©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The CD4-associated Tyrosine Kinase p56 Is Required for Lymphocyte Chemoattractant Factor-induced T Lymphocyte Migration (*)

(Received for publication, April 27, 1995)

Thomas C. Ryan , William W. Cruikshank , Hardy Kornfeld , Tassie L. Collins (1), David M. Center (§)

From the Pulmonary Center, Boston University School of Medicine, Boston, Massachusetts 02118 Division of Pediatric Oncology, Dana-Farber Cancer Institute and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Lymphocyte chemoattractant factor (LCF) is a polypeptide cytokine which induces both cell motility and activation of T lymphocytes. These LCF-induced events demonstrate an absolute requirement for the cell surface expression of CD4. Because many CD4-mediated T lymphocyte activation events have been demonstrated to require the association of the src-related tyrosine kinase p56 with the cytoplasmic domain of CD4, we examined the role of p56 in LCF-induced lymphocyte migration in a murine T cell hybridoma line expressing transfected human CD4. LCF induces the catalytic activity of CD4 associated p56 at chemoattractant concentrations of cytokine. Hybridoma cells that express CD4 with cytoplasmic point mutations which uncouple the CD4-lck association lack both lck enzymatic activity and chemotactic responses to LCF. The enzymatic activity of lck however does not appear to be required for CD4-mediated migratory signal. First, the protein tyrosine kinase inhibitor herbimycin A blocked LCF-induced p56 activation but had no effect on the LCF-induced motile response. Second, T cell hybridomas expressing a chimeric receptor combining the extracellular domain of human CD4 and murine p56 which lacked the kinase domain had a normal LCF-induced motile response. We conclude from these observations that CD4-lck coupling is essential for LCF-induced T lymphocyte migration but the motile response is independent of the enzymatic activity of CD4-associated p56.


INTRODUCTION

Accumulation of T cells in tissue at sites of antigen deposition requires the mobilization, adhesion, transendothelial migration, activation, and proliferation of lymphocytes(1) . Locally produced chemotactic cytokines contribute the major driving force to directed lymphocyte migration during this process via their interaction with cell surface receptors(2) . In addition to their role as chemotactic factors, many cytokines have growth factor activity which may also play a role in the phenotype of the accumulated cells(3) . Despite the fact that a large number of growth factor like-cytokines have chemotactic activity for lymphocytes, relatively little is known about the mechanism by which they transduce the motile signal. This study addresses the mechanism by which the chemotactic cytokine lymphocyte chemoattractant factor functions. LCF()is a CD4 immunocyte-specific chemotactic cytokine of CD8 T cell origin(4, 5, 6) . In addition to the activation of cell migration, LCF induces rises in [Ca] and inositol trisphosphate (IP)(7) . The relationship between these messengers and the induction of motility, however, is yet to be defined. The initiation of all these responses, however, is absolutely dependent upon the cell surface expression of CD4(7, 8) .

Along these lines, the CD4 ligand gp120 induces rises in [Ca], IP generation and motile responses in T cells(9) . In addition, HIV-1 gp120 induces CD4-associated activation of p56(10) , a member of the src-tyrosine kinase family which is tightly associated with CD4(11, 12) . From these observations, we investigated the possible requirement for p56 in CD4-mediated lymphocyte migration induced by LCF.

In the present investigations we examine LCF-induced p56activity in a murine T cell hybridoma line which has been transfected with human CD4. Our experiments demonstrate that LCF induces CD4-associated p56 kinase activity in these cell lines. In order to establish a relationship between the LCF-induced enzymatic activity of the kinase and lymphocyte motility, hybridomas expressing CD4 cytoplasmic domain mutants were also studied. The mutations result in the disruption of the physical coupling of CD4 with p56(13) . Neither the CD4-associated kinase activity nor the migration induced by LCF occurs in cells expressing CD4 mutations incapable of binding p56. The relationship between p56catalytic activity and lymphocyte migration was examined in cells treated with the tyrosine-kinase specific inhibitor, herbimycin A. At concentrations that completely abolished the enzymatic activity, the LCF-induced motile response was unaffected. Finally, the removal of the kinase domain from CD4-lck chimeras expressed in this hybridoma line resulted in migratory responses comparable to full-length kinase controls. These data suggest that the physical association of p56 with CD4, and not the enzymatic activity of the kinase, is the essential component for the LCF-mediated lymphocyte motile response.


