Molecular Cloning of Human Syk - The Journal of Biological Chemistry

3 downloads 113 Views 4MB Size Report
Apr 22, 2006 - Andrew C. Channll, Arthur WeisSn **, Susanne Edelhoff+*, Christine M. ...... Perkins, L. A., Larsen, I., and Pemmon, N. (1992) Cell 70,225-236.
THEJOUFLNAL OF BIOUXICAL CHEMISTRY

Vol. 269,No. 16,Issue of April 22,pp. 12310-12319, 1994 Printed in U.S.A.

0 1994 by The American Society for Biochemistry and Molecular Biology, Inc,

Molecular Cloning of Human Syk A B CELL PROTEIN-TYROSINE KINASE ASSOCIATED WITH THE SURFACE IMMUNOGLOBULIN M-B CELL RECEPTOR COMPLEX* (Received forpublication, August 23, 1993, and in revised form, January 21, 1994)

Che-Leung Law*§, Svetlana P. Sidorenko*, KarenA. ChandranS, Kevin E. DravesS, Andrew C. Channll, Arthur WeisSn**, Susanne Edelhoff+*, ChristineM. DistecheSS, and Edward A. Clark$ From the Departments of $Microbiology and $$Pathology, University of Washington, Seattle, Washington 98195 and the Wepartment of Medicine and the **Howard Hughes Medical Institute, University of California, San Francisco, California 94143

BcellantigenreceptorsaremulticomponentcomB cell-associated adhesion molecule, CD22, is a component of of the surface immunoglobulin and acplexes consisting the humanB cell antigen receptor (BCR)complex. Several feacessory molecules with associating protein-tyrosine ki-tures are shared by the molecules associated with the BCR: B cells they are all members nases. A spleen tyrosine kinase, Syk, in porcine of the Ig superfamily (11); their cytoplasand a 72-kDa protein-tyrosine kiaase, PTK72, in murine mic domains contain the antigenreceptor homology-1 motifis) B cells associate withthe B cellantigenreceptor. that have been implicated to function in antigenreceptor signal Herein, we report the isolation of a full-length cDNA transduction (2); and they allbecome tyrosine-phosphorylated encoding the human homologue of Syk. This cDNA pre- following antigen receptor cross-linking(4,7,12,13).A number of two q-terminal SH2 of Src family kinases (13-17) including Lyn, Fyn, Lck, and Blk dicted a polypeptide consisting domains anda COOH-terminal tyrosine kinase domain. and certain undefined serinekhreonine kinase(s1 (13) alsoasSyk is highly conserved between human and swine and is homologous to the T cell-associated protein-tyrosine sociate with the BCR in the cytoplasm and become activated when the receptor is cross-linked (8, 13). kinase ZAP-70. Both Syk mRNA and protein were detected incells derived from multiple hematopoietic lin- Binding of antigen to the BCR initiates a cascade ofbioproliferation eages. Within the B cell compartment,Sykwas ex- chemical events that eventually result in cellular It is now generally believed that the and/or differentiation. pressed from pro-Bcells to plasmacells. In vitro kinase earliest eventfollowing antigen binding to the antigen receptor assays conducted on the human Syk protein isolated from B cells revealed the presenceof autophosphoryla- is the activationof one or more of the protein-tyrosine kinases tion activity on Syk tyrosine residues. Mosine phos- in the cytoplasm by the sIg.BCR complex (1, 3). The active activity of phorylation of Syk associating with theB cell receptor protein-tyrosine kinaseb)thenup-regulatesthe phospholipase C, resulting in the degradation of membrane complex in human was augmented rapidly after surface SYK locus phosphoinositides into inositol 1,4,5-trisphosphate and diacylimmunoglobulincross-linking.Thehuman was mapped to chromosome9 at band q22. glycerol (18, 191, which in turn induce the releaseof free Ca2+ ions fromintracellular stores and membrane translocation and activation of protein kinase C, respectively (reviewed in Refs. T and B cells recognize their specific antigens withclonotypic 20 and 21). Although the sequence of metabolic events elicited by BCR receptors expressedon their cell surfaces. In both T and B cells, the clonotypic antigen receptors are molecular complexes con- stimulation has begun to be elucidated, the contributions of sisting of multiple components that share a number of struc- individual components of the BCR complex to this pathway are tural homologies (reviewed in Refs. 1-3). In murine and humannot fully understood. A group of homologous protein-tyrosine B cells, the antigen receptor consistsof surface immunoglobu- kinases of -70 kDa have been defined recently that appear to lin (sIg)’ noncovalently associated with heterodimersof Iga and be importantconduits in the signal transduction pathways utiIgp (4-8.) We (9) and others (10) have recently reported thata lized by the T cell receptor and BCR complexes. In T cells, a 70-kDa protein-tyrosine kinase, ZAP-70, associates with theT * This work was supported in part by National Institutes of Health cell receptor B cells

