Discovery of a Novel, Potent, and Src Family-selective Tyrosine Kinase Inhibitor

Here, we have studied the activity of a novel protein-tyrosine kinase inhibitor that is selective for the Src family of tyrosine kinases. We have focused our study on the effects of this compound on T cell receptor-induced T cell activation, a process dependent on the activity of the Src kinases Lck and FynT. This compound is a nanomolar inhibitor of Lck and FynT, inhibits anti-CD3-induced protein-tyrosine kinase activity in T cells, demonstrates selectivity for Lck and FynT over ZAP-70, and preferentially inhibits T cell receptor-dependent anti-CD3-induced T cell proliferation over non-T cell receptor-dependent phorbol 12-myristate 13-acetate/interleukin-2 (IL-2)-induced T cell proliferation. Interestingly, this compound selectively inhibits the induction of the IL-2 gene, but not the granulocyte-macrophage colony-stimulating factor or IL-2 receptor genes. This compound offers a useful new tool for examining the role of the Lck and FynT tyrosine kinases versus ZAP-70 in T cell activation as well as the role of other Src family kinases in receptor function. Here, we have studied the activity of a novel protein-tyrosine kinase inhibitor that is selective for the Src family of tyrosine kinases. We have focused our study on the effects of this compound on T cell receptor-induced T cell activation, a process dependent on the activity of the Src kinases Lck and FynT. This compound is a nanomolar inhibitor of Lck and FynT, inhibits anti-CD3-induced protein-tyrosine kinase activity in T cells, demonstrates selectivity for Lck and FynT over ZAP-70, and preferentially inhibits T cell receptor-dependent anti-CD3-induced T cell proliferation over non-T cell receptor-dependent phorbol 12-myristate 13-acetate/interleukin-2 (IL-2)-induced T cell proliferation. Interestingly, this compound selectively inhibits the induction of the IL-2 gene, but not the granulocyte-macrophage colony-stimulating factor or IL-2 receptor genes. This compound offers a useful new tool for examining the role of the Lck and FynT tyrosine kinases versus ZAP-70 in T cell activation as well as the role of other Src family kinases in receptor function. INTRODUCTIONStudy of the roles of the Src family kinases p56lck and p59fynT in T cell receptor (TcR) 1The abbreviations used are: TcRT cell receptorIL-2interleukin-2IL-2RIL-2 receptorEGF-Repidermal growth factor receptorGM-CSFgranulocyte-macrophage colony-stimulating factorPBLperipheral blood lymphocyte(s)PBSphosphate-buffered salinePMAphorbol 12-myristate 13-acetatePHAphytohemagglutininPAGEpolyacrylamide gel electrophoresis. function has been hampered by a lack of specific pharmacological inhibitors. Here, we describe and utilize a novel, potent, and Src family-selective small molecule inhibitor to further study the role of these kinases in T cell activation.The lck gene, which encodes a lymphocyte-specific, membrane-associated protein-tyrosine kinase of the nonreceptor type(1Perlmutter R.M. Marth J.D. Ziegler S.F. Garvin A.M. Pawar S. Cooke M.P. Abraham K.M. Biochim. Biophys. Acta. 1988; 948: 245-262Google Scholar, 2Marth J.D. Peet R. Krebs E.G. Perlmutter R.M. Cell. 1985; 43: 393-404Abstract Full Text PDF PubMed Scopus (357) Google Scholar), was first identified in retrovirally induced murine T cell leukemias(2Marth J.D. Peet R. Krebs E.G. Perlmutter R.M. Cell. 1985; 43: 393-404Abstract Full Text PDF PubMed Scopus (357) Google Scholar, 3Voronova A.F. Sefton B.M. Nature. 1986; 319: 682-685Crossref PubMed Scopus (161) Google Scholar, 4Adler T.H. Reynolds P.J. Kelley C.M. Sefton B.M. J. Virol. 