Proc. Natl. Acad. Sci. USAVol. 88, pp. 2037-2041, March 1991Biochemistry

Tyrosine phosphatase CD45 is required for T-cell antigen receptorand CD2-mediated activation of a protein tyrosine kinase andinterleukin 2 production

(signal transduction/phosphatidylinositol/second messengers/CD28)

GARY A. KORETZKY*, JOEL Picust, TERRIE SCHULTZ*, AND ARTHUR WEISS**Department of Medicine, Division of Rheumatology, Department of Microbiology and Immunology, Howard Hughes Medical Institute, and tCancer ResearchInstitute, University of California, San Francisco, CA 94143

Communicated by Marian E. Koshland, November 29, 1990 (received for review September 12, 1990)

ABSTRACT CD45, a hematopoietic cell-specific surfaceantigen, has recently been shown to be a protein tyrosinephosphatase. Expression of CD45 is essential for the T-cellantigen receptor to couple with the phosphatidylinositol secondmessenger pathway and for antigen-mediated proliferation ofT lymphocytes. In this report we describe a CD45-deficientmutant of the human T-cell leukemia line Jurkat. CD45expression is required for the activation of a T-cell receptor-associated tyrosine kinase as well as the phosphatidylinositolpathway. Additionally, stimulation ofT lymphocytes by way ofthe accessory molecule CD2 requires the expression of CD45.The mutation in the CD45-deficient cell specifically impairssignal transduction by the T-cell receptor and CD2 becauseactivation events by way of another accessory molecule, CD28,are unimpaired.

Activation of T lymphocytes by way of the T-cell antigenreceptor (TCR) results in the stimulation of at least twosecond messenger pathways. The TCR regulates the phos-phatidylinositol (PI) pathway characterized by activation ofa phospholipase C specific for phosphatidylinositol 4,5-bisphosphate (PIP2) (1, 2). The hydrolysis products of PIP2are inositol trisphosphate, which is responsible for the releaseof calcium from intracellular stores, and diacylglycerol, acofactor in the activation of protein kinase C (PKC) (2, 3).Stimulation of the TCR also activates an as yet unidentifiedprotein tyrosine kinase (PTK) (4-6). The physiologicallyrelevant substrates of the PTK remain unknown; however, acomponent of the TCR itself, the chain, becomes phospho-rylated on tyrosine residues (6). Although there exist con-siderable data from studies using pharmacologic agents orvarious signal transduction mutants (2, 7, 8), the precise roleof these two pathways in the initiation of subsequent biologicevents remains unclear.T lymphocytes can also be activated when "accessory"

receptors interact with their ligands. Thus, stimulation of theT-cell surface molecule CD2 results in the generation ofPI-derived second messengers and the appearance of newphosphotyrosine-containing proteins (9-12). Another surfaceantigen, CD28, found on the majority of T lymphocytes canalso deliver stimulatory signals (13, 14). Interestingly, acti-vation of T cells with monoclonal antibody (mAb) directedagainst CD28 cannot be explained by either of the knownsecond messenger pathways stimulated by TCR activation (2,6, 13), yet CD28 ligation does result in enhanced productionof lymphokines such as interleukin 2 (IL-2).CD45 is expressed on all hematopoietic cells except those

of erythrocyte lineage (15). Previous studies have shown thatunder various conditions T-cell activation can be either

enhanced or inhibited in vitro by mAbs against CD45 (16, 17).Possible mechanisms for the influence of CD45 on T-cellactivation were suggested when the cytoplasmic domain ofCD45 was found to contain two tandem repeats that arehomologous to a tyrosine phosphatase isolated from placenta(18). Moreover, immunoprecipitates of CD45 have intrinsictyrosine phosphatase activity (19).We recently characterized a mutant derived from the

human T-cell leukemia line HPB-ALL that lacks expressionof CD45 (20). In contrast to CD45-positive clones, the TCRof the CD45-negative cells fails to couple to the PI secondmessenger pathway. Reconstitution of CD45 by transfectionof cDNA for murine CD45 restored the ability of the TCR tostimulate the formation of PI-derived second messengers(20). These data, coupled with previous studies demonstrat-ing the importance of CD45 in the proliferative response ofmurine T-cell clones to specific antigen (21), suggested animportant role for CD45 in controlling TCR-mediated signaltransduction.Here we describe the isolation of a CD45-deficient mutant

clone derived from the Jurkat human T-cell leukemia line. Incontrast to HPB-ALL, Jurkat expresses CD2 and CD28 andis a potent producer of IL-2. We show that CD45 is essentialfor the TCR and CD2 to couple efficiently to the PI and PTKsecond messenger pathways and is necessary for TCR- andCD2-mediated production of IL-2. In contrast, CD45 is notnecessary for signal transduction by way of CD28.

