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The Journal of Immunology, 2000, 165: 3226-3231.
Copyright © 00 by The American Association of Immunologists

Serine 6 of Lck Tyrosine Kinase: A Critical Site for Lck Myristoylation, Membrane Localization, and Function in T Lymphocytes1

Koubun Yasuda*, Atsushi Kosugi2,{dagger}, Fumie Hayashi{dagger}, Shin-ichiroh Saitoh*, Masakazu Nagafuku{dagger}, Yoshiko Mori*, Masato Ogata* and Toshiyuki Hamaoka*

* Biomedical Research Center and {dagger} School of Allied Health Sciences, Faculty of Medicine, Osaka University Medical School, Osaka, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Lck is a member of the Src family kinases expressed predominantly in T cells, and plays a pivotal role in TCR-mediated signal transduction. Myristoylation of glysine 2 in the N-terminal Src homology 4 (SH4) domain of Lck is essential for membrane localization and function. In this study, we examined a site within the SH4 domain of Lck regulating myristoylation, membrane localization, and function of Lck. A Lck mutant in which serine 6 (Ser6) was substituted by an alanine was almost completely cytosolic in COS-7 cells, and this change of localization was associated with a drastic inhibition of myristoylation in this mutant. To assess the role of Ser6 of Lck in T cell function, we established stable transfectants expressing various Lck mutants using Lck-negative JCaM1 cells. The Lck mutant of Ser6 to alanine, most of which did not target to the plasma membrane, was not able to reconstitute TCR-mediated signaling events in JCaM1 cells, as analyzed by tyrosine phosphorylation of intracellular proteins and CD69 expression. These results demonstrate that Ser6 is a critical factor for Lck myristoylation, membrane localization, and function in T cells, presumably because the residue is important for N-myristoyl transferase recognition.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Engagement of the TCR leads to a rapid rise in intracellular protein tyrosine phophorylation, followed by a series of other biochemical events that eventually result in gene expression and effector function in T cells (1, 2). It has been demonstrated that the initial TCR signal is mediated by the activation of the Src family tyrosine kinases Lck and Fyn (3, 4). Lck and Fyn phosphorylate motifs known as immunoreceptor tyrosine-based activation motifs (ITAMs),3 found multiple times in the signal-transducing subunits of the TCR complex (1). Once phosphorylated, ITAMs are bound by another tyrosine kinase, ZAP-70, which then activates a variety of other signaling molecules, leading to T cell activation. Because Lck-negative T cells are defective in TCR-mediated signaling and mice lacking a functional lck gene manifest an early arrest in T cell development (3, 5), Lck is essential for TCR signal transduction, which leads to both the activation and development of mature T cells.

Lck contains a unique domain at the NH2 terminus (Src homology 4 (SH4) domain), followed by an SH3 domain, an SH2 domain, and a tyrosine kinase or SH1 domain (6). It has been shown that each of the kinase, SH3, and SH2 domains plays a crucial role in TCR-mediated signal transduction (6, 7). The kinase domain is required for ITAM phosphorylation in the TCR complex, whereas the SH2 and SH3 domains contribute to TCR signaling as adapters for recruitment of signaling proteins. After its synthesis, Lck is targeted to the plasma membrane, where it exerts its functions. Although this distribution could be attributable to the association of Lck with the coreceptor molecules CD4 and CD8, a recent study has demonstrated that Lck expressed in lymphoid and in nonlymphoid cells mostly localized to the plasma membrane irrespective of CD4 expression (8). This suggests that Lck has intrinsic signals for targeting to the plasma membrane.

