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The Journal of Immunology, 2001, 166: 754-764.
Copyright © 2001 by The American Association of Immunologists

A Molecular Framework for Two-Step T Cell Signaling: Lck Src Homology 3 Mutations Discriminate Distinctly Regulated Lipid Raft Reorganization Events1

Viresh P. Patel*, Miriana Moran*, Teresa A. Low* and M. Carrie Miceli2,*,{dagger}

* Department of Microbiology, Immunology, and Molecular Genetics and {dagger} The Molecular Biology Institute, University of California School of Medicine, Los Angeles, CA 90095


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Costimulation by CD28 or lipid-raft-associated CD48 potentiate TCR-induced signals, cytoskeletal reorganization, and IL-2 production. We and others have proposed that costimulators function to construct a raft-based platform(s) especially suited for TCR engagement and sustained and processive signal transduction. Here, we characterize TCR/CD48 and TCR/CD28 costimulation in T cells expressing Lck Src homology 3 (SH3) mutants. We demonstrate that Lck SH3 functions after initiation of TCR-induced tyrosine phosphorylation and concentration of transducers within rafts, to regulate the costimulation-dependent migration of rafts to the TCR contact site. Expression of kinase-active/SH3-impaired Lck mutants disrupts costimulation-dependent raft recruitment, sustained TCR protein tyrosine phosphorylation, and IL-2 production. However, TCR-induced apoptosis, shown only to require "partial" TCR signals, is unaffected by expression of kinase-active/SH3-impaired Lck mutants. Therefore, two distinctly regulated raft reorganization events are required for processive and sustained "complete" TCR signal transduction and T cell activation. Together with recent characterization of CD28 and CD48 costimulatory activities, these findings provide a molecular framework for two signal models of T cell activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Advances in membrane biology have revealed that the plasma membrane is compartmentalized into lipid raft and nonraft microdomains (1). Lipid rafts are enriched in glycosphingolipids, saturated phospholipids, cholesterol, and a subset of cellular proteins and float as laterally associated structures within the otherwise glycerophospholipid-rich plasma membrane (1). Recent evidence indicates that lipid rafts function as preformed modules for effecting signal transduction, cytoskeletal reorganization, protein sorting, and membrane trafficking in many cell types (1).

We and others have suggested that engagement of T cell costimulators induces lipid raft recruitment to the TCR contact site and the construction of a platform that facilitates TCR engagement and processive and sustained signal transduction (2, 3). We proposed that lipid rafts function as sites for the integration of TCR engagement, early TCR signals, and actin cytoskeleton-mediated reorganization of the TCR contact cap (2). These suggestions were based, in part, on our observations that recruitment of lipid raft-associated CD48 to the TCR contact site enhances raft-dependent TCR-{zeta} chain tyrosine phosphorylation, {zeta}:actin cytoskeleton association, F-actin reorganization, morphology changes, and Ag-induced functions (Ref. 2 ; and M. Moran and M. C. Miceli, unpublished observations).

That lipid rafts might organize the TCR contact site for sustained and processive signal transduction is further supported by a number of other recent findings. First, several key TCR signal transducers including Lck, Fyn, LAT, and GPI-linked CD48 preferentially partition within lipid raft membranes (1, 4, 5), whereas putative negative regulators of TCR engagement and activation, such as CD43 and CD45, preferentially partition outside raft microdomains (5, 6, 7). Second, TCR engagement triggers the increased stability and/or concentration and tyrosine phosphorylation of TCR-{zeta} and signal transducers within lipid rafts (7, 8, 9). Furthermore, perturbation of the structural integrity of lipid rafts inhibits TCR-induced protein tyrosine phosphorylation and Ca2+ flux (2, 8, 9). Finally, TCR/CD28 or TCR/CD48 costimulation leads to lipid raft migration to and coalescence at the site of TCR engagement and prolonged stability of TCR-induced protein tyrosine phosphorylation (Ref. 3 ; data presented here and M. Moran and M. C. Miceli, unpublished data).

The relationship between the TCR-induced concentration and phosphorylation of transducers within lipid rafts and the coalescence of lipid rafts at the TCR contact cap has not been elucidated. Furthermore, how TCR/costimulator engagement induces raft membrane reorganization remains uncharacterized. Because Lck is a raft resident protein and a primary mediator of TCR signal transduction, we reasoned that Lck might function in regulating TCR/CD48-mediated lipid raft dynamics at the contact cap. Therefore, we analyzed TCR/CD48 and TCR/CD28 costimulatory activity in T cells expressing Lck Src homology 3 (SH3)3 mutants. Here, we present evidence that the Lck SH3 domain functions downstream of the initiation of TCR signal transduction to facilitate TCR/costimulator-induced lipid raft recruitment to the TCR contact cap, sustenance of TCR/costimulator-induced protein tyrosine phosphorylation, and IL-2 production.

By analyzing the Lck SH3 domain requirements for TCR/costimulator raft-mediated events, we discriminate two distinctly regulated lipid raft reorganization events required for T cell activation. In the first step, TCR-{zeta} and downstream transducers are concentrated and tyrosine phosphorylated within lipid rafts. This step requires Lck kinase activity and remains intact in cells expressing kinase-active/SH3-impaired Lck mutants. The second step involves subsequent recruitment of additional nonliganded lipid rafts to the TCR contact cap and is dependent on costimulation and defective in cells expressing kinase-active/SH3-impaired Lck mutants. Furthermore, expression of these SH3 mutants disrupts sustained TCR signal transduction and IL-2 production, but not apoptosis, shown only to require "partial" TCR signals. These data correlate Lck SH3 activity and lipid raft recruitment with signals and functions that rely on prolonged TCR engagement and processive TCR signal transduction. Together with recent characterization of CD28 and CD48 costimulatory activities (3, 10, 11), our findings provide a molecular and cell biological framework for two signal models of T cell activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mutagenesis of Lck and expression in BI-141 or as GST fusion proteins

