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Biochemical and Biophysical Research Communications 288, 573–578 (2001)

doi:10.1006/bbrc.2001.5814, available online at http://www.idealibrary.com on

rc Kinases Fyn and Lck Facilitate the Accumulation ofhosphorylated CTLA-4 and Its Association with PI-3inase in Intracellular Compartments of T-Cells

ui Hu,*,† Christopher E. Rudd,*,‡,§ and Helga Schneider*,†,§,1

Department of Cancer Immunology and AIDS, Dana–Farber Cancer Institute, 44 Binney Street, Boston, Massachusetts2115; †Department of Medicine and ‡Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115;nd §Department of Haematology, Faculty of Medicine, Imperial College of Science, Technology and Medicine,ammersmith Hospital, London W12 ONN, United Kingdom

eceived September 25, 2001

tail of CTLA-4 (7–10). AP-1 binding occurs in the trans-Guwc

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Src kinases bind to surface receptors and mediateignaling events at the surface of cells. Little is knownegarding whether these kinases can mediate eventsithin intracellular compartments. The T-cell antigenTLA-4 resides primarily in the trans-Golgi network

TGN), and as such could serve as a model to study thentracellular function of src kinases in their ability tohosphorylate the receptor. In this study, we showhat tyrosine kinases p56lck and p59fyn phosphorylatehe cytoplasmic domain of CTLA-4 in T-cells. Most in-erestingly, these kinases are also found in the Golgipparatus, the intracellular compartment where mostf CTLA-4 is localized. Transfection of Lck or Fyn re-ulted in increased phosphorylation of intracellularTLA-4 and recruitment of PI-3 kinase. By contrast,hosphorylation did not influence the presence of theeceptor in the TGN. These data demonstrate that srcinases operate to modulate receptor binding to intra-ellular signaling proteins introducing the possibilityhat intracellular forms of receptors may generaterowth signals. © 2001 Academic Press

CTLA-4 can be distinguished from CD28 due to itsegative regulation of T-cell proliferation (1–3) andue to its unusual accumulation in the cytoplasm of-cells (4, 5). CTLA-4 surface expression is low andightly regulated, occurring as early as 6 h postactiva-ion and reaching its highest level by about 36 h (6).ven at its highest levels, however, CTLA-4 is ex-ressed only about 3% relative to CD28 (6). CTLA-4ossesses two tyrosine residues at 201 and 218 locatedn the motifs YVKM and YFIP, respectively. Clathrindaptor complexes AP-1 and AP-2 bind to the nonphos-horylated form of the YVKM motif in the cytoplasmic

1 To whom correspondence should be addressed. Fax: 44(0) 208 383434. E-mail: [email protected].

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olgi network (TGN) and serves as a homeostatic reg-lator of intracellular concentrations of CTLA-4 (10),hile AP-2 complexes bind to surface forms of the

oreceptor and regulate receptor internalization (7–9).Unlike CD28, CTLA-4 negatively regulates T-cell

esponses (11–13). Antibodies to the receptor inhibitcR/CD28 signaling (11, 14, 15) and CTLA-4 negativeice show extensive lymphadenopathy consistent with

he loss of negative regulation (12, 13). The mecha-isms by which CTLA-4 can generate these intracellu-

ar signals are still unclear. One model proposes thatTLA-4 mediates negative signaling by binding to therotein tyrosine phosphatase SHP-2 (16–18). Previoustudies have shown that src kinases p56lck and p59fyn

hosphorylate CTLA-4 (primarily at the YVKM motif)7, 19, 20) leading to the recruitment of the lipid kinaseI 3-kinase (21) and SHP-2 (16, 18, 22). The AP-2inding site in CTLA-4 overlaps with the binding siteor PI 3-kinase and SHP-2. Tyrosine phosphorylationould therefore facilitate binding of PI3-kinase andHP-2 and displace the AP-2 binding which is indepen-ent of tyrosine phosphorylation. CTLA-4 phosphory-ation by Fyn and Lck is a potential mechanism byhich CTLA-4 intracellular trafficking and CTLA-4

ignal transduction can be regulated.Src kinases bind to surface receptors such as CD4,D8 and TcR in their mediation of signaling events

rom the cell surface (23, 24). However, little is knownegarding whether these kinases can mediate eventsithin the intracellular compartments of cells. CD4-56lck associates and synergizes with the TCRz/CD3omplex in the optimal response of T-cells to antigen25). p56lck has been implicated in functions such ashymic differentiation, lymphokine production, T-cellediated cytotoxicity, endocytosis, and in the positive/

egative regulation of T-cell growth (26–29). Miceacking Lck and Fyn show a complete arrest at the