The N-terminal SH4 domain of Lck is modified by the addition of two different types of lipid, myristate and palmitate, and this lipid modification has been found to be necessary and sufficient for membrane localization of the protein (8, 9). Myristoylation is a cotranslational process in which the 14-carbon fatty acid myristate is attached to the NH2-terminal glycine (Gly2) through a stable amido bond (10). Palmitoylation differs from myristoylation in that the 16 carbon fatty acid palmitate is posttranslationally linked to cysteine residues through a labile thioester bond (10). Although both these lipid modifications are important for membrane localization and function, myristoylation is known to be necessary for palmitoylation, but not vice versa (11). Myristoylation occurs on all members of the Src family kinases, and the reaction is mediated by N-myristoyl transferase (NMT), which is expressed in most eukaryotic cells (12). Recently, it was reported that within the SH4 domain of the Src family kinases there exists sites that influence the myristoylation level of Gly2. The lysine residue at position 7 of the SH4 domain has been shown to be a critical determinant for efficient myristoylation of Src (13). Likewise, lysine 9 in addition to lysine 7 is important for myristoylation of Fyn (14). In fact, these residues are conserved in nearly all Src family kinases (15). However, Lck is the only Src family member that lacks both lysine 7 and 9 (15). Presently, it remains unknown whether the myristoylation level of Lck is lower than that of the other Src family kinases because of the absence of lysine 7 and 9 or whether there is another site regulating this lipid modification in Lck.

In the present study, we addressed the above issues by analyzing several Lck mutants in which individual amino acids within the SH4 domain were substituted. We demonstrated herein that the serine residue at position 6 in the SH4 domain is critical for efficient myristoylation and membrane localization of Lck. We also demonstrated that loss of serine 6 impairs the function of Lck in T cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation of Lck construct

The StuI fragment of the mouse lck cDNA from NT18 (16) was subcloned into the EcoRV site of pBluescript SK vector (Stratagene, La Jolla, CA). The pBluescript SK vector, containing lck cDNA, was then digested with XhoI/NotI, and this XhoI-NotI fragment was subcloned into pMKIT Neo vector, an SR{alpha} promotor-driven expression vector (provided by Dr. K. Maruyama, Tokyo Medical and Dental University, Tokyo, Japan). The Lck mutants were generated from the wild-type lck cDNA in pMKIT Neo vector using the Stratagene Quickchange kit as recommended by the manufacturer. The resulting mutations were verified by sequencing.

Cell culture and transfection

COS-7 cells were cultured in DMEM containing 10% FCS, penicillin (100 U/ml), and streptomycin (100 µg/ml). COS-7 cells cells (1 x 107) were transiently transfected with a plasmid encoding murine Lck or its mutants by the electroporation method, as described previously (17). JCaM1, a mutant Jurkat line that had lost the expression of Lck (3), was cultured in RPMI 1640 medium supplemented with 10% FCS, 50 µM 2-ME, 2 mM [scapl-glutamine, penicillin (100 U/ml), and streptomycin (100 µg/ml). For the generation of stable transfectants, a total of 1 x 107 JCaM1 cells was washed with PBS and resuspended in 1 ml ice-cold HEPES-buffered saline. Then 20 µg of plasmid DNA were added to the cell suspension in a cuvette (Gene Pulser Cuvette, Bio-Rad Laboratories, Richmond, CA), and the electric pulse (250 V, 960 µF) was applied by Gene Pulser (Bio-Rad). Stable transfectants were selected in media supplemented with G418 (Life Technologies, Grand Island, NY) at 1 mg/ml begining 48 h after transfection. Growing colonies were expanded in selective medium and assayed for Lck expression. Clones that express similar amounts of the transfected Lck protein were selected for further study.

Abs and reagents

The following Abs were used: MOL 171 (18); anti-human Lck; OKT3 (American Type Culture Collection, Manassas, VA), anti-human CD3{epsilon}; and H146–968 (19), anti-TCR{zeta}. Anti-phosphotyrosine mAb (4G10) was purchased from Upstate Biotechnology (Lake Placid, NY). FITC-conjugated anti-human CD69 Ab was purchased from PharMingen (San Diego, CA). PMA was purchased from Sigma (St. Louis, MO).