T cells

-+ -+ -+ -+

-+ -+ -+ -'79

"

0

" '86

*

"29~

0

-

- 18'

FIG. 4. Western blot analyses.A, transient expression of human Syk in COS cells (left) and anti-Syk and anti-FTK72 recognize the same protein (right).COS cells were transfected with the full-length human Syk cDNAin theorsense antisense directionor without anyexogenous DNA (Mock). Cells were lysed 72 h after transfection. Lysates from-5 x lo6 COS cells or the murine B cell line 38C13 were immunoprecipitated with normal rabbit serum (- or NRS),anti-Syk serum (+), or anti-PTK72 serum. Immunoprecipitates were resolved by SDS-PAGE, transferred onto Immobilon membranes, and thenprobed with the anti-Syk serum plus '251-proteinA to revealthe mobility of the Syk protein.B , expression of the Syk protein in human and mouse cell lines. C, expression of the Syk protein in normal cells. Lysates from 10 x lo6 B or T cells were used per T cells were either resting or stimulated for 72 h with a combination of monoclonal antibodies to CD3 and CD28. The immunoprecipitation. . . mobilities of molecular mass standards (in kilodaltons) are indicated.

lococcus aureus Cowan 1-activated B cells (Fig. 3B). B cell activation both in vivo and in vitro appears to increase Syk mRNA. The Syk message was also expressed in thymocytes (Fig. 3B ). The very weak mRNA signals obtained from freshly isolated circulating T cells were probably due to contaminating B cells since we consistently could not detect theSyk protein in circulating T cells (Fig. 4C). We generated an antiserum againstSyk to examineprotein expression. The peptide used for antiserum production is located in the3'-end of the kinasedomain, a region that is significantly different from the corresponding region in ZAP-70 (Fig. 1B ), but is identical in human andporcine Syk (Fig. 2 A ) . Fig. 4A shows that this antiserumspecifically detected a protein of 72 kDa in COS cells transiently expressing the full-length human Syk cDNA in the sensedirection, but not when the cDNA was expressed in the antisense direction or in themock transfectants. Moreover, this antiserum recognized the sameprotein precipitated by the anti-PTK72 serum from the mouse B cell line 38C13 anti-Syk serum re(Fig. 4A 1. These data demonstrate that this acted specifically against Syk and thatPTK72 is the mouse homologue of Syk. Syk protein levels in various cell lines correlated with their mRNA expression shown in Fig. 3. Thus, B cell lines of both human andmouse origin expresseda protein of 72 kDa, as did the human T cell lines Jurkat andMOLT-4, but not the

lines HUT-78 and HPB-ALL or the murineT cell lines EL4 and shown). The progenitor and myeloid YAC-1 (Fig. 4B and data not cell lines K562, HL-60, and U937 also expressed detectable Syk protein (data not shown). The expression of the Syk proteinin freshly isolatedtonsillar B cell fractions correlated withthat of the mRNA. Higher levels of Syk protein on a per cell basis were found in low density in vivo activated B cells compared with denseresting B cells (Percoll density > 55%)(Fig. 4C). No Syk proteincould be identified either in resting T cells or in T cells activated by anti-CD3 plus anti-CD28; nevertheless, the 72-kDa Syk protein was readily detectable in thymocytes (Fig. 4C). In both tonsillar B cells and thymocytes, a 37-41-kDa doublet and two additional polypeptides of 31 and 22 kDa, respectively, could also be detected (Fig. 4C). The 3741-kDapolypeptides were probably similar to the partial proteolytic product of Syk described previously (24,27). Human Syk Possesses Protein-tyrosine Kinase Activity-Both porcine Syk (24,25) and murineF'TK72 (26-28,35) have been shown to possess protein-tyrosine kinase activity. In human, we have reported that thePTK72 protein associating with the B cell receptor complex is tyrosine-phosphorylated (9, 13).We conducted in vitro kinase assays on human Syk derived from the CESS B cell line to determineif human Syk also had protein-tyrosine kinase activity. A stringent wash procedure, as