1988; 62: 4113-4122Crossref PubMed Google Scholar). Lck contains a unique N-terminal sequence (5Bolen J.B. Rowley R.B. Spana C. Tsygankov A.Y. FASEB J. 1992; 6: 3403-3409Crossref PubMed Scopus (194) Google Scholar) that directs its specific interaction with the cytoplasmic domains of the CD4 and CD8 glycoproteins(6Turner J.M. Brodsky M.H. Irving B.A. Levin S.D. Perlmutter R.M. Littman D.R. Cell. 1990; 60: 755-765Abstract Full Text PDF PubMed Scopus (488) Google Scholar, 7Shaw A.S. Chalupny J. Whitney J.A. Hammond C. Amrein K.E. Kavathas P. Sefton B.M. Rose J.K. Mol. Cell. Biol. 1990; 10: 1853-1862Crossref PubMed Scopus (208) Google Scholar, 8Shaw A.S. Amrein K.E. Hammond C. Stern D.F. Sefton B.M. Rose J.K. Cell. 1989; 59: 627-636Abstract Full Text PDF PubMed Scopus (257) Google Scholar, 9Rudd C.E. Trevillyan J.M. Dasgupta J.D. Wong L.L. Scholssman S.F. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5190-5194Crossref PubMed Scopus (613) Google Scholar, 10Veillette G. Bookman M.A. Horak E.M. Bolen J.B. Cell. 1988; 55: 301-308Abstract Full Text PDF PubMed Scopus (1122) Google Scholar). This interaction is required for antigen-specific responses of several different T cell hybridomas(11Glaichenhaus N. Shastri N. Littman D.R. Turner J.M. Cell. 1991; 64: 511-520Abstract Full Text PDF PubMed Scopus (348) Google Scholar, 12Zamoyska R. Derham P. Gorman S.D. von Hoegen P. Bolen J.B. Veillette A. Parnes J.R. Nature. 1989; 342: 278-281Crossref PubMed Scopus (154) Google Scholar). Lck has also been reported to associate with the IL-2R β-chain via a distinct interaction(13Hatakeyama M.T. Kono T. Kobayashi N. Kawahara A. Levin S.D. Perlmutter R.M. Taniguchi T. Science. 1991; 252: 1523-1528Crossref PubMed Scopus (504) Google Scholar), although its role in IL-2R function remains unclear. Several reports have demonstrated that Lck plays an important role in both T cell maturation and activation. Loss of Lck expression in the human Jurkat T cell line abrogates its response to anti-TcR antibodies(14Straus D.B. Weiss A. Cell. 1992; 70: 585-593Abstract Full Text PDF PubMed Scopus (924) Google Scholar), and inactivation of the lck gene or overexpression of a dominant negative lck transgene in mice leads to an arrest of thymocyte development at a stage prior to the expression of CD4, CD8. and TcR(15Molina T.J. Kishihara K. Siderovski D.P. van Ewijk W. Narendran A. Timms E. Wakeham A. Paige C.J. Hartmann K.U. Veillette A. Davidson D. Mak T.W. Nature. 1992; 357: 161-164Crossref PubMed Scopus (886) Google Scholar, 16Levin S.D. Anderson S.J. Forbush K.A. Perlmutter R.M. EMBO J. 1993; 12: 1671-1680Crossref PubMed Scopus (297) Google Scholar).Whereas the role of Lck in T cell development and function is well established, the role of Fyn is less well defined. Small amounts of Fyn are found associated with the TcR complex following mild detergent extraction of T lymphocytes(17Samelson L.E. Phillips A.F. Luong E.T. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4358-4362Crossref PubMed Scopus (531) Google Scholar). Furthermore, activation of T cells through the TcR results in increased enzymatic activity of Fyn(18Tsygankov A.Y. Broker B.M. Fargnoli J. Ledbetter J.A. Bolen J.B. J. Biol. Chem. 1992; 267: 18259-18262Abstract Full Text PDF PubMed Google Scholar). Additional evidence that Fyn is involved in lymphocyte activation comes from experiments in transgenic animals. Twenty-fold overexpression of FynT in transgenic thymocytes results in enhanced responsiveness to anti-CD3 antibody as measured by the stimulation of tyrosine phosphorylation in whole cells, Ca2+ accumulation, and M.P. Abraham K.M. Forbush K.A. Perlmutter R.M. Cell. 1991; Full Text PDF PubMed Scopus Google Scholar). Furthermore, overexpression of a of FynT in the thymocytes of transgenic mice T cell M.P. Abraham K.M. Forbush K.A. Perlmutter R.M. Cell. 1991; Full Text PDF PubMed Scopus Google Scholar). mice that lack p59fynT or in or J.A. Cooke M.P. Levin S.D. Perlmutter R.M. Cell. 1992; 70: Full Text PDF PubMed Scopus