Cell fractionation

Cells were suspended in the hypotonic buffer (25 mM HEPES-buffered saline, pH 7.4, 10 mM KCl, 1 mM EDTA) for 10 min at 4°C, and homogenized using a Dounce homogenizer (20 strokes). Intact cells, nuclei, and other debris were pelleted by centrifugation at 270 x g for 5 min. Soluble and particulate fractions were generated following centrifugation at 100,000 x g for 30 min. Equivalent portions of the fractionated protein were resolved by SDS-PAGE and immunoblotted with anti-Lck Ab.

Immunofluorescence staining and confocal microscopy

Cells on the 34-mm tissue culture dish were washed with TBS and fixed with 3.7% formaldehyde in TBS for 8 min at room temperature. After permeabilization by 0.2% Triton X-100, cells were washed with TBS and incubated with the first Abs in TBS containing 1% BSA at the concentration of 20 µg/ml. For the detection of the first Abs, 25 µg/ml of FITC-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA) was used. Confocal microscopy was performed on Zeiss LSM410 model confocal microscopes (Oberkochen, Germany). No immunofluorescence signal was detected in cells stained with negative control reagents (data not shown).

Metabolic labeling

293T cells on the 100-mm tissue culture dish were transfected with Lck constructs using calcium phosphate method. Thirty-six hours after transfection, 293T cells were labeled with 0.5 mCi [3H]myristate (New England Nuclear, Boston, MA) in DMEM containing 5% dialyzed FCS and 5 mM sodium pyruvate for 3 h. The labeled cells were solubilized in Triton X-100 lysis buffer, and the lysates were immunoprecipitated with anti-Lck mAb, MOL171 and analyzed by SDS-PAGE.

Immunoprecipitation and immunoblotting analysis

Immunoprecipitation and immunoblotting analysis were performed as previously described (20). Briefly, the samples were electrophoresed in 8% SDS-PAGE and electroblotted onto polyvinylidene fluoride (PVDF) membrane (Immobilon-P; Millipore, Bedford, MA). Membranes were blocked in PBS containing 5% nonfat dried milk for 2 h, and blotted with anti-Lck mAb, MOL171. Membranes were then washed with PBS-0.1% Tween 20 and incubated with HRP-conjugated goat anti-mouse IgG (Chemicon International, Temecula, CA). After washing with PBS-0.1% Tween 20, membranes were developed using ECL system (Amersham, Buckinghamshire, U.K.).

CD69 expression

To assay for the induction of CD69 expression, 2 x 105 cells of the appropriate Lck-transfected JCaM1.6 clone were stimulated either with immobilized anti-CD3 plus 100 pg/ml of PMA or with 10 ng/ml of PMA in a 24-well plate for 16 h. The cells were then stained with FITC-conjugated anti-CD69 mAbs, and analyzed with a FACSort (Becton Dickinson, Mountain View, CA), as previously described (21). For preparation of anti-CD3 coated plate, purified mAbs were diluted to 10 µg/ml with PBS, and 100 µl was added per microtiter well. Plates were incubated at 37°C for 1 h and then washed three times with PBS before use.

Phosphorylation

JCaM1.6-derived transfectants (5 x 106) were stimulated with purified OKT3 (5 µg/ml) for 2 min at 37°C. Stimulated cells were harvested, washed, and solubilized in Triton X-100 lysis buffer with 1 mM sodium orthovanadate. After nuclei were removed by centrifugation, lysates were mixed with 2x sample buffer. The samples (2 x 105 cell equivalent/lane) were electrophoresed and electroblotted onto PVDF membrane. Membranes were blocked, incubated with 1 µg/ml of 4G10 followed by HRP-conjugated protein A (Amersham). After washing, membranes were developed using the ECL system.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ser6 of Lck is necessary for membrane localization

