VoL 33 (Supp4), No. I, 1993 FPMd in U.S. A. ' -* Molecular Dissection of Lymphocyte Signal Transduction Pathways , + . ' .,t,.:.*. .' ROGER M. -. .:, %L$L . ABsTR~.The earliest biochemical changes in lymph+ ble for coupling these meptos to tbe cell interior. We were tyte activation include the accumulation of phosphotyre particularlyintriguadbytheideathatlym~pro- sine-contahieg proteins. This accumuIation,~catalyzedby tein tyrosine kinases might couple these nceptors to other bio- specific protein tyrosine Llnases, is required for subsequent chemical pathways. Our theory was not daived &om detailed coll activation. Expehental manipulation of these protein knowabout the importance of tyrosine -tion. It tyrosine kiuases results in improved or ddtransit simply seemed that in many respects the signal transduction througb the activation sequence. Analysis of transgenic cescade~tcdbylymphocyteeenwlrfaeereceptorsresembled animals bearing altered kinase genes permits assembly of thekindof~transduction~occurriagwithstimu- a model for signal transduction from the T4an- lation of grrwth EPdor mcpton known to possess intrinsic receptor. (P#liorrRu 33 (Suppl): SeSl!5,1993# prorrintymdoe-activity(5). Withtbis~inmind,~stpdiadavariayoflymphoid cell lines, searching for those with elevated tyroshe kinase activ- I , ity. One of the cell lines-a mouse Moloney murine leukemia ng of the TceU antigen receptor by virus-transformed cell line called LSTRA-was particularly in- of biochemical changes results. The formative. earliest obeeMtble events are changes in the accumulation of The LSTRA cell line was found to contain high levels of a phosphoryhtd suLxtmtes, particularly a set of proteins specifi- specialized protein tyrosine kinase of 56 kD apparent molecular cally pbospboryfotat on tyrosine residues. This accumulation of mass. We were able to use molecular biologic techniques to substrates bcgh within seconds after ligand occupancy of the identify the gene that encodes this molecule (6) and learned that Tcell antigen naptor. Thereafter, changes in the turnover of it is a typical nonreceptor protein tyrosine kinase of the type membnne pbpblipids (Fig. 1) and the accumulation of high exemplitied by p60". 5 macentrations of intmcellular calcium oocur transiently. All The src-family of protein tyrosine kinases all share a similar these almatbns precede c4mges in gene expmdon, especially structure. These molecules ~IEmyrktoylated at their amino the activation of a set of genes important for prol8kation. termhi, which presumably assists their interaction with the inner Included among these are genes enadkg the transcription he leaflet of the plasma membrane. Across the entire stnrdure,the tors,genes encoding lymphokine mcptom, and the lymphokine distinct regions can be identitied (Fii 2). Fia unique amjno genes themselves (1). Thevents together permit lymphoqtes terminal domain stnacture, about 70 amino acids in lee to proceed along a course that ultimately win kad to DNA appeac~toc0lltroldirtctin~011~tllnceptorsbY~ replication and proliferation. Then a set of sn: homology motifs appean, including sequ- Two fbdamental problems exist in undmtanding ?his se- that are involved in the binding of ph ' e itself, and, quence &events. Fust, we have no idea of the causal relation- my,there is a carboxymmina, -%the bmims ships among these various biochemical alterations. Is tyrosine end of the molecule responsible for phosphorylating substrates phoaplrorylstion a cause of these subsequent events? Second, we (7). Included in the kinase domain is a critical site of regulation: have no idCB Itow the receptor mdecuks on the surfaces of a tyrosine near the carboxy terminus that is ordinarily phos- lympboid & effect these biochemical changes. This article phorylated in vivo (8). If the phosphate is stripped from this site, focurcs on that problem. the kinase becomes much more active. Study of this activity can Amysd on tbe T lymphocyte surbce is a set of molecules that be useM in investigating the function of these molecules. traasldn dgmb to the cell interior. Tlusc structurally related There are now eight wdkkuibed members of the src family. mokcukspcr13emodedbygenesthataremembersoftheIg It is important to remember that these molecules are usually supertrrmitgr. .lby include the Tcdl antigen receptor a and specialized for expression in hematopoietic cells. For example, chaias(2),tbeiaociatedCD3com~oftheTdl~the blk kinase identified by Steve DePiderio and his colleagues at Johns Hopkins University (9) is qmsdonly in B lymphocytes. Pllirnalinn colnpiex (3), the conoegtors ad (41 and otaetaruriliary~~Allthesemdecules~rbrnIt performs some unique sig~ItraosdFlction function in those ~nonehasanyobviousmcoesof cells. The hckcnded kinase is cxpmsd primarily