Google Scholar, S. P. Cell. 1992; 70: Full Text PDF PubMed Scopus Google Scholar), that Fyn plays a role in in we that protein-tyrosine kinase inhibits Lck and FynT in at required to ZAP-70, the and kinase A. inhibits whole cell tyrosine phosphorylation and proliferation in T cells with anti-CD3 and selectively inhibits IL-2 gene expression over and IL-2R gene induction in human T this compound to a of TcR not by other of TcR as This compound is a new tool to study the role of Src family protein-tyrosine kinases in lymphocyte and blood from by of whole blood J. PubMed Scopus Google Scholar) and in proliferation experiments in that with in for with of compound and anti-CD3 or was by the of at by the cells at a are reported as an from a of the of proliferation from from experiments in T cell proliferation specific was by in with of with of compound or in a was by and of through a to and the to of was to and the for at in was and the for an at with a and the of was a that of proliferation from a of the of proliferation from versus of compound are as the from experiments T cell proliferation in the lymphocyte was by in and compound in in well of a of at in of was to and the cells for cells a was by the following of of the of that of the are as the from experiments human T cells, used in the whole cell by the T cell as by was T cells, with the cells Jurkat T cells in cells at in a in a new used for Lck and FynT activity and was as D. J.M. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar). was with a with for at and with of of Lck was cells or Lck in cells a expression A.S. Amrein K.E. Hammond C. Stern D.F. Sefton B.M. Rose J.K. Cell. 1989; 59: 627-636Abstract Full Text PDF PubMed Scopus (257) Google Scholar, E.G. Proc. Natl. Sci. U. S. A. 1986; PubMed Scopus Google Scholar). FynT of R. of was in cells the E.G. Proc. Natl. Sci. U. S. A. 1986; PubMed Scopus Google Scholar, Bolen J.B. Klausner R.D. A.S. Mol. Cell. Biol. PubMed Scopus Google Scholar). in and and the by at for at in an Lck antibody was by with a of the N-terminal of antibody was from with the antibody at for at as a to the at and to for at in of and in of kinase and and to in kinase of the was to well of the with an of compound and of a in kinase for at of M.P. Sefton B.M. 1988; Google Scholar) was to the to the of the was from the for and on a and to a and the of the was of compound that of phosphorylation from a of the versus of compound experiments for the activity of Lck the was with on a was to the and was measured a of compound that of activity from a of the of activity versus of was the gel and experiments for both Lck and FynT activity was measured by of from cells from the by of to a that a of by as and with for at to the at and to for at in of and in of kinase and to in kinase was of which was for at in an and the was the was of the compound of to a of and of a in kinase for at the with of and with of the was of M.P. Sefton B.M. 1988; Google Scholar) on which and and was the of compound that of activity from a of the of activity by different of was in and as an complex to in of kinase for at and was measured by the and was on a to the as ZAP-70 kinase Weiss A. Cell. 1992; Full Text PDF PubMed Scopus Google Scholar, van A. Weiss A. J. Google Scholar, Weiss A. Science. PubMed Scopus Google Scholar) was D. W. Scholar). from cells with a human ZAP-70 as for and a was used in a kinase kinase activity was by the of the S. D. K. D. P. A. Mol. Cell. Biol. PubMed Google Scholar), to and a to activity was by phosphorylation cell with as in a new of tyrosine phosphorylation in human blood T cells was measured as at in an at a of in of for with prior to a with of of the was by the of of M.P. Sefton B.M. 1988; Google Scholar) at prior to its for and at of these cell on with a and with M.P. Sefton B.M. 1988; Google