MATERIALS AND METHODSmAbs. The anti-CD45, GAP 8.3, and anti-CD2, OKT11,

hybridomas were obtained from the American Type CultureCollection. Purified anti-CD2 mAb, 9.6, anti-CD28 mAb, 9.3,and anti-CD45 mAb, 9.4, were generously supplied by On-cogen (Seattle). Purified anti-CD2 mAb, 9-1, was generouslysupplied by Bo Dupont (Sloan Kettering Institute for CancerResearch, New York). Anti-CD45, HLE-1, was purchasedfrom Becton Dickinson. C305 (clonotypic anti-TCR) has beendescribed (22).

Cell Lines and the Derivation of a CD45-Deficient JurkatClone. Jurkat (clone E6-1), J.CaM1, and J.CaM2 have beendescribed (22, 23). HBP.45.0 is a CD45-negative variant ofHPB-ALL and HBP.45.1 is a CD45-positive variant of thiscell line (20). J45.01, a CD45-deficient clone of Jurkat, wasisolated after y irradiation (200 rads; 1 rad = 0.01 Gy) ofJurkat followed by complement-mediated lysis with the CD45mAb GAP 8.3 and enrichment of surviving CD45-deficient

Abbreviations: TCR, T-cell antigen receptor; PI, phosphatidylino-sitol; PIP2, phosphatidylinositol 4,5-bisphosphate; PTK, proteintyrosine kinase; IL-2, interleukin 2; mAb, monoclonal antibody; IP,inositol phosphate; MAP-2 kinase, microtubule-associated proteinkinase 2; PMA, phorbol 12-myristate 13-acetate; PKC, proteinkinase C.

2037

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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cells by using magnetic beads coated with biotin (MiltenylBiotec GmbH, Bergisch Gladbach, F.R.G.). CD45-deficientcells finally were isolated with a fluorescence-activated cellsorter and cloned by limiting dilution.Flow Cytometry and Transient Heterokaryon Assay. Cells

were stained with mAbs and analyzed by flow cytometry asdescribed (20). The heterokaryon assay for signal transduc-tion complementation analysis was performed as described(23).In Vitro Phosphatase Assay. Membranes were prepared

from wild-type Jurkat and J45.01 by nitrogen cavitation anddiluted to 5 mg of membrane protein per ml of buffer (20 mMHepes, pH 8.0/2 mM MgCl2/1 mM EDTA/1 mM 2-mercap-toethanol/10% glycerol). Membranes were assayed for tyro-sine phosphatase activity by incubation with 10 mM o-phos-pho-L-tyrosine (Sigma) in assay buffer (100 mM sodiumacetate, pH 6/1 mM EDTA) as described (24).Measurement of [3H]Inositol Phosphates ([3HJIPs). Cells

were labeled with myo-[3H]inositol and stimulated with C305(ascites, 1: 1000) or CD2 mAb (1 ,ug/ml each of 9-1 and 9.6).[3H]IPs were then extracted and quantified as described (25).

Immunoprecipitation and Western Blotting. For analysis ofphosphotyrosine-containing proteins, cells were stimulatedwith medium alone, anti-TCR, anti-CD2, or phorbol 12-myristate 13-acetate (PMA) for the indicated time. Lysateswere assessed for the presence ofphosphotyrosine-containingproteins as described (26). For quantitation of CD45, lysateswere immunoprecipitated with anti-CD45 mAb, 9.4. Sampleswere electrophoresed on 7% SDS/polyacrylamide gels thatwere then blotted with purified 9.4 mAb, 0.5 ,ug/ml.

IL-2 Production and Biolgic Assay. Supernatants from cellcultures were assayed for IL-2 as described (22).

