Lack of Palmitoylation Redirects p59Hck from the Plasma Membrane to p61Hck-positive Lysosomes*

Hck, a protein-tyrosine kinase of phagocytes, is the unique member of the Src family expressed under two alternatively translated isoforms differing in their N-terminal site of acylation: p61Hck has an additional 21-amino acid sequence comprising a single myristoylation motif, whereas p59Hck N terminus has myristoylation and palmitoylation sites. To identify the molecular determinants involved in the targeting of each isoform, they were fused to GFP and expressed in HeLa and CHO cells. p61Hck was associated with lysosomal vesicles, whereas p59Hck was found at the plasma membrane and to a low extent associated with lysosomes. Their unique N-terminal domains were sufficient to target GFP to the corresponding intracellular compartments. Mutation of the palmitoylation site of p59Hckredirected this isoform to lysosomes, indicating that the palmitoylation state governs the association of p59Hck with the plasma membrane or with lysosomes. In addition, both isoforms and the nonpalmitoylated p59Hck mutant were found on the Golgi apparatus, suggesting a role of this organelle in the subcellular sorting of Hck isoforms. Regarding their subcellular localizations, we propose that bi-acylated p59Hck might transduce plasma membrane receptor signals, whereas p61Hck and the nonpalmitoylated p59Hck might control the biogenesis of phagolysosomes, two functions yet proposed for Hck in phagocytes.

Protein-tyrosine kinases of the Src family (Src PTKs) 1 are cytosolic proteins, which are key elements of the signaling cascade from the surface to the interior of the cell. Three of these kinases, Src, Fyn, and Yes, are ubiquitously expressed, whereas Blk, Fgr, Hck, Lck, and Lyn are restricted to hematopoietic cells (Ref. 1; for a review see Ref. 2). Src PTKs are composed of distinct functional regions. The first three domains display high sequence homology among members of the Src family: the Src homology domain 1 (SH1), which lies at the C terminus of the molecule is responsible for the catalytic activity of the kinase. Two other Src homology domains, SH2 and SH3, allow interactions with cellular proteins through recognition of phosphotyrosine-and proline-rich domains, respectively (3,4). The fourth domain is unique to each member of the family and contains the N-terminal motif MGXXX(S/T) that is myristoylated on glycine 2. Most of the kinases are also palmitoylated when displaying a cysteine residue at position 3, 5, or 6 (5). Fatty acylation of Src PTKs has yet been shown to influence their interaction with cell membranes (6,7) and as a consequence their intracellular distribution.
Hck is a kinase of the Src family mainly expressed in phagocytes (8). In human neutrophils, Hck is mainly found in the azurophil granule (lysosomal) compartment and, to a low extent, in the plasma membrane-enriched fraction (9). These different locations correlate with functional roles proposed to Hck in the process of fusion between lysosomes and phagosomes (9 -11) and in signaling of membrane receptors (12)(13)(14)(15). However, two Hck isoforms exist, but the precise function exerted by each of them has not yet been determined. The two isoforms are generated by alternative translation; p61 Hck translation starts at a CTG, 21 codons upstream of the p59 Hck ATG codon (16). The two isoforms are myristoylated on glycine 2, but only p59 Hck has a palmitoylated cysteine at position 3. Therefore, Hck is the unique example among the Src PTKs to be expressed as two isoforms, which naturally differ by their N-terminal domains and by their acylation states. Consequently, one would expect to find Hck isoforms in distinct subcellular compartments. Indeed, p59 Hck cofractionates with the plasma membrane, and p61 Hck cofractionates with lysosomes, but, surprisingly, p59 Hck was also found in lysosomes (17). Thus, despite differences in their N-terminal domains, p59 Hck and p61 Hck can be addressed to the same compartment. Because the location of proteins certainly conditions their functional role, we searched to identify the molecular determinants involved in the membrane addressing of Hck isoforms. To this end, separate expression of p59 Hck and p61 Hck was undertaken in HeLa and CHO cells, and their subcellular distribution was determined. We also studied whether their respective N-terminal domains were sufficient to address the reporter protein GFP to the compartment corresponding to the full-length Hck isoforms. Palmitoylation, in contrast to myristoylation, is a reversible post-translational modification (6). A palmitoylation mutant of p59 Hck in fusion with GFP was constructed to examine the role of palmitate in the subcellular addressing of this isoform.

