Combinatorial Effect of T-Cell Receptor Ligation and CD45 Isoform Expression on the Signaling Contribution of the Small GTPases Ras and Rap1

Abstract

By using ligands with various affinities for the T-cell receptor (TCR) and by altering the contribution of the CD45 tyrosine phosphatase, the effects of the potency of TCR-induced signals on the function of small GTPases Ras and Rap1 were studied. T cells expressing low-molecular-weight CD45 isoforms (e.g., CD45RO) exhibited the strongest activation of the Ras-dependent Elk-1 transcription factor and the highest sensitivity to the inhibitory action of dominant negative mutant Ras compared to T cells expressing high-molecular-weight CD45 isoforms (ABC). Moreover, stimulation of CD45RO⁺, but not CD45ABC⁺, T cells with a high-affinity TCR ligand induced suboptimal Elk-1 activation compared with the stimulation induced by an intermediate-affinity TCR-ligand interaction. This observation suggested that the Ras-dependent signaling pathway is safeguarded in CD45RO⁺ expressors by a negative regulatory mechanism(s) which prohibits maximal activation of the Ras-dependent signaling events following high-avidity TCR-ligand engagement. Interestingly, the biochemical activity of another small GTPase, the Ras-like protein Rap1, which has been implicated in the functional suppression of Ras signaling, was inversely correlated with the extent of Elk-1 activation induced by different-affinity TCR ligands. Consistently, overexpression of putative Rap dominant negative mutant RapN17 or the physiologic inhibitor of Rap1, the Rap GTPase-activating protein RapGAP, augmented the Elk-1 response in CD45RO⁺ T cells. This is in contrast to the suppressive effect of RapN17 and RapGAP on CD45ABC⁺ T cells, underscoring the possibility that Rap1 can act as either a repressor or a potentiator of Ras effector signals, depending on CD45 isoform expression. These observations suggest that cells expressing distinct isoforms of CD45 employ different signal transduction schemes to optimize Ras-mediated signal transduction in activated T lymphocytes.

ACKNOWLEDGMENTS

We thank C. A. Janeway and G. M. Losyev for MAbs 5A and 3D3, C. J. Der for helpful suggestions and RafN4, L. A. Quilliam for Rap constructs and RapGAP, Teresa Brtva and K. L. Guan for RasN17, M. H. Cobb for dominant negative Erk-1 and Erk-2, J. L. Bos for GST-RBD of RalGDS, and C. Dong and E. Serfling for the IL-2–luciferase reporter construct. We also thank Teresa Brtva for critical reading of the manuscript.