Reversible Palmitoylation of the Protein-Tyrosine Kinase ~56'”~*

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 12, Issue of April 25, pp. 8669-86’74.1993 Printed in U. S. A.

Reversible Palmitoylation of the Protein-Tyrosine Kinase ~ 5 6 ’ ” ~ * (Received for publication, December 1, 1992)

Lisa A. PaigeSOlI, Monica J. S. NadlerY, Marietta L. Harrison, John M. Cassady 11, and Robert L. Geahlen** From the Department of Medicinal Chemistry and Pharmacognosy, Purdue University, West Lafayette, Indiana 47907 and the IICollege of Pharmacy, The Ohio State University, Columbus. Ohio 43210

The myristoylated protein-tyrosine kinase, ~ 5 6 ” ~ , is expressed predominantly in T cells where it is believed to play a role in T cell activation. We observed a 56- kDa protein that became metabolically labeled in intact T lymphoid cells that were incubated with either [‘HI myristate or [’Hlpalmitate. This protein was identified as ~ 5 6 ” ~ based on its specific immunoprecipitation with polyclonal antisera to ~ 5 6 ” ~ , by induction of a shift in its electrophoretic mobility following treatment of cells with 12-O-tetradecanoylphorbol- 13-acetate and by co- chromatography with p56lCk on protamine-agarose. Characterization of the two acylation events revealed that, in contrast to the p56”’-associated radioactivity from [SH]myristate-labeled cells, the p56’ck-associated radioactivity from [‘Hlpalmitate-labeled cells was susceptible to cleavage by neutral hydroxylamine and was not blocked by inhibitors of protein synthesis. Pulse- chase analyses revealed that the labeling of ~ 5 6 ” ~ with [‘Hlpalmitate, but not [’Hlmyristate, was reversible. The presence of covalently attached palmitate on ~ 5 6 ’ “ ~ from [‘Hlpalmitate-labeled cells was verified by thin- layer chromatography following acid hydrolysis of the acylated protein. 2-Hydroxymyristate, which is metabolically activated to form a potent inhibitor of protein myristoylation, specifically inhibited the acylation of ~ 5 6 “ “ with [‘Hlmyristate without affecting its labeling with [‘Hlpalmitate. These studies indicate that ~ 5 6 “ ~ is both a cotranslationally myristoylated and posttranslationally palmitoylated protein.

Various members of the src family of protein-tyrosine kinases have been implicated in the regulation of cell growth and differentiation. One such member, the 56-kDa protein encoded by the lck gene, ~56’~, is expressed predominantly in T cells. ~ 5 6 ‘ ~ associates with and is believed to receive and transmit signals involving the CD4 and CD8 surface glyco- proteins (1,2). During T cell activation, CD4 and CD8 interact with class I1 and class I major histocompatibility proteins, respectively. The association of p56” with CD4 and CD8 is mediated through a 32-amino acid region present at the amino

* This research was supported by Public Health Service Grants CA45667 (to R. L. G. and J. M. C.) and GM48099 (to M. L. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

3 Supported by Predoctoral Training Grant 5 T32 GM08298 awarded by the National Institutes of Health.

§Present address: Dept. of Cellular and Development Biology, Harvard University, 16 Divinity Ave., Cambridge, MA 02138.

li The first two authors contributed an equal quantity of work toward the completion of this project.

** To whom correspondence should be addressed. Tel.: 317-494- 1457; Fax: 317-494-6790.

terminus of ~ 5 6 ” ~ (3) and a short sequence present within the cytoplasmic domains of CD4 and CD8 (3-5).

~ 5 6 ” ~ , like other members of the src family, is covalently modified by the 14-carbon saturated fatty acid, myristic acid, coupled to its amino-terminal glycine residue (1, 6, 7). My- ristoylation of ~ 5 6 ~ ~ has been shown through mutational analyses to affect membrane localization of ~ 5 6 ‘ ~ molecules (7). Nonmyristoylated variants of ~ 5 6 “ ~ are unable to stably interact with the plasma membrane and remain predominantly cytosolic (7). Nonmyristoylated ~ 5 6 ’ ~ also exhibits impaired tyrosine kinase activity. This impairment is associated with a decrease in phosphorylation at Tyr-394, the major site of autophosphorylation (7). It is therefore likely that myristoylation directs ~ 5 6 ’ ~ to the appropriate environment for the stimulation of Tyr-394 phosphorylation and subse- quent activation.