Lck SH3 domain mutations were created using the QuickChange Mutagenesis System (Stratagene, La Jolla, CA) using LckF505 as a template. Mutants were sequenced to verify that only the intended mutations were created. Stable transfections were performed by electroporation as described (12). GST-LckSH3 fusion constructs were created by subcloning fragments corresponding to amino acids 1–149 from wild-type or mutant LckF505 into pGEX4T-1 (Pharmacia, Piscataway, NJ) and fusion proteins were expressed and purified according to manufacturer’s recommendations (Pharmacia). Hybridomas producing Abs against TCR-{zeta} (H146-968), CD28 (37.51), CD48 (5-8A10), and CD3{epsilon} (145-2C11, ATCC CRL-1975) were obtained from Dr. Kubo (Cytel, San Diego, CA), Dr. Allison (University of California, Berkeley), Dr. Reiser (Dana-Farber Cancer Institute, Boston, MA), and American Type Culture Collection (Manassas, VA), respectively, and Abs were produced and purified from hybridoma supernatants using standard techniques. Goat anti-hamster (GAH) was purchased from Cappel (Durham, NC). Abs directed against c-Cbl, Sam68, p85 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-Lck was produced against a GST fusion protein comprising Lck amino acids 5-144. Anti-phosphotyrosine Ab, 4G10 was purchased from Upstate Biotechnology (Lake Placid, NY).

Cells and functional analysis

BI-141 is a T cell hybridoma that recognizes beef insulin in the context of I-A{alpha}b{beta}k. FT5.7 is a derivative of the DAP cell line that has been transfected with I-A{alpha}b{beta}k (gift of Dr. Germain, National Institutes of Health). Antigenic stimulation of BI-141 T cells was performed as reported (13). For experiments examining the requirements for duration of TCR engagement, T cell transfectants were stimulated using immobilized anti-CD3 with or without anti-CD28 or anti-CD48 for various periods and subsequently cultured in the absence of stimulus for the duration of the 16- to 20-h experiment. IL-2 was quantitated either by bioassay or by ELISA and cell death was detected using propidium iodide staining and FACS analysis as described (13).

GST fusion protein precipitations

Resting BI-141 T cells (2.5 x 107) were lysed in 0.5% Triton X-100, 50 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 mM PMSF, and 1 mM Na3VO4. Clarified lysates were incubated with 25 µg GST fusion protein coupled to 25 µl of GST-Sepharose 4B beads (Pharmacia) for 1 h at 4°C. Samples were washed in lysis buffer and separated on 10% SDS-PAGE, transferred to nitrocellulose, immunoblotted for c-Cbl, p85, and Sam68, and detected using ECL (Amersham, Arlington Heights, IL).

Lck kinase assay

Lck was transfected into COS cells using the Transfast Reagent (Promega, Madison, WI). Forty-eight hours posttransfection, cells were lysed in TNE buffer (50 mM Tris, 1% Nonidet P-40, 1 mM EDTA) containing 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM PMSF, and 1 mM Na3VO4, and postnuclear lysates were immunoprecipitated with rabbit anti-Lck antiserum. Immunoprecipitations were split in half and used for in vitro kinase assay and determination of Lck expression levels by immunoblotting. Kinase assays were performed as previously described (12).

Raft fractionation

BI-141 T cells were incubated with 5 µg/ml of anti-CD3{epsilon} (2C11), with or without 20 µg/ml anti-CD48 (5-8A10), or media for 30 min at 4°C, and cross-linked with GAH at 37°C for various times. Anti-phosphotyrosine and anti-Lck immunoblots were performed as described (2). For raft fractionation, T cells (4 x 107) were resuspended in buffer A (25 mM Tris, pH7.5, 150 mM NaCl, 5 mM EDTA) containing 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM PMSF, and 1 mM Na3VO4. Cell resuspensions were briefly sonicated, centrifuged at 800 x g for 10 min and supernatants were incubated in 1% Brij58 for 1 h at 4°C. An equal volume of 80% sucrose in buffer A was added to the Brij58 lysates and samples were placed in "Ultra-Clear" centrifuge tubes (Beckman Coulter, Fullerton, CA). Samples were then overlaid with 2 volumes of 30% sucrose in buffer A and 1 volume of 5% sucrose in buffer A and centrifuged at 200,000 x g in a SW55Ti rotor (Beckman Coulter) for 16 h at 4°C. Fractions (400 µl) were harvested from the top (9).

4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-D]pyrimidine (PP2) protection assay

Cells were stimulated with anti-CD3 (10 µg/ml) with or without anti-CD28 (40 µg/ml) or anti-CD48 (40 µg/ml) and GAH. After 5 min PP2 was added to a final concentration of 50 µM and aliquots sampled 0–5 min after PP2 addition were lysed in TNE buffer containing protease inhibitors, but without Na3VO4.

Confocal microscopy

T cells (5 x 104) were stimulated with 5 x 104 Ab-coated beads (50 µg/ml anti-CD3{epsilon} with or without 200 µg/ml anti-CD48) in 25 µl for 30 min at 37°C on poly-L-lysine-coated slides (Carlson Scientific, Peotone, IL). Cells were fixed in 3.7% formaldehyde for 10 min, stained with FITC-cholera toxin B subunit (8 µg/ml; Sigma, St. Louis, MO) for 45 min and analyzed by confocal microscopy on a Carl Zeiss LSM 310 microscope using a x100 objective. Sections (10–15 1 µm) were collected and overlaid to create the composite images.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Mutations in the Lck SH3 domain impair intermolecular ligand binding and affect Lck kinase activity

To address the role of the Lck SH3 domain in TCR signal transduction, we generated two clustered sets of mutations at sites predicted to be important for intramolecular and intermolecular SH3 ligand binding (Fig. 1Go, A and B). We expressed wild-type and mutant Lck SH3 domains as GST fusion proteins and determined their relative abilities to precipitate known Lck SH3 ligands from T cell lysates. As shown in Fig. 1GoC, both SH3 mutants are defective at binding Lck SH3 ligands c-Cbl, Sam68, and the p85 subunit of phosphatidylinositol (PI) 3-kinase, relative to wild-type Lck SH3 GST-fusion protein. The YLDY mutation is severely impaired, whereas the AF mutation maintains modest residual binding activity (Fig. 1GoC).