0006-291X/01 $35.00Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved.

double negative stage indicating that both of theses(

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Vol. 288, No. 3, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

rc-related kinases are needed for pre-TcR signaling30, 31).

In this study we show that src kinases p56lck and59fyn facilitate phosphorylation of intracellular local-zed CTLA-4 and induce the recruitment of PI-3 ki-ase. These data suggest the possibility that src ki-ases can operate in intracellular compartments to

nduce events normally found at the cell surface, ands such may generate intracellular signaling events.

ATERIALS AND METHODS

Cells, reagents and antibodies. The murine T cell hybridomaC27.10 was cultured in RPMI 1640 medium supplemented with 5%

v/v) fetal bovine serum and 50 mM 2-mercaptoethanol. DC27.10 cellsere stably transfected with human CTLA-4 as described (10). Anti-TLA-4 mAb (11D4) has been previously described (32), while anti-TLA-4 mAb (BNI3) was purchased from Immunotech (Marseille,rance). Anti-phosphotyrosine mAb was kindly provided by Dr. T.oberts (Dana–Farber Cancer Institute, Boston, MA), the anti-p85ntiserum by Dr. M. White (Joslin Diabetes Center, Boston, MA).nti-fyn mAb was bought from Transduction Laboratories (Saniego, CA), anti-lck mAb from Santa Cruz Biotechnology (Santaruz, CA).

Immunoprecipitation and immunoblotting. For immunoprecipi-ations, cells were lysed in ice-cold lysis buffer containing 1% Triton-100 (v/v) in 20 mM Tris–HCl, pH 8.3, 150 mM NaCl. The lysisuffer contained 1 mM PMSF, 1 mM Na4VO3, 10 mM NaF and 1 mMa4P2O7. Lysates were incubated for 20 min on ice before centrifu-ation at 15,000g for 15 min at 4°C. Aliquots of 1 ml of clear post-uclear lysates were incubated for 1 h with the indicated monoclonalntibody. Protein A–Sepharose beads (30 ml, Pharmacia), swollennd washed in lysis buffer were added and incubated for 1 h at 4°C.he eluted proteins were separated by SDS–PAGE and transferredo nitrocellulose for immunoblotting. Bound antibody was revealedith the appropriate secondary antibody, and protein was visualizedy enhanced chemiluminescence (ECL, Amersham).

Subcellular fractionation. DC27.10 cells were harvested, resus-ended in 0.25 M sucrose, pH 7.4, homogenized and centrifuged at000 rpm for 15 min to pellet the nuclei and unbroken cells. Cyto-lasmic organelles from the postnuclear supernatant (PNS) wereeparated by a Percoll/metrizamide discontinuous density gradients described (33). Lysosomal and lysosomal/mitochondrial mem-ranes were obtained as described (34). The supernatants containedatrix proteins. The membranes were washed twice with 0.5 MaCl and 0.1 M DTT, and then resuspended in PBS. A cocktail ofrotease inhibitors was added to the lysosomal fractions before lysis1 mM EDTA, 0.1 mM pepstatin A, 0.1 mM leupeptin and 0.1 mMEBSF). The purity of the isolated fractions was tested by immuno-lotting using antibodies for Golgi and lysosomes. In addition, theurity of lysosomes was verified by the lysosomal marker enzyme-hexosaminidase as described (33). Protein determinations wereerformed using the Lowry assay (35).