Recently, we have obtained evidence that ~ 5 6 ’ ~ is also palmitoylated. Modification of proteins with the 16-carbon saturated fatty acid, palmitic acid, is both temporally and chemically distinct from protein myristoylation. Palmitoyla- tion is a posttranslational, reversible process, while myristoylation is cotranslational and irreversible (although some ex- ceptions exist (8)). Additionally, palmitate is attached to proteins via an oxy- or thioester linkage to serine, threonine, or cysteine residues (9-12), while myristate is attached via the more stable amide linkage to the amino-terminal glycine residue (13, 14). Palmitate has been found attached to the structural proteins of enveloped DNA and RNA viruses (15- 17), the insulin (18), transferrin (19), and &adrenergic (20) receptors, p21” and other members of the Ras superfamily (21-25), and CD4, the receptor to which ~ 5 6 “ ~ is associated (26). As the majority of palmitoylated proteins are membrane- associated, it would appear that palmitate serves as a membrane-anchoring device (27). Certain members of the Ras family interact less stably with the plasma membrane if they are not palmitoylated (28). However, some palmitoylated proteins are integral membrane proteins that do not require the attachment of palmitate for membrane insertion (27,29). For the &adrenergic receptor, palmitoylation appears to play a role in mediating the interaction between the receptor and the GTP-binding protein (20), and palmitoylation of the transferrin receptor appears to play a role in regulating receptor-mediated endocytosis (30).

We describe here a characterization of the modification of ~ 5 6 ’ ~ with palmitic acid, which indicates that ~ 5 6 ‘ ~ is modified both by irreversible, cotranslational myristoylation and reversible, posttranslational palmitoylation.

EXPERIMENTAL PROCEDURES

Materi~ki-[~H]Myristic acid (39.3 Ci/mmol) and [3H]palmitic acid (30-60 Ci/mmol) were purchased from Du Pont-New England Nu- clear. 12-O-Tetradecanoylphorbol-13-acetate (TPA)’ was purchased

The abbreviations used are: TPA, 12-O-tetradecanoylphorbol-13- acetate; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis.

c

8669

8670 Palmitoylation of ~ 5 6 ' ~

from LC Services. Cycloheximide, Protein A-Sepharose, bovine serum albumin (BSA), and celite were purchased from Sigma. 2-Hydroxy- myristic acid was purchased from Fluka. C18 reverse phase TLC plates were from Whatman.

Cultured Cells-LSTRA, a T cell lymphoma originally induced in BALB/c mice by the Moloney murine leukemia virus (31), and Jurkat, a human T cell line, were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 12 mM NaHC03, 100 IU/ ml penicillin G, 100 pg/ml streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 50 pM 2-mercaptoethanol, and 12.5 mM HEPES pH 7.4.

Metabolic Labeling Studies-For routine analyses of 3H-myristoylated and 3H-palmitoylated proteins, 5 X lo6 LSTRA cells in 1 ml of medium were labeled with 50 pCi of [3H]myristate or [3H]palmitate for 4 h. Cells were harvested and fractionated as previously described (32). For analyses of the effects of hydroxylamine on radiolabeled proteins, equal amounts of protein were loaded onto duplicate 10% SDS-polyacrylamide gels (33). Following staining and destaining, and prior to fluorography, one gel was soaked overnight in 1 M hydroxylamine, pH 7.0; the other was soaked in 1 M Tris/HCl, pH 7.0.

For analyses of 3H-myristoylated and 3H-palmitoylated proteins by cycloheximide treatment, 5 X lo6 LSTRA cells in 1 ml of medium were treated for 2 min with the indicated concentrations of cycloheximide. Cells were then labeled with 50 pCi of [3H]myristate or [3H] palmitate for 4 h. Cells were harvested, and labeled proteins were analyzed as described above.

Analysis of Bound Fatty Acids-Proteins from [3H]palmitate-labeled cells were separated by SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to polyvinylidene difluoride mem- branes. The region of membrane containing the bound 56-kDa protein, identified by immunoblotting with anti-p56" antibodies, was excised and hydrolyzed in 6 N HCl for 14 h at 110 "C under vacuum. The released fatty acids were extracted in toluene and analyzed by thin-layer chromatography on C18 reverse phase plates developed with a 1:l mixture of acetic acidacetonitrile. Radiolabeled fatty acids were detected by autoradiography.

Immunoprecipitation of p56kk-Acylated ~ 5 6 ' ~ was immunoprecipitated from the postnuclear supernatant of 7 X lo6 LSTRA cells, which were labeled for 4 h with 0.5 mCi of either [3H]myristate or [3H]palmitate in 1 ml of medium. Postnuclear supernatants were obtained by lysing the cells for 15 min at 4 "C in 50 mM Tris/HCl, pH 7.6,0.5% Triton X-100, 150 mM NaCI, 1 mM EDTA, and 10 pg/ ml each of aprotinin and leupeptin. Nuclei were pelleted by centrif- ugation at 1300 X g for 10 min at 4 "C. Preparation of polyclonal antipeptide antisera directed against the COOH-terminal 33 amino acids of ~56"' has been previously described (34). Postnuclear supernatants were incubated for 1 h at 4 "C with 25 pl of Protein A- Sepharose that had previously been incubated with 50 pl of p56" antisera. The resulting immune complex was washed 5 times with washing buffer (lysis buffer minus EDTA) and analyzed by SDS- PAGE and fluorography. To verify the specificity of the immunoprecipitation, excess antigenic peptide (10 mg/ml) was added during the immunoprecipitation to two control samples from cells labeled with either [3H]myristate or [3H]palmitate.