Scholar). a and the of the in the of anti-CD3 the and of was as in of in of the of compound at which was as 1992; Google Scholar). by with the gel and over a T. J. Scholar). a human for and from the was from human P. N. PubMed Scopus Google Scholar). to of was on a gel in and was to and a was to in and with at with in for at for in at and for in at prior to a was and to specific for as 1992; Google Scholar). the from the human IL-2 to and to of the gene and human Jurkat T cell line was as and was measured a experiments in and the are as are of Src in was as of a of used for of p56lck and on a compound first in tyrosine kinase inhibitor was first in for its to tyrosine phosphorylation of by was found to a inhibitor of this p56lck from was with and of in a was and the on an in phosphorylation of the in a was a and the for of was in in the in the of of and a for of Lck in these the was the gel with the of and not results a the sequence from the human TcR not the gel and results and the was for experiments the to for of Lck and FynT. p56lck activity with an of results with p56lck in cells a expression not A.S. Amrein K.E. Hammond C. Stern D.F. Sefton B.M. Rose J.K. Cell. 1989; 59: 627-636Abstract Full Text PDF PubMed Scopus (257) Google Scholar, E.G. Proc. Natl. Sci. U. S. A. 1986; PubMed Scopus Google Scholar). compound was also a inhibitor of a lymphocyte Src family p59fynT the was in the of Lck and FynT further selectivity other Src family protein-tyrosine also Src and demonstrated of not and both less in the of growth factor receptor and for of Lck and FynT was demonstrated was found that and for of ZAP-70 and and kinase not the activity of the ZAP-70 enhanced following phosphorylation at by R. G. T. R. T. EMBO J. PubMed Scopus Google Scholar), we also of ZAP-70 by was in cells with Lck as R. G. T. R. T. EMBO J. PubMed Scopus Google Scholar). of ZAP-70 with the of Lck to a in the specific activity of ZAP-70 for the was to this to of not we also the activity of and tyrosine kinase inhibitors. has been demonstrated to a but kinase J.M. G. A. J.A. 1993; PubMed Scopus Google Scholar, 1992; Scholar). the experiments reported was found to a nanomolar inhibitor of p56lck and p59fynT as well as a inhibitor of the and was also a inhibitor of the ZAP-70 and tyrosine kinases. further the J.M. G. A. J.A. 1993; PubMed Scopus Google Scholar) was for its to the tyrosine kinases. was the inhibitor to other reported tyrosine kinase the novel and demonstrated and selective of the Src family as p56lck and of and are to the reported J. Chem. 1988; of in T of the in of T cells is the stimulation of the tyrosine phosphorylation of Luong E.T. Klausner R.D. L.E. J. Biol. Chem. 1989; Full Text PDF PubMed Google Scholar). have demonstrated that tyrosine kinase are of T cell Ledbetter J.A. L.E. J. 1990; Google Scholar, Ledbetter J.A. Phillips A.F. L.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: PubMed Scopus Google Scholar). was for its to tyrosine phosphorylation in human T cells T cells or with anti-CD3 or with anti-CD3 a with or of the was used to stimulation of tyrosine M.P. Sefton B.M. 1988; Google Scholar). is in induced the tyrosine phosphorylation of a of in human T cells and of and phosphorylation of was by and with at as measured by the from experiments not was with for of anti-CD3-induced tyrosine phosphorylation not of human T cells with this Lck and FynT inhibitor the in whole cell tyrosine phosphorylation following with results are with of these kinases in T cell not a role for other Src family kinases that in T is a inhibitor of T cell tyrosine blood T cells as for at in the and or of a or of the tyrosine kinase inhibitor prior to the of of or an with the and the for tyrosine phosphorylation by with antibody M.P. Sefton B.M. 1988; Google Scholar) as are on the of T by