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  		FIGURE 1. Description and characterization of SH3 domain mutants used in these studies. Schematic representations of Lck (A) and its proposed intramolecular and intermolecular interactions (B). N-terminal myristoylation at G2 and palmitoylation at C3 and C5 are required for Lck lipid raft association. The SH2 and SH3 domains and the SH2-CD linker (represented by // in B) involved in intra- and intermolecular interactions are indicated. Mutations in the SH3, SH2-CD linker, catalytic, and regulatory domains used in this study are depicted in A. The AF and YLDY mutations affect both sites 1 and 2. C, GST fusion proteins containing mutant Lck SH3 domains are impaired in their ability to interact with SH3 domain ligands. T cells lysates were incubated with the indicated GST-LckSH3 domain fusion protein bound to glutathione-Sepharose beads. Precipitated protein complexes were separated on 10% SDS-PAGE and the presence of SH3 domain ligands determined by immunoblotting with anti-sera directed against c-Cbl, Sam68, or p85. D, Mutation of the Lck SH3 domain can affect its enzymatic activity. Relative in vitro Lck kinase activity and cellular protein tyrosine phosphorylation from LckF505 and LckF505SH3 mutant expressing transfectants were calculated from three independent experiments. Kinase activity and protein tyrosine phosphorylation levels were normalized relative to the amount of Lck expressed. Both assays were determined to be sensitive and linear over the range of Lck expressed in these experiments.

 
We superimposed these SH3 domain mutations on the Lck F505 (kinase-active) mutant so that we could examine the effects of disrupting Lck SH3 intramolecular interactions independent of the confounding influences of SH2:Y505 down-regulatory interactions (14, 15). We next determined the effect of Lck SH3 domain mutations on LckF505 kinase activity, because SH3 domain intramolecular and intermolecular interactions are proposed to regulate the enzymatic activity of src family kinases (16, 17).

Lck SH3 mutants were transiently transfected into COS cells and the Lck kinase activity was determined by in vitro kinase assay. The in vitro kinase activity of LckAF/F505 is comparable to that of LckF505, whereas LckYLDY/F505 activity is enhanced relative to LckF505 activity (Fig. 1GoD). To determine whether the measured in vitro Lck kinase activity is relevant to protein tyrosine phosphorylation in intact cells, levels of tyrosine phosphorylated proteins in COS cell transfectants were measured by anti-phosphotyrosine immunoblotting. The relative intensity of a prominent and representative 40-kDa phosphorylated protein from COS cell transfectants was quantitated and is shown in Fig. 1GoD. The level of cellular protein tyrosine phosphorylation within COS cell transfectants is coincident with the measured in vitro kinase activity of the mutant expressed (Fig. 1Go, C and D). Together, these data support models in which Lck SH3 intramolecular and intermolecular interactions regulate kinase activity (16, 17). Furthermore, they provide us with two independent kinase-active/SH3- impaired (AF/F505 and YLDY/F505) Lck mutants for use in characterizing the contributions of Lck SH3 to T cell activation.

The Lck SH3 domain is not required for the initiation of TCR signal transduction, once Lck is activated

To address the contribution of the Lck SH3 domain in TCR-induced signal transduction and functions, LckF505 or SH3-impaired LckF505 mutants were stably expressed in the BI-141 T cell hybridoma. At least four independent transfectants of each type were matched for Lck and TCR expression levels and used for subsequent analysis (data not shown). Transfectants were assessed for baseline levels of protein tyrosine phosphorylation and for their ability to initiate protein tyrosine phosphorylation in response to TCR engagement (Fig. 2Go). Both LckAF/F505 and LckYLDY/F505 transfectants seem to be less regulated by TCR engagement, having higher baseline kinase activities than LckF505 transfectants (Fig. 2Go). Nonetheless, TCR engagement induces additional protein tyrosine phosphorylation (Fig. 2Go). Transfectants expressing kinase-active/SH3-impaired mutants (LckAF/F505 or LckYLDY/F505) initiate TCR-induced protein tyrosine phosphorylation as efficiently, or slightly better than, LckF505 transfectants (Fig. 2Go). Analysis of protein tyrosine phosphorylation at earlier time points ruled out the possibility that LckAF/F505 or LckYLDY/F505 transfectants delay onset of protein tyrosine phosphorylation relative to LckF505 transfectants (data not shown). In keeping with a previous report (18), these data indicate that the Lck SH3 domain is not essential for the initiation of TCR signal transduction, once Lck is activated. Discrepancies between relative LckAF/F505 and LckYLDY/F505 activity in COS cells and T cells may reflect the presence of T cell-specific SH3 ligands that are able to activate LckAF/F505 through its residual site 3 mediated ligand binding activity (Fig. 1Go, C and D vs Fig. 2Go).



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  		FIGURE 2. Transfectants expressing kinase-active/SH3-impaired mutants are not defective in TCR-induced protein tyrosine phosphorylation. BI-141 T cell transfectants were treated with GAH(-) or anti({alpha})-CD3 and GAH(+) for 5 min. Total cell lysates were subjected to SDS-PAGE, transferred to nitrocellulose and immunoblotted with anti-phosphotyrosine ({alpha}-PY). Transfectants expressing kinase-active (F505) or kinase-active/SH3-impaired (AF/F505 or YLDY/F505) Lck mutants were examined. Similar results were obtained using additional independent transfectants.

 
Both CD48 and CD28 costimulation stabilize TCR-induced protein tyrosine phosphorylation and require an intact Lck SH3 domain

We next addressed whether LckF505 (kinase-active) and LckAF/F505 SH3 (kinase-active/SH3-impaired) transfectants were equally able to sustain signals by maintaining TCR-induced protein tyrosine phosphorylation (Fig. 3Go). LckF505 and LckAF/F505 expressing transfectants were stimulated through CD3, CD3 plus CD28, or CD3 plus CD48 for 5 min. Protein tyrosine phosphorylation was measured initially and at several time points after the addition of PP2, an inhibitor of src family tyrosine kinases, to stop de novo protein tyrosine phosphorylation. By monitoring levels of phosphorylated proteins after PP2 addition, we are able to measure the stability of phosphotyrosyl groups on proteins phosphorylated within the first 5 min of activation (3). In LckF505 (kinase-active) transfectants, CD3 plus CD28 and CD3 plus CD48 costimulation stabilizes protein tyrosine phosphorylation beyond levels observed in response to anti-CD3 alone (Fig. 3Go, A and B). These findings indicate that, like CD28 costimulation (3), CD48 costimulation can stabilize TCR-induced protein tyrosine phosphorylation. LckAF/F505 (kinase-active/SH3-impaired) transfectants are unable to stabilize protein tyrosine phosphorylation through CD3 plus CD28 or CD3 plus CD48 costimulation (Fig. 3Go). Consistent with reports in which CD28 is shown to stabilize protein tyrosine phosphorylation using a similar assay, this effect is most pronounced with regard to proteins migrating within the 110- to 125-kDa and 36- to 40-kDa range (3). Under these assay conditions in which kinase inhibitor is added before lysis, costimulation-mediated enhancement of protein tyrosine phosphorylation is apparent after 5 min of TCR/costimulation (Fig. 3Go). However, costimulatory effects become increasingly dramatic when stability of protein tyrosine phosphorylation is monitored after additional incubation at 37°C with PP2 for 2 and 5 min before lysis. These data implicate the Lck SH3 domain in mediating costimulator stabilization of protein tyrosine phosphorylation.