TPA Treatment of Jurkat Cells-For TPA treatments, 7 X lo6 Jurkat cells in 1 ml of medium were labeled with 100 pCi of [3H] myristate or [3H]palmitate for 3.5 h. During the last 30 min of the labeling period, some samples were treated with 100 nM TPA. Cells were harvested and labeled proteins analyzed by SDS-PAGE and fluorography as described above.

Chromatography on Protamine-Agarose-1.5 X lo7 LSTRA cells in 5 ml of medium were incubated with 250 pCi of [3H]myristate or [3H] palmitate for 4 h. Cells were lysed in 50 mM Tris/HCl, pH 7.6, 2.0% Triton X-100, 1 mM EDTA, and 10 pg/ml each of aprotinin and leupeptin. Lysates (0.5 ml) were applied to 0.5-ml columns of protamine-agarose and batch-eluted with lysis buffer containing 0, 200, 400, 600, or 800 mM NaCl. Fractions were analyzed by SDS-PAGE and fluorography as described above or by Western blotting using anti-~56'~ antisera.

Puke-Chase Analyses of 3H-Acylnted Proteins-Pulse-chase analyses were performed on 1.5 X lo7 LSTRA cells in 5 ml of medium. Cells were pulsed for 2 h with 250 pCi of [3H]myristate or [3H] palmitate. At the end of 2 h, cells were washed once with 5 ml of fresh medium and finally resuspended in 5 ml of chase medium (RPMI 1640 medium containing 10 pg/ml cycloheximide and 25 p~ (a 14-fold excess) unlabeled myristate or palmitate). Aliquots (1 ml) of the cell suspension (3 X lo6 cells) were removed at 0, 1,2, 4, and 6 h after initiating the chase. Radiolabeled proteins present in the cell lysates were analyzed as described above.

Treatment of LSTRA Cells with 2-Hydroxymyristate-LSTRA cells (3 X lo6) in 1 ml of medium were treated with 2-hydroxymyristate delivered to cells as a complex with BSA. Fatty acid-free BSA was prepared by the method of Chen (35). 2-Hydroxymyristate was transferred from celite particles to fatty acid-free BSA as previously described (36, 37) except that 1.0 mmol of 2-hydroxymyristate was used per g of celite. 2-Hydroxymyristate was preincubated with cells as described above at a final concentration of 1.0 mM for 1 h prior to a 4-h labeling with 50 pCi of [3H]myristate or [3H]palmitate. Cells were harvested, and radiolabeled proteins were analyzed by SDS- PAGE and fluorography as described above.

RESULTS

In LSTRA cells, the protein-tyrosine kinase p56" is over- expressed approximately 40-fold (38). Incubation of these cells with [3H]myristate led to the metabolic labeling of sev- eral proteins including ~ 5 6 ' ~ and the Pr6Fq protein encoded by the Moloney murine leukemia virus (6, 39) (Fig. 1). Met- abolic labeling studies revealed the existence of a 56-kDa protein in LSTRA cells that also became labeled upon exposure of intact cells to [3H]palmitate (39) (Fig. 1). The Pr6EP protein was not labeled by [3H]palmitate (Fig. 1). Based on these initial observations, we sought to confirm that this dually acylated protein was ~ 5 6 ' ~ and, if so, to further characterize these two acylation events.

Identification of the Acyluted 56-kDa Protein as ~ 5 6 ' ~ - Three different approaches were used to ascertain the identity of this protein. First, polyclonal antisera to the COOH-terminal region of ~ 5 6 " ~ was used, as described under "Experi- mental Procedures," to immunoprecipitate ~ 5 6 " ~ from LSTRA cells that were labeled with either [3H]myristate or

[%JMyristate [3HjPalrnitate

6 6

56-

45-

36-

29-

1 2 3 4 FIG. 1. Acylation of cellular proteins with ['Hlmyristate

and [Hlpalmitate. LSTRA cells were incubated with either [3H] myristate (lunes 1 and 2) or [3H]palmitate (lunes 3 and 4 ) . Radiola- beled proteins present in cell lysates were analyzed by SDS-PAGE and fluorography as described under "Experimental Procedures." The positions of molecular mass standards (in kilodaltons) are indicated. The migration position of the 56-kDa protein is indicated by the arrow. The Pr6.Yw protein, seen only in lysates from cells labeled with [3H]myristate, migrates slightly below the 66-kDa marker.

