targeting of cd45 protein tyrosine phosphatase activity to lipid microdomains on the t cell surface...

0014-2980/02/0909-2578$17.50+.50/0 © 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Targeting of CD45 protein tyrosine phosphataseactivity to lipid microdomains on the T cell surfaceinhibits TCR signaling

Xiao He, Terry A. Woodford-Thomas, Kenneth G. Johnson, Dulari D. Shah andMatthew L. Thomas

Howard Hughes Medical Institute, Department of Pathology and Immunology, WashingtonUniversity School of Medicine, St. Louis, USA

CD45, a transmembrane protein tyrosine phosphatase (PTP), can either positively or nega-tively regulate Src-family protein tyrosine kinase (PTK) activity in vivo. It is proposed thatTCR-initiated signaling requires the segregation of PTP activities from the engaged TCR,based upon the differential membrane compartmentalization on the T cell surface. To testthe importance of CD45 exclusion from lipid microdomains for proper TCR signaling, a chi-meric molecule was generated by fusing the CD45 cytoplasmic region, which contains thePTP domains, to the amino-terminal 12 amino acids of Lck, which target Lck to lipid micro-domains. Using 3A9 T lymphocyte hybridoma (3A9H) cells whose TCR recognizes hen egg-white lysozyme (HEL), Lck-CD45 expression resulted in its targeting to lipid microdomains.The 3A9H cells expressing Lck-CD45 were reduced in their responses to HEL or co-cross-linking of CD3 and CD4, as assessed by IL-2 production and Ca2+ mobilization. Src-familyPTK activity associated with lipid microdomains was also decreased. These results suggestthat the segregation of CD45 from proximal TCR signaling components is necessary for TCRsignaling and that the targeting of CD45 PTP activity to lipid microdomains on the T cell sur-face results in decreased sensitivity of TCR-mediated signaling.

Key words: CD45 / Protein tyrosine phosphatase / Lipid microdomain / TCR / Signal transduction

Received 3/12/01Revised 17/5/02Accepted 13/6/02

[I 22723]

This paper is dedicated to the memory of Dr. Matthew L.Thomas.

Abbreviations: PTP: Protein tyrosine phosphatase PTK:Protein tyrosine kinase aa: Amino acids 3A9H: 3A9 T lym-phocyte hybridoma TX-100: Triton X-100 M I C: Methyl- g -cyclodextrin

1 Introduction

CD45, a transmembrane protein tyrosine phosphatase(PTP), is a cell surface glycoprotein expressed by allnucleated hematopoietic cells [1]. With regards to TCR-mediated signaling, CD45 is generally believed to func-tion as a positive regulator of Lck and Fyn, the Src-familyprotein tyrosine kinase (PTK) members expressed in Tcells, by dephosphorylating the C-terminal inhibitorytyrosine site [2, 3]. However, CD45 has been shown todephosphorylate the activating site of tyrosines locatedwithin kinase domains of Src-family PTK in vitro and invivo [4–7]. CD45 also negatively modulates the Src-

family PTK members, Hck and Lyn, in BM-derived mac-rophages by dephosphorylating the activating tyrosinesite and regulates g 2-integrin-mediated adhesion [8].Therefore, CD45 can serve as either a positive or a nega-tive regulator of Src-family PTK. This results in the poten-tiation or attenuation of their PTK activity, respectively,depending upon the balance of tyrosine phosphorylationat these two sites and the proximity of the kinases toother modulators containing SH2 and SH3 binding sites[9–11].

Specialized lipid microdomains with enriched glycolipidsand cholesterol, often referred to as lipid rafts, exist inthe cytoplasmic membranes of living cells and areinvolved in important cellular functions [12]. These lipidmicrodomains are also found in T cells and are impli-cated in TCR-mediated signaling [13, 14]. Many signal-ing proteins and glycosyl-phosphatidylinositol (GPI)-linked glycoproteins are localized or recruited into lipidmicrodomains associated with the TCR, prior to or assignal transduction occurs [15–18]. Disruption of theselipid microdomains by treatment of T cells with a choles-terol extracting agent, methyl- g -cyclodextrin (M g C),diminishes TCR-mediated signaling [16]. In contrast to

2578 X. He et al. Eur. J. Immunol. 2002. 32: 2578–2587

Lck and other TCR signaling proteins, which are includedin isolated lipid microdomains, CD45 is primarilyexcluded [15, 16]. These results indicate that the specificmembrane compartmentalization of signaling moleculesmay provide an additional level of regulation for precisecellular signaling.