MATERIALS AND METHODS

Murine T Cell Hybridoma Cell Lines

All CD4-expressing murine T cell hybridomas were the generous gift of Dr. Steven J. Burakoff (Dana Farber Cancer Institute Boston, MA)(14) . Briefly, The murine T cell line, By155.16 was infected with the MNC retroviral vector which contains a neomycin resistance gene, a cytomegalovirus promoter, and the gene for human CD4. The MNC-CD4 transfectants were selected (G418) and assessed for CD4 surface expression. All cell lines examined had comparable expression of CD4. In addition to wild type CD4, By155.16 was transfected with CD4 containing cysteine to serine point mutations at positions 420, 422, and 430 (MNC-CS420, CS422, CS430)(13, 15) . Chimeric molecules containing the extracellular domain of CD4 directly ligated to p56 were also expressed in murine cells. All cells were grown in RPMI 1640 medium (Sigma) containing 200 units/ml of penicillin and 200 mcg/ml streptomycin, 2 mM glutamine, 20 mM HEPES, pH 7.4, and 10% fetal bovine serum.

Reagents

Recombinant LCF was produced as described previously(8) . Anti-human CD4 antibody Leu 3A was purchased from Becton Dickinson (San Jose, CA), Anti-murine CD3 antibody 2C11 was the generous gift of Dr. Anne Rothstein of the Boston University School of Medicine. Rabbit anti-mouse antibodies used as a cross-linking reagent for Leu 3A was purchased from Fisher (Pittsburgh, PA). The protein tyrosine kinase inhibitor herbimycin A was the generous gift of Dr. Y. Uehara (National Institute of Health Tokyo, Japan). The p56 antibody used in Western blot analysis was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Chemotaxis

Cell migration was performed as described previously(4, 5) . Migration was assessed by a modification of a Boyden chamber technique, by using a microchemotaxis chamber (Neuroprobe, Cabin John, MD). T lymphocytes (10 10/ml) were loaded in the upper well of the chamber, with 30 µl of various concentrations of antibody as chemoattractant placed in the lower well. The two wells were separated by a nitrocellulose filter paper with a pore size of 8 µm. Migration chambers were incubated for 3 h, after which the filters were fixed in ethanol, stained, and migration was assessed by counting of the number of cells that migrated beyond 50 µm, by light microscopy. Cells treated with herbimycin A for both chemotaxis in vitro kinase analysis were incubated with 3 µg/ml (MeSO) for 18 h, at 37 °C in 5% CO and were assayed for viability by trypan blue exclusion. Cells were >90% viable. All migration was expressed as percentage values of cell migration in control buffer and statistics calculated by the Student's t test. Data are the mean value ± the standard deviation of three or more experiments.

In Vitro Kinase Assay

5 10 murine T cells/sample were removed from culture and suspended in ice-cold serum-free RPMI 1640 medium. Agonist was added to each sample on ice. The samples were then placed in a 37 °C heat block for 2 min. The reaction was stopped with the addition of ice cold wash buffer (150 mM NaCl, 50 mM Tris, pH 7.6) and the cells were pelleted. The supernatants were removed, and the pellets were solubilized in lysis buffer (1% Nonidet P-40, 150 mM NaCl, 50 mM Tris, pH 7.6) containing protease inhibitors for 20 min at 4 °C. The samples were then centrifuged for 10 min at 11,000 g and the supernatants transferred to fresh Eppendorff tubes. Precipitating antibody and Protein A-Sepharose beads were then added with the samples incubated for 1 h at 4 °C. The beads were washed and 50 µl of ice-cold kinase buffer (20 mM Tris, pH 7.4, 10 mM MnCl) was added to each sample. The kinase reaction was started with the addition of 10 µCi/sample of [-P]ATP and an exogenous substrate, acid-denatured enolase. The reaction was stopped with the addition of 4 SDS-Laemmli sample buffer. The samples were boiled and subjected to 10% SDS-PAGE. The gels were dried, subjected autoradiography, and quantitated by densitometry. Under these conditions the incorporation of P label was linear for the duration of agonist exposure.