anti-CD3-induced tyrosine we the proliferation of human T cells in response to different in experiments different and in the of the compound and was results are as an of experiments different human of the activity of an of with anti-CD3 in different and proliferation was induced by in results T cells not of other T cell for human T cells from in a also with an of T cells from and with with an of was with and demonstrated of tyrosine T cell proliferation. less of that the T cell receptor complex a T cell proliferation in response to both and specific in line with its reported J.M. G. A. J.A. 1993; PubMed Scopus Google Scholar). a tyrosine kinase J.M. G. A. J.A. 1993; PubMed Scopus Google Scholar, 1992; Scholar), demonstrated less of T cell proliferation of IL-2 by selectively proliferation of T cells, we this compound for of involved in T cell proliferation. from a and or with and for in the or of or was used at as a IL-2 induction was by both and and the and not by these of Interestingly, IL-2R enhanced in the of a that is an inhibitor of the J. J.D. J. Cell. 1991; Full Text PDF PubMed Scopus Google Scholar), IL-2 and induction as K.A. P. S. S. J. J. 1989; Google Scholar). IL-2R induction was also by this of results that selectively required for the activation of the IL-2 gene and that the to and IL-2R induction of the effects of on gene expression in human human or for in with and in the or of or and and was for as further this we selectively IL-2 over IL-2R gene expression at the of the IL-2 or IL-2R to the gene Jurkat T cell with these and the cells with and for prior to for activity as 1992; Google Scholar). in both the IL-2 and IL-2R induced to in response to and for demonstrated of the IL-2 with an of the IL-2R was not by to of results are with the and that compound is of selectively required for IL-2 gene of IL-2 and IL-2R activity by Jurkat T cells with IL-2 or IL-2R and in for in the or of and and for activity as was to cells with the IL-2 or IL-2R for in the of prior to the cells and for activity as the of with are of this we have the and activity of a novel tyrosine kinase inhibitor that inhibits Lck and FynT, anti-CD3-induced tyrosine and IL-2 gene activation in T this compound selectivity for the Src family over other of tyrosine kinases ZAP-70, and the from identified tyrosine kinase inhibitors. of in and in and selectivity for of the Src family of tyrosine kinases is have reported the of other tyrosine kinase to in T with a was to tyrosine and Ledbetter J.A. Phillips A.F. L.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: PubMed Scopus Google Scholar). required to and the interaction of with on protein-tyrosine kinases 1992; Scholar) have its T. J. S. S. N. J. Biol. Chem. Full Text PDF PubMed Google Scholar) has been also to T cell receptor and activation J.M. A. Phillips J.M. J. 1990; Google Scholar). with prior to receptor with anti-CD3 Lck activity and phosphorylation as well as activation of the IL-2 gene, but was to IL-2 induced by Ca2+ and that the of required to in and in whole cells and its lack of in kinase J.M. G. A. J.A. 1993; PubMed Scopus Google Scholar) with the and we have with protein-tyrosine kinase Bolen J.B. Biophys. 1991; PubMed Scopus Google Scholar), was to selective for Lck as with and was also selective for of Lck with other Src family kinases to the compound an of for of Lck in several of that which we have for for kinase a of the of is also a inhibitor of Src family tyrosine kinases in in our demonstrated less and FynT, ZAP-70, and have reported that is a inhibitor of Src family kinases and was also for of nonreceptor tyrosine and J.M. G. A. J.A. 1993; PubMed Scopus Google Scholar, 1992; Scholar). new has also been as an inhibitor of S. D. P. J. W. S. P. J. R. D. J. Scopus Google Scholar). This compound is less for of Lck and to less selective demonstrates for the growth factor the have been studied for to of protein-tyrosine A. FASEB J. 1992; 6: PubMed Scopus Google Scholar, A. A. Science. 