PERSONAL AUTHOR(S) Volarevic S, Niklinska BB, Burns CM, June CH, Weissman AM, .... cha .an:d in (12) aı, (14) experiments using electron paramagnetic.
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11. TITLE (Include Security Classification) lacking transmembrane
Regulation of TCR signaling by CD45 12. PERSONAL AUTHOR(S)
16. SUPPLEMENTARY NOTATION
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1993
Science
1993 April; Vol.260 pp.541-544
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Weissman AM, Ashnell jD
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Burns CM, June CH,
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Niklinska BB,
Volarevic S,
SUB-GROUP
CD45; T cell
activation;
Phosphatase
~'t~
19. ABSTRACT (Continue on rev~erse ifnecessary and identify by block number)
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studied to date for NO is extremnely high, with jpding constants on the order of I1oil N4 thermre, the kinetics of dissociation are sl5ow; The rate constant nd NO fromt human for the release o hemoglobin is on the of l0-1 s'- at H() o,%Cadnura Fe(lll) ss heme proteins bind NO with muc affinity, with binding constants in the tang of 103 to 10 M-1, and the kinetics of I to 40 s-') dissociation are faster (kicf (1C). Thus, an Fe(lIl) heme protein would be a much better carrier of NO if release of this effector after dilution is required. On the basis of the spectral changes obser-ved (Figs. I and 2), it appears likely that an Fe(IIl) heme protein may be involved in the Rhodnius salivary vasodilator. To further confirm, the importance of d cut the Fe(lIII) heme protelin, scrre experiments using electron paramagnetic resonance (EPR) spectroscopy. Fe(lll) heme proteins have an odd number of electrons that exist in a high-spin fo when penracoordinated and typically gi rise to EPR spectral features at spectrose icsecrs icsplitting factors (g) of 6 and 2 (11, Addition of NO to an Fe (Ill) h~eepr emn produces an even-electron species t t is EPR silent (12). The E]R spectra o hodgandhomgentesbe re and niu saivay eead nfter epsauvregl andhomognate-ns after subsequent exposure to NO e sho-An in Figy. 4 (13). Only a weak ignal was observed for the homogenate fore argon equilibration, but strong sig s at g = 6 and g =2 were observed aft argon equilibration (Fig. 4, B and D) These signals disappeared after subsequ t exposure of the homogenate to NO. e smaillsignai at 3200 G (g = 2.08) tha does not change with argon or NO equ il ration is probably produced by a nonhen -based radical that accounts for -5%. of he intensity of the high-spin Fe(lll) si al. The observation. that the shape of t g = 2.08 signal does
not change after 0equilibration eliminates a nitrosyliro (11) center from consideration because at odd-electron species gives rise to very haracteristic EPR spectral features in prec ely the same region of the EPP. Spectrum 14). This characteristic is shown in Fi 4E, where dithionite has been added the untreated homogenate solution to oduce the Fe(ll)NO species. Chroma focusing of whole-gland homogenates n a Mono P column yielded three maj heme proteins, with isoelectric points (p s) of 7.97, 7.53, and 6.94. The asso roen ha(paet reduce had aparent masses of (u roteins 26.0 2 1, and 23.4 kD and were present in a~mo isnf 192, 57.6, and 44.0 pmol pcr gt~~an ir, resspe giand ir repctively (15). Taken togethIer:, th e three proteins account for at least
90% the total heme protein content of the g rids. Preliminary results indicate that