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  		FIGURE 3. Costimulator-mediated increased stability of TCR-induced phosphorylation requires an intact Lck SH3 domain. Transfectants were left unstimulated (-) or stimulated with anti-CD3, or anti-CD3 and anti-CD28, or anti-CD3 and anti-CD48 followed by cross-linking with GAH. PP2 was added after 5 min and samples were incubated at 37°C for the indicated times after PP2 addition. The decay of protein tyrosine phosphorylation was assayed by anti-phosphotyrosine ({alpha}-PY) immunoblot analysis.

 
The ability of CD28 and CD48 costimulators to increase the stability of tyrosine phosphorylation may be related to sequestration of signaling components within lipid raft microdomains (2, 3). Because CD45 is excluded from raft microdomains (6, 7) concentration of tyrosine-phosphorylated proteins within lipid rafts could result in lowering their accessibility to CD45 phosphatase activity, thus extending the stability of phosphorylation. Therefore, we next examined TCR/CD48-induced lipid raft dynamics in LckF505 and LckAF/F505 transfectants.

Lck SH3 mutations discriminate two distinctly regulated lipid raft-dependent TCR activation events

Lipid rafts can be biochemically isolated based on their relative detergent insolubility and buoyancy on sucrose gradients (1). Recent data analyzing rafts purified in this manner demonstrate that TCR engagement results in the relocalization and/or tyrosine phosphorylation of transducers and adapters, including TCR-{zeta}, to lipid raft microdomains (4, 8, 9). Because lipid rafts have been estimated to contain only 5% of the total plasma membrane proteins (19), the accumulation of proteins in raft fractions represents a dramatic concentration of those proteins within rafts relative to the rest of the plasma membrane.

To determine whether LckAF/F505 efficiently compartmentalizes TCR-induced phosphoproteins, LckF505 (kinase-active) and LckAF/F505 (kinase-active/SH3-impaired) transfectants were compared for their ability to concentrate tyrosine phosphorylated {zeta} and other signal transducers within lipid rafts in response to stimulation with anti-CD3 or anti-CD3 plus CD48 (Fig. 4GoB). Lysates from unstimulated or stimulated LckF505 and LckAF/F505 transfectants were separated into lipid raft and nonraft fractions by sucrose density centrifugation in the absence of tyrosine kinase inhibitor. As shown in Fig. 4GoA, cellular Lck equally distributes in buoyant lipid raft (1, 2, 3, 4) and nonraft (8, 9, 10, 11) fractions isolated from either LckF505 or LckAF/F505 transfectants. This Lck distribution is consistent with previous reports (6) and indicates that mutation of the LckF505 SH3 domain does not affect its ability to partition within lipid rafts. Aliquots of pooled raft (1, 2, 3, 4) or nonraft (8, 9, 10, 11) fractions from cells left unstimulated or stimulated through CD3 or CD3 plus CD48 for 5 min were separated on SDS-PAGE and immunoblotted with anti-phosphotyrosine Ab reactive with phosphorylated pp21{zeta} and pp23{zeta} isoforms. Both LckF505 and LckAF/F505 transfectants successfully concentrate tyrosine phosphorylated {zeta} and other phosphoproteins in lipid rafts after TCR or TCR/CD48 engagement (Fig. 4GoB). Immunoblotting with an Ab (H146–968) reactive with unphosphorylated and minimally phosphorylated TCR-{zeta} demonstrates increased levels of {zeta} in raft fractions after TCR engagement in both LckF505 and LckAF/F505 transfectants (Fig. 4GoC). Decreased relative levels of p18{zeta} and pp19{zeta} in raft fractions from AF/F505 transfectants are offset by increased levels of phosphorylated pp21{zeta} and pp23{zeta} isoforms, consistent with the modest increase in efficiency of protein tyrosine phosphorylation observed in LckAF/F505 relative to LckF505 transfectants. Taken together, these data indicate that LckF505 and LckAF/F505 transfectants effectively tyrosine phosphorylate and redistribute {zeta} and other phosphoproteins within lipid rafts.



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  		FIGURE 4. Both LckF505 (kinase-active) and LckAF/F505 (kinase-active/SH3-impaired) transfectants successfully concentrate Lck, {zeta}, and other phosphoproteins within lipid rafts. A, Lck distribution within lipid raft and nonraft subcellular compartments. Cell lysates from BI-141 cells expressing LckF505 or LckAF/F505 were separated into raft or nonraft subcellular fractions by sucrose density centrifugation. An aliquot of each fraction was resolved on 10% SDS-PAGE and immunoblotted with anti-Lck. B, Distribution of TCR-induced phosphorylated proteins within lipid raft and nonraft subcellular compartments. BI-141 T cells expressing LckF505 or LckAF/F505 were stimulated with anti-CD3 with or without anti-CD48 and cross-linking secondary Ab (GAH) for 5 min. Cell lysates were separated by sucrose density gradient fractionation. Fractions containing equal cell equivalents of lipid raft proteins (1 2 3 4 ) or nonraft proteins (8 9 10 11 ) were pooled, and an aliquot from each pool was resolved using 12.5% SDS-PAGE, and immunoblotted with {alpha}-PY Ab. Total phosphoproteins are shown in the upper panel and phosphorylated TCR-{zeta} is shown in the lower panel. C, Levels of unphosphorylated or minimally phosphorylated {zeta} (p18/pp19) in raft fractions before and after CD3 or CD3 + CD48 costimulation. Raft fractions were probed with H146-968 anti-{zeta} Ab only reactive with minimally or nonphosphorylated {zeta}. Similar results were obtained using additional independently derived LckF505 and LckAF/F505 transfectants.