RESULTSCharacterization of a CD45-Deficient Jurkat Clone. To

address the potential role of CD45 in coupling the TCR to thePTK second messenger pathway and the possible require-ment for CD45 in CD2 and CD28 signal transduction, wemutagenized the human T-cell leukemia line Jurkat andselected for CD45-deficient variants (see Materials andMethods). One of the clones obtained, J45.01, stains brightly(comparable to wild type) with mAb directed against theTCR, CD2, and CD28 but expresses only 8% as much CD45on its surface when compared to the wild-type Jurkat parent,even when a panel of CD45 mAb was used (Table 1).To characterize J45.01 further, Northern blot analysis was

performed on poly(A)+-selected mRNA obtained from Jurkatand J45.01, which revealed grossly equivalent levels of CD45mRNA (data not shown). We therefore assessed the amount ofCD45 protein expressed intracellularly and on the cell surfacein the two clones by Western blotting ofwhole cell lysates witha mAb directed against all isoforms of CD45. In lysates, sub-stantial amounts of CD45 can be detected in Jurkat, whereasthere is no detectable CD45 in J45.01 (Fig. LA). When immuno-

Table 1. Staining characteristics of Jurkat and J45.01Median

fluorescence

Antigen Jurkat J45.01

TCR 68 66CD2 58 58CD28 39 25CD45 99 8

Median fluorescence was measured by flow cytometry (linearized,

A

kDa

205 - _mW

1 6 -

401 B

E 30Ea

255 10 15Membrane protein, 9g

FIG. 1. (A) Western blot of whole cell lysates (1.25 x 106 cellequivalents per lane) with mAb 9.4 for the presence ofCD45 protein.Lane 1, Jurkat; lane 2, J45.01. Molecular mass markers are in kDa.(B) Tyrosine phosphatase activity in membranes from Jurkat (0) andJ45.01 (*).

precipitates from 100 x 106 cells are compared a small amountof CD45 protein is apparent in J45.01 (data not shown), indi-cating that the mutation in J45.01 does not result in the completeabsence of CD45 protein but does result in marked reduction.Membranes derived from CD45-negative variants of the

murine T-cell lymphoma BW5147 possess only 10%o of thetyrosine phosphatase activity seen in membranes from CD45-positive derivatives of BW5147 (24). Because we were able todetect small amounts of CD45 protein in J45.01 by immuno-fluorescence and immunoprecipitation, we analyzed mem-brane preparations from Jurkat and J45.01 for tyrosine phos-phatase activity. In Fig. 1B membranes from the two cloneswere compared in an in vitro assay using o-phospho-L-tyrosineas a substrate. Although a small amount of CD45 protein isseen in J45.01, we can detect little evidence of membrane-associated tyrosine phosphatase activity in J45.01.TCR-Mediated PIP2 Hydrolysis Requires Expression of

CD45 in Jurkat. We tested the ability of the TCR and otheraccessory signaling molecules to couple to their secondmessenger pathways in J45.01 by examining the ability oftheTCR to initiate increases in intracellular free calcium ([Ca2]1i)in the two clones. Fig. 2 (bars 1 and 2) depicts an experimentin which Jurkat and J45.01 were loaded with the calcium-sensitive dye Indo-1 and then stimulated with anti-TCR mAbwhile the Indo-1 fluorescence ratio was monitored in a flowcytometer. The vast majority ofthe Jurkat cells responded byincreasing calcium but J45.01 did not respond. Quantitatingthis response in a fluorimeter revealed that TCR stimulationof Jurkat resulted in a >900 nM increase in [Ca2+]i (baseline,194 nM), whereas TCR stimulation resulted in an increase ofonly 22 nM in [Ca2+]1 (baseline, 154 nM) in J45.01.

arbitrary units). Background fluorescence seen with a control mAbhas been subtracted. The following antibodies were used to detectthe indicated antigens: TCR, Leu4; CD2, OKT11; CD28, 9.3; CD45,HLE-1.

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100

80 iLOC)

0

-0-

0)C

0c

0.U)uz)0a

60

40

20

01 2 3 4 5 6 7

FIG. 2. Summary of responses of heterokaryons between J45.01and various fusion partners (Jurkat, bar 3; J.CaM1, bar 4; J.CaM2,bar 5; HPB.45.0, bar 6; HPB.45.1, bar 7). The % responding cells isthe fraction of cells in the electronic window representing resting[Ca2+]i that moves to the window representing elevated [Ca2+]i after1 min of stimulation with C305 (1:1000 final concentration). Re-sponses of nonfused Jurkat (bar 1) and J45.01 (bar 2) are also shown.

