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The Journal of Immunology, 2004, 172: 4266-4274.
Copyright © 2004 by The American Association of Immunologists

Enrichment of Lck in Lipid Rafts Regulates Colocalized Fyn Activation and the Initiation of Proximal Signals through TCR{alpha}{beta}1

Dominik Filipp*, Bernadine L. Leung*, Jenny Zhang*, André Veillette{dagger} and Michael Julius2,*

* Sunnybrook and Women’s College Health Sciences Center and Department of Immunology, University of Toronto, Toronto, Ontario, Canada; and {dagger} Laboratory of Molecular Oncology, Institut de Recherches Cliniques de Montreal, and Departments of Microbiology and Immunology and Medicine, McGill University, Montréal, Québec, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recent results provide insight into the temporal and spatial relationship governing lck-dependent fyn activation and demonstrate TCR/CD4-induced activation and translocation of lck into lipid rafts and the ensuing activation of colocalized fyn. The prediction follows that directly targeting lck to lipid rafts will bypass the requirement for juxtaposing TCR and CD4-lck, and rescue cellular activation mediated by Ab specific for the constant region of TCR{beta} chain. The present study uses a family of murine IL-2-dependent CD4+ T cell clonal variants in which anti-TCRC{beta} signaling is impaired in an lck-dependent fashion. Importantly, these variants respond to Ag- and mAb-mediated TCR-CD4 coaggregation, both of which enable the coordinated interaction of CD4-associated lck with the TCR/CD3 complex. We have previously demonstrated that anti-TCRC{beta} responsiveness in this system correlates with the presence of kinase-active, membrane-associated lck and preformed hypophosphorylated TCR{zeta}:{zeta}-associated protein of 70 kDa complexes, a phenotype recapitulated in primary resting CD4+ T cells. We show in this study that forced expression of wild-type lck achieved the same basal composition of the TCR/CD3 complex and yet did not rescue anti-TCRC{beta} signaling. In contrast, forced expression of C20S/C23S-mutated lck (double-cysteine lck), unable to bind CD4, rescues anti-TCRC{beta} proximal signaling and cellular growth. Double-cysteine lck targets lipid rafts, colocalizes with >98% of cellular fyn, and results in a 7-fold increase in basal fyn kinase activity. Coaggregation of CD4 and TCR achieves the same outcome. These results underscore the critical role of lipid rafts in spatially coordinating the interaction between lck and fyn that predicates proximal TCR/CD3 signaling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The proximal signaling events emanating from the TCR/CD3 complex involving the tyrosine phosphorylation of multiple substrates have been ascribed to the function of two Src family protein tyrosine kinases, namely p56lck and p59fyn (1). The facility with which lck involvement in proximal TCR/CD3 signaling in MHC class II-restricted T cells is modeled is due in part to the high stochiometry of its interaction with the accessory activation molecule CD4 (2, 3). As CD4-lck and TCR interact with the same or neighboring MHC molecules during the activation process (4), the proximity of lck with immunoreceptor tyrosine-based activation motifs on CD3 chains results in their phosphorylation and the provision of docking sites for downstream signaling elements, supporting initiation of the signaling cascade (5).

In contrast, fyn is not constitutively associated with any known ligand, although 0.1–1% of fyn is associated directly with immunoreceptor tyrosine-based activation motif motifs found within CD3 and {zeta}-chains through partially characterized mechanisms (6, 7, 8, 9, 10, 11). Further, signaling defects in fyn-deficient T cells are less dramatic than those observed in lck-deficient T cells, but do indicate some role in supporting IL-2 production (12, 13). The dispensability of fyn has led some to propose redundant functions for lck and fyn. Evidence derived from in vitro analyses demonstrates that they can phosphorylate some of the same substrates, including the {zeta}-chain (14), and fyn does counteract some of the consequences of the absence of lck during T cell development (15, 16, 17). However, more recent studies characterize lck- and fyn-specific substrates (18). Consistent with these observations, we and others have demonstrated that lck and fyn reside in different subcellular compartments of the plasma membrane (19, 20)).

In CD4+ primary T cells, up to 95% of lck resides in soluble membrane fractions, whereas >98% of fyn concentrates in specialized plasma membrane microdomains, termed lipid rafts (LR),3 which are postulated to function in signaling and membrane trafficking (21, 22, 23, 24). We have demonstrated that Ab-mediated coaggregation of TCR and CD4 resulted in the activation of lck outside LR, followed by its translocation into LR and the ensuing activation of colocalized fyn (19). Thus, in the resting state, LR function to regulate the spatial distribution of these two kinases. This predicts that enrichment of lck in LR would bypass the requirement for coordinated interaction between TCR and CD4-lck and result in the activation of LR-associated fyn.

The analyses described in this study were focused on characterization of the mechanism(s) responsible for the differential responsiveness of an IL-2-dependent, Ag-specific, CD4+ T cell clone to Ag-, anti-CD3-, and anti-TCR-mediated activation (25, 26). Briefly, both IL-2 and CD4 play key roles in mediating the refractoriness of clone 2.5 to anti-TCR stimulation. Although clone 2.5 is lck sufficient, IL-2 was shown to profoundly down-regulate the kinase activity of plasma membrane-associated lck, which is observed in anti-TCR-responsive primary CD4+ T cells (25, 26, 27). This pool of lck is observed in CD4- variants of clone 2.5, which are responsive to anti-TCR, and ablated, along with anti-TCR responsiveness, upon ectopic expression of wild-type CD4, but not C418A/C420A-mutated CD4, which is unable to bind lck (25, 26). Further, IL-2 withdrawal from clone 2.5 also rescued this pool of membrane-associated, kinase-active lck and anti-TCR responsiveness (25, 26). Thus, non-CD4-associated lck exhibiting basal kinase activity appears critical to support anti-TCR responsiveness in this system. In contrast, responsiveness to Ag uses the CD4-associated pool of lck (28, 29). We reasoned that forced expression of lck in clone 2.5 would overcome this IL-2-induced deficit in lck function.

