T Cell Antigen Receptor Ubiquitination Is a Consequence of Receptor-mediated Tyrosine Kinase Activation

Engagement of the T cell antigen receptor results in both its phosphorylation and its ubiquitination. T cell antigen receptor ubiquitination was evaluated in Jurkat, a well characterized human T leukemia cell line. Treatment of cells with the tyrosine kinase inhibitor herbimycin A resulted in an inhibition of receptor ubiquitination. Consistent with this, pervanadate, which increases cellular tyrosine phosphorylation, enhanced receptor ubiquitination. A requirement for receptor-mediated tyrosine kinase activity for ubiquitination was confirmed in cells lacking the tyrosine kinase p56 and also in cells that are defective in expression of CD45, a tyrosine phosphatase that regulates the activity of p56. The need for tyrosine kinase activation for ubiquitination was not bypassed by directly activating protein kinase C and stimulating endocytosis of receptors. These observations establish ubiquitination of the T cell antigen receptor as a tyrosine kinase-dependent manifestation of transmembrane signaling and suggest a role for tyrosine phosphorylation in the ligand-dependent ubiquitination of mammalian transmembrane receptors. Engagement of the T cell antigen receptor results in both its phosphorylation and its ubiquitination. T cell antigen receptor ubiquitination was evaluated in Jurkat, a well characterized human T leukemia cell line. Treatment of cells with the tyrosine kinase inhibitor herbimycin A resulted in an inhibition of receptor ubiquitination. Consistent with this, pervanadate, which increases cellular tyrosine phosphorylation, enhanced receptor ubiquitination. A requirement for receptor-mediated tyrosine kinase activity for ubiquitination was confirmed in cells lacking the tyrosine kinase p56 and also in cells that are defective in expression of CD45, a tyrosine phosphatase that regulates the activity of p56. The need for tyrosine kinase activation for ubiquitination was not bypassed by directly activating protein kinase C and stimulating endocytosis of receptors. These observations establish ubiquitination of the T cell antigen receptor as a tyrosine kinase-dependent manifestation of transmembrane signaling and suggest a role for tyrosine phosphorylation in the ligand-dependent ubiquitination of mammalian transmembrane receptors. INTRODUCTIONFor many transmembrane receptors, including the multisubunit TCR, 1The abbreviations used are: TCRT cell antigen receptorPMAphorbol myristate acetatePAGEpolyacrylamide gel electrophoresis.signaling is initiated by ligand-induced aggregation(1.Metzger H. J. Immunol. 1992; 149: 1477-1487PubMed Google Scholar, 2.Abastado J.-P. Lone Y.-C. Casrouge A. Boulet G. Kourilsky P. J. Exp. Med. 1995; 182: 439-447Crossref PubMed Scopus (99) Google Scholar). The earliest obligate intracellular event following TCR aggregation is the activation of the src-family protein tyrosine kinases, Lck (p56) and Fyn (p59). Lck and/or Fyn phosphorylate TCR subunits resulting in the association of a third tyrosine kinase, ZAP-70 (70-kDa -associated protein), with the TCR and to subsequent activation events(3.Weiss A. Cell. 1993; 73: 209-212Abstract Full Text PDF PubMed Scopus (472) Google Scholar, 4.Weissman A.M. Chem. Immunol. 1994; 59: 1-18Crossref PubMed Scopus (19) Google Scholar, 5.Peri K.G. Veillette A. Chem. Immunol. 1994; 59: 19-39Crossref PubMed Scopus (15) Google Scholar). CD45, a tyrosine phosphatase that dephosphorylates key regulatory residues on Lck and Fyn, is also implicated in the initiation of TCR-mediated signaling(6.Trowbridge I.S. Thomas M.L. Annu. Rev. Immunol. 1994; 12: 85-116Crossref PubMed Scopus (654) Google Scholar).TCRs consist of six different polypeptides, these include the antigen-recognition element, in most cells an α-β heterodimer, and a set of invariant signal transducing subunits. The invariant subunits include CD3-, -, and - and the structurally distinct TCR- subunit, which exists within the TCR as a disulfide-linked homodimer (4.Weissman A.M. Chem. Immunol. 