Palmitoylation of ~ 5 6 “ ~ 8671

[3H]palmitate. Results from this experiment are shown in Fig. 2A. Antipeptide antibodies specifically immunoprecipitated an acylated 56-kDa protein from cells labeled with either [3H] myristate or [3H]palmitate. The immunoprecipitation of these proteins was selectively blocked by the inclusion of an excess of the ~ 5 6 ‘ ~ COOH-terminal peptide from which the antibody was derived.

Second, TPA was used, as described under “Experimental Procedures,” to induce a shift in the electrophoretic mobility of ~56’~ . Previous studies have demonstrated that treatment of T cells with activators of protein kinase C induces phosphorylation of ~ 5 6 ‘ ~ on serine and threonine residues (40- 43). These phosphorylation events produce an apparent shift in the electrophoretic mobility of ~ 5 6 “ ~ to that of a 59-60- kDa protein as analyzed by SDS-PAGE. This shift can be utilized as a diagnostic tool to identify ~ 5 6 “ ~ in a crude cell extract. The shift in the mobility of ~ 5 6 ‘ ~ in response to activators of protein kinase can be observed in LSTRA cells; however, the shift is more apparent in the Jurkat cell line,

A [3wMyristate [3HJPalmitate

Ps-

1 2 3 4

[3HJMyristate [3H]Palmitate

TPA - + - +

66-

45-

36-

1 2 3 4 FIG. 2. Identification of the 56-kDa protein as ~ 5 6 ~ ~ . A,

using polyclonal, antipeptide antisera, ~ 5 6 ” ~ was immunoprecipitated from 0.5% Triton X-100 extracts of LSTRA cells that were labeled with either [3H]myristate (lanes 1 and 2 ) or [3H]palmitate (lanes 3 and 4 ) , as described under “Experimental Procedures.” The migration position of ~ 5 6 “ ~ , which is present in lanes 2 and 4, is indicated. Samples shown in lanes 1 and 3 contained an excess (10 mg/ml) of the peptide from which the antibody was derived. B, Jurkat cells were labeled with either [3H]myristate (lanes 1 and 2 ) or [3H]palmitate (lanes 3 and 4 ) for 3 h prior to incubation for 30 min in the absence (lanes I and 3 ) or presence (lanes 2 and 4 ) of 100 nM TPA. The migration position of p56“ (p56), which shifts to 60 kDa in the +TPA lanes (p60), is indicated. Exposure to x-ray film was 12 weeks for A and 4 weeks for B.

which expresses normal levels of ~56‘~. Fig. 2B shows results from an experiment in which Jurkat cells were labeled with either [3H]myristate or [3H]palmitate in the presence or absence of 100 nM TPA added during the last 30 min of the labeling period. These results demonstrate a shift in the apparent molecular mass of a 56-kDa protein labeled with both [3H]myristate and [3H]palmitate to approximately 60 kDa upon the addition of TPA.

Finally, we took advantage of our previous finding that ~ 5 6 ‘ ~ bound tightly to resins containing immobilized protamine (34). As shown in Fig. 3, [3H]myristate- and [3H]palmitate-labeled 56-kDa proteins co-eluted from protamine- agarose with ~ 5 6 ‘ ~ detected by Western blotting. These data are all consistent with the identification of the myristoylated and palmitoylated 56-kDa protein as ~ 5 6 “ ~ .

Analysis of 3H-Myristoylated and 3H-Palmitoylated Proteins by Treatment with Hydroxylamine-Palmitate attached to proteins can be identified by the susceptibility of the oxy- or thioester linkage to cleavage by base or neutral hydroxylamine. Under these conditions, the amide linkage of N-myristoylated proteins is stable. The chemical stability of the protein-associated radioactivity from [3H]myristate- and [3H] palmitate-labeled LSTRA cells is illustrated in Fig. 4A. Ac- ylated proteins from labeled cells were analyzed as described under “Experimental Procedures.” Lanes 1 and 2 represent extracts from cells labeled with [3H]myristate, while lanes 3 and 4 derive from cells labeled with [3H]palmitate. The migration position of ~ 5 6 “ ~ is denoted. Prior to fluorography, lanes 2 and 4 were treated with 1 M hydroxylamine, pH 7.0. Lanes 1 and 3 were treated for the same period of time with 1 M Tris/HCl, pH 7.0. The label on p56” from the [3H] myristate-labeled cells was refractory to cleavage by hydroxylamine, while the ~56‘~-associated radioactivity from the [3H]palmitate-labeled cells was susceptible to cleavage. Analy- sis by thin-layer chromatography of the radiolabeled fatty acid attached to the 56-kDa protein from [3H]palmitate- labeled cells confirmed the presence of [3H]palmitate (Fig. 4B).

