors. CD45+ and CD45 T cells were pretreated with or without 20 μM LY294002, 0.5 μM U73122, or 0.5 μM U73343 for 30 min before incubation on immobilized CD44 mAb for 2 h. The cells were then fixed and stained with Alexa Fluor 488-conjugated phalloidin. Pictures shown are single 0.3-μm slices of confocal images close to the interacting surface with immobilized mAb. These data are representative of three independent experiments. Scale bar represents 10 μm.

FIGURE 8.

CD44-mediated F-actin rearrangement in the presence of PI3K and PLC inhibitors. CD45+ and CD45 T cells were pretreated with or without 20 μM LY294002, 0.5 μM U73122, or 0.5 μM U73343 for 30 min before incubation on immobilized CD44 mAb for 2 h. The cells were then fixed and stained with Alexa Fluor 488-conjugated phalloidin. Pictures shown are single 0.3-μm slices of confocal images close to the interacting surface with immobilized mAb. These data are representative of three independent experiments. Scale bar represents 10 μm.

Close modal

Overall, the recruitment of CD45 to CD44 clusters induces transient Lck activation that results in a signal that leads to the formation of an F-actin ring and round spreading. In the absence of CD45, unchecked Lck activation induces sustained protein tyrosine phosphorylation and activation of PI3K and PLCγ that results in directed F-actin formation and elongated T cell spreading.

In this study we have shown that CD44 can signal changes to the actin cytoskeleton that promote cell spreading. We have also shown that this signal is modulated by CD45. Immobilization of T cells on CD44 mAb led to the clustering of CD44 and Lck in a CD45-independent manner. However, CD45 was required to regulate the strength of the Src family kinase-mediated signal. CD45 was recruited to the CD44 clusters where no Lck Y394 phosphorylation and reduced protein tyrosine phosphorylation was observed, suggesting that CD45 down-regulates CD44 mediated Lck signaling by dephosphorylation of Lck Y394. This response is consistent with the ability of CD45 to directly dephosphorylate Y394 of Lck in vitro (54) and with the hyperphosphorylation of Y394 in CD45 T cells (20).

CD44 signaling had a different outcome on F-actin formation and cell spreading, depending on the presence or absence of CD45 in BW5147 T cells and in activated primary thymocytes and T cells. Signaling in the presence of CD45, attributable to transient Lck activation, resulted in peripheral F-actin ring formation and predominantly round spreading, whereas in the absence of CD45, CD44 ligation led to directed or localized F-actin rearrangement and elongated, polarized cell spreading. In the CD45 BW5147 T cells this elongated cell spreading was attributed to sustained Lck activation leading to PI3K and PLCγ activation.

The transient and weak vs sustained and robust signaling from the same receptor leading to different outcomes is reminiscent of other key receptors in T cells. For example, a weak TCR signal in the thymus can lead to positive selection, whereas a stronger signal leads to negative selection (55 , 56). Also, costimulatory signals can sustain transient TCR signals (57), the duration of which is important for T cell activation and IL-2 production (58, 59, 60). Interestingly, these signals also rely on Src family kinase activity and are also affected by the presence and absence of CD45.

In addition to the transient and sustained nature of CD44-induced signaling in the CD45+ and CD45 cells, there are other differences that may contribute to the different signaling outcomes. One relates to Lck phosphorylation at Y505 in the CD45 T cells (4). Although this does not prevent Lck from initiating a signal upon CD44 clustering, it may recruit Src homology 2 domain containing proteins that modify the subsequent signal. Secondly, perhaps due to the sustained nature of Lck activation, Fyn is recruited more efficiently to the clusters in the CD45 T cells and so its activity may also contribute to the different signaling outcomes observed between the CD45+ and CD45 cells. Indeed, in T cells, Fyn is known to phosphorylate Pyk2 (61), a focal adhesion-related kinase that has been implicated in cell spreading (reviewed in Ref. 62). Consistent with this observation, CD44 preferentially induces Pyk2 phosphorylation in the CD45 cells (21). Fyn may also link to the actin cytoskeleton via ADAP/SLAP/Fyb, a Fyn associated molecule, which is important in TCR-induced integrin-mediated clustering and adhesion (63). Thus Fyn activation may be important in transmitting the CD44 signal to the cytoskeleton. Fyn recruitment to the clusters lagged Lck recruitment, suggesting that Fyn may be a downstream effector of Lck. This sequential Src family kinase activation has recently been observed upon TCR activation, where TCR and CD4 cross-linking lead to rapid Lck activation, which is required for subsequent Fyn activation (64).