The paradoxical role of CD45 as either a positive or anegative regulator of Src-family PTK may depend uponthe membrane localization of kinases with respect totheir counterpart PTP [9, 19]. On the T cell surface, theimmunological synapse was formed where thesupramolecular-complexes of TCR/CD3/CD4/Lck areclustered upon TCR–peptide–MHC engagement [20].Subsequently, it was found that CD45 is predominantlyexcluded from the immunological synapse [21, 22]. Totest the hypothesis that CD45 exclusion from lipid micro-domains is necessary for proper TCR signaling, a chime-ric protein (Lck-CD45), composed of the lipid modifica-tion sites of Lck and the cytoplasmic region of CD45,was introduced into a CD4+ T cell hybridoma cell line,3A9, using retrovirus-mediated gene transduction. Thebiological functions of Lck-CD45-transduced 3A9hybridoma (3A9H) cells following TCR stimulation wereinvestigated. Our results suggest that the aberrant locali-zation of an active pool of CD45 PTP in lipid microdo-mains results in a TCR signaling dysfunction, whichmight be mediated by Src-family PTK, Lck and Fyn, in3A9H cells.

2 Results

2.1 Generation and expression of the Lck-CD45chimeric protein

A chimeric Lck-CD45 molecule (Fig. 1A) and a catalyti-cally inactive form [Lck-CD45(2CS)], in which the activesite cysteine was mutated to serine in both phosphatasedomains of the CD45 cytoplasmic region, were gener-ated by fusion of the wild-type or the mutated murineCD45 cytoplasmic region, respectively, to the N-terminal12 amino acids (aa), MGCVCSSNPEDD, from murineLck, a sequence known to be required for the plasmamembrane localization and activation of the kinase [23].This region can provide the necessary lipid modificationsignals and has been used to target heterologous pro-teins to lipid microdomains on the cell surface [24]. Theexpression of the Lck-CD45 chimeric molecules wasexpected to target the CD45 cytoplasmic PTP domainsto the region on the cell surface where Lck normallyresides. In another version of this construct, a triple mycepitope tag was added at the C terminus of the chimericmolecules (Lck-CD45-myc) for further detection. Expres-sion of the Lck-CD45 protein and its phosphatase-

inactive counterpart was verified in HeLa cells using theT7 Vaccinia virus overexpression system (data notshown). Subsequently, the Lck-CD45 constructs weresubcloned into a retrovirus vector, GFP-RV, renderingLck-CD45-RV or Lck-CD45-myc-RV or its C-S mutantform [Lck-CD45(2CS)-myc-RV].

The GFP-RV constructs were transfected into thePhoenix-exo retrovirus packaging cells. 3A9H cells weresubsequently infected with supernatants from GFP-RV-transfected Phoenix cells. The expression of Lck-CD45chimeric protein in 3A9H cells after transduction wasobserved at the predicted molecular weight (m.w.) ofapproximately 90 kDa protein by SDS-PAGE and immu-noblot analysis with a CD45 cytoplasmic region specificantiserum (Fig. 1B). In contrast, no bands of similar sizewere detected in the cell lysate from 3A9H cells infectedwith the empty vector. All 3A9H lysates contained similarlevels of endogenous CD45 protein (m.w. ˚ 200 kDa)serving as an internal control. Using confocal micros-copy, anti-myc immunostaining of 3A9H cells trans-duced with Lck-CD45-myc-RV or Lck-CD45(2CS)-myc-RV clearly showed surface localization of the chimericproteins on GFP+ cells (Fig. 1C). In contrast, no mycstaining was observed in GFP+ 3A9H cells transducedwith either GFP-RV alone (data not shown) or the Lck-CD45-RV construct lacking the myc tag.

2.2 Localization of the expressed Lck-CD45chimeric molecule in lipid microdomains

To isolate lipid microdomains, a simplified isolationmethod [25] was adapted. Following the lysis of 3A9Hcells in 0.5% Triton X-100 (TX-100) at 4°C, lipid microdo-mains form insoluble complexes, which can be sepa-rated by centrifugation. The addition of 1% SDS in TX-100 lysis buffer and brief sonication at 4°C allowed therelease of lipid microdomains from the insoluble pellets.Thy-1, an GPI-anchored glycoprotein known to localizeto detergent-resistant lipid microdomains [16, 25], wasused to verify the isolation of lipid microdomains. Indeed,Thy-1 was predominately localized in the TX-100-resistant (insoluble) fraction from 3A9H cells. In contrast,little Thy-1 was detected in the TX-100 soluble fractionfrom 3A9H cells. Following treatment of 3A9H cells withM g C, the amount of Thy-1 localized to lipid microdo-mains was decreased in a concentration-dependentmanner, while the amount of Thy-1 detected in the TX-100-soluble fraction increased conversely (data notshown).