Metabolic Labeling in the Presence of Cycloheximide-As well as being chemically distinct, the process of protein palmitoylation differs temporally from protein myristoylation. Palmitoylation is a posttranslational, reversible process, whereas myristoylation is cotranslational and irreversible. Compounds that block protein synthesis completely inhibit the cotranslational attachment of myristate to proteins, while protein palmitoylation is affected to a much lesser extent. Such a study is illustrated in Fig. 5. LSTRA cells were treated for 2 min with the indicated concentration of cycloheximide prior to labeling the cells with either [3H]myristate or [3H] palmitate. Cells were fractionated, and labeled proteins were analyzed as described under “Experimental Procedures.” The range of cycloheximide concentrations used for this study was shown to inhibit [14C]leucine incorporation into protein in this cell line from 60% (0.1 pg/ml) to 95% (5 pg/ml) (data not shown). At each concentration, cycloheximide had a dra- matic effect on the labeling of ~ 5 6 ” ~ with [3H]myristate. In contrast, [3H]palmitate labeling of ~ 5 6 “ ~ was only moderately affected by treatment with cycloheximide. As some protein palmitoylation would be expected to occur on newly synthe- sized protein, it is not surprising that inhibitors of protein synthesis would also partially inhibit protein palmitoylation. Hydroxylamine treatment of the gel in Fig. 5B indicated that all of the [3H]palmitate incorporated into ~ 5 6 ‘ ~ in the presence of cycloheximide was incorporated in an ester (hydroxylamine labile) linkage (data not shown).

Turnover of the PHJMyristate and PHJPalmitate Label on ~56‘~”Another property that can be used to distinguish between amino-terminal myristoylation and palmitoylation is

8672

A

Palmitoylation of ~ 5 6 ' ~

0 C ?c'p

c t

1 2 3 4 5 1 2 3 4 5 1 2 3 4 5

FIG. 3. Chromatography of 'H-acylated ~ 6 6 ~ ~ on protamine-agarose. 2% Triton X-100 extracts of [3H]myristate- ( B ) or [3H] palmitate-labeled (C) LSTRA cells were bound to protamine-agarose and eluted with buffers containing increasing concentrations of NaC1. Fractions were analyzed by SDS-PAGE and fluorography ( B and C) or by Western blotting with anti-p561k antibodies ( A ) . Exposure to X- ray film was 2 weeks.

the rate of turnover of the myristate and palmitate moieties. Myristoylation is most often irreversible. Therefore, the myristate moiety would be predicted to have a half-life equal to that of the modified protein. Palmitoylation, on the other hand, is in most cases reversible, and the palmitoyl group would have a half-life shorter than that of the protein. The half-life of ~ 5 6 ' ~ has been reported to be 20-30 h in LSTRA cells (38). Using pulse-chase experiments, we could detect the turnover of the [3H]palmitate moiety in as little as 6 h. At this time point, there was relatively little change in the [3H] myristate moiety (Fig. 6).

2-Hydroxymyristate Specifically Inhibits Myristoylation but Not Palmitoylation of ~ 5 6 ' ~ " W e have shown previously that the myristate analog, 2-hydroxymyristate, becomes metabolically activated in cultured cells to form the potent inhibitor of protein myristoylation, 2-hydroxymyristoyl-CoA (32). This compound had no inhibitory effect on protein palmitoylation. Thus, incubation of cells with 2-hydroxymyristate reduced the incorporation of [3H]myristate but not [3H]palmitate into cellular proteins (32). Due to the differential effects produced by this compound on these two acylation events, 2-hydroxymyristate can be used to distinguish between protein myristoylation and palmitoylation. This effect is further illustrated in Fig. 7. Incubation of LSTRA cells with 2-hydroxymyristate specifically reduced the labeling of ~ 5 6 ' ~ with [3H]myristate while having no inhibitory effect on the labeling of the protein with [3H]palmitate.

DISCUSSION

The modification of ~ 5 6 " ~ with myristic acid on its amino- terminal glycine is well documented (1,6,7). We demonstrate in this report that ~ 5 6 ' ~ is also acylated with palmitic acid. We have identified a cellular protein that is labeled with both myristate and palmitate as ~ 5 6 ' ~ by specific immunoprecipitation from cells labeled with either [3H]myristate or [3H] palmitate (Fig. 2 A ) , induction of a shift in electrophoretic mobility in response to TPA (Fig. 2B), and chromatography on protamine-agarose (Fig. 3).

During the course of metabolic labeling, there can be some interconversion of the radiolabeled fatty acids due to the action of enzymes involved in fatty acid elongation and p- oxidation. Upon labeling of the human epidermal carcinoma cell line, A431, with [3H]myristate for 4 h, approximately 50% of the protein-associated radioactivity was present as [3H] palmitate (9). The label may also be incorporated into amino acids. In 3T3 fibroblasts, two-thirds of the myristate radiola- bel was incorporated into proteins in the form of glutamate,