Plasmid Construction and Mutagenesis
The wild-type Hck cDNA was a gift from N. Quintrell (8). The CTG initiation codon of Hck was either deleted or mutated to an ATG codon by polymerase chain reaction amplification of the unique N-terminal * This work was supported in part by the Ministère de l'Education Nationale de la Recherche et de la Technologie, Appel d'offres Microbiologie and by Sidaction. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Cell Culture and Transfection
HeLa and CHO cells were cultured at 37°C, 5% CO 2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 1% L-glutamine, 100 IU/ml penicillin, and 100 g/ml streptomycin. HeLa and CHO cells were seeded in 24-well plates for immunofluorescence experiments (3⅐10 4 and 1⅐10 4 cells/well on glass coverslips, respectively) or in 9-cm Petri dishes (10 6 cells for HeLa cells) for immunoblotting, S100/P100 partitioning, Triton X-100 fractionation, and solubilization for kinase assay. The following day, DNA/calcium phosphate precipitates were added to the cells, which were washed after 16 -18 h and incubated in fresh medium for an additional period of 48 -72 h before analyses.

Isolation of Human Monocytes
White blood cells from healthy donors were isolated by Dextran sedimentation and centrifugation through Ficoll-Hypaque as described previously (11). The interface band containing mononuclear cells was removed, washed twice with ice-cold phosphate-buffered saline (PBS), and the cell pellet was resuspended in cold RPMI 1640 supplemented with 100 units/ml penicillin/streptomycin. Cells were then plated on glass coverslips inserted in 24-well tissue culture plates and incubated for 1 h at 37°C to allow adherence of monocytes. The nonadherent cells were removed, and the cell monolayer was washed vigorously twice with warm RPMI and used for immunofluorescence experiments 3 h later.

Subcellular Localization of Hck by Fluorescence Microscopy
Transfected cells were washed twice with PBS and fixed in 3.7% paraformaldehyde for 30 min at room temperature, and unreacted aldehyde groups were neutralized in 50 mM NH 4 Cl for 1 min. After washing and permeabilization (0.3% Triton X-100, 5 min), cells were washed for 10 min in PBS containing 1% bovine serum albumin. Coverslips were then overlaid as indicated with 25 l of one of the following primary antibodies diluted into PBS containing 1% bovine serum albumin:rabbit anti-human Hck (1:100; Santa Cruz biotechnology, Santa Cruz, CA), mouse anti-human CD63 (1:100; CLB, Amsterdam, Netherlands), mouse anti-human Golgi (CTR 433, 1:2; Ref. 18), and mouse anti-hamster lysosomal glycoprotein lgp95 (E9A, 1:100; Ref. 19). After a 30-min incubation period, the coverslips were washed three times in PBS and incubated for 30 min at room temperature with a 1:100 dilution of affinity purified TRITC-or fluorescein isothiocyanate-conjugated secondary antibodies (Sigma) directed against mouse or rabbit IgG. The coverslips were washed three times in PBS and mounted in Mowiol. The fluorescent staining was visualized under a confocal or a standard microscope.
In some experiments, the Golgi apparatus was disrupted before cell fixation, by treatment with 5 g/ml brefeldin A for 10 min (Sigma). In other experiments, the lysosomal pathway was labeled with 4 mg/ml lysine-fixable tetramethylrhodamine (molecular weight, 10,000; Molecular Probes, Leiden, Netherlands), which was added to HeLa cells 24 h after transfection and left in contact with the cells for 16 h at 37°C in complete medium (20).