Cs 3 V B ~ all three proteins di~lyidentical NObinding properties, which closely match the 131oCxe",: 313 f properties of the whole-gland homogtnates.'C fF~l e ý' ,1'c o 1 7 The summed content oftthese proteins (293 5 P. W R lamp ao1 !c 3D s qir apmol per gland pair) also matc~h content of No, 28; .K'er pair, inca18, 5387 (179) -te'e.e J A S surcd in thd as e of Hg. 6 R E Euiel 1)HI -E Let?1 55. Nitric oxide as recently been identified as an imporsa signal molecule itvolved in (1969). t ulati of vascular tone in vertc8 J S. S~a-r;er efal Pro,- Na! A.,ý SiciUA 89 444 (1099?)1 S Sia~nef e:al, o,: p K2~7 s presence in Rhodni:us saliva brates R "I. 'a Bf25n~ li3 9. E cheon 241.V 'able case of convergent r SUIuesets eoluti .The R eeproteins are H.G~bson. J &Iot Sol 91- ,.l (1ý7Si 10. V S S!ara T, ("7C'ý' Ig 'Uccess b.y well s ed to enhance '3-E-2-78 ii. G. Pamne, 5iochncs:-.n S26. hey are unlo ing the v%--sodilator N dil d in the host bloodstream; r, 12. H HOri, Al~iIca-S5'tc. 3 'Lang, T ei~ J 5,01. Ch-,e.- 285. 15--l- 090C IN exhibits antiplatelet activity and ma pinhibit the formation of thepalt lpairner'eN -Ia, PstEa-in Ed -A,Ca ug (2). Although NO synthesis is related aZ!e ~4.TeP'h~s0~ cha .an:d in (12) aý, (14) o a variety of functions in vertebrates (2), Fioc P. I FcA 71. I' 14. M. ru it has previously been reported only once in another invertebrate (16). It remains to be Olson G. Pa IJ Sot 6 Cý,-e', 254, ',21',0 seen whether the ability to produce NO is a (1979), .D 1. S fer. AY. R. '-'-n. 3, M, T, Yasnim .ra, -1 noic. Holffman.'bd p. 4 phylogenetically old trait or whether it has vg 18. 263 .5+83 E.Caman1 arisen independently in different lineages.
ibeiro, unpublished resu!s
16. M.W. Radornski. .J F. Aarin, S P
REFERENCES AND NOTES 1. J. M. C.Ribeiro, R. Gonzales. 0. Marinaili. Br. J. Pharmacol. 101, 932 (1,990), 2. S. Moncada, A. Mv.J. Palmer, E. A. Higgs, Pha!macol. Rev-. 43, 109 (1991).
ada. Phjcsý.
Tran~s. R. Soc. London Ser, cR334, 1 99. 17. we thank A. Raiisimrig for obiairing EP spectra. Supported by Ni-i g~anis A - l6694 (J.M.C.R) and DK.31C38 (A,) 14 December 1992; accepied 12 February 1993
Regulation of TCR Signaling by CD45 Lacking Transmembrane and Extracellular Domains Siniga Volarevi6, Barbara B. Niklinska, Christopher M. Burns, Carl H. June, Allan M. Weissman, Jonathan D. Ashwell* The CD45 protein is a transmembrane tyrosine phosphatase that is required for normal T cell receptor (TCR)-mediated signaling. A chimeric complementary DNA encoding the intracellular enzymatically active portion of murine CD45 preceded by a short aminoterminal sequence from p6Oc-src was transfected into CD45- T cells. Expression of this chimeric protein Corrected most of the TCR signaling abnormalities observed in the ab-
sence of CD45, including TOR-mediated enhancement of tyrosine kinase activity and Ca 2 l flux. Thus, the enzymatically active intracellular portion of CD45 is sufficient to allow TCR transmembrane signaling.
Stimulation of T cells through the antigen, specific receptor (TCR) initiates increases in tyrosine phosphorylation (1), phosphatidylinositol hydrolysis, and intracellular Ca 2+ concentration (ICa24]1) (2). Studies -coupling
S.Volarevid, B.B.Nikl'inska, C. M. Burns. J_D Ashwell, Biological Response Modifiers Program. Laboratory Of Immune Cell Biology, National Cancer Insltute, National Institutes of Health. Bethesda, MD 20892.
C. H. June, Immune Cell Biology Program, Naval Research Experimental Institute, Bethesda, MO 20889. A.~~~Medical M. Weissman, Immunology Branch, National Cancer Institute. National Institutes of Health, Bethesda. MD 20892. 'To whom correspondence should be addressed.