glutamine, proline, aspartate, and asparagine (9). It is therefore imperative that the chemical nature of the protein- associated radioactivity be assessed. Therefore, we initially sought to characterize the chemical nature of the two acylation events and investigate the timing of the modification, i.e. cotranslational uersus posttranslational. The results of these studies were consistent with ~ 5 6 " ~ being both an N-myristoylated and palmitoylated protein. This conclusion is based on the following observations. (i) The chemical stability of the myristate and palmitate labels on ~ 5 6 ' ~ was consistent with that expected for amide-linked myristate and ester-linked palmitate, i.e. only the palmitate label was susceptible to cleavage by neutral hydroxylamine (Fig. 4A). (ii) The radiolabeled fatty acid attached to ~ 5 6 " ~ recovered from [3H] palmitate-labeled cells was identified as [3H]palmitate (Fig. 4B). (iii) The labeling of ~ 5 6 " ~ with myristate, and not palmitate, was inhibited by the treatment of cells with an inhibitor of protein synthesis, as expected for cotranslational myristoylation and posttranslational palmitoylation (Fig. 5). (iv) The Pr65gw protein encoded by the Moloney murine leukemia virus, a major myristoylated protein in LSTRA cells (39), was labeled in cells incubated with [3H]myristate but not in cells incubated with [3H]palmitate, indicating that little or no conversion of [3H]palmitate to [3H]myristate occurred in LSTRA cells during a 4-h incubation (Fig. 1). (v) The rate of turnover of the palmitate, but not the myristate, on ~ 5 6 " ~ was much more rapid than the rate of turnover of the protein (Fig. 6). (vi) Only the myristoylation of ~ 5 6 ' ~ was inhibited by treatment of cells with 2-hydroxymyristate (Fig. 7).

The site of palmitoylation on ~ 5 6 ' ~ has not yet been deter- mined. The primary sequence of ~ 5 6 ' ~ contains 9 cysteine residues, the most likely sites of palmitoylation. Of these, 4 are found within the first 23 amino acids. The first 32 amino acids of ~ 5 6 " ~ contain a domain essential for binding to CD4 and CD8 (3). In particular, cysteines at positions 20 and 23 have been implicated as crucial for these interactions. It is, therefore, conceivable that one or more of these amino-terminal cysteine residues becomes modified with palmitic acid, which would help to stabilize the protein-protein interactions. While it is possible that palmitoylation of these residues might play a role in these interactions, it has also been proposed that these cysteine residues might serve as a metal binding site (4). The cysteines a t positions 3 and 5 are perhaps the most attractive candidates for palmitoylation, since they would be expected to lie near the membrane interface due to the presence of the myristoyl group on the amino-terminal glycine. In fact, removal by site-directed mutagenesis of either of these cysteine residues results in a protein that is only

Palmitoylation of ~ 5 6 ~ ~ 8673

[3H]Myristate [B~]~a~rnitate A [cycloheximide] @g/ml) 0 5 1 0.5 0.1

A

Hydroxylamine

66-

PS-"

45-

+ i

PS-

illlrp. B

[cycloheximide] (ug/ml) 0 5 1 0.5 0.1

1 2 3 4

1 2 3 FIG. 4. Analysis of fatty acids on 'H-acylated proteins from

LSTRA cells. A, LSTRA cells were incubated in the presence of [3H]myristate (lanes I and 2 ) or [3H]palmitate (lanes 3 and 4 ) . Radiolabeled proteins contained in the cell lysates were fractionated on duplicate 10% SDS-PAGE gels. Prior to fluorography, the gel that contained lanes 2 and 4 was treated overnight in a solution of 1 M hydroxylamine, pH 7.0 (+). The gel that contained lanes I and 3 was treated overnight with 1 M Tris/HCl, pH 7.0 (-). The migration position of ~ 5 6 ' ~ is indicated (p.56). Exposure time for both gels was 3 weeks. B, fatty acids were hydrolyzed from ~ 5 6 " ~ obtained from [3H]palmitate-labeled cells as described under "Experimental Proce- dures" and analyzed by chromatography on C18 reverse phase thin- layer plates. The migration positions of [3H]myristic ( M y r ) and [3H] palmitic (Pal) acid standards are indicated. Exposure time was 1 week.

weakly associated with the plasma membrane (44). This sug- gests that palmitoylation may play an important role in directing the subcellular localization of ~ 5 6 " ~ . Palmitoylation of the neuronal growth cone protein GAP-43 on 2 amino- terminal cysteine residues has been implicated in the localization of GAP-43 specifically to the plasma membrane of growth cones (45, 46). In fact, addition of the amino-terminal sequence of this protein to chloramphenicol acetyltransferase

FIG. 5. Effect of cycloheximide on the labeling of ~ 6 6 ~ with ['Hlmyristate and ['Hlpalmitate. LSTRA cells were incubated for 2 min with the indicated concentrations of cycloheximide prior to labeling for 4 h with either ['Hlmyristate ( A ) or [3H]palmitate ( B ) , as described under "Experimental Procedures." Radiolabeled proteins present in the cell lysates were analyzed by SDS-PAGE and fluorography. The migration position of p56" is denoted (p56). Exposure time for A and B was 4 weeks.

directed chloramphenicol acetyltransferase to sites in growth cones normally occupied by GAP-43 (45). Palmitoylation may likewise stabilize the interactions of p56" with the plasma membrane. A strong association of palmitoylated p56" with the plasma membrane may also provide a molecular mecha- nism to explain the inhibitory effect of p56" on the endocytosis of CD4, the transmembrane protein with which it is associated (47).