Western Blot Analysis

Treated immunoprecipitates were separated on 10% SDS-PAGE gels and electrophoretically transferred to nitrocellulose membranes (Schleicher and Schuell, Keene, NH). The filters were blocked in a solution of 5% non-fat milk in phosphate-buffered saline (PBS: 125 mM NaCl, 8 mM NaHPO, 2 mM NaHPO, 5 mM KCl, pH 7.4), washed, and incubated in p56 antiserum (1:500) in PBS with 0.05% Tween 20 for 2 h. Washed filters were then incubated in goat anti-rabbit-horseradish peroxidase. Following a 1-min exposure to Lumi-GLO substrate (Kirkegaard and Perry Laboratories, Gaithersburg, MD) filters were subjected to chemiluminescense autoradiography.


RESULTS

LCF Induction of p56 in Murine T Cell Hybridoma Cells

Murine T cell hybridomas expressing human CD4 were examined for their ability to induce the catalytic activity of p56 in response to chemotactically active (10M) concentrations of LCF. In addition to the wild type CD4 hybridomas, cells expressing CD4 with selected cysteine to serine mutations were also examined. Alterations in cysteines 420 and 422 have previously been shown to disrupt the lck-binding site on CD4 (15) , while the mutation of cysteine 430, which lies outside of this domain, has no effect on the association of p56 with CD4 (15) . Cells lines were selected for equal expression of CD4 and exposed to LCF. CD4 immunoprecipitates from treated cells were then assayed for kinase activity by the addition of radioactive [P]ATP and the exogenous phosphorylation substrate, enolase. Immunoprecipitates from untreated wild type (data not shown) and CS430 murine hybridoma cells, the cysteine to serine control transfectant, (Fig. 1A, lane 2) demonstrated a background level of kinase and enolase phosphorylation, consistent with previous reports(16, 17) . As described previously(16, 18) ,()a dramatic increase in the autophosphorylation of lck protein was seen following treatment of cells with cross-linked CD4 antibody (Fig. 1A, lane 3). A lower, yet detectable, increase in enolase phosphorylation was also observed following antibody stimulation (Fig. 1B). LCF-induced autophosphorylation of lck was also observed although less pronounced (Fig. 1A, lane 4). Enolase phosphorylation following exposure of cells to LCF, however, was substantially increased (Fig. 1B). It is at present unclear what mechanism might regulate the differential incorporation of P in respective substrates with these two agonists. The association of p56 protein with CD4 was, however, not affected by these treatments as comparable levels of lck protein were immunoprecipitated for each condition (Fig. 1C). LCF-induced kinase activity was observable at 30 s, reached a maximum at 2 min, and began to diminish at 5 min (data not shown). The mutants CS420 and CS422, as anticipated, demonstrated no receptor associated kinase activity in either control or LCF-treated cells (Fig. 2).


