Activation of the Ras-related GTPase Rap1 by thymocyte TCR engagement and during selection.
Signals mediated by activation of the small GTPase Ras play an essential role both in thymocyte development and in TCR-mediated activation of mature T cells. Given the critical requirement of Ras signaling pathways in thymocyte development, and recent indications that Rap1 may negatively regulate Ras-dependent signaling pathways, we examined the possible involvement of Rap1 in thymocyte TCR signaling. We find that Rap1 and proposed regulators of Rap1 (the proto-oncogene product Cbl, Crk family adaptor proteins, and the Rap1 guanine nucleotide exchange factor C3G) are expressed at equivalent levels in both double-negative and double-positive murine thymocytes. Rap1 was transiently activated following TCR stimulation of both total thymocytes and purified double-positive thymocytes, and this activation correlated with tyrosine phosphorylation of Cbl and Cbl association with CrkL. TCR-dependent Rap1 activation was enhanced by co-stimulation through CD28 and could be mimicked by treatment of thymocytes with phorbol ester and calcium. In contrast to mature peripheral T lymphocytes, Rap1 stimulation by CD3 ligation in thymocytes did not require intracellular calcium mobilization. Intriguingly, we found a clear elevation of activated Rap1 in thymocytes undergoing positive selection, suggesting a functional role for Rap1 in thymocyte development and selection.
Amsen
D
D
Division of Immunology, The Netherlands Cancer Institute, Amsterdam.
Kruisbeek
A
A
Bos
J L
JL
Reedquist
K
K
D000818:Animals; D018106:CD28 Antigens / Q000502:physiology; D017252:CD3 Complex / Q000276:immunology; D020013:Calcium Signaling; D017637:Clonal Deletion; D004789:Enzyme Activation; D006801:Humans; D019169:Jurkat Cells; D008213:Lymphocyte Activation; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D050721:Proto-Oncogene Proteins c-cbl; D050737:Proto-Oncogene Proteins c-crk; D011948:Receptors, Antigen, T-Cell / Q000276:immunology*; D015398:Signal Transduction; D013047:Specific Pathogen-Free Organisms; D013601:T-Lymphocytes / Q000201:enzymology* / Q000276:immunology; D044767:Ubiquitin-Protein Ligases; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism*; D018631:ras Proteins / Q000378:metabolism
D018106:CD28 Antigens; D017252:CD3 Complex; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D011948:Receptors, Antigen, T-Cell; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D020827:rap1 GTP-Binding Proteins; D018631:ras Proteins; C067419:CBL protein, human; C493849:Cbl protein, mouse