 
We have suggested that CD48 costimulation recruits lipid rafts to the site of TCR engagement (2). To directly address this issue, we visualized lipid raft distribution in response to CD3 or CD3 plus CD48 stimulation. We discovered that CD48 costimulation, like CD28 costimulation (3), leads to the polarized redistribution of lipid rafts toward the site of TCR engagement (Fig. 5GoA and M. Moran and M. C. Miceli, unpublished observations). To investigate the role of Lck SH3 activity in raft redistribution to the TCR contact cap, we used FITC-cholera toxin to visualize the lipid raft constituent GM1 after T cell exposure to anti-CD3 or anti-CD3 plus CD48 coated microspheres. Whereas both neo (control) and LckF505 (kinase-active) transfectants efficiently relocalize lipid rafts toward the T cell:anti-CD3 plus CD48 coated microsphere interface, transfectants expressing LckF505 SH3 mutants do not (Fig. 5Go). Representative patterns of raft redistribution (clustering) and the percentages of cells exhibiting polarized raft clustering are shown in Fig. 5Go, A and B, respectively. Analysis of single-section fluorescent images demonstrates redistribution of cell surface, and not intracellular, GM1 (data not shown). Raft redistribution is observed 30 min after CD3 + CD48 costimulation, but not as early as 10 min. None of the transfectants redistribute rafts in response to microspheres coated with anti-CD3 or anti-CD48 alone (data not shown). These data implicate Lck SH3 activity in mediating signals required for the costimulation-dependent recruitment of lipid rafts to the TCR contact site. Furthermore, they further correlate migration of lipid rafts to the TCR contact site with the stabilization of protein tyrosine phosphorylation (3).



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  		FIGURE 5. The Lck SH3 domain is required for lipid raft recruitment to the site of TCR contact in response to CD3 plus CD48-mediated costimulation. A, Transmission light images (left) and fluorescent microscopy images (right) of BI-141 transfectants stimulated with anti-CD3 with or without anti-CD48-coated microspheres are shown. Lipid rafts were visualized by FITC-labeled cholera toxin B subunit. Fluorescent images are composite image sections collected by confocal microscopy and transmission light images are single section images with the bead:cell interface in focus. B, The percent of cells that relocalized the raft constituent GM1 toward the bead:cell interface was quantitated. Between 37 and 60 conjugates were counted for each sample shown in A.

 
IL-2 production, but not apoptosis, relies on complete TCR signal transduction and requires two raft-mediated activation events

Because sustained TCR signal transduction and costimulation have been implicated in regulating the functional outcome of TCR engagement, we were interested in determining whether each of these raft-mediated TCR activation events were equally important for all downstream functions. To this end, we defined functions reliant on "sustained and complete" or "partial" TCR signal transduction in BI-141 T cells. We have previously reported that TCR-induced IL-2 production and apoptosis have different requirements for Lck kinase and SH2 activities (13). Indeed, BI-141 transfectants expressing kinase-inactive or SH2 domain-impaired LckF505 are defective in processively phosphorylating {zeta} and downstream signal transducers and producing IL-2 in response to Ag, although they remain fully competent at undergoing apoptosis (12, 13). These data likely reflect a differential dependence of downstream functions on processive and sustained "complete" TCR/Lck signal transduction.

To further address this issue, we investigated the relationship between duration of TCR engagement and functional outcome in BI-141 T cells. We varied the duration of TCR engagement by stimulating BI-141 T cells with plate-bound anti-CD3 and after a given time transferred them to a fresh well in the absence of stimuli for the remainder of the assay period. After 20 h of culture, supernatants were assayed for IL-2 and cells were analyzed for viability. Significant levels of apoptosis were detected within the first hour of TCR engagement. By 6 h of continual engagement as much as 60% of the maximal death response was observed (Fig. 6GoA). In contrast, IL-2 production required more than 6 h of TCR engagement (Fig. 6GoA). Therefore, a shorter length of TCR engagement is required for commitment to apoptosis than is required for commitment to IL-2 production in BI-141 T cells. These data corroborate our suggestion that apoptosis in BI-141 T cells requires "partial" TCR/Lck signal transduction, whereas IL-2 production requires "complete" TCR/Lck signal transduction. Furthermore, they are in keeping with reports demonstrating a similar hierarchy of apoptosis and IL-2 responses in the A.E7 and other T cell clones in response to "partial" and "complete" agonist Ags and in primary T cells under circumstances in which CD28 costimulation is or is not present (20, 21). Together, these data contribute to the growing body of evidence indicating that complete TCR activation requires processive and sustained signal transduction and that the extent to which this signal reaches "completion" can influence the functional outcome of TCR engagement.



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  		FIGURE 6. A, TCR-mediated cell death and IL-2 production require "partial" and "complete" TCR signals, respectively. BI-141 T cells were stimulated with plate-bound anti-CD3 for the indicated times and then cultured in the absence of anti-CD3 for a total of 20 h. Data are shown as percent of the maximal response observed after 20 h of stimulation. B, Lck SH3 domain mutations do not affect the ability of BI-141 T cells to undergo TCR-induced apoptosis. Lck transfectants were treated with plate-bound anti-CD3 mAb for 16 h. Cells were assayed for apoptosis by propidium iodide staining and FACS analysis. C, Lck SH3 domain mutations impair the ability of LckF505 to enhance anti-CD3-induced IL-2 production. Transfectants were stimulated with immobilized anti-CD3 for 18 h and assayed for IL-2 production using ELISA. D, Second-site mutations within Lck SH3 domain diminish the ability of LckF505 to enhance Ag-induced IL-2 production. Beef insulin was presented to Lck or neo transfectants by FT5.7 APCs. After 16 h, supernatants were assayed for IL-2 production. Two independent transfectants of each type were assayed in B and D and averages of TCR-induced apoptosis and IL-2 production are shown. Results similar to those shown in C were obtained using additional independent transfectants .