Molecular Cloning of Human Syk

12316

(A) anti-slgM cross-linking

Reprecipitation

c

30”1’3 5’10’

-200

CD22>

-200 -

anti-phosphotyrosine Syk blot

.97

*

- 97 -

- 68-

.68

-200

” - r

0

- 97 - 68

-

anti-Syk blot

Syk >

Reprecipitation

- 23 -

-200

72 kDa band

- 97

+

- 68 v! c9

r a

- 43

-

t’

pH 1.9

+

FIG.5. Protein-tyrosine kinase activity in human Syk.A (left), in vitro kinase assays. Lysates from the CESS cell line were precipitated with normal rabbit serum (NRS), anti-Syk serum, anti-Lyn serum, or anti-Fyn serum. Immunoprecipitates were then subjected to high and low salt washes asdescribed under “Materials andMethods” before in vitro kinase assays. The resulting phosphoproteins were resolved by SDS-PAGE. A (right),reprecipitation of the 72-kDa phosphoprotein. In separate experiments, CESScell lysates were precipitated with anti-Syk antibody immobilized on Sepharose 4B and subjected to high and low salt washes andin vitro kinase assays. The72-kDa phosphoprotein wasthen releasedby a low pH buffer (100 mM citric acid (pH 2.3)); reprecipitated with normal rabbit serum(NRS), anti-Syk serum, or anti-PTK72 serum; and then resolved by SDS-PAGE. The mobilities of molecular mass standards (in kilodaltons) are indicated. B, phosphoamino acid analysis of the 72-kDa phosphoprotein derivedfrom the anti-Syk immunoprecipitate in vitro kinase assay shown inA (left).

detailed under “Materials and Methods,” was performed on various immunoprecipitates to minimize potential associating proteins before in vitro kinase assays. Under theseconditions, human Syk precipitates gave a predominant phosphorylated protein of 72 kDa that could be reprecipitated by either the anti-Syk or anti-PTK72 serum, while the Lyn and Fynprecipitates also gave phosphorylated proteins of sizes corresponding to the respective kinases (Fig. 5A). Moreover, phosphoamino acid analysis of the 72-kDa phosphoprotein revealedthat phos-

. 29 FIG.6. Increased tyrosine phosphorylation of Syk in sIgM complex upon IgM stimulation. In A, Daudi cells were incubated a t 37 “C with anti-CDS mAb G10-1for 10 min (control ( C ) )or with antiIgMmAb 4B8 for various periods of time. Cells were then lysed in digitonin lysis buffer. The sIgM complex was immunoprecipitated with anti-IgM mAb 4B8 plus protein A-Sepharose and resolved by SDSPAGE. Proteins were then transferred onto Immobilon membranes and probed with either anti-phosphotyrosine mAb 6G9 or the anti-Syk serum. InB,anti-IgM-stimulated Daudicells were lysed in digitoninlysis buffer. The BCR complexes were immunoprecipitated with anti-IgM mAb 4B8 and labeled by in vitro kinase assay. TheBCR complexes were then disrupted with Nonidet P-40 lysis buffer and reprecipitated with either anti-Syk immobilized on Sepharose 4B beads or a control antibody immobilized on Sepharose 4B beads. The on beads lanes show the proteins removed from the disrupted BCR complexes, whereas the lysate lanesshow the materials remaining after reprecipitation. Anti-CD8 mAb was used in the control lane. The mobilities of molecular mass standards (inkilodaltons) are indicated.

phorylationwas exclusively on tyrosine residues (Fig. 5B). Taken together, these results showed that humanSyk is also a protein-tyrosine kinase. BCR-associated Syk Is Phosphorylated on prosine upon B Cell Activation-Increases in tyrosine phosphorylation and enzymatic activity of Syk or PTK72 have been reported in both porcine and murinesplenocytes stimulated by sIg cross-linking (25-28). However, in these studies, total cellular Syk was ex-