~ 5 6 " ~ is, to our knowledge, the first example of a protein

8674 Palmitoylation of ~ 5 6 " ~

Pal

1 2 3 4 5 FIG. 6. Pulse-chase analysis of [sH]myristoyl- and ['Hlpal-

mit0yLp56"~. LSTRA cells were incubated for 2 h with f'H]myristate ( M y r ) or [3H]palmitate (Pal) and then chased with a 14-fold excess of unlabeled fatty acid for 0, 1, 2, 4, or 6 h (lanes 1-5, respectively). Radiolabeled proteins present in cell lysates were analyzed by SDS-PAGE and fluorography. The migration positions of ~ 5 6 " ~ are indicated. Exposure time was 4 weeks.

92

69

46

30

1 2 3 4 FIG. 7. Effect of 2-hydroxymyristate on the labeling of

~56"' with ['Hlmyristate and ['Hlpalmitate. LSTRA cells were incubated for 1 h in the absence (lanes I and 4 ) or presence (lanes 2 and 3 ) of a 1 mM 2-hydroxymyristate-BSA complex and then incubated for 4 h with 50 pCi of [3H]myristate (lanes 1 and 2 ) or [3H] palmitate (lanes 3 and 4 ) . Cells were harvested, and radiolabeled proteins were analyzed by SDS-PAGE and fluorography. Exposure time was 4 weeks.

that is both irreversibly myristoylated and reversibly palmitoylated. This is in contrast to the transferrin receptor, which is both reversibly myristoylated and palmitoylated (48). The slowest migrating of the major myristoylated and palmitoylated proteins present in LSTRA cells is also a protein that undergoes reversible myristoylation and palmitoylation (see, for examples, the 95-kDa proteins in Figs. 1, 4A, and 5). Preliminary studies using anti-transferrin receptor antibodies indicate that this protein is, in fact, the transferrin receptor? This indicates that LSTRA cells have in place mechanisms for both the reversible and irreversible myristoylation of cellular proteins.

In summary, we have provided evidence that p56" is acyl-

ated with palmitic acid. An examination of the sequences of other myristoylated, src family protein-tyrosine kinases re- veals that products of the lyn, fyn, and yes genes also have cysteine residues near their myristoylated amino termini, suggesting the possibility that these kinases may also be palmitoylated proteins. Further work will be required to de- termine if these proteins are, in fact, posttranslationally acylated.

REFERENCES 1. Rudd, C. E., Trevillyan, J. M., Dasgupta, J. D., Wong, L. L., and Schloss-

2. Veillette, A., Bookman, M. A., Horak, E. M., and Bolen, J. B. (1988) CeU man, S. F. (1988) Proc. Natl. Acad. Sci. U. S. A. 85,5190-5194

5.5. m-m 3. Shaw. A. S.. Amrein. K. E.. Hammond. C.. Stern. D. F.. Sefton. B. M., and

- - , - - - - - -

Rose, J. K. (1989)'Cell59,627-636 ' '

4. Shaw, A. S., Chalupny, J., Whitney, A., Hammond, C., Amrein, K. E., Kavathas. P.. Sefton. B. M.. and Rose. J. K. (1990) Mol. Cell. Biol. 10,

. .

5.

6.

7.

9. 8.

10. 11.

12. 13. 14.

15.

16. 17.

18.

20. 19.

21. 22.

23.

24.

25.

26. 27. 28. 29.

30.

31. 32.

33. 34.

35. 36. 37. 38. 39.

40. 41. 42.

43.

44.

45.

1853-1862 '

M., and Littman, D. R. (1990) Cell 60.755-765

Proc. Natl. Acad. Sci. U. S. A. 81, 7679-7682

Turner, J. M., Brodsky, M. H., Irving, B. A,, Levin, S. D., Perlmutter, R.

Marchildon, G. A., Casnellie, J. E., Walsh, K. A., and Krebs, E. G. (1984)

Abraham, N., and Veillette, A. (1990) Mol. Cell. Biol. 10, 5197-5206 daSilva, A. M., and Klein, C. (1990) J. Cell B~ol. 111, 401-407 Olson, E. N., Towler, D. A., and Glaser, L. (1985) J. Biol. Chem. 260,

Magee, A. I., and Courtneidge, S. A. (1985) EMBOJ. 4,1137-1144 Mcllhinney, R. A., Pelly, S. J., Chadwick, J. K., and Cowley, G. P. (1985)

James, G., and Olson, E. N. (1990) Biochemistry 29,2623-2634 Olson, E. N., and Spizz, G. (1986) J. Biol. Chem. 261.2458-2466

Towler, D. A., Gordon, J. I., Adams, S. P., and Glaser, L. (1988) Annu. Rev.