in granulo- ~-&totbeb.in~*thr)mcy.IIhw cytes (10). The Ick gene, which we oz&bUy doned hmLSIRA cells, is expressed only in lymphoid cells, particularly T lympho- nlrtively-- ~OnStbaS~~~ . -- cytes (6). Thus, the Ick gene is an iderl candidate for a signal tmdudon molecule tbat might link bphoid all surface nceptorstothecdlintaior. But what pmbe function does SsbioteIn tyroshe kim iWm M. -, M.D. PkD.. bppnmt of Immuad- perf~ntl?LcktntnscrigtsaccumulatetoespeciallyhighleveIs~ gy,Uaivanity dWabb@n SEQa dMcdicinc, He.Scamm Bdd& Md thymacytes.ThisJugsesSsthattheIckgenepcrfwmssomefun8 Stop SUH, Sattle, WA 98195. -.ispatbyrLRstfme,tseNM(CMWS682)mdbythcHawud tion critical to T lymphocyte development. Moreover, the 14 H~M~Iartitutc gene is exm@ the eqligt time @a?one mCBll@@ifyT4 II PERLMUTTER None of this theorizing advances our understanding of what the Ick gene actually does; however, it suggests that p56" may PIP2 turnover behave as a signal transduction molecule involved in the control of replication or those early activation events in T lymphocytes / co2+influr Lyrnphokine that give rise to proliferation. Recent studies demonstrate that expression p56'* physically associates with the CD4/CD8 coreceptor mole- cules on the T-cell surface. These observations argue that p56lCk may be brought into the T-cell receptor complex at the time of antigen recognition and may be directly involved in transmitting signals from that antigen receptor complex. Studies by our group and others have defined the mechanism Hours Post-Stimulation of association of CD4 and CD8 with p56Ickand have shown that cysteine residues near the N terminus of ~56'~and the C termi- Fig. 1. Early metabolic events after T-cell activation. Shown is a nus of these coreceptors are crucial for this interaction. Intermp hypothetical time line for activation listing some of the major early tion of any of these four cysteine residues completely destroys biochemical changes that occur after T-cell receptor stimulation. NFAT the association (14). is a transcription factor complex involved in regulating IL2 expression. Thus, the amino terminal domain of p56lCkis involved in ODC is ornithine decarboxylase. PIP2 is phosphatidylinositol-bis-phos- permitting the molecule to associate with the T-cell antigen phate. IL-2 rcp is interleukin-2-receptor. receptor complex by its interaction with the coreceptors, CD4 and CD8. This observation gives rise to a fairly simple hypothesis: Under ordinary circumstances, p56lCkis in some sort of equilib- rium state interacting with this coreceptor. At the time of antigen recognition, p56lCkbecomes involved in the complex and be- comes activated, presumably by dephosphorylation of a carboxy terminal tyrosine residue. Indeed, several groups have demon- strated that the CD45 phosphotyrosine phosphatase can dephos- phorylate tyrosine 505 and hence activate p56Ickin precisely the fashion previously explored using mutant Ick cDNA introduced into fibroblasts (15). To test this model, we hoped to artificially augment or decrease Fig. 2. Structural concept of a typical src-family protein tyrosine the amount of p56lCkactivity present in otherwise normal T kinase. Shown is a schematic representation of p56lCkillustrating the lymphocytes, that is, in transgenic mice. Such experiments have positions of the amino-terminal and "src-homology" (SH3, SH2) do- numerous advantages over conventional transfection studies. mains. See text for details. First, the signaling molecules are expressed in a normal cellular context, obviating the use of cell lines that invariably exhibit abnormal signaling properties. Second, all potentially relevant precursors in the thymus and throughout the set of maturation lymphocyte subsets can be simultaneously examined. It is not stages defining T-cell development. clear that all lymphocyte subsets can be cultured in vitro with The structure of p56Ick closely resembles that of a number of the same efficiency. The advantage of using this in vivo system oncoproteins, suggesting that it may be involved in controlling is that all potentially relevant lymphocyte subsets can be simul- replication. Indeed, it is possible to demonstrate directly that the taneously targeted. Finally, the significance of signaling pathways lck gene is an oncogene that can confer on certain cells the ability in lymphocyte development is readily addressed in transgenic to grow in the absence of appropriate growth factors and appro- mice, and there are no satisfactory in vitro models for lymphocyte priate substrate contacts. For example, we have used transcrip development. tional regulatory sequences from the mouse metallothionein To perform these experiments, it was necessary to characterize promoter to drive the expression of normal and mutated Ick