As reported previously, Thy-1, Lck, Fyn and CD4 werepredominantly localized in lipid microdomains on the cellsurface (data not shown). However, certain cell surface

Eur. J. Immunol. 2002. 32: 2578–2587 Expression of CD45 protein tyrosine phosphatase in lipid microdomains 2579

Fig. 1. Generation and expression of Lck-CD45 chimeric protein. (A) Schematic diagram of the Lck-CD45 chimeric molecule. ALck-CD45 chimeric molecule was generated by fusing the murine CD45 cytoplasmic region, which contains 2 PTP domains, tothe 12 N-terminal aa (MGCVCSSNPEDD) of Lck. The lipid modification sites are underlined. In another version of this construct,a triple myc tag was added at the C-terminal Lck-CD45 construct (Lck-CD45-myc). A mutated form of Lck-CD45-myc [Lck-CD45(2CS)-myc], in which the active site cysteine was mutated to serine in both phosphatase domains, was generated as well.(B) Anti-CD45 immunoblot analysis of the total cell lysates from the GFP-RV vector-, Lck-CD45-, Lck-CD45-myc- or Lck-CD45(2CS)-myc-transduced 3A9H cells. (C) Confocal microscopy analysis of the Lck-CD45-, Lck-CD45-myc- or Lck-CD45(2CS)-myc-transduced 3A9H cells by anti-myc staining. The experiment was repeated three times, and one representativeis shown.

transmembrane proteins, such as CD45 (Fig. 2) andCD71 (data not shown), were primarily excluded fromlipid microdomains. Certain signaling molecules, such asSHP-1 and Csk, were distributed in both lipid microdo-mains and other regions of the cell surface (data notshown). These data demonstrated that this simplifiedTX-100 precipitation and SDS-release method can beused as an alternative method to the standard sucrose

gradient centrifugation for lipid microdomain isolation.Indeed, the partitioning and localization of Thy-1, Lckand CD4 in the TX-100-insoluble fraction correspondedwell to the enrichment of them at the interface of 6% and30% in sucrose gradients following equilibrium densitygradient centrifugation. The localization of Lck, CD4 andFyn to lipid microdomains was also disrupted by M g Ctreatment (data not shown).


Fig. 2. Localization of the Lck-CD45 chimeric protein in lipidmicrodomains. Immunoblot analysis of endogenous CD45,Lck-CD45, Lck-CD45-myc or Lck-CD45(2CS)-myc in theTX-100-soluble and -insoluble fractions isolated from 3A9Hcells transduced with different GFP-RV constructs. Theexperiment was repeated five times, and one representativeis shown.

Fig. 3. Ca2+ mobilization of the transduced 3A9H cells. GFP+

3A9H (6×106) cells transduced with empty GFP-RV (solidline), Lck-CD45 (dotted line), Lck-CD45-myc (dashed line) orLck-CD45(2CS)-myc (dotted-dashed line) were pre-loadedwith Fura-2AM, and stimulated with biotinylated anti-CD3(1 ? g) and anti-CD4 (1 ? g) Ab. Streptavidin (25 ? g) wasadded at 30 s. [Ca2+]i was measured by fluorimetry. Theexperiment was repeated nine times, and one representativeis shown.

The Lck-CD45 chimeric proteins in GFP+ transduced3A9H cells (the myc tagged form migrating slightlyslower than the untagged form) was predominantly local-ized in the TX-100-insoluble fraction, as expected(Fig. 2). Similarly, this localization was verified with equi-librium density gradient centrifugation and disrupted bythe treatment of M g C (data not shown). Taken together,these data strongly support the contention that the CD45cytoplasmic region can be forcedly localized to lipidmicrodomains on the 3A9H cell surface through theunique N-terminal sequence of Lck.

2.3 Inhibition of Ca2+ mobilization in the Lck-CD45-transduced T hybridoma cells

To examine whether Ca2+ mobilization after TCR engage-ment was affected by the Lck-CD45 protein expression,GFP+ transduced 3A9H cells were activated by incuba-tion with biotinylated anti-CD3 and anti-CD4 Ab, fol-lowed by co-cross-linking with streptavidin. Ca2+ mobili-zation was decreased by 30% and 38% in Lck-CD45- orLck-CD45-myc-transduced cells, respectively, after TCRstimulation, compared to that induced in 3A9H cellstransduced with GFP-RV vector (Fig. 3). To determinewhether the phosphatase activity of CD45 was requiredfor the observed decrease in Ca2+ mobilization, a phos-phatase inactive form of Lck-CD45-myc [Lck-CD45(2CS)-myc] was also expressed in 3A9H cells. Fol-lowing the co-cross-linking of the CD3 and CD4 recep-tors, the change in intracellular Ca2+ ([Ca2+]i ) was verysimilar to that in cells infected with vector alone. All fourdifferent transfectants showed similar Ca2+ mobilization

after treatment with ionomycin. The 3A9H cells trans-duced with three different constructs, Lck-CD45, Lck-CD45-myc or Lck-CD45(2CS)-myc, expressed similarlevels of GFP as assessed by FACS analysis (data notshown). In addition, the 3A9H cells, either nontrans-duced or transduced with empty vector or different Lck-CD45 constructs, were shown to express similar levelsof CD3, CD4 and CD45 on the cell surface, as measuredby FACS analysis (data not shown). These data suggestthat the expression of Lck-CD45 attenuated signalinginitiated by CD3 and CD4 co-cross-linking and that thephosphatase activity of the CD45 cytoplasmic regionwas required for this inhibitory effect.