Toward this end, a series of clone 2.5 infectants were generated that overexpressed matched levels of either wild-type lck (Wtlck) or a C20S/C23S mutated lck (double-cysteine lck (Dclck)), which is unable to associate with CD4. The Wtlck and Dclck infectants expressed comparable levels of plasma membrane-associated, kinase-active lck, hypophosphorylated TCR{zeta}, and preformed TCR{zeta}-{zeta}-associated protein of 70 kDa (ZAP70) complexes, yet anti-TCR responsiveness was rescued in the Dclck infectants, exclusively. The Ab specific for the constant region of TCR{beta} chain (TCRC{beta}) mediated responsiveness of the Dclck infectants correlated with the presence of kinase-active fyn comparable to the basal levels of fyn activity observed in primary resting CD4+ T cells. The basis for Dclck, but not Wtlck, to mediate this activity relates to their distinct subcellular localization. Membrane partitioning revealed that LR function to physically segregate fyn and lck in clone 2.5, with 90–95% of endogenous lck residing in soluble membrane fractions, whereas >98% of fyn is LR-associated. Overexpression of Wtlck supersaturated endogenous CD4, with the balance of excess lck localizing to LR. Overexpression of Dclck resulted in a >30-fold increase in the non-CD4-associated pool of lck, with the majority localizing to LR, suggesting a direct role for lck in supporting fyn activation. The relevance of this observation was demonstrated in clone 2.5 upon coaggregation of CD4 and TCR, which resulted in the activation of lck outside LR, its translocation into LR, and the ensuing activation of colocalized LR-associated fyn. These results characterize the spatial and temporal roles of lck and fyn in TCR-proximal signaling and underscore the importance of LR in coordinating the interaction of these kinases either directly or through intermediates.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Six- to 8-wk-old C57BL/6 male mice were obtained from National Cancer Institute (Bethesda, MD) and maintained in a specific pathogen-free animal facility at Sunnybrook and Women’s Research Institute.

Abs and reagents

Polyclonal rabbit anti-murine lck, fyn, and ZAP-70 were prepared as described previously (30, 31, 32). Murine-specific mAb for TCRC{beta}, H57.597 (33), and CD3{epsilon}, 145.2C11 (34) were purified on protein A-conjugated Sepharose 4B (Amersham Pharmacia Biotech, Piscataway, NJ). The murine CD4-specific mAb H129 (35) was purified on mouse anti-rat Ig{kappa}-conjugated Sepharose 4B (Amersham Pharmacia Biotech). Phosphotyrosine-specific mAb, 4G10 (36), was purchased from Upstate Biotechnology (Lake Placid, NY). The {zeta}-chain-specific mouse mAb G3 (37) was provided by Dr. H.-S. Teh (University of British Columbia, Vancouver, British Columbia, Canada). Goat anti-mouse IgG-HRP, anti-actin Ab, streptavidin, Brij 58, cholera toxin B-HRP, and enolase were purchased from Sigma-Aldrich (St. Louis, MO). Protein A-HRP was purchased from Bio-Rad (Hercules, CA).

T cell clones and primary T cells

The CD4+ T cell clones infected with either the empty murine stem cell virus (MSCV)-based internal ribosome entry site (IRES)-enhanced green fluorescent protein virus (MIEV) vector or the MIEV encoding for either Wtlck or Dclck have been described previously (38). Clonal variants were maintained in serum-free IMDM containing 3 U/ml rIL-2 supernatant (26) and 1% soy bean lecithin. Primary CD4+ lymph node T cells were isolated as described previously (19).

Proliferation assays

Soluble anti-TCRC{beta} or an OVA-derived peptide, residues 143–157, was added at the indicated concentration to cultures containing 3 x 104 T cell clones and 5 x 105 irradiated (2000 rad) syngeneic splenocytes. Cultures were pulsed with 1 µCi of [3H]TdR (Amersham Pharmacia Biotech) at 40 h and harvested onto Unifilter plates (Canberra Packard, Concord, Ontario, Canada) 6 h later. Thymidine uptake was assessed by liquid scintillation spectroscopy.

Cell fractionation

Membrane isolation was performed as previously described (25, 39). Briefly, cells were pelleted in a borosilicate tube and then resuspended at 20 x 106/ml in ice-cold hypotonic extraction buffer containing 25 mM HEPES (pH 7), 1 mM MgCl2, 1 mM EGTA, 100 µg/ml aprotinin, and 100 µg/ml leupeptin. Cells were incubated on ice for 40 min, then subjected to 20 shears using a 30-gauge needle on a 1-ml syringe barrel. The sheared material was examined microscopically to verify cell lysis, and nuclear material was removed by centrifugation. The supernatant was transferred to Eppendorf tubes and centrifuged at 12,000 x g for 10 min. The pellet contains the heavy membrane fraction (HMF), and the supernatant contains the cytosol. The HMF was washed once and then resuspended at 40 x 106 cell equivalents/ml in extraction buffer containing 0.1% Triton X-100. This suspension was solubilized with gentle agitation and then centrifuged at 12,000 rpm for 10 s to remove detergent-insoluble material. The supernatant contains the detergent-soluble HMF.

Cell lysis, immunoprecipitations, and immunoblotting

T cell clonal infectants were lysed at 20 x 106/ml in TNE buffer containing 50 mM Tris (pH 8), 20 mM EDTA, 200 µM Na3VO4, 50 mM NaF, 1% Nonidet P-40, 20 µg/ml leupeptin, and 20 µg/ml aprotinin for Lck immunoprecipitations. For ZAP and TCRC{beta} immunoprecipitations, cells were lysed in buffer containing 10 mM Na4P2O7, 140 mM NaCl, 10 mM Tris, 400 µM Na3VO4, 10 mM NaF, 0.9% digitonin, 20 µg/ml leupeptin, 20 µg/ml aprotinin, 100 µg/ml Pefabloc, and 50 µg/ml tosyl-L-lysine chloromethyl ketone, adjusted to pH 7.4. For fyn immunoprecipitations, cells were lysed in buffer containing 20 mM Tris (pH 8), 150 mM NaCl, 200 µg/ml Na3VO4, 0.9% digitonin, 20 µg/ml leupeptin, 20 µg/ml aprotinin, and 1 mM PMSF.