12317

Molecular Cloning of Human Syk

m

0

5

cc

w

3

s



P E B P E B

- 23

- 9.4 - 6.6 -

-

- 4.4

- 2.3

- 2.0

-0.5

SYK

P I

13-1

9

2 -1

I

..........

e 35

9 FIG.7. Organization andchromosomallocation of human SYK gene. A, Southern blot analysis of genomic DNA. Ten pg of genomic DNA from the B cell lines NALM-6 and Ramos weredigestedwith

amined, making it difficult to assess €he degree of tyrosine phosphorylation and enzymatic activity of Syk that is BCRassociated. Therefore, we examined tyrosine phosphorylation of Syk associated with the human sIgM.BCR complex in response to sIgM stimulation. We stimulated Daudi cells with eitheranti-CD8 or anti-IgM mAb, isolated sIgM.BCR complexes from digitonin cell lysates a t various time points, and then determined both the amount of Syk and the extent of tyrosine phosphorylation of proteins in the sIgM.BCR complex. Cross-linking of sIgM elicited arapid increase intyrosine phosphorylation of a 72-kDa protein, with identical mobility to Syk, in the sIgM complex (Fig. 6A). This was accompanied by an increased tyrosinephosphorylation of CD22 (150-160 kDa), as previously reported (9).Sequential immunoprecipitation experiments revealed that only a single species of 72-kDa phosphoprotein, Syk, associated with the sIgM complex (Fig. 6B). This wasconfirmed by protease V8 digestion; the 72-kDa phosphoprotein detected in thesIgM complex and the reprecipitated Syk protein shown in Fig. 6B gave identical peptidemaps (data not shown). These suggested that theSyk molecules associated with the sIgM complex weretyrosine-phosphorylated upon sIgM cross-linking. Moreover, the amount of Syk protein associated with the sIgM complex in the Daudi cell line remained relatively constant during the course of stimulation, suggesting that the increase in tyrosine phosphorylation was not simply due to recruitment of Syk molecules into the sIgM complex (Fig. 6A). Organization and ChromosomalLocation of Human SYK Locus-To characterize thelocus encoding human Syk,we conducted Southern analysis on genomic DNA isolated from cell lines derived from various hematopoietic lineages using the 2.8-kb full-length human Syk cDNA as a probe. Fig. 7A shows that the pre-B cell line NALM-6 and the mature B cell line Ramos gave identicalrestriction fragment patternswhen their DNAs were digested with BamHI, EcoRI, or PstI. The same patterns of restriction fragments were also obtained from all other hematopoietic cell lines examined using these enzymes (data not shown). This suggested that Syk is probably encoded by a simple locus that does not undergo somatic gene rearrangement. Moreover, with additional restriction enzymes, we have not identified any restriction fragment length polymorphism in the SYK locus (data not shown). This is consistent with a single allele being responsible for controlling the expression of human Syk. We estimate that the exons encoding human Syk spanned -20 kb of genomic DNA. We also mapped the location of the human SYK gene on metaphase chromosomes by fluorescence in situ hybridization using the same full-length Syk cDNA clone as a probe. Of 55 cells examined, 35 (63.6%) showed signals on both chromatids of one or both of chromosome 9 a t band q22 (Fig. 7B ). There was no significant hybridization to other chromosomes. DISCUSSION

We have isolated a full-length cDNA encoding human Syk, examined theexpression pattern of Syk in various hematopoietic lineages, andconducted initial structural characterization of the humanSyk protein. We have alsofound that the human SYK gene is a single locus -20 kb long that apparentlydoes not undergosomaticgene rearrangement.The SYK gene was BamHI ( B ) ,EcoRI (E),or PstI ( P ) and resolved on a 0.7% agarose gel. After Southern blotting onto nylon membrane, the DNA was probed with the full-length human Syk cDNAinsert(see Fig. 1). The mobilities of molecular massstandards (in kilobases) are indicated. B, assignment of the SYK locus to human chromosome 9 band q22 as detected by fluorescence in situ hybridization and distributionof 35 signals of hybridization on a diagram of chromosome 9.