2.4 Inhibition of IL-2 production from the Lck-CD45-transduced 3A9 T hybridoma cells

As a more physiological readout to examine the effect ofLck-CD45 expression on 3A9H cells, the ability of thesecells to produce IL-2 upon activation was examined. The3A9H cells respond to the specific Ag, hen egg-whitelysozyme (HEL), in the context of H-2k. When 3A9H cellswere stimulated with HEL in the presence of the APC, IL-2 production from the Lck-CD45-transduced cells wasdiminished compared to that produced by 3A9H cellstransduced with empty vector (Fig. 4A). Similarly, IL-2production from Lck-CD45-transduced cells was alsodecreased upon stimulation with plate-bound anti-CD3Ab compared to the IL-2 produced from GFP-RV-transduced 3A9H cells (Fig. 4B). In contrast, the parental3A9H cells and 3A9H cells transduced with either empty


Fig. 4. IL-2 production of the transduced 3A9H cells. Supernatants were collected from the untransduced 3A9H cells (solid line),or GFP-RV vector (dotted-dashed line) or Lck-CD45 (dashed line) transduced 3A9H cells stimulated for 18 h: (A) with increasingdoses of HEL and APC, (B) with increasing doses of plate-bound anti-CD3 Ab and (C) with increasing doses of ionomycin andPMA (4 ng/ml). The biological activity of IL-2 in the supernatants was measured by [3H]thymidine incorporation using CTLL cells.The experiment was repeated four times.

vector or Lck-CD45-RV produced similar amounts of IL-2 when they were stimulated with PMA and ionomycin(Fig. 4C). Taken together, these results strongly suggestthat the defect in IL-2 production occurred at an earlysignaling event(s), which was proximal to the activationof PKC and the triggering of Ca2+ mobilization.

2.5 Inhibition of lipid microdomain-associatedPTK activity in the Lck-CD45-transduced 3A9T hybridoma cells

To directly assess PTK activity associated with lipidmicrodomains isolated from GFP-RV-transduced cells,an in vitro kinase assay was performed, using an optimalpeptide substrate, GAEEEIYAAFFAKKK, for Lck kinaseassay [26]. The PTK activity of lipid microdomains iso-lated from stimulated Lck-CD45-myc-transduced 3A9Hcells was inhibited by approximately 50%, as comparedto the PTK activity from the 3A9H cells transduced withempty vector (Fig. 5A). In contrast, the lipidmicrodomain-associated PTK activities were compara-ble in 3A9H cells transduced with GFP-RV and Lck-CD45(2CS)-myc. All lipid microdomain preparationscontained similar levels of Lck and Fyn (Fig. 5B). Theinhibition was only observed in Lck-CD45-myc-transduced 3A9H cells and not in the catalytically inac-tive Lck-CD45(2CS)-myc-transduced cells, indicatingthat the phosphatase activity of the CD45 cytoplasmicregion was necessary for the inhibition. The Src-familyPTK, Lck and Fyn, localized in lipid microdomains, werelikely substrates of Lck-CD45.

3 Discussion

CD45 can regulate members of the Src-family PTK inboth positive and negative manners. In BM-derived mac-rophages, CD45 is found to co-localize with g 2-integrinand Lyn to adhesion sites and down-regulate Hck andLyn kinase activity, thus serving as a negative regulatorof integrin-mediated adhesion [8]. However, in T cells,CD45 also functions as a positive regulator of Src-familyPTK members by dephosphorylating the inhibitory phos-photyrosyl site located in the C-terminal regions of Lckand Fyn. CD45-deficient T cell lines cannot initiate cellu-lar signaling upon TCR engagement, validating theessential role played by CD45 in T cell activation [2]. Onemechanism to explain this paradoxical function of CD45and its net effect on Src-family PTK activities is basedupon the dynamic redistribution of CD45 and its subcel-lular localization with respect to other cellular mediatorsin different biological contexts [9, 11].

The targeting of Lck to lipid microdomains is necessaryfor T cell signaling events to proceed. A non-raft-associated transmembrane chimera containing theCD16 extracellular region, the CD7 transmembraneregion and un-S-acylated Lck was excluded from lipidmicrodomains on the cell surface, did not co-localizewith the TCR and was not able to restore a full TCR sig-naling in Lck-deficient cells [27]. The rapid phosphoryla-tion and activation of Lck and Fyn are the earliest bio-chemical events detectable during T activation. Thisrapid phosphorylation event is observed in lipid microdo-mains upon engagement of the TCR [21, 28]. Severalreports showed that lipid microdomains on lymphocytesare enriched for Src-family PTK, yet contain little or virtu-