Postnuclear extracts were isolated and quantitated using a commercially available protein determination kit (Pierce, Rockford, IL). Twenty-five microgram protein equivalents were resolved on 8% SDS-PAGE and transferred to nitrocellulose (Schleicher & Schuell, Keene, NH), and the filters were blocked as described below. Alternatively, lck, fyn, ZAP-70, or TCRC{beta} was immunoprecipitated from the indicated protein equivalents and then washed three times before resolution by SDS-PAGE.

To determine the proportion of cellular lck complexed with CD4, lck was sequentially and quantitatively precipitated from 25 µg of protein derived from the postnuclear membrane fraction of cell lysates using mAb specific for CD4 and purified polyclonal anti-Lck covalently coupled to Sepharose 4B, respectively.

Antiphosphotyrosine blots were blocked in 3% gelatin, developed in 1% gelatin containing 4G10 mAb, and revealed with HRP-conjugated, polyclonal, goat anti-mouse IgG. Lck, fyn, or ZAP-70 immunoblots were blocked with 5% milk containing specific serum and revealed using protein A-HRP. Anti-{zeta}-chain immunoblots were blocked in gelatin with G3 mAb and revealed using goat anti-mouse HRP. Actin immunoblots were also blocked in 5% milk containing anti-actin serum, and revealed using HRP-conjugated goat anti-mouse HRP. Immunoblots were developed using ECL reagents (Amersham, Arlington Heights, IL).

Coaggregation assay

T cell clonal variants (2 x 106) were precoated with 1 µg/ml biotinylated anti-TCRC{beta} and 0.3 µg/ml biotinylated anti-CD4 for 30 min at 4°C. Cells were washed twice with ice-cold washing buffer (PBS supplemented with 3% FCS), and the pelleted cells were resuspended in 20 µl of washing buffer and prewarmed to 37°C for 1 min. Ab-mediated coaggregation of TCR and CD4 was achieved by addition of streptavidin to a final concentration of 50 µg/ml at 37°C for the indicated times.

Isolation of LR

Primary CD4+ lymph node T cells (5 x 106) or 2 x 106 T cell clonal variants were lysed in a total volume of 250 µl of TKM buffer containing 50 mM Tris (pH 7.4), 25 mM KCl, 5 mM MgCl2, 1 mM EDTA, 0.5% Brij 58, 20 µg/ml leupeptin, 20 µg/ml aprotinin, and 100 µg/ml Pefabloc. After incubation on ice for 30 min, the lysates were mixed with 250 µl of 80% sucrose in TKM buffer, transferred to centrifuge tubes, and sequentially overlaid with 4.3 and 0.2 ml of 36%, then 5% sucrose solution to a total volume of 5 ml. The mixture was subjected to equilibrium density gradient centrifugation at 250,000 x g for 16–20 h at 4°C in a SW55 Ti rotor (Beckman Coulter, Fullerton, CA). Fractions (0.5 ml) were collected from the top of the gradient and sequentially numbered 1–10. Using a dot-blot apparatus, 20 µl of the indicated fractions were transferred to nitrocellulose (Schleicher & Schuell) and probed for GM1 ganglioside (GM1) distribution as a specific marker for LR using cholera toxin B (CTB)-HRP. To assess the subcellular partitioning of lck and fyn subsequent to equilibrium density gradient centrifugation, 30 µl of fractions were mixed with 10 µl of 4x loading buffer, boiled for 5 min, and loaded onto 9% SDS-PAGE, followed by transfer to a polyvinylidene difluoride (PVDF) membrane. The membranes were probed with anti-lck, stripped, and reprobed with anti-fyn.

Immunoprecipitation of lck and fyn from LR and soluble membrane fractions was performed as follows: 400 µl of cholera toxin-positive fractions and soluble fractions from the indicated samples were diluted 1/5 in fyn lysis buffer (see above) and immunoprecipitated with anti-fyn for 5 h at 4°C. Beads were pelleted, and supernatants were transferred to fresh tubes. The supernatants were adjusted for lck precipitation by supplementing with Tris (pH 8), EDTA (pH 8), and NaF to final concentrations of 50, 20, and 50 mM, respectively (see TNE buffer above). Lck was immunoprecipitated overnight at 4°C. Fyn and lck immunoprecipitates were used for immune complex kinase assays (see below).

Immune complex kinase assays

Lck immunoprecipitates were subjected to in vitro kinase assays as described previously (25, 39). Fyn immunoprecipitates were washed four times in their corresponding lysis buffer and then twice in kinase buffer containing 20 mM Tris (pH 7.2), 10 mM MgCl2, and 10 mM MnCl2. Washed fyn immunoprecipitates were supplemented with 10 µl of fyn kinase buffer supplemented with 0.5 µg of freshly acid-denatured enolase, 1 µM cold ATP, and 10 µCi of [{gamma}-32P]dATP. After incubation at 37°C for 30 min, 30 µl of 1.3x reduced sample buffer was added. Lck and fyn immunoprecipitates were boiled for 5 min, centrifuged, and resolved on 10% SDS-PAGE. Gels were transferred to a PVDF membrane and immunoblotted for lck and fyn, respectively. The same membranes were then exposed to x-ray film for 1–3 days.