Cell Lysis and Immunoblotting
72 h after transfection, HeLa cells (3.10 6 cells) were washed in PBS, lysed in 1 ml of reducing sample buffer (21), and boiled for 5 min. 1 ⁄10 of total proteins were electrophoresed through 8% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane, which was incubated with anti-Hck antibodies (1:2,000, Santa Cruz biotechnology, Santa Cruz, CA), and revealed by ECL as described previously (9).

Hck Tyrosine Kinase Assay
Proteins were solubilized in a buffer containing 2% Nonidet P-40, 3 g/ml leupeptine, 1 g/ml pepstatine, 1 M aprotinine, 500 M PMSF, and 100 M sodium vanadate. Hck was immunoprecipitated and its kinase activity was assayed using acid-denatured rabbit muscle enolase (Roche Molecular Biochemicals) as a nonspecific substrate and 10 Ci of [␥-32 P]ATP (6,000 Ci/mmol) as described previously (17). Proteins were then separated on 8% SDS-polyacrylamide gel electrophoresis, and Hck-dependent phosphorylation of enolase was observed by using the Image QuaNT programm on a Molecular Dynamic Storm840 imager.
The percentage of insoluble GFP fluorescence was calculated as follows: % P100 ϭ (P100 Ϫ P(o)100)/(S100 Ϫ S(o)100 ϩ P100 Ϫ P(o)100), where P100 and S100 are fluorescence values measured in the crude membrane fraction and in the cytosol, respectively, and P(o)100 and S(o)100 are the corresponding values obtained from parallel experiments performed with mock-transfected HeLa cells.
The percentage of insoluble GFP fluorescence was calculated as follows:

RESULTS
p59 Hck Is Associated with the Plasma Membrane-The cellular localization of p59 Hck and p61 Hck was studied here by using engineered cDNAs (Fig. 1) that allowed separate expression of each isoform in fusion with GFP ( Fig. 2A) First, we show that GFP in fusion with Hck isoforms did not interfere with the kinase activity (Fig. 2B) because these chimera were able to perform autophosphorylation and to phosphorylate the exogenous substrate enolase. In HeLa cells transiently expressing GFP, nuclei were strongly fluorescent and a diffuse staining was present in the cytoplasm (Fig. 3). Cells expressing p59 Hck -GFP showed a staining of the plasma membrane but not cells expressing p61 Hck -GFP (Fig. 3).
To further demonstrate that p59 Hck was stably associated with cell membranes, cells were fractionated, and the majority of the p59 Hck -GFP fluorescence was found in the membrane pellet P100 fraction (76 Ϯ 14%, n ϭ 3). In addition, part of p59 Hck -GFP fluorescence (39 Ϯ 3%, n ϭ 5) was found in the cold Triton X-100-insoluble fraction (Fig. 4) where detergent-resistant glycolipid-enriched membrane domains (GEMs) and cytoskeletal components are present (22,23). When the Triton X-100 extraction was performed at 37°C to solubilize proteins of GEMs but not those of the cytoskeleton (23), the fluorescence associated with the detergent insoluble fraction returned to background levels (Fig. 4). Moreover treatment of the cells with ␤-methyl-cyclodextrin, a chemical agent that destabilizes GEMs by chelating cholesterol (24), allowed the redistribution of about 50% of the fluorescence from the detergent-insoluble to the detergent-soluble fraction (Fig. 4). These results from fluorescence microscopy and cell fractionation indicate that p59 Hck is expressed at the plasma membrane and that part of it is present in GEM structures rather than in the cytoskeleton compartment.