Fig. 5. Lipid microdomain-associated PTK activity fromstimulated 3A9H cells. (A) In vitro PTK activity of lipid micro-domains isolated by equilibrium density gradient centrifuga-tion from 3A9H cells transduced with different GFP-RV con-structs after anti-CD3 and anti-CD4 cross-linking. (B) Immu-noblot analysis of Lck and Fyn from the same lipid microdo-mains isolated as (A). The experiment was repeated threetimes and a representative immunoblot is shown.

ally undetectable levels of CD45 [13, 15, 16]. Thus differ-ential membrane compartmentalization may be respon-sible for the segregation of CD45 from Lck and Fyn onthe T cell surface. In this study, we directly tested theeffect of forced expression of the catalytically activeCD45 cytoplasmic region in lipid microdomains on thesurface of T hybridoma cells, which have normal levels ofthe endogenous CD45 proteins.

Lipid modification of Lck and Fyn is required for targetingthem to lipid microdomains. The myristylation of the Gly2in the N-terminal sequence of Lck has been shown to benecessary for the membrane recruitment and activationof Lck. Cys3 and Cys5 are the palmitoylation sites [23].We generated a Lck-CD45 chimera by fusing the CD45cytoplasmic region to the 12 N-terminal aa of Lck(Fig. 1A). Therefore, it was expected that the lipid modifi-cation signal in the 12 N-terminal aa of Lck would targetthe CD45 cytoplasmic region to the area on the cell sur-face where Lck molecules normally localize. Indeedwhen the localization of Lck-CD45 chimeric protein wasexamined, the Lck-CD45 chimeric protein was predomi-

nantly localized in lipid microdomains on the 3A9H cellsurface (Fig. 1C and 2). The effect of Lck-CD45 proteinexpression on the biological functions of 3A9H cells wasalso examined. Both Ca2+ mobilization and IL-2 produc-tion in Lck-CD45-transduced 3A9H cells were signifi-cantly decreased after stimulation by either following Abco-cross-linking of surface CD3 and CD4, or more physi-ologically, by stimulation with Ag in the presence of APC(Fig. 3 and 4). The extent of inhibition of Ca2+ mobilizationin Lck-CD45-transduced 3A9H cells was correlated tothe expression levels of GFP and Lck-CD45 chimericproteins as well as to the amount of Lck-CD45 proteinslocalized in the lipid microdomains (data not shown). Fur-thermore, the PTK activity in lipid microdomain prepara-tion was decreased from Lck-CD45-transduced 3A9Hcells (Fig. 5). Importantly, the inhibitory effects on Ca2+

flux and lipid microdomain-associated PTK activityobserved with Lck-CD45 chimeric protein expressionwere dependent upon the PTP activity of the CD45 cyto-plasmic region. The responses of 3A9H cells transducedwith the enzymatically inactive form of Lck-CD45 (2CS)to co-cross-linking of CD3 and CD4 were similar to theresponses of cells transduced with vector alone (Fig. 3and 5).

CD45 has potent phosphatase activity and a broad sub-strate specificity in vitro. It has been shown that in thecases for Lck and Lyn, CD45 can dephosphorylate boththe activating and negative regulatory sites (phosphoty-rosyl 394 and 505 in Lck) in vivo [6, 7]. By comparing thecontrol and different Lck-CD45-transduced cells, nochanges in the basal level of total protein tyrosine phos-phorylation from cell lysates or lipid microdomains wereobserved. Furthermore, after anti-CD3/CD4 co-cross-linking, there were no changes in the kinetics of inducedtyrosine phosphorylation of either total cytoplasmic pro-teins or proteins in lipid microdomains, or immunopre-cipitates by anti-Lck serum from total proteins or lipidmicrodomains. Finally, our preliminary experiment usingantiserum specific for phosphotyrosyl 505 of Lck did notdetect any difference in phosphorylation status of Tyr505of Lck in lipid microdomains isolated from stimulated3A9H cells transduced with different Lck-CD45 con-structs (data not shown). Since the transduced cellshave normal levels of endogenous CD45, the phospho-tyrosyl 505 of Lck still could be primed (dephosphory-lated) at the edge of lipid microdomains, as in untrans-duced cells, before or on the TCR engagement, allowingTCR-mediated signaling to initiate. Only in the trans-duced cells can Lck-CD45 proteins co-migrate with Lckand other signal proteins into lipid microdomains todiminish TCR-initiated signaling. Although the phospho-tyrosyl 394 of Lck is a likely substrate of Lck-CD45 intransfected 3A9H cells, other proximal localized sub-strates, including the phosphorylated ITAM of the Ag–


TCR/CD3 complex are also potentially dephosphory-lated by CD45 [29]. In both scenarios, dephosphorylationof PTK or PTK substrates catalyzed by CD45 could resultin diminished TCR signaling. To fully elucidate the molec-ular mechanism(s) of the inhibitory effect of Lck-CD45,the phosphorylation status of the activating and inhibi-tory regulatory tyrosine sites in Lck and Fyn, the phos-phorylation status of the CD3 ITAM and regulatory tyro-sine sites in ZAP70, the kinase activity of Lck, Fyn andZAP70 in lipid microdomains need to be examined. Theavailability of phosphorylated Tyr394 of Lck-specific Abwill greatly accelerate the study in this area.

