Functional analysis of immunoreceptor tyrosine-based activation motif (ITAM)-mediated signal transduction: the two YxxL segments within a single CD3zeta-ITAM are functionally distinct.
Functional analysis of the immunoreceptor tyrosine-based activation motif (ITAM) derived from the membrane-proximal ITAM of CD3zeta demonstrates that mutations at either the tyrosine or leucine residues in the N-terminal YxxL segment of the ITAM abolish all signal transduction functions of this ITAM. In contrast, mutations at the tyrosine or leucine residues in the C-terminal YxxL segment abrogate signals for interleukin (IL)-2 production but do not prevent tyrosine phosphorylation of the N-terminal tyrosine of the ITAM, lck association with the ITAM, activation of phospholipase C-gamma1 or calcium mobilization. Cross-linking of chimeric receptors containing a C-terminal YxxL leucine mutation induces tyrosine phosphorylation of ZAP70 but without stable binding to the phosphorylated ITAM. These results indicate that the two YxxL segments in an ITAM are functionally distinct and that both are essential for ZAP70 binding and IL-2 production. Furthermore, tyrosine phosphorylation of ZAP70 per se is not sufficient to trigger the downstream events leading to IL-2 production. Substitution of an alanine for the bulky side chain at the Y+1 position of the N-terminal YxxL segment reduces the receptor cross-linking requirement necessary to achieve cellular activation and the absolute dependence on lck in this process. Our results reveal that both the number of ITAM as well as the specific amino acid residues within a single ITAM determine the extent of chimeric receptor cross-linking required to trigger tyrosine phosphorylation-dependent signaling events.
Sunder-Plassmann
R
R
Department of Medicine, Harvard Medical School, Boston, USA. Raute.Sunder-Plassmann@macmailgw.dfci.harvard.edu
Lialios
F
F
Madsen
M
M
Koyasu
S
S
Reinherz
E L
EL
D000595:Amino Acid Sequence; D000818:Animals; D017252:CD3 Complex / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D066298:In Vitro Techniques; D007376:Interleukin-2 / Q000096:biosynthesis / Q000235:genetics; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051379:Mice; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010748:Phosphopeptides / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D017354:Point Mutation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011971:Receptors, Immunologic / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014443:Tyrosine / Q000378:metabolism; D051746:ZAP-70 Protein-Tyrosine Kinase; D019061:src-Family Kinases / Q000378:metabolism
D017252:CD3 Complex; D007376:Interleukin-2; D010748:Phosphopeptides; D011971:Receptors, Immunologic; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human; C497262:Zap70 protein, mouse; D019061:src-Family Kinases