Densitometry

Densitometric analysis of immunoblots was performed on a GS800 densitometer (Bio-Rad) using Quantity One quantitation software (Bio-Rad). Phosphoenolase signals obtained from the volume analysis of densitometric data are expressed in arbitrary units after normalization to total protein signals of the respective kinases. All densitometric values obtained were calculated from nonsaturated signals.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Forced expression of Dclck rescues mAb-mediated TCR signaling in clone 2.5

The involvement of non-CD4-associated lck in proximal TCR signaling was assessed using a set of CD4+ clonal variants of clone 2.5 ectopically expressing matched levels of Wtlck, Dclck, or the empty MIEV vector. Levels of TCR and CD4 expression in these variants were comparable in each infectant (not shown). As illustrated in Fig. 1A, overexpression of Dclck efficiently rescues anti-TCRC{beta} responsiveness in comparison with the empty vector control infectant. The Wtlck infectant exhibits an intermediate response, 3- to 30-fold lower than that observed in the Dclck infectants over the dose range of soluble anti-TCRC{beta} tested. Similar results were obtained in response to immobilized anti-TCRC{beta} stimulation (not shown). In contrast, all three variants responded comparably to Ag-mediated activation, demonstrating that TCR{alpha}{beta} signaling is functional in this context (Fig. 1B).



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  		FIGURE 1. Forced expression of Dclck rescues anti-TCRC{beta} responsiveness. Clone 2.5 (3 x 104) infected with the empty MIEV vector ({blacksquare}) or MIEV containing Wtlck ({square}) or Dclck ({circ}) were stimulated with the indicated concentrations of anti-TCRC{beta} (A) or OVA143–157 peptide (B) in the presence of irradiated syngeneic filler cells. Cultures were incubated for 40 h, pulsed with 1 µCi of [3H]TdR, and harvested onto UniFilter plates 6 h later. Thymidine uptake was assessed by liquid scintillation spectroscopy. The values represent the mean of triplicate wells, with 1 SD indicated. The background counts in unstimulated cultures were 134, 79, and 122 cpm for empty, Wtlck-containing, and Dclck-containing variants, respectively.

 
Densitometric analysis of lck immunoblots derived from lysates of the three variants reveal comparable levels of lck expression in the Wtlck and Dclck infectants that were, on the average, 4.9- and 4.6-fold higher, respectively, than the endogenous level of lck expressed in the empty MIEV infectant (Fig. 2A). The proportions of CD4-associated and non-CD4-associated lck were determined by sequentially and quantitatively precipitating CD4, then lck, from lysate derived from each of the three variants, followed by densitometric analysis of lck immunoblots. As illustrated in Fig. 2B, 90% of lck is CD4-associated in the empty MIEV infectant. The CD4-associated pool of lck is roughly 4-fold higher in the Wtlck infectants in comparison with Dclck infectants or those carrying the empty MIEV vector. The amounts of non-CD4-associated lck in variants expressing Wtlck and Dclck are 9- and 37-fold higher, respectively, in comparison with the variant expressing the empty MIEV, in which the non-CD4-associated pool represents 10% of the total lck (Fig. 2B). These results indicate that lck binding sites on CD4 in clone 2.5 are not saturated, that the level of overexpression of Wtlck achieves saturation of these CD4 binding sites, and that, consistent with the inability of Dclck to associate with CD4, all the ectopic Dclck is found in the non-CD4-associated pool. These results are consistent with the model proposing the requirement for non-CD4-associated lck in mAb-mediated TCR signaling in this system (25, 39). Artificially enlarging this pool of lck in the Dclck infectant restores anti-TCR responsiveness (Fig. 1A), and the intermediate response of the Wtlck infectant is consistent with the intermediate increased level of non-CD4-associated lck.



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  		FIGURE 2. Forced expression of Dclck amplifies the non-CD4-associated pool of lck. A, Twenty-five microgram protein equivalents from each of the three categories of clonal variant were resolved by 8% SDS-PAGE and transferred to nitrocellulose. The blot was probed with anti-lck (top panel), stripped, and then probed with anti-actin (bottom panel). The histogram shows the quantification of total cellular levels of lck normalized to the actin signal in each infectant, normalized to the empty MIEV infectant, which was assigned a value of 1. B, One hundred microgram protein equivalents from each category of infectant were subjected to four sequential immunoprecipitations with anti-CD4 (top panel) and then anti-lck (bottom panel). The immunoprecipitates were resolved on 8% SDS-PAGE and transferred to nitrocellulose. The blots were probed with anti-lck. The histogram shows quantification of CD4-associated and non-CD4-associated pools of lck in the three categories of infectant where the total lck content in the empty vector infectant was assigned a value of 1.

 
Our previous studies have correlated the presence of kinase active, plasma membrane-associated lck, and preformed TCR{zeta}-ZAP-70 complexes with permissive anti-TCRC{beta} signaling (25, 39). To determine whether this basal composition of the TCR complex is necessary and sufficient to support anti-TCR responsiveness, the kinase activity of lck as well as the level of phospho-TCR{zeta} associated with the TCR complex in the trilogy of infectants were examined.

Comparable lck kinase activity and basal composition of the TCR complex in Wtlck and Dclck infectants

Previous studies have demonstrated that the nonmembrane (cytosolic)-associated pools of lck in both anti-TCRC{beta}-responsive and nonresponsive clonal variants are catalytically active. In contrast, differential lck kinase activity, which correlated with anti-TCRC{beta} responsiveness, was restricted to the membrane pools of lck (25, 39). We therefore restricted our analyses to the membrane pools of lck in the three categories of infectants. A comparable increase in the total level of lck kinase activity was observed in both the Wtlck and Dclck infectants compared with the variant expressing the empty MIEV vector (Fig. 3A). However, when normalized for lck levels, the specific kinase activity of lck toward an exogenous substrate enolase was the same within each of the three infectants (not shown).