1994; 59: 1-18Crossref PubMed Scopus (19) Google Scholar). The minimal signal transducing element of the TCR is the immunoreceptor tyrosine-based activation motif (ITAM)(7.Cambier J.C. Immunol. Today. 1995; 16: 110Abstract Full Text PDF PubMed Scopus (256) Google Scholar). monomers have three ITAMs, and each CD3 subunit has one. ITAMs include two tyrosine residues 10 or 11 amino acids apart that are potential phosphorylation sites. The subunit is a particularly prominent substrate for tyrosine phosphorylation; up to 5% of subunits are phosphorylated on multiple tyrosines upon TCR engagement(8.Baniyash M. Garcia-Morales P. Luong E. Samelson L.E. Klausner R.D. J. Biol. Chem. 1988; 263: 18225-18230Abstract Full Text PDF PubMed Google Scholar, 9.Koyasu S. McConkey D.J. Clayton L.K. Abraham S. Yandava B. Katagiri T. Moingeon P. Yamamoto T. Reinherz E.L. J. Biol. Chem. 1992; 267: 3375-3381Abstract Full Text PDF PubMed Google Scholar, 10.Frank S.J. Cenciarelli C. Niklinska B.B. Letourneur F. Ashwell J.D. Weissman A.M. J. Biol. Chem. 1992; 267: 13656-13660Abstract Full Text PDF PubMed Google Scholar, 11.Cenciarelli C. Hou D. Hsu K.-C. Rellahan B.L. Wiest D.L. Smith H.T. Fried V.A. Weissman A.M. Science. 1992; 257: 795-797Crossref PubMed Scopus (183) Google Scholar).In addition to being a substrate for tyrosine phosphorylation when cross-linked by antibody or mitogen(11.Cenciarelli C. Hou D. Hsu K.-C. Rellahan B.L. Wiest D.L. Smith H.T. Fried V.A. Weissman A.M. Science. 1992; 257: 795-797Crossref PubMed Scopus (183) Google Scholar), TCRs also are ubiquitinated. The covalent modification of proteins with chains of ubiquitin, a highly conserved 76-amino acid polypeptide, plays a central role in the targeting of abnormal proteins and a number of regulatory cytosolic and nuclear proteins for degradation in the 26 S proteasome(12.Finley D. Chau V. Annu. Rev. Cell Biol. 1991; 7: 25-69Crossref PubMed Scopus (421) Google Scholar, 13.Rechsteiner M. Cell. 1991; 66: 615-618Abstract Full Text PDF PubMed Scopus (205) Google Scholar, 14.Varshavsky A. Cell. 1992; 69: 725-735Abstract Full Text PDF PubMed Scopus (389) Google Scholar, 15.Jentsch S. Annu. Rev. Genet. 1992; 1992: 179-207Crossref Scopus (450) Google Scholar, 16.Ciechanover A. Cell. 1994; 79: 13-21Abstract Full Text PDF PubMed Scopus (1587) Google Scholar). Ubiquitination occurs via a multienzyme process involving families of enzymes termed E1-E3. E1 (ubiquitin-activating enzyme) is involved in the ATP-dependent charging of ubiquitin. The high energy thiol-ester bond between E1 and ubiquitin is transferred to an E2 (ubiquitin-conjugating enzyme). E2s either by themselves or in conjunction with E3s (ubiquitin protein ligases), transfer ubiquitin monomers or multiubiquitin chains to target proteins, where isopeptide linkages are formed with lysine residues.The signals that lead to ubiquitination of most naturally occurring substrates are unknown. The N-end rule established a relationship between the N-terminal amino acid of certain proteins and susceptibility to ubiquitination(14.Varshavsky A. Cell. 1992; 69: 725-735Abstract Full Text PDF PubMed Scopus (389) Google Scholar). For the cyclins and c-Jun, specific internal polypeptide sequences have been implicated in targeting for ubiquitination(17.Glotzer M. Murray A.W. Kirschner M.W. Nature. 1991; 349: 132-138Crossref PubMed Scopus (1886) Google Scholar, 18.Treier M. Staszewski L.M. Bohmann D. Cell. 1994; 78: 787-798Abstract Full Text PDF PubMed Scopus (846) Google Scholar), and for cyclins as well as IκBα, serine phosphorylation also plays a role in this process (19.Deshaies R.J. Chau V. Kirschner M. EMBO J. 1995; 14: 303-312Crossref PubMed Scopus (188) Google Scholar, 20.Lahav-Baratz S. Sudakin V. Ruderman J. Hershko A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9303-9307Crossref PubMed Scopus (156) Google Scholar, 21.Alkalay I. Yaron A. Hatzubai A. Orian A. Ciechanover A. Ben-Neriah Y. