LFA-1 to LFA-1 signals involve zeta-associated protein-70 (ZAP-70) tyrosine kinase: relevance for invasion and migration of a T cell hybridoma.
We previously showed that LFA-1-dependent in vitro invasion and in vivo migration of a T cell hybridoma was blocked in cells overexpressing a truncated dominant-negative zeta-associated protein (ZAP)-70. The truncated ZAP-70 also blocked LFA-1-dependent chemotaxis through ICAM-1-coated filters induced by 1 ng/ml stromal cell-derived factor-1, but not LFA-1-independent chemotaxis induced by 100 ng/ml stromal cell-derived factor-1. This suggested that LFA-1 engagement triggers a signal that amplifies a weak chemokine signal and that dominant-negative ZAP-70 blocks this LFA-1 signal. Here we show that cross-linking of part of the LFA-1 molecules with Abs causes activation of free LFA-1 molecules (not occupied by the Ab) on the same cell, which then bind to ICAM-2 on other cells. This causes cell aggregation that was also blocked by dominant-negative ZAP-70. Thus, an LFA-1 signal involving ZAP-70 activates other LFA-1 molecules, suggesting that the chemokine signal can be amplified by multiple cycles of LFA-1 activation. The chemokine and the LFA-1 signal were both blocked by a phospholipase C inhibitor and a calpain inhibitor, suggesting that one of the amplified signals is the phospholipase C-dependent activation of calpain. Finally, we show that both Src-homology 2 domains are required for inhibition of invasion, chemotaxis, and aggregation by the truncated ZAP-70, suggesting that ZAP-70 interacts with a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) sequence. Remarkably, this is not an ITAM in the TCR/CD3 complex because this is not expressed by this T cell hybridoma.
Soede
R D
RD
Division of Cell Biology, The Netherlands Cancer Institute, Amsterdam.
Driessens
M H
MH
Ruuls-Van Stalle
L
L
Van Hulten
P E
PE
Brink
A
A
Roos
E
E
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism / Q000494:pharmacology; D015703:Antigens, CD / Q000502:physiology; D001666:Binding Sites, Antibody; D002154:Calpain / Q000037:antagonists & inhibitors / Q000502:physiology; D002448:Cell Adhesion / Q000276:immunology; D015815:Cell Adhesion Molecules / Q000502:physiology; D002449:Cell Aggregation / Q000187:drug effects / Q000276:immunology; D002465:Cell Movement / Q000187:drug effects / Q000276:immunology*; D004306:Dose-Response Relationship, Immunologic; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006825:Hybridomas / Q000187:drug effects / Q000201:enzymology / Q000276:immunology* / Q000378:metabolism; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000502:physiology; D016169:Lymphocyte Function-Associated Antigen-1 / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D051966:Phospholipase C gamma; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000502:physiology*; D051381:Rats; D015398:Signal Transduction / Q000276:immunology*; D013267:Stilbenes / Q000494:pharmacology; D013601:T-Lymphocytes / Q000187:drug effects / Q000201:enzymology* / Q000276:immunology* / Q000378:metabolism; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000502:physiology; D051746:ZAP-70 Protein-Tyrosine Kinase
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D015815:Cell Adhesion Molecules; D004791:Enzyme Inhibitors; C065289:ICAM2 protein, human; D007140:Immunoglobulin Fab Fragments; D007527:Isoenzymes; D016169:Lymphocyte Function-Associated Antigen-1; D010446:Peptide Fragments; D013267:Stilbenes; C041525:3,3',4,5'-tetrahydroxystilbene; D011505:Protein-Tyrosine Kinases; D051746:ZAP-70 Protein-Tyrosine Kinase; C497262:Zap70 protein, mouse; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D002154:Calpain