267: PubMed Scopus Google Scholar). selectivity for protein-tyrosine kinases over other of kinases as kinase kinase or kinases. less is the of these on T cell kinases and function. of tyrosine kinase are by the lack of for of and of kinase our the that is the and selective inhibitor of Src family tyrosine kinases as Lck and FynT reported to of to was in of our was at anti-CD3-induced T cell activation was less at the proliferation induced by and and we found that was at IL-2 gene expression or IL-2R gene results that Lck and FynT a specific role in IL-2 gene expression required for T cell but not in the induction of the or IL-2R genes. anti-CD3-induced expression of IL-2 and the effects of to specific of a complex with and the complex to and inhibits the J. J.D. J. Cell. 1991; Full Text PDF PubMed Scopus Google Scholar). This results in the of that to TcR as IL-2 and K.A. P. S. S. J. J. 1989; Google Scholar). inhibits Ca2+ in T cells not was that and for of in T and This that a from the TcR to expression and that but not inhibits this of this other and the by which this other to of in T cell tyrosine phosphorylation and was to of kinase This in to of the compound and its the but also a of the of the inhibitor with not complex for of at of the compound to with is that the of found the cell T.W. Mol. Cell. Biol. Scholar) to the of the inhibitor in are to the of Lck by and this of and its to as pharmacological in the of T a in the of tyrosine kinase as to study the role of Lck and FynT in T cell INTRODUCTIONStudy of the roles of the Src family kinases p56lck and p59fynT in T cell receptor (TcR) 1The abbreviations used are: TcRT cell receptorIL-2interleukin-2IL-2RIL-2 receptorEGF-Repidermal growth factor receptorGM-CSFgranulocyte-macrophage colony-stimulating factorPBLperipheral blood lymphocyte(s)PBSphosphate-buffered salinePMAphorbol 12-myristate 13-acetatePHAphytohemagglutininPAGEpolyacrylamide gel electrophoresis. function has been hampered by a lack of specific pharmacological inhibitors. Here, we describe and utilize a novel, potent, and Src family-selective small molecule inhibitor to further study the role of these kinases in T cell activation.The lck gene, which encodes a lymphocyte-specific, membrane-associated protein-tyrosine kinase of the nonreceptor type(1Perlmutter R.M. Marth J.D. Ziegler S.F. Garvin A.M. Pawar S. Cooke M.P. Abraham K.M. Biochim. Biophys. Acta. 1988; 948: 245-262Google Scholar, 2Marth J.D. Peet R. Krebs E.G. Perlmutter R.M. Cell. 1985; 43: 393-404Abstract Full Text PDF PubMed Scopus (357) Google Scholar), was first identified in retrovirally induced murine T cell leukemias(2Marth J.D. Peet R. Krebs E.G. Perlmutter R.M. Cell. 1985; 43: 393-404Abstract Full Text PDF PubMed Scopus (357) Google Scholar, 3Voronova A.F. Sefton B.M. Nature. 1986; 319: 682-685Crossref PubMed Scopus (161) Google Scholar, 4Adler T.H. Reynolds P.J. Kelley C.M. Sefton B.M. J. Virol. 1988; 62: 4113-4122Crossref PubMed Google Scholar). Lck contains a unique N-terminal sequence (5Bolen J.B. Rowley R.B. Spana C. Tsygankov A.Y. FASEB J. 1992; 6: 3403-3409Crossref PubMed Scopus (194) Google Scholar) that directs its specific interaction with the cytoplasmic domains of the CD4 and CD8 glycoproteins(6Turner J.M. Brodsky M.H. Irving B.A. Levin S.D. Perlmutter R.M. Littman D.R. Cell. 1990; 60: 755-765Abstract Full Text PDF PubMed Scopus (488) Google Scholar, 7Shaw A.S. Chalupny J. Whitney J.A. Hammond C. Amrein K.E. Kavathas P. Sefton B.M. Rose J.K. Mol. Cell. Biol. 1990; 10: 1853-1862Crossref PubMed Scopus (208) Google Scholar, 8Shaw A.S. Amrein K.E. Hammond C. Stern D.F. Sefton B.M. Rose J.K. Cell. 1989; 59: 627-636Abstract Full Text PDF PubMed Scopus (257) Google Scholar, 9Rudd C.E. Trevillyan J.M. Dasgupta J.D. Wong L.L. Scholssman S.F. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5190-5194Crossref PubMed Scopus (613) Google Scholar, 10Veillette G. Bookman M.A. Horak E.M. Bolen J.B. Cell. 1988; 55: 301-308Abstract Full Text PDF PubMed Scopus (1122) Google Scholar). This interaction is required for antigen-specific responses of several different T cell hybridomas(11Glaichenhaus N. Shastri N. Littman D.R. Turner J.M. Cell. 1991; 64: 511-520Abstract Full Text PDF PubMed Scopus (348) Google Scholar, 12Zamoyska R. Derham P. Gorman S.D. von Hoegen P. Bolen J.B. Veillette A. Parnes J.R. Nature. 1989; 342: 278-281Crossref PubMed Scopus (154) Google Scholar). Lck has also been reported to associate with the IL-2R β-chain via a distinct interaction(13Hatakeyama M.T. Kono T. Kobayashi N. Kawahara A. Levin S.D. Perlmutter R.M. Taniguchi T. Science. 1991; 252: 1523-1528Crossref PubMed Scopus (504) Google Scholar), although its role in IL-2R function remains unclear. Several reports have demonstrated that Lck plays an important role in both T cell maturation and activation. Loss of Lck expression in the human Jurkat T cell line abrogates its response to anti-TcR antibodies(14Straus D.B. Weiss A. Cell. 1992; 70: 585-593Abstract Full Text PDF PubMed Scopus (924) Google Scholar), and inactivation of the lck gene or overexpression of a dominant negative lck transgene in mice leads to an arrest of thymocyte development at a stage prior to the expression of CD4, CD8. and TcR(15Molina T.J. Kishihara K. Siderovski D.P. van Ewijk W. Narendran A. Timms E. Wakeham A. Paige C.J. Hartmann K.U. Veillette A. Davidson D. Mak T.W. Nature. 1992; 357: 161-164Crossref PubMed Scopus (886) Google Scholar, 16Levin S.D. Anderson S.J. Forbush K.A. Perlmutter R.M. EMBO J. 1993; 12: 1671-1680Crossref PubMed Scopus (297) Google Scholar).Whereas the role of Lck in T cell development and function is well established, the role of Fyn is less well defined. Small amounts of Fyn are found associated with the TcR complex following mild detergent extraction of T lymphocytes(17Samelson L.E. Phillips A.F. Luong E.T. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4358-4362Crossref PubMed Scopus (531) Google Scholar). Furthermore, activation of T cells through the TcR results in increased enzymatic activity of Fyn(18Tsygankov A.Y. Broker B.M. Fargnoli J. Ledbetter J.A. Bolen J.B. J. Biol. Chem. 1992; 267: 18259-18262Abstract Full Text PDF PubMed Google Scholar). Additional evidence that Fyn is involved in lymphocyte activation comes from experiments in transgenic animals. Twenty-fold overexpression of FynT in transgenic thymocytes results in enhanced responsiveness to anti-CD3 antibody as measured by the stimulation of tyrosine phosphorylation in whole cells, Ca2+ accumulation, and M.P. Abraham K.M. Forbush K.A. Perlmutter R.M. Cell. 1991; Full Text PDF PubMed Scopus Google Scholar). Furthermore, overexpression of a of FynT in the thymocytes of transgenic mice T cell M.P. Abraham K.M. Forbush K.A. Perlmutter R.M. Cell. 1991; Full Text PDF PubMed Scopus Google Scholar). mice that lack p59fynT or in or J.A. Cooke M.P. Levin S.D. Perlmutter R.M. Cell. 1992; 70: Full Text PDF PubMed Scopus Google Scholar, S. P. Cell. 1992; 70: Full Text PDF PubMed Scopus Google Scholar), that Fyn plays a role in in we that protein-tyrosine kinase inhibits Lck and FynT in at required to ZAP-70, the and kinase A. inhibits whole cell tyrosine phosphorylation and proliferation in T cells with anti-CD3 and selectively inhibits IL-2 gene expression over and IL-2R gene induction in human T this compound to a of TcR not by other of TcR as This compound is a new tool to study the role of Src family protein-tyrosine kinases in lymphocyte function.