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  		FIGURE 3. Membrane lck activity and preformed pp21{zeta}-TCR and pp21{zeta}-ZAP-70 complexes are insufficient to support anti-TCR responsiveness. A, Membrane fractions of each of the three categories of infectant were prepared as described in Materials and Methods. Lck was immunoprecipitated from 50 µg protein equivalents of the solubilized membrane fractions and subjected to immune complex kinase assay (top panel). The amount of lck immunoprecipitated was quantified by immunoblotting (bottom panel). B, Five hundred microgram protein equivalents from each clonal variant was subjected to anti-TCRC{beta} immunoprecipitation and resolved on 12.5% SDS-PAGE. The top panel shows pp18{zeta} and pp21{zeta} associated with the anti-TCRC{beta} immunoprecipitates revealed with phosphotyrosine-specific mAb 4G10. The blot was stripped and reprobed with {zeta}-chain-specific mAb (bottom panel). The positions of p21, p18, and p16{zeta} are indicated. C, Anti-ZAP70 immunoprecipitation was performed as described in B. The amount of ZAP70 immunoprecipitated and its associated phosphotyrosyl content are shown in the top and middle panels, respectively. The bottom panel shows the coimmunoprecipitated pp18{zeta} and pp21{zeta} revealed with phosphotyrosine-specific mAb 4G10.

 
A pivotal lck-dependent event in TCR-mediated proximal signaling involves the tyrosine phosphorylation of TCR {zeta}-chains and the subsequent recruitment and activation of ZAP70 kinase (1). Importantly, primary resting T cells constitutively express tyrosine-phosphorylated TCR {zeta}-chains, which are associated with ZAP70 (25, 40, 41). Thought to reflect a priming of the TCR complex mediated by low affinity interactions with self-MHC/peptide, the presence of these preformed TCR{zeta}-ZAP70 complexes predicates anti-TCR responses (25, 27, 40, 41). We therefore assessed the basal composition of the TCR complex in the three categories of infectant toward characterizing possible differences underpinning their differential anti-TCRC{beta} responsiveness.

As illustrated in the top panel of Fig. 3B, comparable levels of pp21{zeta} coimmunoprecipitate with TCRC{beta} from lysates of Wtlck and Dclck infectants. Consistent with the role of lck in phosphorylating TCR{zeta}, both Wtlck and Dclck infectants contain higher levels of TCR-associated pp21{zeta} than do empty vector control cells. Importantly, the anti-{zeta} immunoblot of the same samples revealed comparable levels of p16{zeta}, thus indicating that the overall composition of the TCR/CD3 complex is not altered in any of the three clonal infectants (Fig. 3B, bottom panel). Consistent with this result, levels of pp21{zeta} associated with ZAP-70 were comparable in Wtlck and Dclck infectants and were significantly higher than those observed in empty vector control cells (Fig. 3C). The levels of tyrosine-phosphorylated ZAP-70 in both Wtlck and Dclck infectants were significantly higher than those observed in ZAP-70 immunoprecipitated from control cells (Fig. 3C). Thus, overexpression of either form of lck rescues a basal composition of the TCR complex that has been shown to predicate anti-TCR responsiveness. However, as the response of Wtlck infectants to anti-TCR is not fully rescued (Fig. 1A), the presence of pp21{zeta} and preformed pp21{zeta}-ZAP-70 complexes may be necessary, but are not sufficient.

Anti-TCR responsiveness correlates with increased fyn kinase activity

Analysis of the effects of ectopic expression of Wtlck and Dclck on fyn physiology provided the first insight into the mechanism supporting the differential capacity of these two forms of lck to rescue anti-TCR responsiveness in clone 2.5. As illustrated in Fig. 4, immune complex kinase assays revealed that ectopic expression of Dclck resulted in a 10-fold increase in auto/trans-phosphorylated fyn compared with levels observed in either Wtlck or empty vector infectants. Insight into the mechanism underpinning the differential capacity of Wtlck and Dclck to alter fyn physiology was derived from membrane partitioning experiments.



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  		FIGURE 4. Forced expression of Dclck induces increased basal fyn activity. Fyn was immunoprecipitated from 300 µg protein equivalents from each of the categories of infectant and subjected to an immune complex kinase assay (top panel). The histogram shows the specific kinase activity of fyn expressed as a ratio between auto/trans-phosphorylated fyn signal (pY-Fyn) normalized to the total fyn content. Fyn-specific kinase activities were normalized to that observed in empty MIEV infectant, which was assigned a value of 1.

 
Dclck colocalizes with fyn in LR and correlates with the induction of basal fyn kinase activity

Isolation of LR and analysis of lck and fyn distribution in the three categories of infectants provided insight into the differential capacity of the two forms of lck to affect fyn physiology. As illustrated in Fig. 5, LR served to segregate lck and fyn in the empty vector control. Ninety-five percent of lck was found in the soluble membrane fractions, whereas 98% of fyn was associated with LR. Ectopic Dclck, in the majority of LR, colocalized with fyn, whereas ectopic expression of Wtlck resulted in an increase in LR-associated lck, consistent with the level of non-CD4-associated lck observed in these infectants (Figs. 5 and 2B). Thus, the distinct membrane compartmentalization of Wtlck and Dclck and the ensuing preferential colocalization of Dclck with fyn provide a physical basis for the distinct impacts of these two forms of lck on fyn physiology. This was formally demonstrated through immune complex kinase analyses of fyn present in LR and soluble fractions derived from the three categories of infectants.



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  		FIGURE 5. Colocalization of lck and fyn in LR of Dclck infectants. Cells (2 x 106) of each of the three categories of infectant were lysed in TKM buffer containing 0.5% Brij 58 and subjected to sucrose equilibrium density gradient centrifugation. Using a dot-blot apparatus, 20 µl of each gradient fraction was transferred to nitrocellulose and probed with CTB-HRP (top panels). Fifteen microliters of each gradient fraction was resolved on 9% SDS-PAGE, transferred to a PVDF membrane, and probed with anti-fyn (middle panel) or anti-lck (bottom panel), respectively. Numbers represent the proportion of total lck detected in the LR and soluble fractions in each category of infectant.