12318

Molecular Cloning of Human Syk

mapped to chromosome 9 band region q22 (Fig. 7B), a region pathway in which Syk may participate in B cells. In the prewhere the loci relating to fructose intolerance (36, 37) and cursor B cell compartment, the candidate receptor signaling nevoid basal cell carcinoma syndrome have been mapped (38). pathways inwhich Syk may function include the pro- and pre-B No other genesinfluencing hematopoiesishave been previously cell antigen receptors, both of which have been implicated in mapped to this region. regulating the antigen-independent phase of B cell developBoth cDNA and peptide sequence analyses confirmed that ment (49-51). Alternatively, it is possible that Syk may functhe Sykprotein is highly conserved between human and swine tion in signal transduction pathways mediated by cell-surface and is a homologue of the T cell-associated protein-tyrosine receptors that are broadly expressed during B cell ontogeny kinase ZAP-70 (Figs. 1and 2). During the course of this work, such as CD19 or CD72 (11). a sequence derived from a PCR product encoding human Syk Experiments conducted on the Sykproteinisolated from was published (291, and it is identical to thesequence predicted CESS cells demonstrated that humanSyk possesses autophosby the cDNA isolated in thisstudy. Both Syk and ZAP-70 have phorylation activity (Fig. 5).Syk is part of the sIgM.BCR coma unique structural feature: they possess two SH2 domains, but plex in human(Fig. 6and Ref. 9) and other mammalian species no SH3 domain, in contrast to the Src family and the newly (25,281. Experiments in this study demonstrated that upon B defined AtMtk family (39). In addition,no NH,-terminal myris- cell activation ,by cross-linking sIgM, there was a very rapid toylation site wasidentified in eitherSyk or ZAP-70 (Fig. 1).It increase in tyrosine phosphorylation of the Syk molecules asis therefore appropriate todefine a new family of non-receptor sociated with the sIgM complex (Fig. 6). Tyrosine phosphoprotein-tyrosine kinases constituted by Syk and ZAP-70 and rylation of Syk has been reported to be accompanied by a contheir homologues distinct from both the Src and AtMtkfami- comitant augmentation of its enzymatic activity (25, 26, 28). lies. Interestingly, tandem arrangementof two SH2 domains in The kinase(s) phosphorylating Syk, and hence regulating its the absence of any SH3 domain is also found in a growing enzymatic activity in the BCR complex, is still unidentified. number of protein-tyrosine phosphatases, e.g. protein-tyrosine Recently, the protein-tyrosine kinase inhibitor genistein has phosphatases 1C (40, 411, 1D (Syp) (42, 43), 2C (44) and the been shown to reduce both the activation of Syk and the inproduct of the Drosophila gene csw (45). crease in intracellular Ca2+elicited by sIgM cross-linking in A protein-tyrosine kinase of 72 kDa (PTK72) defined in mu- porcine splenocytes, whereas inhibitorsof protein kinase C and rine B lymphocytes is capable of associating with sIgcomplexes Ca2+chelators hadno effect on these events(25). This supports (28, 35). Although both murine F'TK72 and porcine Syk have the idea that activation of Syk is a signal transduction event been found in sIg complexes, it has not been entirely clear if more proximal to the antigen receptor than that of protein PTK72 is in fact the murine homologue of Syk. The anti-Syk kinase C activation and Ca2+flux. Thus, it is possible that Syk serum generated in this study and theanti-PTK72 serum rec- may be phosphorylated by Src family kinases associated with ognized the same proteins in both human and mouse cells the BCR complex such asLyn or Fyn, which are also amongthe (Figs. 4A and 5 A ) . The human Syk cDNA could also hybridize first protein-tyrosine kinases to be activated subsequent to to mouse genomic DNA and RNA isolated from murine B cells sIgM cross-linking (13-17). Alternatively, Syk may autophos(data notshown). We have also recently reportedthat