SCIENCE
*
VOL. 260 - 23 APRIL
1993
of T cell lines that are deficient in expression of CD45 have established that this transinembrane molecule, -whose intracyto-
plasmic domains have intrinsic tyrosine phosphatase activity (3, 4), Participates in the TCR to these activation events (5, 6). Studies of CD45-deficient A-I el ie amrn ainso ellnYC1 famrn vrat have rhnwn thnt CD45 exprees-icn iý ;nversely related to spontaneous tyrosine popoyaino ubro usrts popoyaino ubro usrts including the ý chain of the TCR. In contrast to the typical rapid lCa2'l, elevation exhibited by the YAC- I wild-type
541
(WVT) cells, stimulation of the CD45- ccis with monroclonal antibodies (mAb) to CD3 resulted in delayed, asynchronous JCa` 1 oscillations (6). Experiments wvere done to determine if expression of the intracellular portion of CD45 alone is sufficient to restore normal TCR-mediated signaling in CD45 -T cells, Since the in %ivo biological activity of CD45 migght be dependent upon its location in the plasma membrane, a cDNA that encoded a chimeric molecule [the NH,terminal first 15 amino acids of p60-~ (Ste) and the enzymarically active intracellular portion of murine CD451 wkas created (myriCD45, Fig. ]A). The first 14 amino acids of Src constitute a sequence that results in its own myristylation, and can direct heterologous proteins to the plasma membrane (7). Two independent CD45- mutants. NI anj \12, wvere transfected, and stable neomycin-resistant transfectants from both N I (NI.SlOO) and N2 (N2SI and N2.S4) cells were obtained. Expression of the TCR, as judged by flow cytometry, w~as comparable in all of the cells (6, 8). Polymerase chain reaction (PCR) amplification with oligomers specific for the Ste myrisrylation sequence (5') and COOH-terminal CD45-sequence (3') detected chimeric transcripts from N2.Sl and NI.SII0i', but not WT, N I, N2, or N2.S4 cells (8). To assess the amount of myr-iCD45 protein, detergent lysates Of Cells Were analyzed by immunoblorting with an antiserumn to the. cytoplasmic domains of CD45 (-1). Fulllength CD45 wvas detected only in \WT Fig. 1. Expression cf myr-iCD45 in YAC-1 transfectants. (A) An
A myr-iC045 S 1 I PTPase
cells. In agreement with the PCR .nl'i, this antiserumn detected a protein %kith t hc predicted molecular si:e of thie rn' 5 r-iCD45 chimeric molecule (-83 kD) in N2.SI , but not NI, N2, or \21.S4 cell lys~ites (Fig. IB). A- few nonspecific bands of larger molecular si:c were detected but did nor correlate with expression of the chimenic mulecule. A arAb to the Nfl--terminAl portion of Ste was used to imniunioprecipitate and for immunoblotting and detected a molecule of the predicted si:e for myriCD45 in detergent lysistes cf N2.51 and NI.SICO cells but not the untransfected ceils or N2.S4 (Fig. IC). Also shown is a control T cell hybridomna that was transfected with pp60-"c (9); anti-Ster detected pp60`-i1 but not the higher molecular si:e baind that represents myr-iCD43. NMyriCD45 was also detected in total membrane preparations5 Made from N2.SI and NI .Sl0OJ cells, indicating that at least a portion of this molecule was directed to membranes (S), although the subcellular distribution of myr-iCD45 is not yet characterized. Finally, the m.Ab to Src specifically precipitated large and similar amounts of tyrosine phosphatase activity from N l.S100 and N2.SI but not NI or N2 cells, indicating that the myr-iCD45 chimera is enzymatically active (Fig. 10). Loss of CD45 expression in YAC- I cells results in spontaneous ryrosine hyperphosphorylation of a number of proteins (6). To determine if the myr-iCD45 chimeric protein would substitute for WT CD45 protein in regulation of ryrosine phosphorylarion, B
ts
t2 COOH PTPg1asei
f
a c:DNA insert encoding the myr-iCD4-5 chimeric molecule was prpae (18) Thi iss aý schematic representation of the myr-iCD45 chimeric protein. The
,.D
-
106-
----
-.-rnyt-icm~
8-0-sponding
. fluirsth15 aminraidst(imyistyla the cludin tion~30 re sit) f - Sc *P03 shown, as well as the two 0045 phosphatase domains. (B) Immunoibtot of YAC-1 derivatives immunoprecipitated with antiserum to intracellular 0045 and blotted with the same serum (21). (C) lmmunoiprecipitation and immunoblot of YAC-1 derivatives with a mAb to residues 7 to 15 of Src (D710. NCt/BCB Repository). 5B2 is a T cell hybridoma that expresses v-src (9). (D) Tyrosine phosphatase activity of immunoprecipitated myr-iCD45 was measured in vitro by release of 32p from labeled substrate, as mscdionss (_2&). The described (6) except fcr standard ofdphaeeprmetlpit ~ ~ ~ ~ ~ r~
stndrddeiaios uliat epeimntl f
ont
ae
2 20-: 0 15-
a
5
~
0L
542
SCIENCE
-
1. deitin P ~
Zsie
shown. 0045 immunoprecipilaled from WVTcells with a mAb to the external domain of 0045 (6) had a A~ cpm of 29,700 in the same experiment. These results are representative of three independent experiments.