Figure 1: A, in vitro kinase assay of LCF-induced wild type CD4-associated p56 kinase activity. 5 10 CS430 murine T cells were incubated with LCF, detergent solubilized, and immunoprecipitated with anti-receptor antibody. The immunoprecipitates were assayed for kinase activity and separated by SDS-PAGE. The gels were dried and subjected to autoradiography for 12-18 h. lck represents autophosphorylated p56 protein, en denotes acid denatured enolase. Lane 1, precipitation with Protein A alone. CD4 immunoprecipitation from T cell hydridomas were either untreated (lane 2), treated with cross-linked Leu 3A (lane 3), or treated with LCF (lane 4). B, densitometric analysis of LCF-induced kinase activity. Incorporation of labeled phosphate into both CD4-associated lck protein (Top) and enolase (Bottom) was quantitated by densitometry using the Molecular Dynamics Computing Densitometer. C, p56 Western blot of LCF-treated murine T cell hybridomas kinase activity. 5 10 CS430 murine T cells were incubated with LCF, detergent solubilized, and immunoprecipitated with anti-receptor antibody. The immunoprecipitates were assayed separated by SDS-PAGE transferred to nitrocellulose. The filters were first blocked in 5% milk in phosphate-buffered saline, then incubated in p56 antiserum followed by goat anti-rabbit-horseradish peroxidase, and finally subjected to chemiluminescense autoradiography. Arrow indicates p56 protein. Lane 1, precipitation with Protein A alone. CD4 immunoprecipitation from T cell hydridomas were either untreated (lane 2), treated with cross-linked Leu 3A (lane 3), or treated with LCF (lane 4).




Figure 2: In vitro kinase assay of LCF-induced mutant CD4-associated p56 kinase activity. 5 10 CS420 murine T cells were treated as in Fig. 1. Lane 1, precipitation with Protein A alone. CD4 immunoprecipitation from T cell hydridomas were either untreated (lane 2), treated with cross-linked Leu 3A (lane 3), or treated with LCF (lane 4).



Murine T Cell Hybridomas Migrate in Response to LCF

We next investigated these murine T cell hybridomas for their ability to migrate in response to LCF. Cells which express either wild type CD4 (open square) or CS430 CD4 (closed diamond) exhibited dose dependent migration following exposure of cells to micromolar-picomolar LCF concentrations (Fig. 3). The hybridoma cells, which express either CS420 (open diamond) or CS422 (closed square) CD4, failed to respond to any dose of LCF (Fig. 3). These results suggested that the binding of the tyrosine kinase was essential to CD4-mediated LCF-induced T cell migration. In order to confirm the CD4 specificity of the migration inactive clones, the cells were examined for their ability to migrate in response anti-CD3 antibody. We have previously shown (8) that the parental and mock transfected hybridoma lines migrate in response to CD3 antibody. Fig. 4shows that all four of the murine hybridoma lines expressing different forms, but equal amounts, of CD4 migrated in a similar dose-dependent manner following exposure to anti-CD3 antibody. These experiments demonstrate that the impaired motility observed in the CS420 and CS422 clones in response to LCF was restricted to alterations in CD4 and suggests that the CD4 signaling pathway induced during lymphocyte migration is independent of the signal transduction pathway utilized for anti-CD3-induced motility.


Figure 3: LCF-induced murine T cell migration. 10 10/ml murine T cells were incubated in a modified Boyden chamber in the presence of 1 pM to 0.1 µM of LCF for 3 h. The results are expressed as a percentage of the migration of untreated T cells. , MNC-CD4 T cells; , CS430 T cells; , CS420 T cells; , CS422 T cells. Asterisks denote a significant difference in migration from control at p = 0.05.




Figure 4: Anti-T cell receptor antibody-induced T cell migration. Murine T cell migration was assayed as in Fig. 3using 2C11 antibody. , MNC-CD4 T cells; , CS430 T cells; , CS420 T cells; , CS422 T cells.



Effect of Tyrosine Kinase Inhibitor, Herbimycin A, on LCF Induction of Murine T Cell Hybridomas Migration and p56 Kinase Activity

A number of groups have demonstrated that functions associated with lck interaction with CD4 are independent of the kinase activity of lck(10, 20, 21) . Along these lines we have recently demonstrated that divalent, uncross-linked anti-CD4 antibodies, incapable of inducing lck kinase activity do induce motile responses dependent on physical association of CD4 with p56. LCF induction of the kinase enzymatic activity, however, led us to determine whether LCF-induced lymphocyte migration requires the enzymatic activity of p56 activity. In these experiments CS430 hybridoma cells were exposed to the kinase inhibitor herbimycin A. Herbimycin A has a bimodal effect on tyrosine kinases, first directly decreasing the activity of the kinase(22, 23, 24) and then acting to increase the degradation rate of the steady state levels of the protein(25) . At 18 h, a time when there is complete inhibition of enzymatic activity (Fig. 5) but minimal protein degradation(23) , the motility induced by LCF (Fig. 6) and CD3 antibodies (Fig. 7) was identical to that observed in untreated cells ( Fig. 3and Fig. 4). To further understand the role of lck enzymatic activity in CD4-mediated migration, we used two T cell hybridoma cell lines expressing two different CD4-lck chimeras.