a 72-kDa phorylateitself and activate its enzymatic activity. Experiphosphoprotein found associated with the human sIgM.BCR ments are currently in progress to identify the specific kicomplex could be recognized by an antiserum raised against nase(s) that can phosphorylate human Syk and regulate its murine PTK72 (91, and thisprotein was confirmed to be Syk in enzymatic activity. this study(Fig. 6).Together, these observations strongly argue Partial proteolytic products of Syk of 37-40 kDa could be that murine PTK72 is the murinehomologue of Syk. detected readily in thymocytes and freshly isolated tonsillar B Two major species of the Syk message, 5.6 and 2.8 kb, were cells (Fig. 412). These proteolytic fragmentshave been dedetected in a variety of cell types (Fig. 3). Since the 2.8-kb scribed in both porcine Syk (46) and murine PTK72 (35) to species was similar insize to thefull-length cDNA (Fig. 11, the consist of a n enzymatically active kinase domain. Such partial larger hybridizing species probably represented a partially pro- proteolysis may potentially provide a means by which Syk can cessed message. Compared with its T cell homologue, ZAP-70, become disengaged from receptor complexes once its function human Syk has a relatively broader cellular distribution. Ex- at the membrane is fulfilled. The protease(s) andmechanisms pression of both ZAP-70 mRNA and protein appears to be re- controlling this process have not yet been elucidated. stricted only to T cells and some natural killer cells (23).' In The lack of myristoylation signals in both Syk and ZAP-70 contrast, Syk was expressed in two acute lymphoblastic leuke- indicates that their localization to the plasma membrane must mia T cell lines, freshly isolated thymocytes, the progenitor cell be mediated by another mechanism. ZAP-70 can associate only line K562, and the myeloid cell lines HL-60 and U937, in ad- with thetyrosine-phosphorylated (-homodimer of the CD3 comdition to itsexpression in all B cell types examined (Figs.3 and plex, but not with thenonphosphorylated (-chain (22, 23, 52). 4). Similarly, porcine Syk (24, 46) and murine PTK72 (35) are The ability of SH2 domains to mediate interaction with phosalso expressed in hematopoietic cells outside of the B lineage. phoproteins has been demonstrated in a wide variety of signal This broader spectrumof tissue distribution suggests that Syktransduction pathways involving protein tyrosine phosphorylprobably functions in multiple receptor-mediated signal trans- ation (53, 54). The association of ZAP-70 with the activated T duction pathways in addition to thatof the BCR. This is sup- cell receptor complex is dependent on the interaction of both ported by recent reports that, in a mast cell line, Syk/€'TK72 SH2 domains of ZAP-70 with both phosphorylated tyrosine interacts with FcrRI and becomes activated whenFcrRI is residues within each antigen receptor homology-1 motif of the cross-linked (47), whereas in the human monocyte cell line (-chain (55). Thus, it is likely that membrane localization of HL-60, stimulation of Fcy receptors results inactivation of Syk Syk isalso mediated by the binding of both of its SH2 domains tyrosine phosphorylation (48). Within the B lineage, Syk ex- to phosphoproteins present in the sIgM.BCR complex. Candipression was not restricted only to IgM' B cells, but was also dates for these phosphoproteins include the Srcfamily kinases expressed in progenitorB cells, class-switchedB cells, and present in the sIg complexes, e.g. Lyn, Lck, Fyn, and Blk, or antibody-producing plasma cells (Fig. 4). Thus, the sIgM.BCR other accessory proteins, e.g. Iga, Igp, andCD22. Using glutasignal transduction pathway is not only the signal transduction thione S-transferasefusion proteins of Iga andIgp, Clark et al. (56) did not detect the association of a 72-kDa protein with either Igaor Igp. We have generatedfusion proteins of the SH2 A. C. Chan and A. Weiss, unpublished results.

