Activation of the Ras-related GTPase Rap1 by thymocyte TCR engagement and during selection

Eur J Immunol. 2000 Oct;30(10):2832-41. doi: 10.1002/1521-4141(200010)30:10<2832::AID-IMMU2832>3.0.CO;2-D.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CD28 Antigens / physiology
  • CD3 Complex / immunology
  • Calcium Signaling
  • Clonal Deletion
  • Enzyme Activation
  • Humans
  • Jurkat Cells
  • Lymphocyte Activation*
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Proto-Oncogene Proteins / metabolism
  • Proto-Oncogene Proteins c-cbl
  • Proto-Oncogene Proteins c-crk
  • Receptors, Antigen, T-Cell / immunology*
  • Signal Transduction
  • Specific Pathogen-Free Organisms
  • T-Lymphocytes / enzymology*
  • T-Lymphocytes / immunology
  • Ubiquitin-Protein Ligases*
  • rap1 GTP-Binding Proteins / metabolism*
  • ras Proteins / metabolism

Substances

  • CD28 Antigens
  • CD3 Complex
  • Proto-Oncogene Proteins
  • Proto-Oncogene Proteins c-crk
  • Receptors, Antigen, T-Cell
  • Proto-Oncogene Proteins c-cbl
  • Ubiquitin-Protein Ligases
  • rap1 GTP-Binding Proteins
  • ras Proteins
  • CBL protein, human
  • Cbl protein, mouse