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10599-10603Crossref PubMed Scopus (388) Google Scholar, 22.Chen Z. Hagler J. Palombella V.J. Melandri F. Scherer D. Ballard D. Maniatis T. Genes & Dev. 1995; 9: 1586-1597Crossref PubMed Scopus (1160) Google Scholar).The TCR is distinguished from most ubiquitination substrates by its long half-life and by being ubiquitinated in response to a specific external stimulus. TCR ubiquitination occurs on multiple subunits and on multiple intracellular lysines, with mono- and multiubiquitinated species detectable(23.Hou D. Cenciarelli C. Jensen J.P. Nguyen H.B. Weissman A.M. J. Biol. Chem. 1994; 269: 14244-14247Abstract Full Text PDF PubMed Google Scholar). As with tyrosine phosphorylation, the subunit is the most prominent substrate for this modification, likely due to the nine intracellular lysines in each monomer, compared with two or three for each of the other invariant subunits. Fundamental to understanding TCR ubiquitination is a determination of the events that couple receptor engagement to this modification. Using Jurkat (24.Weiss A. Stobo J.D. J. Exp. Med. 1984; 160: 1284-1299Crossref PubMed Scopus (381) Google Scholar), a well characterized human T leukemia cell line, we address the relationship between receptor occupancy and ubiquitination. Our findings establish a relationship between receptor ubiquitination and early signaling events mediated by TCR engagement and the activation of protein tyrosine kinases.MATERIALS AND METHODSCell Lines and AntibodiesCell lines were maintained in complete medium containing RPMI 1640 (Biofluids) and 8% fetal calf serum(25.Rellahan B.L. Jensen J.P. Weissman A.M. J. Exp. Med. 1994; 180: 1529-1534Crossref PubMed Scopus (30) Google Scholar). The cell line JCaM1.6 (26.Straus D.B. Weiss A. Cell. 1992; 70: 585-593Abstract Full Text PDF PubMed Scopus (924) Google Scholar) was obtained from the American Tissue Culture Collection (Rockville, MD) and was subcloned by limiting dilution. J45.01 and J45.LB3.3 were provided by Gary Koretzky and Sue of J. Thomas M.L. Weiss A. Nature. PubMed Scopus Google Scholar, G. T. Weiss A. J. Immunol. 1992; 149: Google Scholar). expression human Lck and the for was provided by and Ashwell of of was as for Jurkat B.L. Jensen J.P. Weissman A.M. J. Exp. Med. 1994; 180: 1529-1534Crossref PubMed Scopus (30) Google Scholar). of with and were for to in is a antibody the subunit of the CD3 of the J.C. Reinherz E.L. J. Exp. Med. PubMed Scopus Google Scholar). antibody was a from of a to amino acids of human was as S.J. Weissman A.M. Klausner R.D. J. Biol. Chem. Full Text PDF PubMed Google Scholar). L.E. Luong Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) was provided by Samelson of The antibody has been L.E. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). has been D. Cenciarelli C. Jensen J.P. Nguyen H.B. Weissman A.M. J. Biol. Chem. 1994; 269: 14244-14247Abstract Full Text PDF PubMed Google A was in was formed by a of and and to cells was in where were in of complete medium with of for 10 or cells were with with phosphatase S.J. Cenciarelli C. Niklinska B.B. Letourneur F. Ashwell J.D. Weissman A.M. J. Biol. Chem. 1992; 267: 13656-13660Abstract Full Text PDF PubMed Google Scholar). Cell were in that also and and phosphatase as S.J. Cenciarelli C. Niklinska B.B. Letourneur F. Ashwell J.D. Weissman A.M. J. Biol. Chem. 1992; 267: 13656-13660Abstract Full Text PDF PubMed Google Scholar). for to were with to protein for were with of that was to the that was used and the was were to in containing to on or cell were transferred to either & or to For transfer to were in in containing for and an in to with antibody by with protein A and as D. Cenciarelli C. Jensen J.P. Nguyen H.B. Weissman A.M. J. Biol. Chem. 1994; 269: 14244-14247Abstract Full Text PDF PubMed Google Scholar). with and was as S.J. Cenciarelli C. Niklinska B.B. Letourneur F. Ashwell J.D. Weissman A.M. J. Biol. Chem. 1992; 267: 13656-13660Abstract Full Text PDF PubMed Google Scholar). Cell of receptor expression was as S.J. Cenciarelli C. Niklinska B.B. Letourneur F. Ashwell J.D. Weissman A.M. J. Biol. Chem. 1992; 267: 13656-13660Abstract Full Text PDF PubMed Google Ubiquitination with events to TCR aggregation are for Jurkat cells were for 10 in the or the of the TCR two antibody tyrosine phosphorylation of T. Samelson L.E. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). were with with a was used human from other TCR in A.M. Samelson L.E. Klausner R.D. Nature. PubMed Scopus Google Scholar, B. A. C. J. Med. 1988; PubMed Scopus Google Scholar). with the in the of phosphorylated two evaluated by with a number of ubiquitinated TCR species were The most prominent of these and of which and The species is on which is of is as a this species is not well due to by addition to other TCR are also ubiquitinated in response to receptor C. Hou D. Hsu K.-C. Rellahan B.L. Wiest D.L. Smith H.T. Fried V.A. Weissman A.M. Science. 1992; 257: 795-797Crossref PubMed Scopus (183) Google Scholar) and to the in tyrosine kinase activation is for Jurkat cells were with herbimycin A. tyrosine kinase inhibitor the degradation of tyrosine as Lck and M. Samelson L.E. Weiss A. Immunol. 1992; PubMed Scopus Google Scholar). A not or receptor resulted in a in phosphorylation receptor ubiquitination was was by this of cells with herbimycin which not phosphorylation, on TCR herbimycin A results suggest a relationship between TCR ubiquitination and TCR-mediated tyrosine kinase this the of on TCR ubiquitination were increases cellular tyrosine phosphorylation by protein tyrosine and has been to TCR-mediated C. Proc. Natl. Acad. Sci. U. S. A. 1992; PubMed Scopus Google Scholar, J.P. Koretzky Abraham J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar, J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, V. D. B. P. B. J. 1994; PubMed Scopus Google Scholar). Jurkat cells were with pervanadate, a in cellular tyrosine phosphorylation was in cell of TCR engagement with TCRs were from the subunit of the TCR was also to The of with was that with and and were with to evaluated for ubiquitination by resulted in the of ubiquitinated the of ubiquitination with was that with and not As with tyrosine phosphorylation, and on ubiquitination. multiple with and resulted in increases in ubiquitination from to to conjunction with the herbimycin A these findings suggest that tyrosine kinase activation plays a role in TCR ubiquitination and that receptor engagement the of Lck for TCR address tyrosine involved in TCR-mediated signaling are for we evaluated the Jurkat cell line is defective in TCR-mediated signaling due to of D.B. Weiss A. Cell. 1992; 70: 585-593Abstract Full Text PDF PubMed Scopus (924) Google Scholar). JCaM1.6 was subcloned to TCR Jurkat and evaluated for both TCR phosphorylation and ubiquitination compared with Jurkat, phosphorylation was as was receptor ubiquitination establish of Lck is for the of ubiquitination in Lck expression was by of with an expression The of Lck expression in two and was by with of Lck resulted in of phosphorylation of Lck TCR ubiquitination a requirement for Lck in this of ubiquitination on of Jurkat and cells were in the or the of as in the to and as for was with either or as of of Lck expression in and of The of Lck and a to an of this in JCaM1.6 (26.Straus D.B. Weiss A. Cell. 1992; 70: 585-593Abstract Full Text PDF PubMed Scopus (924) Google Scholar) are by from cells were in each TCRs from Jurkat and of with Lck were evaluated as in Lck TCR Engagement and C for kinase activation plays an role in a number of events to TCR of TCR-mediated tyrosine kinase activation that directly the receptor is the activation of protein kinase C. of this kinase the phosphorylation of CD3 subunits on serine residues and results in enhanced receptor Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, Y. Samelson L.E. Klausner R.D. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Exp. Med. PubMed Scopus Google Scholar). Treatment of T cells with the protein kinase TCR-mediated tyrosine kinase activation and protein kinase C. results in the serine phosphorylation of CD3 subunits and an intracellular of receptors, in the of TCR Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, Y. Samelson L.E. Klausner R.D. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Exp. Med. PubMed Scopus Google receptor endocytosis is in when compared with Jurkat a and with and with TCR-mediated signaling and results in an intracellular of with that with Jurkat and and established that results in receptor in the of of and on TCR ubiquitination were evaluated in this cell line. resulted in receptor ubiquitination in serine phosphorylation of TCR subunits and receptor is not to in ubiquitination of and which Jurkat cells in a A. J. D. Stobo J. Proc. Natl. Acad. Sci. U. S. A. 1984; PubMed Scopus Google Scholar), also to ubiquitination in when were cross-linked with Consistent with the requirement for tyrosine kinase activation for of Jurkat with and/or in the of TCR engagement resulted in TCR ubiquitination tyrosine phosphorylation kinase C activation to ubiquitination. cell expression of TCRs in Jurkat and cells in response to of and were with the for and to were with or a antibody in two by with and by cells were with the to by and with Ubiquitination the addition to tyrosine kinases, TCR-mediated signaling also the tyrosine phosphatase transmembrane phosphatase is to in the initiation of signaling by key regulatory residues on Lck and Fyn I.S. Thomas M.L. Annu. Rev. Immunol. 1994; 12: 85-116Crossref PubMed Scopus (654) Google Scholar). cells are defective in signaling and not the in TCR phosphorylation in response to receptor J. Thomas M.L. Weiss A. Nature. PubMed Scopus Google Scholar, Thomas M.L. Cell. Full Text PDF PubMed Scopus Google Scholar, S. Niklinska B.B. H. Ashwell J.D. J. Exp. Med. 1992; PubMed Scopus Google Scholar). a Jurkat ligand-induced TCR ubiquitination was of TCR and cellular As with the Lck and not in ubiquitination As when these cells were with G. T. Weiss A. J. Immunol. 1992; 149: Google Scholar), TCR ubiquitination was that TCR ubiquitination is not on tyrosine also on the of a tyrosine phosphatase that regulates kinase of ubiquitination on TCRs from Jurkat cells or a were with as for of Jurkat and J45.01 to a of J45.01 was with either or that TCR ubiquitination to tyrosine kinase activation and is not the of the of TCR by requirement is not bypassed by stimulating serine phosphorylation and of receptors. which results in an in tyrosine phosphorylation, is to in of TCR in the of receptor an in tyrosine phosphorylation, of receptor to to in ubiquitination. the that the of ubiquitination with is that with receptor occupancy that specific signals in response to TCR engagement in stimulating a of TCR ubiquitination. These signals a manifestation of TCR or a different or of phosphorylation when tyrosine are by TCR receptor and cell are tyrosine of the of that are ubiquitinated in response to S. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar, A. C. 1994; PubMed Google Scholar). the of of with ligand-dependent S. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar), and for the tyrosine kinase inhibitor results in ligand-dependent A. C. 1994; PubMed Google Scholar). other mammalian transmembrane that either signal by to tyrosine kinase activation J.P. EMBO J. 1993; 12: PubMed Scopus Google Scholar) or that tyrosine kinase activity S. Y. Y. 1995; PubMed Scopus Google Scholar) are ubiquitinated in an with these observations suggest that tyrosine phosphorylation likely plays an role in ligand-dependent ubiquitination of a number of mammalian transmembrane by which receptor ubiquitination in response to tyrosine kinase activation is by the phosphorylation of enzymes with in and/or in the in tyrosine phosphorylation of an E2 was to with enhanced J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). ubiquitinated by enzymes that that as is with enzymes this have been of ubiquitination in the of transmembrane to has been that transmembrane are in to to receptor need to in the of in in a in the 26 S as a the degradation of the signaling of receptors. results obtained with the receptor are S. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar), for transmembrane receptor has a relationship between ligand-induced ubiquitination and degradation been The of herbimycin ubiquitination and degradation of the not the of ligand-dependent that transmembrane in by Z. A. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google of the of ubiquitinated receptors, the of ubiquitin of the of to on intracellular with signaling and between receptors. For the TCR, Fyn, and are the proteins that by ubiquitination. or not ubiquitination is a in ligand-dependent receptor is likely that this modification a of the of transmembrane receptors. INTRODUCTIONFor many transmembrane receptors, including the multisubunit TCR, 1The abbreviations used are: TCRT cell antigen receptorPMAphorbol myristate acetatePAGEpolyacrylamide gel electrophoresis.signaling is initiated by ligand-induced aggregation(1.Metzger H. J. Immunol. 1992; 149: 1477-1487PubMed Google Scholar, 2.Abastado J.-P. Lone Y.-C. Casrouge A. Boulet G. Kourilsky P. J. Exp. Med. 1995; 182: 439-447Crossref PubMed Scopus (99) Google Scholar). The earliest obligate intracellular event following TCR aggregation is the activation of the src-family protein tyrosine kinases, Lck (p56) and Fyn (p59). Lck and/or Fyn phosphorylate TCR subunits resulting in the association of a third tyrosine kinase, ZAP-70 (70-kDa -associated protein), with the TCR and to subsequent activation events(3.Weiss A. Cell. 1993; 73: 209-212Abstract Full Text PDF PubMed Scopus (472) Google Scholar, 4.Weissman A.M. Chem. Immunol. 1994; 59: 1-18Crossref PubMed Scopus (19) Google Scholar, 5.Peri K.G. Veillette A. Chem. Immunol. 1994; 59: 19-39Crossref PubMed Scopus (15) Google Scholar). CD45, a tyrosine phosphatase that dephosphorylates key regulatory residues on Lck and Fyn, is also implicated in the initiation of TCR-mediated signaling(6.Trowbridge I.S. Thomas M.L. Annu. Rev. Immunol. 1994; 12: 85-116Crossref PubMed Scopus (654) Google Scholar).TCRs consist of six different polypeptides, these include the antigen-recognition element, in most cells an α-β heterodimer, and a set of invariant signal transducing subunits. The invariant subunits include CD3-, -, and - and the structurally distinct TCR- subunit, which exists within the TCR as a disulfide-linked homodimer (4.Weissman A.M. Chem. Immunol. 1994; 59: 1-18Crossref PubMed Scopus (19) Google Scholar). The minimal signal transducing element of the TCR is the immunoreceptor tyrosine-based activation motif (ITAM)(7.Cambier J.C. Immunol. Today. 1995; 16: 110Abstract Full Text PDF PubMed Scopus (256) Google Scholar). monomers have three ITAMs, and each CD3 subunit has one. ITAMs include two tyrosine residues 10 or 11 amino acids apart that are potential phosphorylation sites. The subunit is a particularly prominent substrate for tyrosine phosphorylation; up to 5% of subunits are phosphorylated on multiple tyrosines upon TCR engagement(8.Baniyash M. Garcia-Morales P. Luong E. Samelson L.E. Klausner R.D. J. Biol. Chem. 1988; 263: 18225-18230Abstract Full Text PDF PubMed Google Scholar, 9.Koyasu S. McConkey D.J. Clayton L.K. Abraham S. Yandava B. Katagiri T. Moingeon P. Yamamoto T. Reinherz E.L. J. Biol. Chem. 1992; 267: 3375-3381Abstract Full Text PDF PubMed Google Scholar, 10.Frank S.J. Cenciarelli C. Niklinska B.B. Letourneur F. Ashwell J.D. Weissman A.M. J. Biol. Chem. 1992; 267: 13656-13660Abstract Full Text PDF PubMed Google Scholar, 11.Cenciarelli C. Hou D. Hsu K.-C. Rellahan B.L. Wiest D.L. Smith H.T. Fried V.A. Weissman A.M. Science. 1992; 257: 795-797Crossref PubMed Scopus (183) Google Scholar).In addition to being a substrate for tyrosine phosphorylation when cross-linked by antibody or mitogen(11.Cenciarelli C. Hou D. Hsu K.