To explore how individual amino acids within the Lck SH4 domain affect the localization and function of Lck, we generated two Lck mutants in which either Ser6 or Ser7 were substituted by an alanine (A6 and A7, Table IGo). As controls, we also generated A2 and S3 Lck mutants (Table IGo), in which the site of myristoylation (Gly2) and that of palmitoylation (Cys3) were replaced by an alanine and a serine, respectively. Localization of the Lck mutants was first analyzed by transient expression in COS-7 cells and subcellular fractionation (Fig. 1GoA). Wild-type Lck was almost totally membrane associated, whereas the A2 mutant was completely cytosolic. Mutation of Cys3, the site known to be the major determinant for palmitoylation, resulted in a decrease of Lck localization to the membrane fraction, which is consistent with previous findings (11, 22). Interestingly, although the A6 mutant contained both Gly2 and Cys3, it was mainly found in the cytosolic fraction, whereas the A7 mutant was detected in the membrane fraction as per wild-type Lck. This result was confirmed by immunofluorescence and confocal microscopy (Fig. 1GoB). Previously, it has been shown that Lck expressed in nonlymphoid cells localizes to the plasma membrane and the perinuclear region (8, 22), and this staining pattern was similarly observed in COS-7 cells expressing S3, A7, and wild-type Lck. In contrast, the A2 and A6 mutants were found diffusely throughout the cytoplasm. These results clearly demonstrated that the Ser at position 6 is essential in targeting Lck to the plasma membrane.


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  		Table I. Schematic representation of mutant Lck constructs1

 


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  		FIGURE 1. Subcellular localization of mutant Lck expressed in COS-7 cells. COS-7 cells were transiently transfected with the Lck cDNA constructs indicated. A, Cells were disrupted by homogenization in hypotonic buffer and then separated into soluble cytosolic (S) and membrane pellet (P) fractions by ultracentrifugation. Equal aliquots from the two fractions were then resolved by 8% SDS-PAGE, transferred onto a PVDF membrane and probed with anti-Lck Abs. B, Cells were fixed with formaldehyde, permeabilized, and then stained for expression of Lck. Cells were observed by confocal microscopy.

 
Replacement of Ser6 in Lck results in reduced myristoylation

Because myristoylation is known to be critical for plasma membrane targeting of Lck (15), we next analyzed whether the A6 mutant can be myristoylated. 293T cells were transfected with wild-type, A2, and A6 Lck constructs, and labeled with [3H]myristate. Equivalent levels of labeling were observed in each transfectant as analyzed by SDS-PAGE using whole cell lysates (Fig. 2GoA). In contrast to wild-type Lck, which was efficiently labeled with [3H]myristate, [3H]myristate incorporation was not detected in the A2 mutant and drastically reduced in the A6 mutant. Calculation of relative myristoylation showed that mutation of Ser6 reduced myristoylation of Lck by 66% (Fig. 2GoB), suggesting that the defect of the A6 mutant in membrane association is due to its reduced level of myristoylation.



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  		FIGURE 2. Myristoylation of wild-type and mutant Lck. A, 293T cells transfected with wild-type and mutant Lck constructs were labeled with [3H]myristate. The labeled cells were solubilized with lysis buffer, and the lysates were immunoprecipitated with anti-Lck mAb and analyzed by SDS-PAGE. To check that equivalent levels of Lck were present in each immunoprecipitate, aliquots of the samples were probed with anti-Lck Abs. To ensure equivalent levels of [3H]myristate labeling, aliquots of whole cell lysates from each of the labeled cells were analyzed by SDS-PAGE. The proteins migrating at 56 kDa (indicated by an open arrowhead) are likely to be Lck, whereas the proteins migrating at 40 kDa (indicated by a filled arrowhead) cannot be specified. B, Quantitation of [3H]myristate-labeled wild-type and mutant Lck by densimetry. Data are from two independent experiments.

 
Replacement of Ser6 in Lck results in a defect of TCR{zeta} phosphorylation in COS-7 cells

We next examined the function of the Lck mutants expressed in COS-7 cells. COS-7 cells were transiently transfected with cDNAs encoding the various Lck mutants and TCR{zeta} cDNA. As shown in Fig. 3Go, wild-type Lck and the A7 mutant were able to phosphorylate TCR{zeta} chains, whereas the A2 mutant was not. The A6 mutant induced faint tyrosine phosphorylation of TCR{zeta}. Immunoblotting analysis revealed that the expression levels of TCR{zeta} were comparable between wild-type Lck and these mutants. Thus, the result indicated that the A6 mutant, which did not stably associate with the plasma membrane, failed to phosphorylate TCR{zeta} expressed in COS-7 cells.