We characterized further the mutation in J45.01 by using atechnique for assigning signal transduction mutants intocomplementation groups (23). Indo-1-loaded mutants arefused in a transient heterokaryon assay to other mutantslabeled with a fluorescein-conjugated mAb directed against acell surface antigen. Heterokaryons are detected by gating onfluorescein isothiocyanate-positive, Indo-1-loaded cells in aflow cytometer and responses of the heterokaryons can bemonitored. We fused J45.01 to two previously isolated Jur-kat-derived CD45-positive signal transduction mutants,J.CaM1 and J.CaM2 (23), and to the CD45-negative,HBP.45.0 (20). None of these cells alone is capable ofresponding to TCR stimulation. Jurkat fused to J45.01 resultsin a heterokaryon capable of responding to TCR stimulationwith an increase in [Ca2+], (Fig. 2). J.CaM1 and J.CaM2reconstitute signaling when fused to J45.01, establishing thatthese mutations lie in separate complementation groups.Conversely, HPB.45.0 does not reconstitute signal transduc-tion in J45.01. Since these two CD45-deficient cells do notcomplement each other, the signaling defect in J45.01 is likelyto be attributable to its deficiency in CD45 expression. Theability of other cells from HPB-ALL origin to complementJ45.01 was shown by the fusion ofHBP.45.1 (CD45-positive)to J45.01. This latter fusion reconstituted signaling by way ofthe Jurkat-derived partner (J45.01) because the stimulusused, C305, is a clonotypic mAb directed against the TCR ofJurkat (22).The increase in [Ca2]j, seen after TCR stimulation is

thought to be due to activation of the PI second messengerpathway, resulting in increases in soluble IPs. As expected,stimulation of the TCR in Jurkat resulted in large increases inIPs, whereas no such increase was seen in J45.01 (Fig. 3).Thus, CD45 expression is essential for the TCR to couple tothe PI pathway in Jurkat, consistent with our previousfindings with HPB-ALL (20).CD2-Mediated PIP2 Hydrolysis Requires CD45 Expression.

Stimulation of CD2 by way of the combination of two mAbs(9-1 and 9.6) results in the activation of the PI secondmessenger pathway (9-11), but this response depends uponthe expression of a functional TCR (11). The experimentdepicted in Fig. 3 demonstrates that in Jurkat and J45.01surface expression of CD45 is essential for CD2 to couple tothe PI second messenger pathway. Thus, in addition to an

WUO0°n 400-

C/ /0"

200-

05 15 30Time after stimulation, min

FIG. 3. Soluble IP generation in Jurkat (solid symbols) and J45.01(open symbols) after stimulation of the TCR (squares) or CD2(circles). Data points are means of triplicates. Standard errors of themean were <10o.

intact TCR complex, at least one other molecule, CD45, isnecessary for CD2 to initiate PI-derived signals.TCR- and CD2-Mediated PTIK Activity Requires CD45

Expression. Stimulation of the TCR or CD2 activates a PTK(4, 12). Some evidence suggests that the two signal trans-duction pathways are causally linked (6, 27). Having cellsdeficient in CD45 enabled us to address the role of thistyrosine phosphatase in the stimulation of the PTK. Wholecell lysates obtained from unstimulated and stimulated Jurkator J45.01 were analyzed by Western blotting for phosphoty-rosine-containing proteins. There is a rapid appearance ofnewly tyrosine phosphorylated proteins in lysates obtainedfrom the wild-type clone after either TCR (Fig. 4A) or CD2(Fig. 4B) stimulation. Little, if any, PTK activity is seen inlysates from J45.01. Stimulation with control mAbs did notresult in PTK activity in either cell (data not shown). ThusCD45 expression is required for TCR- and CD2-mediatedPTK activity.To address whether there is a global defect in PTK activity

in the CD45-deficient clone, we studied MAP-2 kinase, a42-kDa protein found in many cell types, including T cells(28), which has been shown to be phosphorylated on tyrosineresidues following PKC activation (29). Stimulation ofJurkatand J.CaM signal transduction mutants with PMA (whichdirectly activates PKC) activates MAP-2 kinase independentof TCR signaling and correlates well with the appearance ofa 42-kDa tyrosine phosphoprotein (A.W., unpublished data).Stimulation of Jurkat with PMA induced a new doublet of42-44 kDa, which likely represents MAP-2 kinase (Fig. 4C,lane 3). TCR stimulation also results in the phosphorylationof this protein among the many induced tyrosine phosphopro-teins in Jurkat (lane 2), most likely due to TCR activation ofthe PI pathway with subsequent PKC activation. AlthoughTCR stimulation did not stimulate phosphorylation of the 42-to 44-kDa doublet in J45.01 (lane 5), PMA stimulation, whichdirectly activates PKC, did result in the phosphorylation ofthis protein (lane 6). This indicates that there is PTK activityin the CD45-deficient clone; however, the TCR-associatedPTK is uncoupled from the TCR in these cells.TCR- and CD2- but Not CD28-Mediated IL-2 Production