Both p59 Hck and p61 Hck Are Associated with the Golgi Apparatus-In addition, an intense juxta-nuclear staining was obtained with both isoforms (Fig. 3). Staining of the Golgi apparatus with the specific antibodies CTR433 (18) and TRITCconjugated secondary antibodies revealed its colocalization with p59 Hck -GFP or p61 Hck -GFP (Fig. 5). Although p59 Hck -GFP was undoubtedly associated with the Golgi, confocal microscopy was necessary to ascertain the association of p61 Hck with the Golgi (Fig. 5, G-I). Similarly, the Golgi apparatus of human monocytes that naturally express Hck, was stained with anti-Hck antibodies (Fig. 5, J-L).
p61 Hck Is Associated with Perinuclear Vesicles of the Lysosomal Pathway-The juxta-nuclear localization of p61 Hck -GFP was not restricted to the Golgi because it was also found on surrounding vesicles (Fig. 5, G-I, arrowheads), which persisted when the Golgi apparatus was redistributed after cell treatment with brefeldin A (data not shown). In a previous study we have shown that Hck was partially localized on lysosomes of phagocytes (17). To verify whether the above vesicular structures belonged to the endosome-lysosome pathway, transfected HeLa cells were incubated for 16 h with rhodamine-dextran without chase of the fluorescent marker to stain the whole endocytic pathway. The vesicles stained by p61 Hck -GFP, which contained rhodamine-dextran were close to the nucleus, as expected for late endosomes-lysosomes (Fig. 6, A-C), on the contrary vesicles positive for rhodamine-dextran adjacent to the plasma membrane, the location of early endosomes, were not labeled by Hck. In addition, p61 Hck -GFP partially colocalized with CD63, a marker of the late endosome-early lysosome compartment (Fig. 6, D-F) (25). Finally, to confirm the association of p61 Hck -GFP with lysosomes, we took advantage of a subset of CHO cells that contain large lysosomes identified by the presence of the lysosomal marker lgp95 (19) (Fig. 7, A-C). p59 Hck -GFP was clearly associated with the plasma membrane of these cells (Fig. 7, D-I), and in some cases (Ͻ5% of the cells), an association of this isoform with lgp95-positive lysosomes was observed (Fig. 7, G-I).
The Unique Domains of p59 Hck and p61 Hck Are Sufficient to Target Each Isoform to Their Respective Subcellular Localization-Next, we investigated the role of the unique domains in the subcellular targeting of p59 Hck and p61 Hck . For this purpose, chimeric cDNA comprising the unique domain of p59 Hck or p61 Hck , fused at their C terminus with the GFP gene (p59U-Hck-GFP and p61U-Hck-GFP, respectively) were constructed (Fig. 1). Observation by direct fluorescence microscopy of HeLa cells transfected p59U-Hck-GFP or p61U-Hck-GFP revealed an overall cellular staining identical to that obtained with the corresponding full-length constructs (Fig. 8). Double labeling experiments with the appropriate antibodies indeed confirmed that both p59U-Hck-GFP and p61U-Hck-GFP were present on the Golgi apparatus (Fig. 8) and that p61U-Hck-GFP was on the CD63 positive endosomal-lysosomal compartment (Fig. 8).
The plasma membrane localization of p59U-Hck-GFP was also clearly visible by direct fluorescence, and Triton X-100 solubi- FIG. 5. Both Hck isoforms are associated with the Golgi apparatus. p59 Hck -GFP or p61 Hck -GFP were visualized by direct fluorescence on standard microscope (D-F) or confocal microscope (A-C and G-I). The Golgi apparatus was stained by CTR433 antibodies revealed by TRITC-conjugated secondary antibodies. Colocalization (arrows) of p59 Hck -GFP and p61 Hck -GFP with the Golgi apparatus appeared in yellow in merged images. p61 Hck -GFP was also present on vesicles surrounding the Golgi apparatus (arrowheads). Freshly isolated human monocytes were fixed and permeabilized, and double staining was performed using Hck antibodies and CTR433 antibodies revealed by fluorescein isothiocyanate-and TRITC-conjugated secondary antibodies, respectively. Part of cellular Hck colocalized (arrows) with the Golgi and part of Hck were present on cytoplasmic vesicles as observed by standard fluorescence microscopy (arrowheads).

FIG. 6. p61 Hck -GFP is associated with lysosomes in HeLa cells.