ESULTS AND DISCUSSION

Given the accumulation of CTLA-4 in the TGN (4,0), and the ability of src kinases to phosphorylateTLA-4 (7, 19, 20), it was of interest whether src ki-ases could regulate the phosphorylation in this intra-ellular compartment. The YVKM motif has beenound to regulate the release of CTLA-4 from the TGN

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nduce phosphorylation of CTLA-4 in the murine T-cellybridoma DC27.10 stably transfected with humanTLA-4 (10). Phosphorylation of CTLA-4 by p56lck and59fyn in cells was demonstrated by transfection withonstitutively active forms of p56lck and p59fyn (p56lck505,59fyn528). As shown by anti-phosphotyrosine blotting,oth kinases induced significant levels of tyrosinehosphorylation of CTLA-4 when compared withector-transfected cells (Fig. 1A, lanes 2 and 3). Ex-ression levels of p56lck505 or p59fyn528 are shown bymmunoblotting of the cell lysate with anti-lck or anti-yn mAb (Fig. 1A, lower panel). These data confirmhat Lck and Fyn share an ability to phosphorylateTLA-4 in T-cells.It was next of interest to distinguish between surface

nd intracellular forms of phosphorylated CTLA-4.urface CTLA-4 was distinguished from intracellu-

ar CTLA-4 by preincubating intact cells with anti-TLA-4 antibody at 4°C followed by washing to removexcess antibody, detergent lysis and immunoprecipita-ion. Following this, lysates were subjected to a roundf preclearing with protein A–Sepharose, followed by aecond precipitation to purify intracellular CTLA-4. Aseen in Fig. 1B (lanes 3 and 4), most of the CTLA-4hich was phosphorylated resided intracellularlyhen compared with phosphorylated surface CTLA-4

lanes 1 and 2). This finding indicates that Fyn and Lckan phosphorylate CTLA-4 in intracellular stores. Al-hough it cannot be excluded that a portion of thentracellular material was derived from the cell sur-ace, phosphorylated CTLA-4 has previously beenhown to block AP-2 complex binding and internaliza-ion of the receptor (8, 9).

Given the fact, that src kinases phosphorylate intra-ellular localized CTLA-4, we next assessed whetherhis phosphorylation can modulate PI-3 kinase bind-ng. As shown in Fig. 1C, accompanying the intracel-ular phosphorylation of CTLA-4 (upper panel) was theecruitment of associated p85 of PI-3 kinase (loweranel; lane 2 vs 1). Immunoblotting against cell lysateserved as a control. These data demonstrate that srcinases can modulate receptor binding to PI-3 kinasen the intracellular compartments of T-cells.

To assess in more detail the subcellular localizationf phosphorylated intracellular CTLA-4, membranesere subfractionated on a Percoll/metrizamide discon-

inuous density gradient to purify Golgi-enriched, mi-ochondrial and lysosomal vesicles, as described underaterials and Methods (10, 36). In addition to theolgi-enriched fraction, two populations of lysosomesere defined according to their density in the Percoll/etrizamide gradients, one light and another heavier

ysosomal fraction that co-sediments with the mito-hondria. Immunoblotting with antisera against aolgi specific 58Kd protein confirmed the identity of

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as found in the two lysosomal fractions. In addition,he purity of lysosomes was verified by the lysosomalarker b-hexosaminidase (data not shown). Phosphor-

lated CTLA-4 from DC27.10 cells transfected with56lck505 or p59fyn528 could be detected in both Golgi-nriched and lysosomal fractions (Fig. 2A, lanes 2, 3nd 6, 7). Within the lysosomal fraction, CTLA-4 wasound primarily in the light lysosomal fraction (Fig. 2A,anes 3 and 7). Lck and Fyn were also found to beocalized in the Golgi-enriched compartments (loweranels, lanes 2 and 6). Significantly, to our knowledge,his is the first documented report of src kinase local-zation in the Golgi apparatus. Little, if any phosphor-lated CTLA-4 was detectable in the Golgi-enrichedraction of CTLA-4 expressing cells when contrastedith the phosphorylated form of CTLA-4 from p56lck505

ransfectants (lane10 vs 12). The identity of the proteinas confirmed by an anti-CTLA-4 blot (lane 14). Nota-ly, the p85 subunit of PI-3 kinase was found in theolgi and lysosomal fractions, the same compartmentshere phosphorylated CTLA-4 was localized (Fig. 2B,

anes 2, 3). These data demonstrate that src kinasesodulate binding of intracellular receptors to signal-