-C. Rellahan B.L. Wiest D.L. Smith H.T. Fried V.A. Weissman A.M. Science. 1992; 257: 795-797Crossref PubMed Scopus (183) Google Scholar), TCRs also are ubiquitinated. The covalent modification of proteins with chains of ubiquitin, a highly conserved 76-amino acid polypeptide, plays a central role in the targeting of abnormal proteins and a number of regulatory cytosolic and nuclear proteins for degradation in the 26 S proteasome(12.Finley D. Chau V. Annu. Rev. Cell Biol. 1991; 7: 25-69Crossref PubMed Scopus (421) Google Scholar, 13.Rechsteiner M. Cell. 1991; 66: 615-618Abstract Full Text PDF PubMed Scopus (205) Google Scholar, 14.Varshavsky A. Cell. 1992; 69: 725-735Abstract Full Text PDF PubMed Scopus (389) Google Scholar, 15.Jentsch S. Annu. Rev. Genet. 1992; 1992: 179-207Crossref Scopus (450) Google Scholar, 16.Ciechanover A. Cell. 1994; 79: 13-21Abstract Full Text PDF PubMed Scopus (1587) Google Scholar). Ubiquitination occurs via a multienzyme process involving families of enzymes termed E1-E3. E1 (ubiquitin-activating enzyme) is involved in the ATP-dependent charging of ubiquitin. The high energy thiol-ester bond between E1 and ubiquitin is transferred to an E2 (ubiquitin-conjugating enzyme). E2s either by themselves or in conjunction with E3s (ubiquitin protein ligases), transfer ubiquitin monomers or multiubiquitin chains to target proteins, where isopeptide linkages are formed with lysine residues.The signals that lead to ubiquitination of most naturally occurring substrates are unknown. The N-end rule established a relationship between the N-terminal amino acid of certain proteins and susceptibility to ubiquitination(14.Varshavsky A. Cell. 1992; 69: 725-735Abstract Full Text PDF PubMed Scopus (389) Google Scholar). For the cyclins and c-Jun, specific internal polypeptide sequences have been implicated in targeting for ubiquitination(17.Glotzer M. Murray A.W. Kirschner M.W. Nature. 1991; 349: 132-138Crossref PubMed Scopus (1886) Google Scholar, 18.Treier M. Staszewski L.M. Bohmann D. Cell. 1994; 78: 787-798Abstract Full Text PDF PubMed Scopus (846) Google Scholar), and for cyclins as well as IκBα, serine phosphorylation also plays a role in this process (19.Deshaies R.J. Chau V. Kirschner M. EMBO J. 1995; 14: 303-312Crossref PubMed Scopus (188) Google Scholar, 20.Lahav-Baratz S. Sudakin V. Ruderman J. Hershko A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9303-9307Crossref PubMed Scopus (156) Google Scholar, 21.Alkalay I. Yaron A. Hatzubai A. Orian A. Ciechanover A. Ben-Neriah Y. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10599-10603Crossref PubMed Scopus (388) Google Scholar, 22.Chen Z. Hagler J. Palombella V.J. Melandri F. Scherer D. Ballard D. Maniatis T. Genes & Dev. 1995; 9: 1586-1597Crossref PubMed Scopus (1160) Google Scholar).The TCR is distinguished from most ubiquitination substrates by its long half-life and by being ubiquitinated in response to a specific external stimulus. TCR ubiquitination occurs on multiple subunits and on multiple intracellular lysines, with mono- and multiubiquitinated species detectable(23.Hou D. Cenciarelli C. Jensen J.P. Nguyen H.B. Weissman A.M. J. Biol. Chem. 1994; 269: 14244-14247Abstract Full Text PDF PubMed Google Scholar). As with tyrosine phosphorylation, the subunit is the most prominent substrate for this modification, likely due to the nine intracellular lysines in each monomer, compared with two or three for each of the other invariant subunits. Fundamental to understanding TCR ubiquitination is a determination of the events that couple receptor engagement to this modification. Using Jurkat (24.Weiss A. Stobo J.D. J. Exp. Med. 1984; 160: 1284-1299Crossref PubMed Scopus (381) Google Scholar), a well characterized human T leukemia cell line, we address the relationship between receptor occupancy and ubiquitination. Our findings establish a relationship between receptor ubiquitination and early signaling events mediated by TCR engagement and the activation of protein tyrosine