HeLa cells expressing p61 Hck -GFP were examined by confocal microscopy. Cells having internalized lysine-fixable rhodamine-dextran for 14 h are shown in A-C. Colocalization of p61 Hck -GFP with rhodaminedextran positive endo-lysosomal vesicles appeared in yellow (C). Colocalization of p61 Hck with rhodamine-dextran positive vesicles was close to the nucleus (arrows), there was no colocalization of p61 Hck -GFP with endosomes near the plasma membrane (arrowheads). Staining of the late endosome-early lysosome compartment was realized using anti-CD63 antibodies (E) revealed by TRITC-conjugated secondary antibodies. Colocalization of p61 Hck -GFP with CD63 positive vesicles appeared in yellow (F). lization experiments confirmed its association with GEMs (33% of the fluorescence in the detergent-insoluble fraction), whereas only 4% of p61U-Hck-GFP was found in this fraction. These findings support the conclusion that localization of each isoform of Hck to distinct subcellular compartments directly depends on determinants present in their unique domains, which differ by the additional 21 amino acids present in the longer isoform and by their state of mono-(p61 Hck ) or biacylation (p59 Hck ).
A Mono-acylated Mutant of p59 Hck Localizes to the Same Compartments as p61 Hck -To study the reason why p59 Hck is found in the lysosomal fraction in neutrophils and is sometimes associated with lysosomes in CHO cells, the role played by the acylation state was examined. To this aim, we constructed a mutant of p59 Hck (p59 Hck C3S-GFP) in which the cysteine residue at position 3, which is normally palmitoylated, was substituted for a serine (Fig. 1), a substitution previously shown to abolish palmitoylation of p59 Hck (26). Observation by direct fluorescence microscopy of HeLa cells expressing the p59 Hck C3S-GFP mutant showed the disappearance of the plasma membrane labeling previously seen with p59 Hck -GFP (Fig. 9, A and D). Furthermore, whereas about 40% of p59 Hck -GFP was present in the Triton X-100-insoluble fraction (Fig. 4), only 7% Ϯ 2 (n ϭ 3) of p59 Hck C3S-GFP remained in this fraction.
Similarly to p59 Hck and p61 Hck , the p59 Hck C3S-GFP mutant was found on the Golgi apparatus (Fig. 9, A-C). A punctuated staining was also clearly visible around the Golgi apparatus. By labeling the cells with either dextran-rhodamine (not shown) or CD63 antibodies, we observed that these vesicles corresponded to the endosome-lysosome compartment (Fig. 9, D-F). In addition, when p59 Hck C3S-GFP was expressed in CHO cells, a co-staining with the lysosomal marker, lgp95 was clearly observed in most of the cells (Fig. 9, G-I). These results demonstrate that p59 Hck C3S is targeted to the same compartments than p61 Hck and that the acylation state plays a critical role in the subcellular localizations. DISCUSSION In this study, we show that chimeras between p59 Hck or p61 Hck and GFP transfected into different cell lines are directed to separate subcellular compartments. Both isoforms are located on the Golgi apparatus. p59 Hck is mainly associated with the plasma membrane and to a low extent with lysosomes, FIG. 7. Association of p61 Hck -GFP with lysosomes and p59 Hck -GFP with the plasma membrane and lysosomes in CHO cells. CHO cells expressing indicated cDNA were observed by standard fluorescence microscopy. p61 Hck -GFP (A) was associated with the membrane of large lysosomes stained with anti-lgp95 antibodies (B). Colocalization of these two proteins appeared in yellow (arrows in C). p59 Hck -GFP was associated with the plasma membrane. Lysosomes were stained with lgp95 antibodies (E and H), and arrowheads show lysosomes not stained by p59 Hck -GFP in the merged image (F). Occasionally p59 Hck colocalized with lysosomal vesicles positive for lgp95 (I, arrows).