Requires CD45 Expression. Jurkat cells produce the lympho-kine IL-2 when stimulated by way of the TCR, CD2, or theaccessory molecule CD28. As discussed above, the TCR andCD2 appear to utilize similar proximal signal transductionpathways. On the other hand, the functional activity ofCD28cannot be attributable to PI- or PTK-derived second mes-sengers (6, 13). The role of CD45 in IL-2 production wascompared by testing the ability of Jurkat and J45.01 to

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2 3 4 5 6 7 8 9 10 11 12 13 141s . .s ____S x = _ - ................. t .;__6

., .;....6.__x__F;::

Table 2. CD45 is essential for TCR- and CD2- but notCD28-mediated IL-2 production

IL-2 production,units/ml

Stimulus Jurkat J45.01

C305 + PMA 6 <29.6 + 9-1 + PMA 11 <2lonomycin + PMA 14 109.3 + ionomycin + PMA 43 46

Light IL-2 production was calculated as described (23). The indicatedchain stimuli were used as follows: C305, 1:1000 final concentration of

ascitic fluid; PMA, 50 ng/ml; 9.6 + 9-1, each used at 1 j&g/ml; 9.3,1:10,000 final concentration of ascitic fluid; ionomycin, 1 ,uM.

0' 5r" 30r" 1' 2' 5 t 5'J45.01

7 8 9 10 11 12

33

24

16

0" 30 2' 5' 15 30 0" 30 2' 5' 1 5' 30Jurkat J45.01

2 3 4 5 6C

kDa

11084

47 -

I pp42

33

24

160 C305 PMA 0 C305 PMA

Jurkat J45.01

FIG. 4. (A) Appearance of phosphotyrosine-containing proteinsin lysates of Jurkat (lanes 1-7) and J45.01 (lanes 8-14) after TCRstimulation with C305 (ascites, 1: 200) for times as indicated (secondsor minutes). Molecular mass markers in kDa. A band representinglight chain from the stimulating antibody is indicated by an arrow. (B)Appearance of phosphotyrosine-containing proteins in lysates ofJurkat (lanes 1-6) and J45.01 (lanes 7-12) after CD2 stimulation withmAbs 9-1 and 9.6 (1 ,ug/ml) for times as indicated (seconds orminutes). (C) Appearance of phosphotyrosine-containing proteinsafter a 2-min stimulation of Jurkat (lanes 1-3) and J45.01 (lanes 4-6)with medium alone (0) (lanes 1 and 4), C305 (lanes 2 and 5), or PMA(50 ng/ml) (lanes 3 and 6). Molecular mass markers are in kDa. Adoublet that likely represents microtubule-associated protein kinase2 (MAP-2 kinase) (pp42) is indicated by a bracket.

produce IL-2 in response to various stimuli (Table 2). Whenthe TCR is bypassed with phorbol ester and calcium iono-phore, Jurkat and J45.01 make significant quantities of IL-2.Stimulation of the TCR or CD2 induces IL-2 production only

in Jurkat. However, CD28 stimulation augments IL-2 pro-duction similarly in both clones. Thus, signal transduction bya receptor not coupled to the PI or PTK pathways is notregulated by expression of the CD45 tyrosine phosphatase.

DISCUSSIONThere has been a great deal of interest in the potential role ofCD45 in lymphocyte activation. We have taken a geneticapproach to address this by isolating and characterizingmutants of two T-cell leukemia lines, HPB-ALL (20) andJurkat, which have markedly reduced expression ofthe CD45tyrosine phosphatase. We demonstrate that the TCR of ourJurkat-derived mutant, as our previously described HBP-ALL mutant, fails to couple to the PI second messengerpathway. The site of the mutation in J45.01 remains un-known. The presence of endogenous CD45 mRNA mayreflect an alteration in sequence resulting in a nonfunctionaltranscript or nonfunctional protein. Alternatively, there maybe other requirements for CD45 expression such as anobligatory associated protein. We obtained only low-levelexpression of murine CD45 in the HPB-ALL mutant thatfailed to express endogenous CD45 mRNA (20) and have, asyet, been unable to reconstitute the Jurkat mutant by trans-fection. Nevertheless, several lines of evidence suggest thatthe signaling defect in this cell is due to its loss of CD45. (i)Our selection only demanded that the cell be CD45 deficient.Our first signal transduction assay was performed only afterwe had obtained a low expressing clone. (ii) One ofthe cloneswe obtained had an intermediate amount of surface CD45(about 20% of wild type). In preliminary assays, stimulationof the TCR in this clone resulted in intermediate productionof soluble IPs when compared to the wild-type Jurkat (datanot shown). (iii) In our transient heterokaryon assay wefound that signaling mutants that had wild-type levels ofCD45 on their surface complemented the signaling mutationin J45.01, whereas the HPB-ALL CD45-negative mutantcould not.We have extended our previous findings on the role of