Schmidt, M. F. G., Brecha, M., and Schlessinger, M. J. (1979) Proc. Natl.

Schmidt, M. F. G. (1982) Virology 116,327-338 Magee, A. I., Koyama, A. H., Malfer, C., Wen, D., and Schlessinger, M. J.

(1984) Biochim. B~ophys. Acta 798,156-166 Magee, A. I., and Siddle, K. (1988) J. Cell. Biockm. 37,347-357 Omary, M. B., and Trowbridge, I. S. (1981) J . Bml. Chem. 256,4715-4718 ODowd, B. F., Hnatowich, M., Caron, M. G., Lefkowitz, R. J., and Bouvier,

Buss, J. E., and Sefton, B. M. (1986) Mol. Cell. BWL 6,116-122 Tamanoi, F., Hsueh, E. C., Goodman, L. E., Cobitz, A. R., Detrick, R. J.,

Clarke, S., Vogel, J. P., Deschenes, R. J., and Stock, J. (1988) Proc. Natl. Brown, W. R., and Fujiyama, A. (1988) J. Cell. Biochem. 36,261-273

Gutierrez, L., Magee, A. I., Marshall, C. J., and Hancock, J. (1989) EMBO Acad. Sci. U. S. A. 85,4643-4647

Hancock, J. F., Magee, A. I., Childs, J. E., and Marshall, C. J. (1989) Cell J. 8, 1093-1098

Crise. B.. and Rose. J. K. (1992) J. Biol. Chem. 267.13593-13597 57, 1167-1177

3784-3790

EMBO J. 4,1145-1152

Riochem. 57,69-99

A d . Set. U. S. A. 76,1687-1691

M. (1989) J. Biol. Chem. 264,7564-7569

McIlhinney, R. A. (1990) ?rem& Biochem. Sci. 15,387-391 Hancock, J. F., Paterson, H., and Marshall, C. J. (1990) Cell 63, 133-139 Deschenes. R. J.. Resh. M. D.. and Broach. J. R. (1990) Curr. b i n . CeU

id. 2, iloailn '

Alverez, E., Girones, N., and Davis, R. J. (1990) J. BioL Chem. 265,16644-

Glynn, J. P., Bianco, A. R., and Goldin, A. (1964) Cancer Res. 24,502-508 Paige, L. A., Zheng, G., DeFrees, S. A., Cassady, J. M., and Geahlen, R. L.

Laemmli, U. K. (1970) Nature 227,680-685 Wang, Q., Srinivas, P. R., Harrison, M. L., and Geahlen, R. L. (1991)

Chen, R. F. (1967) J. Biol. Chem. 242,173-181 Spector, A. A., and Hoak, J. C. (1969) Anal. Biochem. 32,297-302 Spector, A. A. (1986) M e t M Enzymol. 128,320-339

Voronova, A. F., Buss, J. E., Patschinsky, T., Hunter, T., and Sefton, B. Hurley, T. R., and Sefton, B. M. (1989) Oncogene 4,265-272

Casnellie, J. E., and Lamberts, R. J. (1986) J. Biol. Chem. 261,4921-4925 Casnellie, J. E. (1987) J. Biol. Chem. 262,9859-9864 Veillette, A., Horak, I. D., and Bolen, J. B. (1988) Oncogene Res. 2, 385-

Marth, J. D., Lewis, D. B., Cooke, M. P., Mellins, E. D., Gearn, M. E., 401

Samelson, L. E., Wilson, C. B., Miller, A. D., and Perlmutter, R. M. (1989) J. Immunol. 142,2430-2437

Turner, J. M., Brodsky, M. H., Irving, B. A., Levin, S. D., Perlmutter, R. M., and Littman, D. R. (1990) Cell 60,755-765

Zuber. M. X., Strittmatter, S. M., and Fishman, M. C. (1989) Nature 341,

16655

(1990) BiochemLstry 29, 10566-10573

Biochem. J. 279,567-574

M. (1984) Mol. Cell. Biol. 4,2705-2713

46. Skene, J. H. P., and Virag, I. (1989) J. Cell Biol. 108,613-624 47. Pelchen-Matthews, A., Boulet, I., Littman, D. R., Fagard, R., and Marsh,

'hem.

345-348

M. (1992) J. Cell Biol. 117.279-290

* M. J. S. Nadler and R. L. Geahlen, unpublished observations. 48. Adam; M.,' Turbide, C., and Johnstone, R. M. (1988) Arch. Bioc

Rwphys. 264,553-563

Documents

Reversible Palmitoylation of the Protein-Tyrosine Kinase ~56'”~*