FIG. 8. The unique domain of p59 Hck or p61 Hck is sufficient to target GFP to the corresponding subcellular localization. Localization of p59U-Hck-GFP and p61U-Hck-GFP was examined by standard fluorescence microscopy. p59U-Hck-GFP was associated with the plasma membrane (A). Colocalization of p59U-Hck-GFP or p61U-Hck-GFP with the Golgi apparatus (B and E) appears in yellow in the merged images (C and F). Colocalization of p61U-Hck-GFP with lysosomes was assessed after staining of lysosomes with anti-CD63 antibodies plus TRITC-conjugated secondary antibodies. Arrows show p61U-Hck-GFP vesicles doubly labeled with anti-CD63 antibodies (I).
whereas p61 Hck is specifically on the lysosomal compartment. Furthermore, we also demonstrate that the differential localization of Hck isoforms solely rely on their unique N-terminal domains, their acylation state is a critical determinant for addressing Hck to the plasma membrane or to lysosomes. These findings may have important implications for the functions of each Hck variant.
Addition of GFP at the C-terminal end of Hck isoforms was used to improve the microscopic detection. It did not affect their kinase activity nor their cellular localization because the distribution of p59 Hck -GFP and p61 Hck -GFP was identical to that of p59 Hck and p61 Hck expressed in HeLa cells and visualized by using anti-Hck antibodies (data not shown). Hck was expected to be present at the plasma membrane because it has been shown to be activated by several membrane receptors and co-immunoprecipitated with some of them (14,15,27,28); however, this localization has not yet been clearly demonstrated. Indeed, immunofluorescence microscopy did not allow to detect Hck at the plasma membrane of neutrophils, probably because low levels of the kinase are present in the "granulefree, plasma membrane-enriched fraction" (17), a fraction that also contains Golgi and endoplasmic reticulum markers (29). In addition, co-fractionation of Hck and caveolin in transfected fibroblasts (26) suggested that Hck could be a plasma membrane-and/or a Golgi vesicle-associated protein because caveolin is present in these two compartments (22,23). Here we demonstrate by direct fluorescence microscopy that p59 Hck -GFP is present at the plasma membrane and on the Golgi apparatus. In addition, we show that the palmitoylation mutant p59 Hck C3S disappeared simultaneously from the plasma membrane and from the cold Triton X-100 insoluble fraction (GEMs; Ref. 30) but remained associated with the Golgi apparatus. Thus part of bi-acylated p59 Hck is in GEMs of the plasma membrane. Actually, Hck can be activated by lipoarabinomannans, molecules from mycobacteria that have a phosphatidylmyo-inositol anchor and interact with GEMs (31). Furthermore association of Hck with plasma membrane receptors (14,15,27,28) among which GPI-anchored receptors (32) particularly concentrated in GEMs has been reported. We suggest that biacylated p59 Hck could be the isoform involved in the signaling of plasma membrane receptors, thereby in chemotaxis, because some of Hck associated receptors are implicated in the adhesion-migration process (33,34).
We have previously demonstrated that Hck is present on lysosomes of human neutrophils and macrophages (11,17), both isoforms of Hck being present on neutrophil lysosomes (17). In this study we show by colocalization with the lysosomal markers CD63 in HeLa cells and lgp95 in CHO cells and with the marker of the endosomal-lysosomal pathway rhodaminedextran that expression of p61 Hck -GFP in nonphagocytic cells leads to its association with the lysosomal compartment. This localization again correlates with the acylation state of the kinase isoforms because mono-acylated forms of Hck, either the native p61 Hck isoform or the palmitoylation mutant p59 Hck C3S, are found on such structures.
Although addition of a myristate on Src family tyrosine kinases is an irreversible co-translational event, palmitoylation is a reversible and regulated post-translational mechanism (6,35). Thus, as observed for other Src PTKs (36,37), at least two pools of p59 Hck exist in a cell: a mono-acylated (myristoylated) one and a bi-acylated (myristoylated and palmitoylated) one. We propose that the part of p59 Hck that co-fractionates with lysosomes in human neutrophils (17) or the part of p59 Hck -GFP that is present on lysosomes in CHO cells (this report) or HeLa cells (data not shown) represents the nonpalmitoylated form of p59 Hck . Similarly, c-Src that is naturally only myristoylated, is associated with synaptic vesicles in neuronal cells (38,39), with endosomes in fibroblasts (40), or with intracellular vesicles in osteoclasts (41). Mutation of the palmitoylation site of Fyn also resulted in its redistribution from the plasma membrane to yet unidentified punctuated intracellular structures (37).