Figure 5: Effect of herbimycin A on in vitro kinase assay of LCF-induced CD4 associated p56 kinase activity. Kinase activity was assayed as in Fig. 1, following 18 h of incubation with 3 µg/ml herbimycin A. Lane 1, precipitation with Protein A alone. CD4 immunoprecipitation from T cell hydridomas were either untreated (lane 2), treated with cross-linked Leu 3A (lane 3), or treated with LCF (lane 4).




Figure 6: Effect of tyrosine kinase inhibitor herbimycin A on LCF-induced T cell migration. Murine CS430 T cell migration was assayed as in Fig. 3following an 18 h incubation with 3 µg/ml herbimycin A. , with treatment; , without.




Figure 7: Anti-T cell receptor antibody-induced T cell migration. Murine T cell migration was assayed as in Fig. 6using 2C11 antibody. , MNC-CD4 T cells; , CS430 T cells; , CS420 T cells; , CS422 T cells.



Chimeric CD4-lck T Cell Hybridoma Migration

Chimeric CD4-lck containing either full-length lck protein or kinase domain-deleted lck protein were expressed in the By155.16 T cell line. Cells were selected for stable and equivalent surface expression of these constructs and examined for their LCF-induced migratory response. Fig. 8shows that hybridoma cells expressing full-length CD4-lck responded to LCF in a dose-dependent manner similar to that of the CD4 wild type control. Cells expressing CD4-lck which lacks the catalytic domain of the kinase demonstrated comparable motility. Thus T cells in which the kinase activity was eliminated either by herbimycin A treatment or construction of kinase deficient chimeric molecules have normal motile responses to LCF. These data indicate that while LCF induces CD4-associated lck kinase activity and that induced migration requires lck association with CD4, LCF-induced cell migration is independent of the catalytic activity of the kinase.


Figure 8: LCF-induced murine T cell migration. 10 10/ml murine T cells were incubated in a modified Boyden chamber in the presence of 10 pM to 0.1 µM of LCF for 3 h. The results are expressed as a percentage of the migration of untreated T cells. , By155.16 parent line T cells; , MNC-CD4 T cells; , full-length CD4-lck chimeric T cells; , kinase-deleted CD4-lck chimeric T cells.




DISCUSSION

CD4 is required as an essential component of the cell surface signaling complex engaged during LCF-induced motility. The evidence for this relationship rests with CD4 transfection experiments in which we have previously shown that the LCF-induced rises in [Ca] and inositol trisphosphate (IP) are CD4 dependent(7) , as is the motile response (8, Fig. 3). Further, LCF-induced chemotactic responses in monocytes (5) and eosinophils (6) are also directly dependent upon the expression of cell surface CD4, and Fab fragments of anti-CD4 monoclonal antibody inhibit all LCF-induced functions on these cells(5, 6, 7, 8) . The data presented in this paper extend these observations to show that LCF induces CD4-associated p56 kinase activity and therefore provides further evidence for a direct signaling role of CD4 in LCF-induced functions.