 
To determine the role of the Lck SH3 domain in inducing functions dependent on "partial" and "complete" TCR signals, BI-141 transfectants were tested for their ability to undergo apoptosis and to produce IL-2 in response to TCR stimulation (Fig. 6Go, B–D). Transfectants expressing Lck mutants that are either kinase inactive (LckR273/F505) or SH3 impaired (LckAF/F505, LckYLDY/F505) are not defective in apoptosis (Ref. 12 and Fig. 6GoB). In contrast, transfectants expressing either of the SH3-impaired Lck mutants produced significantly less IL-2 than LckF505 (kinase-active) transfectants in response to anti-CD3 or Ag (Fig. 6Go, C and D). LckAF/F505 or LckYLDY/F505 transfectants demonstrate impaired IL-2 production relative to LckF505 transfectants despite comparable or increased initiation of protein tyrosine phosphorylation after TCR engagement (Figs. 2Go and 4Go). These findings highlight the importance of Lck SH3 domain contributions downstream or independent of initiation of tyrosine kinase activity. Furthermore, these data correlate Lck SH3 activity and lipid raft recruitment with signals and functions that rely on prolonged TCR engagement and processive TCR signal transduction.

CD28 or CD48 TCR costimulation shortens the duration of TCR engagement required for commitment to IL-2 production

Previous reports indicate that CD28 functions to decrease the duration of TCR engagement required for commitment to T cell activation by increasing the potency of TCR engagement through a mechanism involving raft mediated amplification and sustenance of TCR induced signals (3). Therefore, we next investigated whether CD48 or CD28 costimulation decreases the duration of TCR engagement required for commitment to IL-2 production in BI-141 T cells and what influence LckSH3 mutant expression has on this costimulatory activity. To this end, BI-141 transfectants expressing neo, LckF505 (kinase-active), LckAF/F505 (kinase-active/SH3-impaired), or LckYLDY/F505 (kinase-active/SH3-impaired) were stimulated through their TCRs alone or in the presence of CD48 or CD28 coengagement for varying times ranging from 4 to 16 h. T cells were subsequently transferred to wells in the absence of stimulation for the duration of the 16-h assay and supernatants assayed for IL-2 production at the 16-h time point. As shown in Fig. 7Go, either CD48 or CD28 costimulation can significantly shorten the duration of TCR engagement required for commitment to IL-2 production in neo or LckF505 transfectants. Alternatively, transfectants expressing LckAF/F505 or LckYLDY/F505 make very little IL-2 even after 16 h of continuous CD3 or CD3 plus costimulator engagement. Low levels of residual costimulatory activity in cells expressing SH3 mutants may result from contributions of a subset of endogenous Lck not affected by expression of mutant Lck SH3. These findings further support the concept that T cell functions, such as IL-2 production, which require processive and sustained TCR signal transduction are particularly sensitive to costimulation and intact Lck SH3 activity. Furthermore, they correlate requirements for raft clustering, sustenance of protein tyrosine phosphorylation and decreased duration of engagement and provide support for suggestions that raft clustering at the TCR contact cap functions to increase the potency of TCR signal transduction.



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  		FIGURE 7. CD28 or CD48 TCR costimulation shortens the duration of TCR engagement required for commitment to IL-2 production and require Lck SH3 activity. Transfectants were stimulated with plate-bound anti-CD3 (0.3 µg/ml) alone or with either anti-CD28 (3 µg/ml) or anti-CD48 (3 µg/ml) for the indicated times and then cultured in the absence of stimulus for the remainder of 16 h culture period. IL-2 production was quantitated using ELISA. Similar results were obtained using additional independent transfectants.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recent models of src family kinase regulation propose that intramolecular interactions between the Lck SH3 domain and the SH2-CD linker repress kinase activity by imposing constraints on the catalytic domain (17, 22). Interactions between Lck SH2 domain and a phosphorylated tyrosine (Y505) in the carboxyl-terminal tail are proposed to function similarly to down-regulate Lck kinase activity. Dephosphorylation of LckY505 or disruption of SH2:Y505 or SH3:SH2-CD linker interactions through intermolecular SH2 or SH3 ligand binding are predicted to up-regulate Lck kinase activity (16, 17). The regulatory function of Lck SH2:Y505 interactions is well supported experimentally (23, 24). However, experimental support for SH3:SH2-CD linker regulation of Lck activity is more limited (10, 18, 25). Here, we demonstrate that mutation of Lck SH3 (LckYLDY/F505) ligand-binding sites affect Lck kinase activity. This finding is consistent with previous reports in which mutation of a SH3 domain or SH2-CD linker leads to increased activity in Lck (18, 25) or other src family members (25, 26). However, our data demonstrate this phenomenon on an already "constitutively active" LckF505 background, supporting the suggestion that disruption of src family SH3 intramolecular interactions up-regulates kinase activity above levels achieved through disruption of intramolecular SH2:phosphotyrosine binding (17, 26).

We expressed these Lck mutants in BI-141 T cells to determine the role of Lck SH3 in mediating TCR signal transduction. We demonstrated that once Lck is activated, Lck SH3 functions downstream of the initiation of TCR-induced protein tyrosine phosphorylation to regulate the costimulation-dependent migration of lipid rafts to the TCR contact cap, stabilization of protein tyrosine phosphorylation and induction of IL-2 production. These findings are consistent with previous reports implicating the Lck SH3 domain in generating TCR signal(s) downstream of the initiation of protein tyrosine phosphorylation that are required for Ag-induced IL-2 production (18, 27, 28). However, our studies extend previous findings by defining a role for Lck SH3 in controlling TCR/costimulation-induced lipid raft migration to the TCR contact cap, stabilization of TCR protein tyrosine phosphorylation, and shortening of the required duration of TCR engagement. Furthermore, our data establish critical relationships among Lck SH3 activity, raft coalescence at the contact cap, sustained signal transduction, and IL-2 production. Expression of kinase active/SH3 impaired mutants does not interfere with the concentration and phosphorylation of transducers within lipid rafts or with apoptosis, which only requires "partial" TCR signals. However, the expression of kinase-active/SH3-impaired mutants does disrupt costimulation-mediated recruitment of lipid rafts to the site of TCR engagement and stabilization of protein tyrosine phosphorylation, and IL-2 production. These data correlate Lck SH3 activity, lipid raft recruitment, and sustained signals with functions that rely on prolonged TCR engagement and processive and sustained TCR signal transduction. In keeping with data presented here, recent characterization of peripheral T cells from asmase-/- (raft-deficient) mice demonstrates that anti-CD3/anti-CD28 induced proliferation, but not anti-CD3-induced susceptibility to apoptosis, is impaired relative to wild type mice (29, 30).