 
As illustrated in Fig. 6A, the capacity of LR-associated fyn from Dclck infectants to phosphorylate enolase was enhanced >6-fold compared with that of fyn derived from empty vector control cells when normalized to fyn content. In contrast, LR-associated fyn isolated from Wtlck infectants exhibited a 2-fold increased capacity to phosphorylate enolase compared with the control, consistent with the lower level of enrichment of Wtlck in LR of this infectant. Of note, the kinase activity of the small pool of fyn present in the soluble membrane fractions, probably that associated with chains of the CD3 complex, was not increased in infectants of either form of lck relative to that in control cells (Fig. 6A).



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  		FIGURE 6. Colocalization of Dclck and fyn results in enhanced fyn kinase activity. A, Fyn immunoprecipitates from LR (R) and soluble membrane fractions (S) derived from each of the three categories of infectant were subjected to an immune complex kinase assay. The phosphoenolase signals (pY-E) were normalized to total fyn content and represent the specific kinase activity of fyn (histogram). Bars representing R and S fractions from the same sample are grouped and aligned with electrophoretic tracks. B, Lysates from 5 x 106 primary CD4+ lymph node cells were subjected to equilibrium density gradient centrifugation, and 15 µl of each fraction was used to reveal the presence of GM1 (top panel), lck (middle panel), and fyn (bottom panel) by immunoblotting with CTB-HRP, anti-lck, and anti-fyn, respectively. C, Fyn immunoprecipitates from LR (R) and soluble membrane fractions (S) derived from 2.5 x 106 primary CD4+ lymph node cells (CD4+) and 1.2 x 106 Dclck infectants were subjected to an immune complex kinase assay (upper panel) or immunoblotted with anti-fyn (lower panel).

 
The distinct subcellular localization of lck and fyn observed in the empty vector control variant of clone 2.5 accurately reflects normal physiology. As recently described (19), LR also function to segregate the majority of fyn from the majority of lck in primary resting CD4+ T cells. Specifically, as illustrated in Fig. 6B, >98% of fyn localized to LR, and >90% of lck was found in soluble membrane fractions. As illustrated in Fig. 6C, the basal kinase activity associated with fyn derived from either LR or soluble membrane fractions prepared from primary CD4+ T cells and Dclck infectants was comparable, and normalization of LR-associated fyn kinase activities to respective fyn levels revealed that specific kinase activities were within 25%.

Thus, overexpression of Dclck in clone 2.5 results in a recapitulation of a basal level of fyn kinase activity, also observed in primary resting CD4+ T cells, that, in turn, correlates with anti-TCR responsiveness. We recently demonstrated that during the process of TCR/CD4-mediated activation of primary CD4+ T cells, lck functions as a mobile signaling element that is activated within seconds after TCR/CD4 coaggregation outside of LR, followed by its translocation into LR where the activity of colocalized fyn is dramatically up-regulated. The question follows whether the phenotype observed in Dclck infectants of clone 2.5, in which the pool of LR-associated lck was artificially increased, is mimicked in circumstances that do support the activation of clone 2.5.

Coaggregation of CD4 and TCR in clone 2.5 induces colocalization of lck and fyn, and fyn activation

As previously described, although refractory to anti-TCRC{beta} stimulation, clone 2.5 responds to both Ag- and mAb-mediated TCR-CD4 coaggregation (25, 39). In contrast to anti-TCRC{beta}, these latter modes of activation use the pool of CD4-associated lck (25, 39). To determine whether mimicking Ag-mediated activation of clone 2.5 results in alterations of fyn physiology analogous to those achieved through ectopic expression of Dclck, the consequences of TCR-CD4 coaggregation were assessed.

As illustrated in Fig. 7A, the spatial distribution of lck and fyn in clone 2.5 was analogous to that observed in primary CD4+ T cells (Fig. 6B). Global tyrosine phosphorylation of cellular substrates peaked at 20 s after coaggregation of TCR and CD4 (Fig. 7B). These phosphorylation events correlated with the redistribution of lck. As illustrated in Fig. 7C, within the first 20 s after mAb-mediated coaggregation of TCR and CD4, the amount of LR-associated lck increased by a factor of 1.5–2 (Fig. 7E), peaking at 60 s and decreasing to prestimulation levels by 180 s. As the total lck signal detected at each time point varied within 10%, the altered distribution of lck reflects its translocation from the soluble membrane fractions into LR (Fig. 7D). As shown in Fig. 8A, TCR/CD4 coaggregation resulted in the sequential activation of lck, then fyn. Lck was activated within the first 20 s in the soluble membrane fractions, after its translocation to LR where its activity peaked at 60 s at levels 3-fold more than those in the non-TCR-CD4 aggregated control. This, in turn, was paralleled by the first detectable increase in LR-associated fyn activity, which increased 3-fold over control levels at 60 s and continued to rise to 10-fold over control levels at 180 s (Fig. 8B). Importantly, at this time point, the activity of LR-associated lck had decreased to control levels (Fig. 8A). Of note is that the kinetics of activation of the small pool of fyn localized in the soluble membrane fractions preceded those of LR-associated fyn and occurred immediately after the first detectable rise in lck activity within the soluble membrane fractions (Fig. 8). These results recapitulate those recently described in primary resting CD4+ T cells (19) and establish the physiological relevance of the phenotype observed in clone 2.5 infectants of Dclck: specifically, the capacity of LR-associated lck to regulate the function of colocalized fyn and rescue TCR signaling.