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  		FIGURE 3. Induction of TCR{zeta} phosphorylation by wild-type and mutant Lck expression in COS-7 cells. COS-7 cells were transiently transfected with vectors encoding the various Lck mutants in the presence of TCR{zeta} cDNA. At 48 h after transfection, the cells were lysed, and the lysates were immunoprecipitated with anti-TCR{zeta} mAb. The immunoprecipitates were blotted with anti-phosphotyrosine mAb, 4G10. To check that equivalent levels of TCR{zeta} were present in each immunoprecipitate, aliquots of the samples were probed with anti-TCR{zeta} Abs. To check that equivalent levels of Lck were present in each transfectant, aliquots of whole cell lysates were probed with anti-Lck Abs.

 
Effect of replacement of Ser6 in Lck on T cell function

To assess the role of Ser6 of Lck in T cell function, we established JCaM1-derived transfectants expressing wild-type Lck or the various Lck mutants. JCaM1, a mutant Jurkat cell line, expresses a defective form of Lck due to a splicing defect in the lck gene (3). We asked whether the Lck mutants can reconstitute TCR-mediated signaling events in JCaM1 T cells. JCaM1 cells were transfected with the various mutant Lck constructs and stable clones expressing levels of transfected Lck similar to that of wild-type Lck were selected for analysis. At least two cell lines were established from each mutant Lck construct, and similar results were obtained with them.

Localization of the Lck mutants in JCaM1-derived transfectants was analyzed by subcellular fractionation. As shown in Fig. 4Go, the result of subcellular fractionation from stable clones was almost identical with that from transiently transfected COS-7 cells. Replacement of Ser6 shifted Lck into the soluble fraction, whereas that of Ser7 did not. Over 80% of the protein was detected in the soluble fraction in the A6 mutant. Intriguingly, a slower mobility form of Lck in SDS-PAGE was clearly detected for the A2 and A6 mutants. This form of Lck was also increased in COS-7 cells expressing the A2 and A6 mutant (Fig. 1GoA). It is currently unknown whether the generation of this form of Lck is due to lack of myristoylation or differential phosphorylation. Immunofluorescence analysis using the stable clones did not show a clear picture as shown in Fig. 1GoB, presumably because a truncated form of Lck produced in JCaM1 cells affected the staining pattern for transfected Lck proteins (data not shown).



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  		FIGURE 4. Subcellular localization of wild-type and mutant Lck expressed in JCaM1-derived transfectants. JCaM1 cells were stably transfected with the Lck cDNA constructs indicated. Localization of mutant Lck was analyzed as described in Fig. 1GoA.

 
Using these transfectants, we analyzed the tyrosine phosphorylation of intracellular proteins after TCR cross-linking. Jurkat, JCaM1, and JCaM1-derived transfectants were stimulated with anti-CD3{epsilon} mAb OKT3, and whole cell lysates were analyzed by immunoblotting with anti-phosphotyrosine mAb (Fig. 5Go). Tyrosine phosphorylation of proteins likely to be Cbl (120 kDa), Vav (100 kDa), SLP-76 (76 kDa), ZAP-70 (70 kDa), LAT (36 kDa), and TCR{zeta} (23 kDa) was almost abrogated in JCaM1 cells, but these phosphorylated proteins were clearly recovered in the transfectant expressing wild-type Lck. Importantly, the recovery of these phosphorylated proteins was not observed in the A2, S3, and A6 mutants. The inhibition of tyrosine phosphorylation of cellular proteins in the S3 mutant is surprising in view of the fact that 30% of the protein was localized to the plasma membrane (Fig. 4Go). Reconstitution of the A7 mutant in JCaM1 cells restored the tyrosine phosphorylation of cellular proteins.