Molecular Cloning of Human Syk domains for human Syk and are currently utilizing these fusion proteins to identify molecules with which Syk may interact. Acknowledgments-We thank Dr. Stephen J. Klaus for comments on this manuscript. We also thank Dr. Tucker W. LeBien (Department of Laboratory Medicine and Pathology, University of Minnesota) for providing the acute lymphoblastic leukemia B cell line BLIN-1 and its subclone, B9,and Dr. Christoph Turck (Howard Hughes Medical Institute protein core facility, University of California, San Francisco) for synthesizing the peptide used in this study. REFERENCES Cambier. J. C., and Campbell. K. S. (1992)FASEB J. 6, 3207-3217 Malissen, B., and Schmitt-Verhulst,A. (1993) CumOpin. Zmmunol. 5,324-333 Reth, M. (1992)Annu. Rev. Immunol. 10,97-121 Campbell, K. S., and Cambier, J. C. (1990)EMBO J . 9, 441448 Hombach, J., Tsubata, T., Leclercq, L., Stappert, H., and Reth, M. (1990) Nature 343, 76C-762 6. Hombach, J.,Leclercq, L., Radbruch, A,, Rajewsky, K., and Reth, M. (1988) EMBO J . 7,3451-3456 7. Van Noesel, C. J. M., Borst, J., De Vries, E. F. R., and Van Lier, R. A. W. (1990) Eur. J . Immunol. 20,2789-2793 8. Van Noesel, C. J. M., Van Lier, R.A.W., Cordell, J. L., Tse, A. G . D., Van Schijndel, G . M. W., De Vries, E. F. R., Mason, D. Y., and Borst, J. (1991)J . Immunol. 146, 1-8 9. Leprince, C., Draves, K. E., Geahlen, R. L., Ledbetter, J. A,. and Clark, E. A. (1993)Proc. Natl. Acad. Sci. U. S. A. 90, 32363240 10. Peaker, C. J. G., and Neuberger, M. S. (1993) Eur. J . Immunol. 23, 13581363 11. Barclay, A. N., Birkeland, M. L., Brown, M. H.. Beyers, A. D., Davis, S. J., Somoza, C., and Williams, A. F. (1993) The Iaucocyte Antigen Facts Book, 1st Ed., Academic Press, Inc., San Diego, CA 12. Campbell, K. S., Hager, E. J., Friedrich, R. J., and Cambier, J. C. (1991)Proc. Natl. Acad. Sci. U. S. A. 88, 3982-3986 13. Leprince, C., Draves, K. E., Ledbetter, J. A,, Torres, R. M., and Clark, E. A. (1992)Eur. J . Immunol. 22,2093-2099 14. Burkhardt, A. L., Brunswick, M., Bolen, J. B., and Mond, J. J. (1991) Proc. Natl. Acad. Sci. U. S. A. 8 8 , 741C-7414 15. Brunswick, M., Burkhardt,A., Finkelman, F., Bolen, J., and Mond, J. J. (19921 J. Immunol. 149,2249-2254 16. Campbell, M. A,, and Sefton, B. M. (1992) Mol. Cell. Biol. 12, 2315-2321 17. Yamanashi, Y., Fukui, Y., Wongsasant, B., Kinoshita, Y., Ichlmori, Y., Toyoshima, K., and Yamamoto, T.(1992) Proc. Natl. Acad. Sci. U. S. A. 89, 1118-1122 18. Coggeshall, K. M., McHugh, J. C., and Altman,A. (1992)Proc. Natl. Acad. Sci. U. S. A . 89, 566M664 19. Carter, R. H., Park, D. J., Rhee, S. G., and Fearon, D. T. (19911 Proc. Natl. Acad. Sci. U. S. A . 88, 2745-2749 20. Bemdge, M. J. (1993) Nature 61,315-325 21. Nishizuka, Y. (1984)Nature 308.693497 22. Chan, A. C., Irving, B. A., Fraser, J. D., and Weiss, A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,91669170 23. Chan, A. C., Iwashima, M., Turck, C. W., and Weiss, A. (1992)Cell 71,649462 24. Taniguchi, T., Kobayashi, T., Kondo, J., Takahashi, K., Nakamura, H., Suzuki, J., Nagai, K., Yamada, T.,Nakamura, S., andyamamura, H. (1991)J . Biol. Chem. 266, 15790-15796 25. Yamada, T., Taniguchi, T., Yang, C., Yasue, S., Saito, H., and Yamamura, H. (1993)Eur. J. Biochem. 213,455-459 1. 2. 3. 4. 5.