Recently, a study showed that CD45 negatively regulatescytokine receptor signaling by dephosphorylating JAKkinases [30]. This further supports the possibility thatCD45 can inhibit the functions of signal proteins thoughdephosphorylation of important regulatory tyrosine siteswithin those molecules. Two groups reported that target-ing the PTP domain of SHP-1, a PTP which can bind toZAP70 and inhibit TCR signaling [31], to lipid microdo-mains in Jurkat cells by fusing with the 14 N-terminal aaof Lck or the 36 N-terminal aa of linker for activation of Tcells (LAT), respectively [32, 33]. In both cases, the distalevents of TCR signaling, such as CD69 induction, Ca2+

influx, NFAT activation and IL-2 production were inhib-ited and the inhibitory effect of chimeric SHP-1 wasdepending on the PTP activity. In LAT/SHP-1 transfec-tants, the phosphorylation of proximal signal proteins,such as TCR ´ and ZAP70, was normal; while the phos-phorylation of signal proteins downstream of LAT weredecreased. Because a similar chimeric protein of LAT/SHP-2 had no effect on TCR signaling, the inhibition byLAT/SHP-1 is specific. Furthermore, the two Cys, thepalmitoylation sites in the LAT N-terminal sequence,were necessary for the functions of LAT/SHP-1 since amutation of them to Ala abolished both the lipid microdo-mains targeting and the TCR signaling inhibitory effects.These results, together with our results, support thenotion that the specific membrane compartmentalizationof PTK, PTP (including CD45 and SHP-1) and other sig-naling molecules during TCR activation may provide anadditional level of regulation for precise TCR signaling.

The results presented here support the model of CD45localization and its substrate(s) accessibility proposedby Thomas and Brown [9]. This does not exclude theimplications of the receptor topology model proposed byShaw, Dustin and van der Merwe [34, 35], which empha-sizes that the sizes of the extracellular domains of CD45,CD43 and LFA-1 are too large to be accommodated inthe space formed within the contact area between T cellsand APC. The mechanism underlining the exclusion ofCD45 from lipid microdomains in cells is unclear. Theinteraction of CD45 with CD45AP [36], its ability to inter-

act with certain cytoskeletal components [37] and therearrangement of cytoskeleton proteins occurring uponT cell activation [38] may be involved. In addition, theregion(s) of CD45 that is responsible for the exclusion ofCD45 from lipid microdomains also remains to be identi-fied.

In this study, we demonstrate that inclusion of CD45 PTPactivity into lipid microdomains will diminish TCR-initiated signaling. Therefore the segregation of CD45transmembrane PTP from proximal TCR signaling com-ponents after priming Src-family PTK is essential for sus-tained TCR-mediated signaling.

4 Materials and methods

4.1 Cell lines and reagents

The 3A9H cells, specific for an epitope of HEL peptide46–61 in the context of I-Ak, provided by M. Dustin (Wash-ington University, St. Louis, MO), were cultured in RPMI con-taining 10% fetal calf serum (RPMI–10%FCS) and 2 mM L-glutamine. CH27 cells, a B cell lymphoma line (H-2k), pro-vided by P. Allen (Washington University), were cultured inRPMI–10%FCS, and used as APC for HEL (Sigma, St. Louis,MO). Phoenix-exo fibroblast cells, provided by K. Murphy(Washington University), were maintained in DMEM-10%FCS and 2 mM L-glutamine. To extract cholesterol fromeukaryotic plasma membranes, 3A9H cells were incubatedwith increasing concentrations of M g C (Sigma), at 37°C for20 min.

Rabbit antisera to Lck, Fyn, SHP-1 and the recombinantCD45 cytoplasmic region were as described previously [3,31, 36], rabbit anti-mouse CD4 IgG (Santa Cruz Biotechnol-ogy, Santa Cruz, CA), mouse anti-transferrin (CD71; Zymed,South San Francisco, CA), anti-c-myc (9E10) and anti-Csk(Transduction Laboratories, Lexington, KY), rat anti-CD90(Thy-1.2) Ab (30-H12; American Type Culture Collection,Rockville, MD) were used in immunoblot analysis. A biotiny-lated anti-c-myc Ab (9E10), from BabCo (Richmond, CA),was used for immunostaining. Biotinylated anti-mouse CD3Ab (145-2C11), CD4 Ab (GK1.5; both from PharMingen, SanDiego, CA) and streptavidin (Jackson Immuno-Research,West Grove, PA) were used for immuno-crosslinking.