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  		FIGURE 7. The mAb-mediated coaggregation of CD4 and TCR in clone 2.5 induces colocalization of lck and fyn in LR. A, Subcellular distribution of lck and fyn was assessed by probing LR and soluble membrane fractions derived from lysates subjected to equilibrium density centrifugation with CTB-HRP (top panel), anti-lck (middle panel), and anti-fyn (bottom panel). B, Cells (5 x 105) were precoated with 1 µg/ml biotinylated anti-TCRC{beta} and 0.3 µg/ml biotinylated anti-CD4, followed by the addition of 50 µg/ml streptavidin for 20, 60, and 180 s. The global phosphotyrosyl content in lysates derived from these samples was revealed by immunoblotting with phosphotyrosine-specific mAb 4G10. C, Cells (2 x 106) were precoated with 1 µg/ml biotinylated anti-TCRC{beta} and 0.3 µg/ml biotinylated anti-CD4, followed by the addition of streptavidin for 20, 60, and 180 s. GM1, lck, and fyn contents were assessed in fractions 1–3 and 8–10, representing LR and soluble membrane fractions, respectively. D, Quantification of lck redistribution after TCR-CD4 coaggregation. Numbers represent the proportion of total lck revealed in LR and soluble membrane fractions. E, Quantification of lck enrichment in LR. The proportion of lck in the nonaggregated control was assigned the value 1.

 


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  		FIGURE 8. The mAb-mediated TCR-CD4 coaggregation in clone 2.5 induces the activation of lck, then fyn. Lck (A) and fyn (B) immunoprecipitates from LR (R) and soluble membrane fractions (S) derived from noncoaggregated control samples and samples coaggregated for the indicated times, as described in Fig. 7, were subjected to immune complex kinase assays. The phosphoenolase signals (pY-E) for lck (A, top panel) and fyn (B, top panel) were normalized to total lck (A, bottom panel) and fyn (B, bottom panel) contents, respectively. Specific kinase activity is expressed as a ratio between pY-E and total lck (A, histogram) and total fyn (B, histogram). Levels of specific kinase activities in the LR and soluble membrane fractions in noncoaggregated samples were assigned a reference value of 1. Bars representing R and S fractions from the same sample are grouped and aligned with electrophoretic tracks in both panels.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although both genetic and biochemical evidence support the fundamental roles of Src family tyrosine kinases lck and fyn in the initiation of membrane-proximal signals emanating from the TCR/CD3 complex (1, 18), there is a paucity of information regarding whether or how their activation and delivery of function are coordinated. The present study provides insight into the role of lck as a mobile signaling element, whose subcellular localization indirectly predicates anti-TCR-mediated activation through its regulation of the function of colocalized fyn. The results presented characterize the distinct membrane compartmentalization of lck and fyn in unstimulated cloned T cells. The majority of cellular lck resides in soluble membrane fractions and is physically separated from the majority of fyn, which resides in LR. Either artificially targeting lck to LR through ablating its capacity to interact with CD4 or modeling Ag-mediated activation through mAb-mediated coaggregation of TCR and CD4, shown to induce the translocation of lck from the soluble membrane compartment into LR, achieves the same result: the activation of colocalized fyn.

A plausible function of kinase-active, membrane-associated lck is the induction of basal TCR{zeta} phosphorylation. The {zeta}-chain can be a substrate for lck (42, 43), and thymocytes isolated from lck+ mice contain pp21{zeta}, whereas those from lck- animals do not (43). Importantly, pp21{zeta}-ZAP-70 complexes are also observed in functional primary CD4+ T cells (25, 27). Thought to reflect the consequences of intrathymic selection (27) and interaction with self-MHC/peptide in the periphery, which are required for longevity of naive CD4+ peripheral T cells (25, 44, 45), this basal composition of the TCR/CD3 complex promotes T cell sensitivity and responsiveness to foreign Ag (27). Thus, primary resting T cells appear primed to respond, and lck plays a fundamental role in achieving this state.

The object of the present study was to provide convergent results through artificially increasing the pool of lck in anti-TCR-unresponsive clone 2.5 toward rescuing the physiologically relevant basal composition of the TCR/CD3 complex and testing the prediction that it predicates anti-TCR responsiveness. The unexpected result was that comparable levels of membrane-associated, kinase-active lck and hypophosphorylated TCR{zeta} constitutively associated with ZAP-70 were achieved through ectopic expression of either Wtlck or Dclck, but full responsiveness to anti-TCR was rescued only by Dclck. Thus, although necessary, the basal composition of TCR/CD3 described is not sufficient for anti-TCR responsiveness.

The key observation is that although Dclck amplified the non-CD4-associated pool of lck, exclusively, as expected, this ectopic form of lck was found predominantly localized to LR. The overexpressed Wtlck saturated lck binding sites on CD4, and its contribution to the LR-associated pool of lck was significantly less than that observed for Dclck. The consequence of the distinct subcellular localizations of these two forms of lck was dramatic. As observed in primary CD4+ T cells (19), fyn was found to localize almost exclusively to LR in clone 2.5 and empty vector infectants of clone 2.5. The level of colocalization of lck and fyn necessary to support the full sequelae of anti-TCR responsiveness in clone 2.5 was achieved only in Dclck infectants and correlated with the induction of basal fyn kinase activity comparable to that observed in anti-TCR-responsive primary CD4+ T cells.