In conjunction with previous work, the physical association between the src family tyrosine kinase p56 and CD4 is essential for LCF-induced lymphocyte migration. The activation of T cell motile response mediated via CD4 is independent of the catalytic activity of the receptor-coupled kinase. These findings are consistent with recent reports which have identified a kinase independent role for p56 during T cell activation (10, 20, 21) and our own recent analysis demonstrating that uncross-linked anti-CD4 induced migration is independent of the enzymatic activity of p56. While kinase activity is not required for the induction of T cell migration, p56 binding of other intracellular proteins (e.g. GTPase p32, c-raf, phosphatidylinositol 3-kinase, phospholipase C) as an adaptor molecule, may explain the requirement for lck-CD4 association in the motile response(26, 27, 28, 29) . Binding to the src homology domains (30) SH2 and SH3 of p56 appear most likely to provide a recruitment site for these molecules to the CD4-lck complex. The ability of cells, expressing CD4-lck chimeras which lack the catalytic domain, to migrate in response to LCF strongly suggests the involvement of SH2/SH3 binding intermediates in the signal transduction mechanism of lymphocyte motility.

For the motile response, one of the principle events is the induction of cellular shape changes through the reorganization of the cytoskeletal structures(2) . Along these lines, the second messengers generated by phospholipase C, IP, and [Ca] are both released in response to LCF (7) and have been reported to regulate actin-binding proteins (31) and changes in the cytoskeleton(32) , as well as the activation of their down stream effector enzyme protein kinase C(33) . The link to the cytoskeleton could be the lipid kinase, phosphatidylinositol-3 kinase which associates with src kinases, by binding to SH3 domains(34, 35) and has recently been shown to play a role in cell shape change(36) . Alternatively, there might be a direct link between actin-binding proteins, which have been demonstrated to contain SH3 domains(37, 38) to the CD4-coupled p56 complex.

Our data also suggest an explanation for the observations that the CD4 ligands HIV-1 gp120 and anti-CD4 antibodies appear to require cross-linking to induce some, but not all CD4 related activation pathways and cellular functions. The lymphocyte chemoattractant factor is a 14-kDa protein which exists as a bioactive tetramer of 56 kDa in supernatants of conconavalin A or histamine-stimulated peripheral blood mononuclear cells(39, 40) . We subsequently demonstrated that the cDNA for LCF codes for a 14kDa protein, which autopolymerizes to 56 kDa. There is an absolute requirement for a polymerized form of LCF for the biologic activities(8) . Thus, cross-linking of other ligands such as anti-receptor (CD4) antibody or multimeric gp120(9) , might mimic the natural state of LCF.

However, the relationship of CD4 ligands to CD4 must be more complex than a simple cross-linking phenomena as noted for a number of other receptors(41) . Fab of anti-CD4 antibodies do not induce motility; however, intact dimeric antibody induces comparable migration as seen with LCF or gp120. In addition, further cross-linking of divalent anti-CD4 antibody does not further augment the motile response. Intact antibody also down-modulates subsequent activation via CD3, but unlike LCF it does not induce IL-2R nor major histocompatability complex Class II molecules(5) . In addition, intact antibody does not induce any of the traditional intracellular signal transduction pathways including phosphatidyl inositol turnover, rises in [Ca], nor p56 enzymatic activity(19) . The inability of uncross-linked anti-CD4 antibodies to induce p56 kinase activity but their ability to induce motile responses highlights the dissociation between a proposed ``adaptor'' function for lck which appears to be essential for communication with motility-dependent cytoskeletal structures and functions associated with lck kinase activity. More extensive aggregation of surface CD4 by LCF, cross-linked anti-CD4 antibodies, and gp120 appear to have the ability to induce cell activation and the intracellular signal transduction systems noted above.

We propose, therefore, that the src homology domains of lck, independent of the activation of the kinase, act to recruit intracellular molecules which initiate changes in cytoskeletal structure essential for LCF-induced T lymphocyte migration. Therefore, the induction of p56 kinase activity by LCF and cross-linked antibodies suggests a role for the catalytic activity in LCF-induced cell activation events other than motility.


FOOTNOTES

*
This work was supported by Grant HL32802 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 617-638-4860; Fax: 617-536-8093.

The abbreviations used are: LCF, lymphocyte chemoattractant factor; [CA], intracellular calcium; IP, inositol trisphosphate; gp120, glycoprotein 120; PAGE, polyacrylamide gel electrophoresis.