12319

26. Burg, D. L., Hamson, M. L., and Geahlen, R.L. (1993) J . B i d . Chem. 268, 2304-2306 27. Hutchcroft, J. E., Hamson, M. L., and Geahlen, R. L. (1991) J . Biol. Chem. 266,1484614849 28. Hutchcroft, J. E., Hamson, M. L., and Geahlen, R. L. (1992) J. Biol. Chem. 267.86134619 29. Kolanus, W., Romeo, C., and Seed, 8. (1993) Cell 74, 171-183 30. Lemieux, N., Dutrillaux, B., and Viegas-Pequignot. E. (1992) Cytogenet. Cell Genet. 59,311-312 31. Hanks, S. K., and Quinn, A. M. (1991)Methods Enzymol. 200, 3-2 32. Hanks, S. K., Quinn, A. M., and Hunter, T. (1988)Science 241, 42-52 33. Casnellie, J. E., Hamson, M. L., Hellstrom, K. E., and Krebs, E. G . (1982)J . Biol. Chem. 267, 13877-13879 34. Koch, C. A., Anderson, D., Moran, M. F., Ellis, C., and Pawson, T. (1991) Science 252, 66-74 35. Zioncheck, T. F., Harrison, M. L., Isaacson, C. C., and Geahlen, R. L. (19881J . B i d . Chem. 263, 19195-19202 36. Cross, N. C., de Franchis,R., Sebastio, G . , Dazzo, C., Tolan, D. R., Gregori, C., Odievra, M., Vidailhet, M.,Romano, V., Mascali, G., Romano, C., Musumeci, S., Steinmann, B., Gitzelmann, R., and Cox, T.M.(1990)Lancet 335,306309 37. Tolan, D. R., and Penhoet, E. G . (1986)Mol. Biol. & Med. 3, 245-264 38. McKusick, V. A. (19921 Mendelian Inheritance in Man, 10th Ed., Johns Hopkins University Press, Baltimore 39. Desiderio, S . (1993)Nature 361,202-203 40. Shen, S. H., Bastien, L., Posner, B. I., and Chretien, P. 11991) Nature 362, 736739 41. Plutzky, J., Neel, B. G., and Rosenberg, R. D. (1992) Proc. Natl. Acad. Sci. U. S. A . 89, 1123-1127 42. Vogel, W., Lammers, R., Huang, J., and Ullrich, A. (1993)Science 269, 16111614 43. Feng, G.-F., Hui, C.-C., and Pawson, T. (1993) Science 259, 1607-1611 44. Ahmad. S., Banville, D., Zhao, Z., Fischer, E. H., and Shen, S. H. (1993)Proc. Natl. Acad. Sci. U. S. A. 90,2197-2201 45. Perkins, L. A., Larsen, I., and Pemmon, N. (1992)Cell 70,225-236 46. Taniguchi. T., Kitagawa, H., Yasue, S., Yanagi, S., Sakai, K., Asahi, M., Ohta, S., Takeuchi, F., Nakamura, S., and Yamamura, H. (1993) J . Biol. Chem. 268,2277-2279 47. Hutchcroft, J.E., Geahlen, R. L., Deanin, G. G., and Oliver, J. M. (1992)Proc. Natl. Acad. Sci. U. S. A . 89,9107-9111 48. Agarwal, A,, Salem, P., and Robbins, K. C. (1993)J. B ~ o l Chem. . 268, 1 5 9 0 s 15905 49. Nomura, J., Matsuo, T., Kubota, E., Kimoto, M., and Sakaguchi,N. (1991) Int. Immunol. 3, 117-126 50. Matsuo, T., Nomura, J., Kuwahara, K., Igarashi, H., Inui, S., Hamaguchi, M., Kimoto. M., and Sakaguchi, N. (1993)J . Immunol. 150,376-775 51. Brouns, G . S., De Vries, E. F. R., Van Ncesel, C. J. M., Mason, D. Y., Van Lier, R. A. W., and Borst, J. (1993)Eur. J . Immunol. 23, 108%1097 52. Wange, R. L., Malek, S. N., Desiderio, S., and Samelson, L. E. (1993)J . Biol. Chem. 268, 19797-19801 53. Songyang, Z., Shoelson, S. E., Chaudhuri, M., Gish, G., Pawson, T., Haser, W. G., King, F., Roberts, T., Ratnofsky, S., Lechleider, R. J.. Neel, B. G . , Birge, R. B., Fajardo, J. E., Chou, M.M., Hanafusa, H., Schaiihausen, B., and Cantley, L. C. (1993) Cell 72, 767-778 54. Cantley, L. C.,Auger, K. R., Carpenter, C., Duckworth, B., Graziani, A,, Kapeller, R., and Soltoff, S. (1991) Cell 64, 281-302 55. Iwashima, M., Irving, B. A,, van Oers, N. S. C., Chan, A. C., and Weiss, A. (1994) Science 263, 11361139 56. Clark, M. R., Campbell, K. S., Kazlauskas,A., Johnson, S.A,, Hertz,M., Potter, T. A,, Pleiman, C., and Cambier, J. C. (19921 Science 258, 123-126