We have previously proposed that Hck could be involved in the process of mobilization fusion of lysosomes with phagosomes in human neutrophils and macrophages (9 -11). Indeed, upon phagocytosis-induced mobilization of lysosomes, Hck translocates to phagosomes and is activated solely in lysosomes and not in the plasma membrane-enriched fraction (9). In contrast, when phagocytosis is not coupled to fusion of lysosomes with phagosomes (i.e. ingestion of mycobacteria (10) or mannosylated latex beads (11)), Hck does not translocate to the phagosomes and is not activated. Now we suggest that p61 Hck and nonpalmitoylated p59 Hck , both of which are present in the lysosomal compartment, could be involved in the process of phagolysosome biogenesis.
When Hck is not acylated, it is exclusively found in the cytosol (26). Myristoylation directs both Hck isoforms to the Golgi and lysosomes but not to the endoplasmic reticulum (data not shown) stained with the well characterized antiserum of Louvard et al. (42), indicating a targeting to specific organelles. Myristoylation is necessary but probably not sufficient for this specific membrane anchoring because all the myristoylated proteins are not necessarily associated with the Golgi apparatus and lysosomes. Other information contained in the common FIG. 9. Mutation of the palmitoylation site of p59 Hck redirects the protein to lysosomes. HeLa cells expressing p59C3SHck-GFP and stained with either anti-Golgi (B) or anti-CD63 (E) plus TRITCconjugated secondary antibodies were examined by standard microscopy (A-C) or confocal microscopy (D-F). Colocalization of p59C3SHck-GFP with Golgi apparatus and lysosomal vesicles appears in yellow in the merged images (C and F). CHO cells expressing p59C3SHck-GFP were stained with anti-lgp95 antibodies revealed by TRITC-conjugated secondary antibodies and examined by standard fluorescence microscopy. Colocalization of p59C3SHck-GFP with lysosomal vesicles appears in yellow in the merged image (C). part of p59 Hck and p61 Hck unique domains are likely to participate in this subcellular addressing; for example, the first 10 amino acids of Lck contain the acylation sites and other putative targeting information sufficient to address reporter proteins to the Golgi apparatus and the plasma membrane (43).
In addition to their localization at the plasma membrane, Fyn and Lck are associated with the Golgi of transfected cells (44,45). The Golgi localization of Hck has a physiological relevance because the kinase was found on this organelle in human monocytes. It is tempting to speculate a role for the Golgi in the intracellular sorting of Hck isoforms. After synthesis in the cytoplasm on free ribosomes where co-translational myristoylation takes place (46,47), both isoforms would be targeted to the Golgi membrane and then sorted to their respective localizations (26,45,48). Mono-acylated forms of the kinase would then be directed to lysosomal compartments, whereas addressing of p59 Hck to the plasma membrane would depend on a palmitoyltransferase activity present in the Golgi (49,50). Therefore, sorting of Hck isoforms from the Golgi to their respective localizations implicates the existence of Hck-positive vesicles originating from the Golgi en route to lysosomes or to the plasma membrane.
In conclusion, Hck is the unique protein kinase of the Src family expressed under two alternatively translated isoforms differing by their acylation state. The bi-acylated form p59 Hck is addressed to the plasma membrane, whereas the monoacylated isoform p61 Hck and nonpalmitoylated p59 Hck form are targeted to lysosomes. This differential addressing might reflect the need of the same kinase at different sites. Hck is mainly expressed in phagocytes, which ensure host defense by chemotactic migration to the site of infection and expression of their bactericidal responses such as exocytosis of lysosomes. Because Hck has been implicated in these two functions, we suggest that p59 Hck could participate in phagocyte migration, whereas p61 Hck and the nonpalmitoylated form of p59 Hck could regulate phagolysosome biogenesis.