4.2 Generation of the Lck-CD45 chimeric constructs

The Lck-CD45 chimeric molecule was generated inpBluescript SK plasmid (Stratagene, La Jolla, CA) by ligationof the XhoI fragment of the murine CD45 cytoplasmic regioncontaining two PTP domains, to the 12 N-terminal aa(MGCVCSSNPEDD) from the murine Lck using the oligonu-cleotide 5’-ATGGGCTGTGTCTGCAGCTCAAACCCTGAAG-ATGAC-3’. A triple myc tag (5’-GAACAAAAGCTGATTAGC-


GAAGAGGACCTG-3’×3) was added to the C terminus ofthe Lck-CD45 to obtain Lck-CD45-myc. An enzymaticallyinactive form of the Lck-CD45 chimeric construct [Lck-CD45(2CS)-myc] was generated by mutation of the activesite cysteine to serine in both phosphatase domains of theCD45 cytoplasmic domain.

4.3 Transduction of 3A9 T hybridoma cells using GFP-RV retrovirus vector

The GFP-RV retroviral vector was provided by K. Murphy.XhoI-digested Lck-CD45, Lck-CD45-myc or Lck-CD45(2CS)-myc DNA fragments were ligated, respectively,into GFP-RV to produce Lck-CD45-RV, Lck-CD45-myc-RVor Lck-CD45(2CS)-myc-RV. Phoenix-exo packaging cellswere transfected using the Ca2+ phosphate precipitationmethod, following the online protocol (http://www-leland.stanford.edu/group/nolan/NL-phnxr.html). The 3A9Hcells were infected twice with retroviral supernatants fromtransfected Phoenix-exo cells on day 2 and day 3. Infected3A9H cells were sorted for GFP expression on day 7 usingMoFlo (Cytomation, Fort Collin, CO) to a purity of G 95%GFP+ cells.

4.4 Confocal microscopy

Retrovirally transduced, GFP+ 3A9H cells (1×105) with eitherLck-CD45-RV, Lck-CD45-myc-RV or Lck-CD45(2CS)-myc-RV were fixed with 2% paraformaldehyde (Sigma) in PBS(pH 7.4) for 10 min at room temperature (RT), followed bywashing once with PBS. The fixed cells were deposited onpoly-L-lysine coated glass slides at 1,200 rpm for 10 minusing a Cytospin 3 (Shandon, Pittsburgh, PA). Cells werepermeabilized with 0.5% TX-100 (Sigma) in PBS for 4 min atRT and washed once with PBS. Slides were incubated withbiotinylated anti-c-myc Ab, diluted at 1:500 with 1% BSA inPBS, and incubated at RT for 1 h, followed by washing oncewith PBS. Slides were then incubated with Texas Red-conjugated Streptavidin (Molecular Probes, Eugene, OR),diluted at 1:200 with 1% BSA in PBS, at RT for another hour,followed by one PBS wash. Images were obtained using aZeiss LSM510 confocal microscope (Carl Zeiss, Inc. Thorm-wood, NY).

4.5 Preparation of TX-100 lysate and equilibriumdensity gradient centrifugation

The 3A9H cells were lysed with TX-100 as previouslydescribed [25]. Briefly, 3A9H cells (4×107) were washed oncewith PBS and once in TKM buffer (50 mM Tris-HCl, pH 7.4,25 mM KCl, 5 mM MgCl2 and 1 mM EGTA) at 4°C and werelysed in 800 ? l of ice-cold TKM lysis buffer (containing 4 mMleupeptin, 0.2 mg/ml aprotinin, 1 mM PMFS, 200 ? M NaVO4,5 mM iodoacetamide, 10 mM NaF, 10 mM Na2MoO4, all fromSigma, and 0.5% TX-100) for 20 min on ice. The lysates

were microcentrifuged at 12,000×g for 5 min at 4°C to pelletnuclei and TX-100-insoluble complexes. The supernatantwas recovered as the TX-100-soluble fraction. The pelletwas solubilized in 800 ? l of TKM buffer containing 1% SDSand protease/phosphatase inhibitors by sonication on icefor 10 s, followed by centrifugation at 12,000×g for 5 min at4°C. The supernatant (TX-100-insoluble fraction) was col-lected and stored it at –20°C.

The 3A9H cell lysates were also subjected to equilibriumdensity gradient centrifugation. Briefly, 1 ml of 1% TX-100whole cell lysate was adjusted to 40% sucrose by adding1 ml of 80% sucrose in TKM, and overlaid with 6 ml of 30%sucrose in TKM, followed by 3.5 ml of 5% sucrose in TKMand centrifugated at 35,000 rpm for 12 h at 4°C in a Beck-man SW41 rotor. Fractions (1 ml) were collected from thetop of the gradient and subjected to immunoblot analysis.