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  		FIGURE 5. Tyrosine phosphorylation of cellular proteins in Jurkat, JCaM1, and JCaM1-derived transfectants expressing wild-type and mutant Lck. Cells were stimulated either with normal mouse IgG (NMIgG) or with anti-CD3{epsilon} mAb (OKT3) for 2 min, and lysed in lysis buffer. Postnuclear lysates were electrophoresed in 10% SDS-PAGE under reducing conditions and analyzed by immunoblotting with anti-phosphotyrosine mAb, 4G10. Molecular sizes (kDa) are shown on the left.

 
The expression of cell surface marker CD69, a marker for late activation events in T cell activation, is up-regulated following anti-TCR or PMA stimulation, and is known to require the activation of Ras (23). To investigate the ability of the various Lck mutants to reconstitute late signaling events in JCaM1 cells, stable clones were stimulated with OKT3 or PMA and the expression of CD69 was analyzed (Fig. 6Go). Wild-type Lck induced expression of CD69 as expected. Both the S3 and A7 mutants were able to induce the expression of CD69, although the levels of induced expression in S3 mutant-transfected cells were reduced compared with wild-type Lck. The A2 and A6 mutants were completely unable to induce CD69 expression following TCR stimulation. However, if the initial Lck activation following TCR stimulation was bypassed by PMA stimulation, all transfectants were able to express CD69 on their cell surface (Fig. 6Go, right).



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  		FIGURE 6. CD69 expression in JCaM1-derived transfectants expressing wild-type and mutant Lck. Cells were stimulated either with immobilized anti-CD3{epsilon} (OKT3) plus 100 pg/ml of PMA or with 10 ng/ml of PMA. The cells were stained with FITC-conjugated anti-CD69 mAbs, and analyzed by flow cytometry. Dotted lines and solid lines represent cells without and with staining, respectively.

 
Taken together, the experiments using JCaM1-derived transfectants demonstrated that Ser at position 6 is important for membrane association of Lck in T cells, and the Lck protein which has a mutation at this site cannnot reconstitute early and late signaling events after TCR stimulation in JCaM1 cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Various members of the Src family of protein tyrosine kinases have been implicated in the regulation of cell growth and differentiation. Lck is a member of the Src family kinases expressed predominantly in T cells, and plays a pivotal role in T cell activation and in T cell differentiation. It is well documented that in all members of the Src family kinases, including Lck, myristic acid is attached to the NH2-terminal glysine through an amide linkage, and this lipid modification is essential for membrane localization and function (10, 15). Recently, additional amino acids in the SH4 domain of the Src family kinases have been shown to contribute to myristoylation at glysine 2 (13, 14). Replacement of lysine 7 in Src or that of lysine 7 and 9 in Fyn resulted in a drastic reduction in myristoylation, indicating that these residues are critical determinants for efficient myristoylation of the Src family kinases. Indeed, nearly all Src family kinases contain lysine 7 in their N-terminal SH4 domain (15). However, Lck is the only Src member that lacks both lysine 7 and 9. It was speculated that serine 6 in Lck could have a regulatory role in myristoylation like lysine 7 in the other Src family kinases, because this residue is conserved in all known yeast proteins that are myristoylated and substitution of this serine with an alanine greatly decreases their affinity for NMT, an enzyme responsible for N-myristoylation (12). However, the direct evidence for the importance of the serine 6 in Lck has not been presented until now. Our data clearly demonstrated that the serine 6 in Lck is essential for efficient myristoylation. Our data also showed that replacement of serine 6 impairs membrane localization of Lck, resulting in a defective function in T cells.

