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

Signals mediated by activation of the small GTPase Pas play an essential ro le both in thymocyte development and in TCR-mediated activation of mature T cells. Given the critical requirement of Ras signaling pathways in thymocy te development, and recent indications that Rap1 may negatively regulate Ra s-dependent signaling pathways, we examined the possible involvement of Rap 1 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 le vels in both double-negative and double-positive murine thymocytes. Rap1 wa s transiently activated following TCR stimulation of both total thymocytes and purified double-positive thymocytes, and this activation correlated wit h tyrosine phosphorylation of Cbl and Cbl association with CrkL. TCR-depend ent Rap1 activation was enhanced by co-stimulation through CD28 and could b e mimicked by treatment of thymocytes with phorbol ester and calcium. In co ntrast to mature peripheral T lymphocytes, Rap1 stimulation by CD3 ligation in thymocytes did not require intracellular calcium mobilization. Intrigui ngly, we found a clear elevation of activated Rap1 in thymocytes undergoing positive selection, suggesting a functional role for Rap1 in thymocyte dev elopment and selection.