4.6 Immunoblot analysis

The 3A9H cells (2×106) were lysed in 40 ? l of NP40 (Amer-sham, Arlington Heights, IL) lysis buffer (containing 4 mMleupeptin, 0.2 mg/ml aprotinin, 1 mM PMFS, 200 ? M NaVO4,5 mM iodoacetamide and 1% NP40) for 20 min on ice. Thelysate was microcentrifuged at 12,000×g for 20 min at 4°Cto pellet nuclei. The supernatant was collected. The total celllysates, the TX-100-soluble or insoluble fractions from equalnumbers of 3A9H cells were resolved by SDS-PAGE gelunder nonreducing (for Thy-1 immunoblotting) or reducing(with 7% 2-ME) conditions. Following immunoblotting wasperformed as described [31, 36].

4.7 [Ca2+]i measurement

GFP+ transduced 3A9H Cells (6×106) were loaded with 3 mMFura-2AM (Molecular Probes) in RPMI–10%FCS for 40 minat 37°C. Cells were washed once with cold PBS and incu-bated on ice for 20 min with 0.2 ml of biotinylated anti-CD3and -CD4 Ab at 5 ? g/ml in PBS. [Ca2+]i measurement wasperformed and intracellular Ca2+ concentrations calculatedas described [39].

4.8 IL-2 bioassay

GFP+ transduced 3A9H (2×105) cells were incubated withserial dilutions of HEL and 105 APC/well in 200 ? l of RPMI-10% FCS in 96-well flat-bottom culture plates at 37°C for18 h. To stimulate 3A9H cells with cross-linked Ab, anti-CD3Ab was serially diluted in PBS and 100 ? l aliquots/well wasused to coat plates by incubation overnight at 4°C. Afterwashing twice with PBS, 3A9H cells were added to wells in200 ? l of RPMI-10% FCS and incubated at 37°C for 18 h.3A9H cells were also stimulated with serial dilutions of iono-mycin in the presence of PMA (4 ng/ml, both from Calbio-chem, La Jolla) for 18 h. Supernatants (100 ? l) from unstim-


ulated or stimulated cells were collected, and cultured with5×103/well of IL-2-dependent CTLL cells for 18 h. The cul-tures were labeled with 0.25 ? Ci [3H]thymidine (Amersham)for 8 h, then the amount of incorporated radioactivity deter-mined. Reactions were carried out in triplicates.

4.9 In vitro kinase assay and immunoblot analysis ofPTK in lipid microdomains

TX-100-insoluble fractions were prepared from 1×108 GFP+

transduced 3A9H cells by equilibrium density gradient cen-trifugation as described above. Fractions at the interfaces(1.2 ml) between 5% and 30% sucrose in TKM were col-lected and diluted with equal volume of ice-cold TKM fol-lowed by centrifugation at 100,000×g at 4°C for 1 h. The pel-lets were resuspended in 200 ? l of kinase buffer (50 mMPIPES, pH 6.5, 2 mM MnCl2, 5 mM DTT, 0.1 mg/ml BSA).The kinase reaction was carried out, as described [27], byincubation of 20 ? l of lipid microdomain preparation with10 ? M ATP, 5 ? Ci [ + -32P]-ATP and 1 mM of a Lck substratepeptide, GAEEEIYAAFFAKKK, in 40 ? l at 30°C for 10 minand stopped by the addition of 20 ? l of 30% phosphoricacid. Kinase reaction mixture (15 ? l) was spotted onto p81paper squares followed by extensive washing in 0.5% phos-phoric acid and a quick rinse with acetone. The filters wereair dried and the amount of incorporated radioactivity deter-mined. Reactions were carried out in triplicates.

Aliquots (25 ? l) from the same lipid microdomain preparationwere subjected to Lck and Fyn immunoblot analysis.

Acknowledgments: We thank Drs. Ken Murphy for provid-ing retroviral vector, Phoenix-exo packaging cells and usingMoFlo, Andy Chan for using the spectrofluorimeter, MikeDustin for 3A9H cells, Paul Allen for CH27 and CTLL cells,and our colleagues for help and discussions. We also thankDrs. Rick Brown, Paul Allen, Hai-Tao He and Kerry Campbellfor critical review of this manuscript. This work was sup-ported by grants A126363 and GM56455 (MLT) and traininggrants AI07163 and AI09816 (XH) from the NIH. M. L. T. wasan investigator of HHMI.

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Correspondence: Xiao He, Immunobiology Program, Insti-tute for Cancer Research, Fox Chase Cancer Center, 7701Burholme Ave., Philadelphia, PA 19111, USAFax: +1-215-728-2412e-mail: X–He — fccc.edu

Present addresses: T. A. Woodford-Thomas, Donald Dan-forth Plant Science Center, St. Louis, Missouri, USA; K. G.Johnson and D. D. Shah, Department of Medicine, Washing-ton University School of Medicine, St. Louis, Missouri, USA


targeting of cd45 protein tyrosine phosphatase activity to lipid microdomains on the t cell surface...

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