Protein myristoylation is catalyzed by NMT, and is now recognized to be a widespread and functionally important modification of proteins (12). Several viruses have been shown to require myristoylation of structural and nonstructural proteins for viral assembly and replication. Mutations that render NMT inactive are lethal in yeast Saccharomyces cerevisiae and in fungus Candida albicans. In mammalians, two different NMT isoforms have been characterized (24). The substrate specificity of NMT has been determined in in vitro assay by combining more than 100 synthetic peptides and S. cerevisiae NMT (25). According to this study, the serine 6 increases the affinity for the peptide for NMT by several orders of magnitude, possibly by formation of a hydrogen bond with a negatively charged residue present in NMT’s peptide binding site. It has also been shown that the peptide substrate specificities of fungal and human NMTs have diverged, suggesting the possiblity to design peptide-based inhibitors of fungal NMTs that could function as antifungal agents. Because the peptide sequence of the SH4 domain for NMT in Lck is quite different from that in the other Src family kinases and N-myristoylation is absolutely required for membrane localization and function of Lck in T cells, it may be possible to develop peptide-based immunosuppressive agents capable of inhibiting Lck function in T cells by way of competing for N-myristoylation. Studies are now in progress to analyze whether NMT could be a target for immunosuppressive drugs.

It is well established that Lck is noncovalently associated with CD4 or CD8, and this association is important for optimal T cell activation. These membrane proteins have been believed to guide membrane association of Lck. However, a recent study demonstrated that neither CD4, CD8, nor other T cell-specific proteins are required for the plasma membrane localization of Lck (8). Rather, Lck has intrinsic signals for targeting to the plasma membrane. Several experimental systems have now shown that the lipid modifications, myristoylation and palmitoylation, in the SH4 domain of Lck are necessary and sufficient for membrane localization (8, 9). The result using COS-7 cells in this study (Fig. 1Go, A and B) are in line with these observations. Although both myristoylation and palmitoylation are necessary to confer localization of Lck to the plasma membrane, palmitoylation is considered to be a second signal to myristoylation because it has been found that blocking myristoylation prevents palmitoylation (26). The results using A2 and A6 Lck mutants in this study indicated a preferential role of myristoylation for membrane localization of Lck.

Earlier study demonstrated the importance of myristoylation at glysine 2 of Lck in T cell function (27). Recently, Kabouridis et al. (28) reported that palmitoylation of Lck at cysteine 3 and 5 is essential for its signaling function in T cells. These lipid modifications, myristoylation and palmitoylation, not only allow migration of Lck to the plasma membrane, but confer its localization to glycolipid-enriched membrane (GEM) microdomains, or lipid rafts (9, 29). A recent flood of information points to the GEM domain as the critical site for TCR-mediated signal transduction (17, 30, 31, 32, 33). In T cells, molecules essential for transduction of the TCR signal, such as Lck and LAT, are shown to localize to the GEM domain by way of palmitoylation (29, 34). Indeed, the S3 Lck mutant, in which cysteine 3 was replaced by a serine, failed to phosphorylate intracellular proteins in JCaM1 cells (Fig. 5Go), although it was expressed in part at the plasma membrane (Fig. 4Go). Investigating the regulatory mechanism for the lipid modifications of signaling molecules may contribute to a better understanding for the role of the GEM microdomain in TCR signal tarnsduction.


   Acknowledgments
 
We thank Dr. Makio Iwashima for JCaM1 cells; Dr. Yasuhiro Minami for mouse lck cDNA; Miki Enomoto for technical assistance; and Douglas R. Liddicoat for a critical review of the manuscript.


   Footnotes
 
1 This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan (11670319), by a Research Grant from the Ryoichi Naito Foundation for Medical Research, and by a grant provided by the Ichiro Kanehara Foundation. Back

2 Address correspondence and reprint requests to Dr. Atsushi Kosugi, School of Allied Health Science, Faculty of Medicine Osaka University, 1-7, Yamada-oka, Suita, Osaka 565-0871, Japan. Back

3 Abbreviations used in this paper: ITAM, immunoreceptor tyrosine-based activation motif: SH, Src homology: NMT, N-myristoyl transferase; PVDF, polyvinylidene fluoride. Back

Received for publication February 14, 2000. Accepted for publication June 26, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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