CD45 in T-cell signal transduction with the observation thatCD45 expression is also necessary for the TCR to efficientlyactivate a PTK. These data are compatible with the sugges-tion that TCR-mediated PIP2 hydrolysis may be causallylinked and dependent upon the prior activation of a PTK (6,27). Since CD45 has intrinsic tyrosine phosphatase activity(19), a model for how this molecule may play its essential rolein T-cell activation is suggested. Although there do not existdefinitive data on the identity of the TCR-associated PTK,two candidate molecules are Ick and fyn (30, 31). Both ofthese kinases belong to the src family of PTKs and appear tobe regulated, at least in part, by the state of phosphorylationof critical tyrosines (32). CD45 may exert its effect bydephosphorylating the TCR-associated kinase on a criticalresidue rendering it able to be activated and interact with its

AkDa

110-84 -

47 -

33 -

24 -

16 -

o" 5" 30" 1' 2' 5' 15'Jurkat

BkDa110-84

1 2 3 4 5 6-.._

___tm M

47- -_ -

WP wwwl TL--r--L .'_-

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substrates. By allowing PTK activity to occur, a cascade ofevents ensues, including the activation of the PI secondmessenger pathway, perhaps by direct phosphorylation ofphospholipase C. It is possible that there exist other impor-tant substrates for CD45. One potential candidate is the;chain of the TCR itself, whose state of phosphorylation mayinfluence the ability of the TCR to transduce its signals.

It is important to note that PTK activity is not completelylost in our CD45-deficient clone. When J45.01 is stimulatedwith phorbol ester (Fig. 4C, lane 6), a 42- to 44-kDa doubletappears on the phosphotyrosine blot. This band likely rep-resents MAP-2 kinase (28), which has been shown to bephosphorylated on tyrosines after threonine phosphorylationby PKC (29). Thus, there appears to be a specific subset ofPTKs in T lymphocytes that'require CD45 for their activity.

Signaling by way of the accessory molecule CD2, whichalso couples to the PI and PTK second messenger pathways,requires the expression of CD45. We and others have shownthat for CD2 to transduce its signals, a functional TCR mustbe present on the cell surface (11). In addition to componentsof the receptor complex itself, we now show that CD45 mustbe present for CD2 to transduce activation signals. Ourresults, however, do not establish that the critical substratefor CD45 is the same in TCR- and CD2-mediated signaltransduction.

In addition to the inability of the TCR or CD2 to couple tothe known proximal signal transduction pathways in ourCD45-deficient cells, receptor-mediated distal biologicevents in these cells are disrupted. Thus, although J45.01 iscapable of producing IL-2 in response to calcium ionophoreand phorbol ester (Table 2), stimulation of neither the TCRnor the CD2 results in IL-2 production.

Finally, CD28, which does not couple to the PI or PTKsecond messenger pathways in Jurkat, functions normally inthe CD4S-deficient cells. These data are consistent with ourprevious finding that a heterologous receptor, the humanmuscarinic receptor type I, when transfected into the CD45-negative HPB-ALL mutant, still functionally activates the PIsecond messenger pathway (20). These findings togetherindicate that CD45 loss does not cause a global defect inT-cell signal transduction and links the tyrosine phosphatasemore specifically to the TCR signaling apparatus.

G.A.K. is a Scholar of the Rosalind Russel Arthritis Center(University of California, San Francisco) and is suported in part bythe Arthritis Foundation. J.P. is supported in part by the AmericanCancer Society. This work was supported in part by NationalInstitutes of Health Grant 5ROlGM39553-02 (A.W.).

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Biochemistry: Koretzky et al.

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