T. C. Ryan, W. W. Cruikshank, and D. M. Center, manuscript submitted.


REFERENCES
  1. Roitt, I. (1991) Essential Immunology, 7th Ed., Blackwell Scientific Publisher, Oxford
  2. Berman, J. S., Beer, D. J., Theodore, A. C., Kornfeld, H., Bernardo, J., and Center, D. M. (1990)Am. Rev. Respir. Dis. 142, 238-257 [Medline] [Order article via Infotrieve]
  3. Center, D. M., Berman, J. S., Kornfeld, H., Theodore, A. C. and Cruikshank, W. W.(1993) Chest103,88S-91S [Medline] [Order article via Infotrieve]
  4. Berman, J. S., Cruikshank, W. W., Center, D. M., Theodore, A. C., and Beer, D. J. (1985)Cell Immunol. 95, 105-112 [Medline] [Order article via Infotrieve]
  5. Cruikshank, W. W., Berman, J. S., Theodore, A. C., Bernardo, J., and Center, D. M. (1987)J. Immunol. 138, 3817-3823 [Abstract/Free Full Text]
  6. Rand, T., Cruikshank, W. W., Center, D. M., and Weller, P. F.(1991)J. Exp. Med. 173, 1521-1528 [Abstract]
  7. Cruikshank, W. W., Greenstein, J. L., Theodore, A. C., and Center, D. M.(1991) J. Immunol. 146, 2928-2934 [Abstract/Free Full Text]
  8. Cruikshank, W. W., Center, D. M., Nisar, N., Wu, M., Natke, B., Theodore, A., and Kornfeld, H.(1994)Proc. Natl. Acad. Sci. U. S. A 91, 5109-5113 [Abstract]
  9. Kornfeld, H., Cruikshank, W.W., Pyle, S.W., Berman, J. S., and Center, D.M.(1988) Nature 335, 445-448 [CrossRef][Medline] [Order article via Infotrieve]
  10. Goldman, F., Jensen, W. A., Johnson, G. L., Heasley, L., and Cambier, J.(1994) J. Immunol. 153, 2905-2917 [Abstract/Free Full Text]
  11. Veillette, A., Brookman, M.A., Horak, E.M., and Bolen, J. B.(1988)Cell 55, 301-308 [Medline] [Order article via Infotrieve]
  12. Rudd, C. E., Trevilyan, J.M., Dasqueta, J. D., Wong, L.L., and Shlossmann, S. F.(1988)Proc. Natl. Acad. Sci.U. S. A. 85, 5190-5194 [Abstract]
  13. Sleckman, B. P., Shin, J., Igras, V. E., Collins, T. L., Stominger, J. L., and Burakoff, S. J. (1992)Proc. Natl. Acad. Sci. U. S.A.89,7566-7570 [Abstract]
  14. Sleckman, B., Peterson, A., Jones, W. K., Foran, J. A., Greenstein, J. L., Seed, B., and Burakoff, S.(1987)Nature 328, 351-353 [Medline] [Order article via Infotrieve]
  15. Collins, T. L., Uniyal, S., Shin, J., Stominger, J. L., Mittler, R. S., and Burakoff, S. J. (1992)J. Immunol. 148, 2159-2162 [Abstract/Free Full Text]
  16. Bramson, H. N, Casnellie, J. E., Nachod, H., Regan, L., and Sommers, C.(1991) J. Biol. Chem. 266, 16219-16225 [Abstract/Free Full Text]
  17. Rudd, C. E., Janssen, O., Prasad, K. V. S., Rabb, M., Da Silva, A., Telfer, J. C., and Yamamoto, M.(1993)Biochim. Biophys. Acta 1155, 239-266 [CrossRef][Medline] [Order article via Infotrieve]
  18. Veillette, A., Bookman, M. A., Horak, E. M., Samelson, L. E., and Bolen, J. B.(1989) Nature 338, 257-259 [CrossRef][Medline] [Order article via Infotrieve]