Recent studies indicate that membrane and protein reorganization events at the TCR contact cap are essential for successful T cell activation (31). In response to APCs presenting antigenic peptide, the T cell:APC contact site assembles into topologically and spatially distinct regions (32, 33). Initial TCR engagement occurs at the periphery of the APC:T cell contact area. Within 20 min of TCR engagement, MHC/peptide-bound TCR complexes and membrane-associated intracellular Lck, Fyn, and protein kinase C{theta} (PKC{theta}) cluster in the center of this specialized junction, referred to as the immunological synapse (32, 33, 34). They are surrounded by a ring of relatively taller (ICAM-bound) LFA-1 surface adhesion proteins and the intracellular cytoskeletal protein talin. It has been suggested that bulky CD43 and CD45 phosphatase need to be excluded from the T cell:APC contact to accommodate TCR/Ag-MHC binding and sustained protein tyrosine phosphorylation (13). The formation of a stable central cluster at the heart of the synapse is determinative for the induction of naive peripheral T cell activation (32, 33). Therefore, the organized immunological synapse is the likely site in which processive TCR signal transduction is orchestrated.

The molecular basis of synapse formation and function remains elusive, although related experiments have suggested that the liganded engagement of CD28 (3, 35), CD48 (2, 36), CD2 (37), and/or LFA-1 (35) costimulators may facilitate receptor recruitment, sorting, and cytoskeletal interactions at the TCR contact site and processive and sustained TCR signal transduction. Furthermore, inhibitor studies implicate PI 3-kinase and actin-myosin motor activities in the costimulation-induced flow of membrane lipids and proteins toward the contact cap and, thus, the formation of the immune synapse (3, 35). CD48 or CD28 costimulation-induced raft coalescence at the contact cap is temporally coincident with stable synapse formation (data presented here and (3, 33)). Furthermore, lipid rafts specifically include several proteins known to concentrate within the core of the immune synapse (i.e., Fyn, Lck, and TCR) and exclude proteins that segregate from the center of the synapse (CD45 and CD43) (1, 5, 6, 7). Together, these findings are suggestive of a role for rafts in immune synapse formation and stabilization.

We and others have previously suggested that raft recruitment to the TCR contact site might facilitate the stability of protein tyrosine phosphorylation through the exclusion of CD45 phosphatase activity (2, 3, 7). Raft/nonraft partitioning of CD45 phosphatase activity and TCR tyrosine phosphorylated substrates could explain why costimulators that induce raft recruitment to the TCR contact site also increase the stability of protein tyrosine phosphorylation. In keeping with this suggestion, Lck SH3 mutants that interfere with raft recruitment and coalescence also interfere with sustained TCR protein tyrosine phosphorylation and IL-2 production. Additionally, as TCR signal transducers are localized within growing lipid raft platforms, sustained Lck kinase activity may become increasingly reliant on SH3 intermolecular interactions to maintain kinase activity. Indeed, the liganded displacement of SH2 or SH3 intramolecular interactions may be the primary mechanism for Lck kinase activation within aggregated lipid rafts devoid of CD45. These suggestions are consistent with recent findings that the CD28 cytoplasmic tail activates Lck through direct interaction with Lck SH3 (10) and that CD28 engagement can induce lipid raft recruitment to the contact cap, prolong the stability of protein tyrosine phosphorylation, and sustain TCR signals (3).

We have yet to determine whether the requirement for Lck SH3 in costimulation-mediated raft recruitment reflects its activity as an adapter for localizing a particular SH3 ligand. The Lck SH3 has been reported to associate with several TCR signal transducers, including the p85 subunit of PI 3-kinase, PKC{theta}, HS1, CD2, CD28, Ras-GAP, Cdc2, Sam68, c-Cbl, and extracellular signal-related kinase (ERK) (10, 38, 39, 40, 41, 42, 43, 44, 45, 46). The Lck SH3 ligands ERK, PI 3-kinase, and PKC{theta} have been previously implicated in lipid raft reorganization, immune synapse formation, actin cytoskeletal reorganization, costimulatory activity, and/or downstream TCR signal transduction and, therefore, represent prime effector candidates (3, 18, 34, 47). Although previous studies have implicated Lck SH3 (18) and lipid rafts (48, 49) in ERK activation, the relationship between ERK activation and raft clustering has yet to be determined. Indeed, it is possible that ERK activity is required for the migration of rafts to the contact cap or that large raft platforms are required for efficient and sustained ERK activation. Finally, initial ERK activation may facilitate raft coalescence at the contact cap, which, in turn, sustains ERK activity during T cell activation. Experiments addressing the role of each of these proteins in TCR raft reorganization events are underway.

Recent experiments have demonstrated an essential role for lipid raft membrane compartmentalization in T cell activation (2, 3, 4, 8, 9). However, the relationship between the concentration and phosphorylation of transducers within lipid rafts and the costimulation-dependent migration of rafts to the contact cap has not been previously established. Here we demonstrate that these events are distinctly regulated and that both reorganization events are required for "complete," but not "partial," TCR signal transduction. Indeed, requirements for both the concentration and phosphorylation of transducers within rafts and raft clustering for IL-2 production are particularly evident under circumstances in which either the initial signal strength (i.e, in neo vs LckF505 transfectants) or the duration of TCR are limiting. Although neo transfectants can cluster rafts, initial signal strength is lower than what is observed in LckAF/F505 or LckF505 transfectants. Signal initiation is more intense in LckAF/F505 transfectants; however, signal amplification/sustenance is defective due to impaired raft clustering. LckF505 transfectants have both strong signal initiation and intact raft clustering mechanisms. That IL-2 production is lower in LckAF/F505 transfectants relative to neo or LckF505 transfectants (Fig. 7Go) despite high signal initiation highlight the importance of costimulation mediated raft clustering in processive signal transduction. The fact that recruitment of lipid rafts is impaired in cells expressing Lck SH3 mutants and that recruitment affects lipid rafts extending beyond the area of direct Ab-coated microsphere contact, indicate that signals generated through liganded lipid raft-associated CD48/TCR lead to the active recruitment of additional nonliganded rafts to the contact cap. Together with other recent findings (3, 10), these data support a model in which costimulators function by recruiting additional lipid rafts to the contact cap and facilitating processive and sustained TCR signals.