Immobilization of T cells on a planar surface containing TCR Abs has been used as a model system to mimic T cell activation occurring via TCR engagement and has been shown to generate an actin ring (65, 66). Formation of this actin ring in response to the TCR signal was dependent upon tyrosine kinase activation and the raft resident adaptor molecule, LAT (65). Although there are some similarities between CD44 and TCR signaling in that they both involve Lck and Fyn and can lead to actin ring formation, CD44 signaling did not induce LAT phosphorylation (data not shown). Although LAT was not phosphorylated upon CD44 ligation, PLCγ1 was activated in the CD45 T cells and this was important, together with PI3K for disruption of the F-actin ring and elongated cell spreading. Although the activation of both PLCγ1 and the PI3K pathway was Src family kinase dependent, the activation of the PI3K pathway was not CD44-specific as incubation of CD45 T cells on BSA also induced AKT phosphorylation (data not shown). Phosphorylation may be due to the basal level of Src family kinase activity present in these cells. Nevertheless, activation of the PI3K pathway alone was insufficient to induce actin rearrangement or cell spreading on BSA.

In order for cells to migrate, cell elongation and polarization has to occur. Fanning et al. (35) recently reported that activated human lymphocytes, which exhibit polarized cell spreading, do migrate on immobilized CD44 Ab. In neutrophils and dictyostelium, PI3K has been shown to play a major role in establishing cell polarity and directional migration (67, 68). Whether this finding is also true for lymphocytes remains to be established (69). Consistent with our data, PLCγ1 has recently been shown to be important for integrin-mediated cell spreading and elongation in endothelial cells and was also required for subsequent cell motility (70). In addition to cell polarization, a certain amount of cell adhesion is needed for migration, however adhesion has to be finely regulated as either too little or too much adhesion can prevent migration. Given that CD45 can negatively regulate CD44-mediated T cell adhesion and is redistributed upon directional cell migration in neutrophils (71), CD45 may also modulate T cell migration.

Actin ring formation has been reported to occur upon TCR stimulation (65, 66) and TCR induced actin rearrangement is crucial for immune synapse formation between a T cell and an APC upon Ag recognition (72, 73). It is possible that CD44 signaling at the immune synapse may augment this type of actin rearrangement. Although CD44 has been reported to have costimulatory activity (74), the localization of CD44 at the immune synapse and its contribution toward actin rearrangement at the immune synapse are not known. In this study we have shown that CD44 signaling resulting in F-actin rearrangement and cell spreading is mediated by Src family kinases and modulated by CD45. Unlike its accepted role in the dephosphorylation of Lck at Y505, which is required for its effective participation in TCR signaling events, we show a role for CD45 in the dephosphorylation of Lck at Y394, which prevents sustained Lck activation thereby modulating actin rearrangement and cell spreading in T cells.

We thank Dr. Fumio Takei for providing soluble ICAM, and the University of British Columbia Imaging Facility and Dr. Robert Nabi for use of the confocal microscopes.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by funds from Natural Sciences and Engineering Research Council of Canada and from the Canadian Institutes of Health Research.

4

Abbreviations used in this paper: PLC, phospholipase C; DN, double negative; LN, lymph node; LAT, linker for activation of T cell.

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