  19. Ledbetter, J. A., June, C. H., Grosmaire, L. S., and Rabinovitch, P. S.(1987) Proc. Natl. Acad. Sci.U. S. A. 84, 1384-1388 [Abstract]
  20. Collins, T. L., and Burakoff, S. J.(1993)Proc. Natl. Acad. Sci. U. S.A.90,11885-11889 [Abstract]
  21. Xu, H., and Littman, D. R.(1993)Cell 74, 633-643 [Medline] [Order article via Infotrieve]
  22. Uehara, Y., Fukazawa, H., Murakami, Y., and Mizuno, S.(1989)Biochem. Biophys. Res. Commun. 163, 803-809 [Medline] [Order article via Infotrieve]
  23. June, C. H., Fletcher, M. C., Ledbetter, J. A., Schieven, G. L., Siegel, J. N., Phillips, A. F., and Samelson, L. E.(1990)Proc. Natl. Acad. Sci. U.S.A.87,7722-7726 [Abstract]
  24. Veillette, A., Dumont, S., and Fournel, M.(1993)J. Biol. Chem. 268, 17547-17553 [Abstract/Free Full Text]
  25. Uehara, Y. Murakami, Y., Sugimoto, Y., and Mizuno, S.(1989)Cancer Res. 49, 780-785 [Abstract]
  26. Tefler, J. C., and Rudd, C. E.(1991)Science 254, 439-441 [Medline] [Order article via Infotrieve]
  27. Thompson, P. A., Ledbetter, J., Rapp, U. R., and Bolen, J. S.(1992) Cell Growth Diff. 2, 609-617 [Abstract]
  28. Prasad, K. V. S., Janssen, O., Kapeller, R., Raab, M., Cantley, L. C., and Rudd, C. E. (1993)Proc. Natl. Acad. Sci. U. S.A 90, 7366-7370 [Abstract]
  29. Weber, J. R., Bell, G. M., Han, M. Y., Pawson, T., and Imboden, J. B.(1992) J. Exp. Med. 176, 373-379 [Abstract]
  30. Pawson, T., and Schlessinger, J.(1993)Curr. Biol.3,434-436
  31. Adams, R. J., and Pollard, T. D.(1989)Nature 340, 565-568 [CrossRef][Medline] [Order article via Infotrieve]
  32. Shelanski, M. L. (1989)Ann.N.Y. Acad. Sci. 568, 121-124
  33. Thorp, K. M., Southern, C., and Matthews, N.(1994)Immunology 81, 546-550 [Medline] [Order article via Infotrieve]
  34. Liu, X., Marengere, L. E. M., Kock, C. A., and Pawson, T.(1993)Mol. Cell. Biol. 13, 5225-5232 [Abstract]
  35. Pleiman, C. M., Clark, M. R., Timson Gauen, L. K., Winitz, S., Coggeshall, K. M., Johnson, G. L., Shaw, A. S., and Cambier, J. C.(1993)Mol. Cell. Biol. 13, 5877-5887 [Abstract]
  36. Herman, P. K., Stack, J. H., and Emr, S. D.(1992)Trends Cell Biol. 2, 363-368 [Medline] [Order article via Infotrieve]
  37. Drubin, D. G., Mulholland, J., Zhu, Z., and Botstein, D.(1990) Nature 343, 288-290 [CrossRef][Medline] [Order article via Infotrieve]
  38. Weng, Z., Taylor, J. A., Turner, C. E., Brugge, J. S., and Seidel-Dugan, C.(1993) J. Biol. Chem. 268, 14956-14963 [Abstract/Free Full Text]
  39. Center, D. M., Cruikshank, W. W., Berman, J. S., and Beer, D. J.(1983) J. Immunol. 131, 1854-1859 [Abstract/Free Full Text]
  40. Berman, J. S., McFadden, R. G., Cruikshank, W. W., Center, D. M., and Beer, D. J.(1984) J. Immunol. 133, 1495-1504 [Abstract/Free Full Text]
  41. Metzger, H.(1992) J. Immunol. 149, 1477-1487 [Free Full Text]

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