A "two (raft-mediated) signal model of T cell activation"

The two signal model of T cell activation is a central tenet of cellular immunology proposed as a mechanism for regulating peripheral T cell activation and tolerance induction. This model posits that two independent signals are required for TCR activation: "signal one" supplied by the TCR and "signal two" provided by a costimulator (50). Generation of signal one without signal two results in T cell inactivation (apoptosis or anergy), whereas generation of both signals results in IL-2 production and T cell activation (and in some instances rescue from apoptosis/anergy) (50, 51). Whereas CD28 is the best-studied costimulator in this regard, the recent description of dramatic T cell activation defects in CD48-deficient mice highlight the importance of CD48 costimulatory activity (11). Furthermore, similarities in the abilities of CD48 and CD28 costimulators to recruit lipid rafts to the contact cap stabilize TCR-induced protein tyrosine phosphorylation and decrease the duration of TCR engagement required for IL-2 production and their reliance on Lck SH3 activity indicate that they function similarly, but likely not identically, to modulate TCR signals ((3, 10) and data shown here). The two-signal model is well supported by experiments analyzing the functional effects of engaging the TCR in the presence or absence of costimulation. However, elucidation of a costimulatory signal distinct from signal 1 has proven elusive. Recent experiments indicate that costimulators may not send a second independent signal, but rather, may function to enable the TCR to send a sustained and processive signal (3, 10, 31). Here, we propose a "two (raft-mediated) signal model of T cell activation" reconciling these findings (Fig. 8Go).



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  		FIGURE 8. A two-signal model for T cell activation that relies on distinctly regulated raft-mediated signals one and two. In the first step, TCR engagement induces the concentration and tyrosine phosphorylation of TCR-{zeta} and signal transducers within lipid rafts. TCR signal transduction is initiated and functions that rely on "partial" TCR signals are triggered (death in the BI-141 T cell studied) (signal one). In the second step, TCR/costimulator coengagement leads to an Lck SH3 and lipid raft-mediated signal resulting in the migration of lipid rafts to the site of TCR engagement (signal two). Coalescence of rafts at the TCR contact cap generates a platform that facilitates processive and sustained TCR signal transduction required for induction of functions that rely on "complete" TCR signals (IL-2 production in the BI-141 T cell studied). Raft membranes are depicted in gray, whereas nonraft membranes are depicted in white. See text for additional discussion of this model.

 
In the first step induced by TCR engagement, TCR-{zeta} and downstream transducers are concentrated (or stabilized) and tyrosine phosphorylated within lipid rafts (signal one). This step requires Lck kinase activity, but remains intact in cells expressing kinase-active/SH3-impaired Lck mutants. Completion of the first step allows for the generation of early protein tyrosine phosphorylation and the induction of functions reliant on partial TCR signals including anergy and/or apoptosis. The second step involves the subsequent costimulation-dependent recruitment of additional lipid rafts to the TCR contact cap (signal two). In the case of CD48/TCR costimulation, this second signal may result from CD48/TCR-mediated raft clustering and actin cytoskeletal reorganization. How transmembrane CD28 interfaces with lipid rafts is unclear (52), thus how this step is induced by CD28 costimulation remains speculative. This second "raft recruitment" step is defective in T cells expressing kinase-active/SH3-impaired Lck mutants and is required for the generation of sustained TCR signals and functions dependent on "complete" TCR signals such as IL-2 production. We propose that Lck SH3-regulated/actin-cytoskeleton-dependent raft recruitment leads to the concentration and raft-based sorting of TCR, costimulators, and accessory molecules within the immune synapse. Furthermore, we propose that the construction and stabilization of a raft-based platform(s) at the TCR contact cap facilitates sustained and processive TCR signal transduction. This likely results from a number of predicted outcomes of raft recruitment and cross-linking at the TCR contact cap: 1) the promotion of MHC peptide access to the TCR due to raft exclusion of highly extending and glycosylated CD43 and CD45; 2) increased interaction between apposing T cell:APC membrane protein pairs due to increased lipid raft membrane rigidity and increased LFA-1 binding (53); 3) the concentration and activation of raft-associated tyrosine kinases through intermolecular displacement of SH2 and SH3 kinase down-regulatory interactions; 4) the exclusion of phosphatase activity; 5) the polarized recruitment and concentration of downstream signal transducers to raft-associated proteins or lipids); 6) the integration of signal transduction pathways; and 7) the induction of actin-cytoskeletal reorganization events that stabilize the immune synapse.

The studies presented here demonstrate that two distinctly regulated lipid raft reorganization events are required for complete T cell activation. They provide the molecular framework for a model of TCR/costimulator-mediated T cell activation. Future studies will test and further refine this model to determine the molecular mediators of each of these raft reorganization events and elaborate how different T cell costimulators modulate these events to facilitate TCR activation.


   Acknowledgments
 
We thank C. J. Guidos, J. F. Miller, J. Zack, C. Chung, and K. Kozak for critical review of this manuscript, and M. Schibler for assistance with the confocal microscopy.


   Footnotes
 
1 This work was supported by Grant 2R01 CA65979 from the National Institutes of Health and Grant 012939 from the Arthritis Foundation. V.P.P. was additionally supported by the Microbial Pathogenesis Training Grant 5-T32-AI-07323. T.A.L. is supported by the Eugene V. Cota Robles Award from the University of California Office of the President. Back

2 Address correspondence and reprint requests to Dr. M. Carrie Miceli, Department of Microbiology, Immunology, and Molecular Genetics and The Molecular Biology Institute, University of California, Los Angeles School of Medicine, Los Angeles, CA 90095-1570. Back

3 Abbreviations used in this paper: SH3, Src homology 3; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-D]pyrimidine; ERK, extracellular signal-related kinase; GAH, goat anti-hamster; PI, phosphatidylinositol; PKC{theta}, protein kinase C{theta}. Back

Received for publication March 2, 2000. Accepted for publication October 11, 2000.


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 Materials and Methods
 Results
 Discussion
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