As there was a significant increase in LR-associated lck in Wtlck infectants, its inability to significantly affect the kinase activity of colocalized fyn supports a threshold hypothesis for lck-dependent fyn activation and homeostatic regulation of LR-associated lck and fyn activity. Net specific activity of these Src kinases results from the opposing activities of two key effector molecules that regulate the phosphorylation status of the negative regulatory C-terminal tyrosine residues. The C-terminal Src kinase (Csk) phosphorylates lckY505 and fynY528, supporting intramolecular interactions with Src homology 2 domains on the respective kinases that, in turn, support folding of the kinases into a closed or off conformation (46, 47, 48). The CD45 phosphatase opposes this action (1). Until recently, the key regulatory function of CD45 was considered predominantly in the context of soluble membrane fraction-associated kinases, as CD45 appeared to be excluded from LR (49, 50, 51, 52). However, recent evidence demonstrates that up to 5% of CD45 is associated with LR in unstimulated T cells (53). Our previous report (19) coupled with the present study support the conclusion that fyn is the predominant target of raft-associated CD45. Fyn-mediated phosphorylation of Csk-binding protein/ phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG) on residue Y314 (54, 55) results in the recruitment of Csk (20, 55). Thus, a balanced interaction of CD45 and Csk would maintain the basal kinase activity of LR-associated fyn. Of note is the recent demonstration that CD45 can also dephosphorylate PAG on Y314 (56). The prediction follows that this CD45-mediated loss of PAG-associated Csk would also contribute to fyn activation. This balance defines the basal level of fyn kinase activity observed in both unstimulated primary resting T cells and anti-TCR-responsive clonal variants reported in this study. The introduction of kinase-active lck into LR perturbs this balance and profoundly impacts on the activity of LR-associated fyn. Importantly, as described in the present study, the differential persistence of LR-associated fyn kinase activity suggests that the interdependence of lck and fyn is unidirectional. Specifically, although fyn activation is preceded by the translocation of activated lck into LR, active fyn appears not to impact on the activity of colocalized lck. Thus, this LR-associated homeostatic regulatory mechanism(s) is kinase specific.

Artificially amplifying the pool of LR-associated lck reflects a physiologically relevant step in the process of T cell activation. As recently demonstrated in primary CD4+ T cells (19), TCR-CD4 coaggregation in clone 2.5 demonstrates that lck functions as a mobile signaling element, moving from soluble membrane fractions into LR and supporting the subsequent activation of colocalized fyn. Although the time frame of the analyses performed in the present study precluded the ability to confirm this observation in Ag-mediated stimulation of clone 2.5, recent evidence supports the conclusion that the consequences of mAb-mediated coaggregation of TCR and CD4 accurately reflect those induced upon Ag-mediated activation. Specifically, Ag recognition involving TCR, CD4, and CD28 was necessary and sufficient for the sustained autophosphorylation of lck on Y394, which predicates its optimal activation, and its translocation into the immunological synpase, which is known to depend on the coalescence of LR (24, 28, 57, 58, 59, 60). Further, the results presented highlight the potential role of CD4 in functioning as a gatekeeper for lck, and thus regulating lck-dependent fyn activation. A recent report demonstrates that TCR, CD4, and associated lck reside outside LR in unstimulated T cells and are induced to redistribute into rafts upon Ag stimulation (29). The role of palmitoylation in these translocation events as well as in the differential localization of Dclck and Wtlck to LR reported in this study remain to be determined.

The distinct effects mediated by lck and fyn are now being ascribed to arrays of kinase-specific targets. Evidence to date supports the conclusion that Pyk2 and Fyb are fyn-specific substrates. Pyk2 is a member of the focal adhesion family of protein tyrosine kinases that undergoes fyn-dependent tyrosine phosphorylation after TCR stimulation (61). Recent evidence has linked Pyk2 to the activation of mitogen-activated protein kinase cascades (62). In addition, Pyk2 has been implicated in CD28 as well as IL-2 activation pathways (63, 64, 65). Fyb, also known as SLAP-130 (66, 67), also undergoes fyn-dependent tyrosine phosphorylation in response to TCR stimulation (68). The Src homology 2 domains of both fyn and SH2-domain containing leukocyte protein of 76 kDa bind to phosphorylated Fyb at distinct sites. This trimolecular complex, when coexpressed in Jurkat cells, results in the synergistic up-regulation of TCR-induced IL-2 transcription (68). T cells from Fyb-deficient mice exhibit profound defects in IL-2 production and TCR-mediated up-regulation of CD25, establishing Fyb as an essential positive regulator of T cell activation downstream of fyn (69, 70, 71).

The interdependency of lck and fyn activation reported in this study is supported by previous studies. Fyn is hyperactive in Jurkat cells overexpressing constitutively active lckY505F, and mAb-mediated aggregation of CD4 in control Jurkat cells resulted in lck activation and subsequently fyn activation (72). Taken together, these results characterize a pathway from TCR to fyn, culminating in IL-2 transcription. Taken in context with the results presented in this study and the recent characterization of lck-dependent fyn activation in primary CD4+ T cells (19), this pathway can now be extended through the linking of TCR/CD4/lck to fyn activation.

In conclusion, characterization of the lck-dependent mechanism underpinning responsiveness to anti-TCR in this study, although focused on a specific clonal system, reveals a general mechanism underpinning the initiation of proximal signals emanating from the TCR complex. Although functioning independently, the activation of lck and fyn is interdependent and tightly regulated. The role of CD4 in impeding lck mobility and therefore membrane localization appears critical in this process, as does membrane architecture. The results underscore the importance of targeting Src family kinase function in the activation process and the distinct roles played by lck and fyn.


   Acknowledgments
 
We thank G. Knowles and C. Cantin for cell sorting.


   Footnotes
 
1 This work was supported by grants from the Canadian Institutes of Health Research (FRN9735 and MT10702) and a Canadian Institutes of Health Research studentship (to B.L.L.). Back

2 Address correspondence and reprint requests to Dr. Michael Julius, Sunnybrook and Women’s Health Sciences Center, Room A3 33, 2075 Bayview Avenue, Toronto, Ontario, Canada M4N 3M5. E-mail address: michael.julius{at}swri.ca Back

3 Abbreviations used in this paper: LR, lipid raft; ZAP70, {zeta}-associated protein of 70 kDa; Csk, C-terminal Src kinase; CTB, cholera toxin B; Dclck, double-cysteine lck; GM1, GM1 ganglioside; HMF, heavy membrane fraction; MIEV, MSCV-based IRES-enhanced green fluorescent protein virus; PVDF, polyvinylidene difluoride; Wtlck, wild-type lck; MSCV, murine stem cell virus; IRES, internal ribosome entry site; TCRC{beta}, constant region of TCR{beta} chain; PAG, phosphoprotein associated with glycosphingolipid-enriched microdomain. Back

Received for publication October 7, 2003. Accepted for publication January 23, 2004.


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