niC3%ih
kinetics similar to WVT and with an even greater increase in mean lCa-'), than seen in WVT cells. This enhanced response by N2.SI and Nl.SIOO cells wvas reproduced in three independent experiments and is due to an increase in the fraction of recells (60% of WVT cells versus 8 0% of myr-iCD45 transfectants) (Fig. 3B) as well as higher Ica`~), in the responding population. WVhether the difference in the Oanti-LFAaxmlstmlte an -1J ewenW mat-t axmlsiuatdlaewe V and myr-iCD45--transfected1 cells was a reflection of some propert that differs between whole and truncated CD45 requires the analysis of more transfectants. yoiepopo Aciainidcd rylation of the TCR ý subunit, another ca,!y event in TCR signaling, was also
Intracelfular CD45
Z Nj U?
'
Z
expression vector withN211T777;NIS10clsrsoddt
t
z(within
z
Cell *VOL.
constitutiv ne tyt m c~~flI~ iae (Fit,~ 2.A). lfusr~ftt~ and immumobhrin wýith a " -,L'to phý photyrosine detected a nu'nlcr of fivixrphosphornilateJ speics in the ( 045- NI cell and the G418ýrvei'tnt N. cS4cell thait does niot express the myr iCD45 chirneric molecule. The mc't prominent ýptcics inschided a doublet at '1 and 23 kI), .:)d [lan~is at 38, 56, anid 71 1.. In conr'rnt, eX'ress;i1n of mvr-iCD45 resulted in a return Of a phosphoryrosine profile si~ rto the WT cells, with the exception of the prominent band at 56 k0 in N2.SI cells. The reason fkir this difference betweeen the two inyr-iCD45-expressing cells is not k-nown. The doubler at 21 and 23 1.0 migrated simrnialy to rhcsphorylated ý chain of thie TCR in YAC-l cells, which is spontineoukly hvpet-phosphorylared in the absence of CD45 (6). Thle state of ý phiosphorylaýt ion" was d'irec tl dVete:rMined bv imimunoprecipi.tarion with serwni to ý and iininunciblotting with anti-phosphotyrosine antibodies (Fig. 2B). No phospho-4 wvas detected in WT cells, whereas the CD45- NI and N2.S4 cells expressed thecir characteristic phospho-4 doublet. Although a small amount was detected, ý phosphor')lation was tminimal in both of the myr-iCD45-expressing cells, N2.SI and NI.SlCOV (Hg. 2B). Fluctuations in Ica:'] that can be induced by stimulation through the TCR were assessed. WT cells had an early increase in ICa'I~ after stimulation (6) (Fig. 3A). CD45- N I and N2.S4 Cell$ responded with a smniil and delayed ICa , l, increase, which reflects delayed and asynchronous Ca 2 + oscillations in these cells after stimulation with mrAbs to CIJ3 (anti2S n D)(6.Temy-C4C .S anndD to ' ded c hell mrespo (6S). N
260 & 23 APRIL 1993
examined (Fig. 4). Unstimulated \VT cells ad a small amount of spontaneous ý ryrohophrlaio tatWa rpil sn hshr-ainta a ail 5 min) enhanced by treatment with anti-CD3. TCR ý from the CD45- cells was already hyperphosphorylated and did
not exhibit ony increas~e uplon stimulation.
CD45RB but not CU4 5RB it~i.If 06). If
Expression of the ni r-tCD-13 !,,e vfsiate for 33 rn~n CAt30,C and Ecý;prri teaciton by addndý 550 of _1% t~tdc~ coal in 20 raHiepes bjf'er, pH4 74 A'-:er cenlr'fugalron. Ine Sýp inatant: Aas cox~nt' na soinillalon counter. 23 C Cenciarellt et al. S:,nc 257. 7c5 (I9C.7) 24 P S Rabinovi-rc and C H J.Lne, FisCn 6C.r 3n.%So'7-