FIG. 1. (A) In vivo phosphorylation of CTLA-4 by the protein-tytably transfected with CTLA-4 were transfected with vector (lane 1),p56lck505) (lane 3). Cells were lysed, immunoprecipitated with CTLA-4qual amounts of cell lysates of each of these transfectants were anaespectively. (B) Amounts of intracellular and cell surface expressedransfected with p56lck505 or p59fyn528 was precipitated by preincubatind immunoprecipitation. Lysates were subjected to a round of pre-curify intracellular CTLA-4. Precipitates were blotted with anti-pTntracellular CTLA-4; (C) Src kinase p56lck facilitates PI-3 kinase bin56lck505 was precipitated as described above. Precipitates were blotanel). Lane 1, surface CTLA-4; lane 2, intracellular CTLA-4.

575

ng proteins introducing the possibility that intracellu-ar forms of receptors may generate growth signals.

In summary, our findings demonstrate for the firstime that src kinases and CTLA-4 are colocalized in theolgi apparatus. These data suggest that src kinases

an phosphorylate and induce PI-3 kinase binding toTLA-4 in the intracellular compartments of T-cells.revious studies have shown that these kinases canhosphorylate the co-receptor, but at the cell surface7, 19, 20), in a manner analogous to CD28 (37). How-ver, CTLA-4 can be distinguished from CD28 by theact that it is primarily an intracellular antigen (4).ne question concerns the molecular basis for this

ntracellular accumulation, although it has been re-orted to be linked to a tyrosine-containing motif in theTLA-4 cytoplasmic tail (4). Interestingly, we found

hat this YVKM motif is also the principal site of phos-horylation by Lck and Fyn (Fig. 1). While tyrosinehosphorylation of CTLA-4 is a potential mechanismy which intracellular trafficking of CTLA-4 can beegulated, we failed to observe a noticeable differencen surface expression of the receptor in src kinaseransfected and nontransfected cells (data not shown).

ine kinases p56lck and p59fyn in T-cells. Upper panel: DC27.10 cellsstitutively active Fyn (p59fyn528) (lane 2), or constitutively active Lckb (lanes 1–3) and immunoblotted with anti-pTyr mAb. Lower panel:

ed for the presence of Lck and Fyn with anti-lck and anti-fyn mAbs,sphorylated CTLA-4. Surface CTLA-4 from CTLA-4 expressing cellsintact cells with anti-CTLA-4 antibody at 4°C, followed by washinging with protein A–Sepharose, followed by a second precipitation tomAb (lanes 1–4). Lanes 1 and 2, surface CTLA-4; lanes 3 and 4,g. Surface CTLA-4 from CTLA-4 cells transiently transfected withwith anti-pTyr mAb (upper panel) and anti-p85 antiserum (lower

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herefore a more likely interpretation is that src ki-ase mediated PI3-kinase recruitment can generate

ntracellular signals that provide basal level signals.his event would occur independently of CTLA-4 liga-ion at the cell surface. Alternatively, the trafficking ofntracellular ligands CD80/CD86 through intracellularesicles may allow for transient interactions that facil-tate signaling. Previous studies have shown that liga-ion of PI3-kinase associated receptors can activate theatalytic activity of the enzyme (38, 39). Further stud-es will be required to determine the relevance forntracellular CTLA-4 phosphorylation in CTLA-4 me-iated negative signaling and intracellular proteinrafficking.

FIG. 2. (A) Intracellular phosphorylation of CTLA-4 by p56lck andith p56lck505 or p59fyn528 and subcellular fractionation of intracellularach fraction was precipitated with anti-CTLA-4 mAb and immun

ransfectants were either left untreated (lanes 9, 10 and 13, 14) or tntracellular membranes was performed as described under Materia

Ab (lanes 9–14) and immunoblotted with anti-pTyr mAb (lanes 9olgi-enriched fractions. Lower panel: Equal amounts of cell lysatesnd anti-fyn mAbs, respectively. (B) Intracellular localization of PI-3ubcellular fractionation of intracellular membranes was performedith anti-p85 antiserum (lanes 1–4).

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3

interacts with the lipid kinase phosphatidylinositol 3-kinase by a

3


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src kinases fyn and lck facilitate the accumulation of phosphorylated ctla-4 and its association...

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