A Diacylglycerol-Protein Kinase C-RasGRP1 Pathway Directs Ras Activation upon Antigen Receptor Stimulation of T Cells

Abstract

Ras GTPases are on/off switches regulating numerous cellular responses by signaling to various effector molecules. In T lymphocytes, Ras can be activated by two Ras exchange factors, SOS and RasGRP1, which are recruited through the adapters Grb2 and LAT and via the second-messenger diacylglycerol (DAG), respectively. Mitogen-activated protein (MAP) kinase phosphorylation patterns induced by active Ras can vary and contribute to distinct cellular responses. The different consequences of Ras activation by either guanine exchange factor are unknown. DAG also recruits and activates the kinase protein kinase Cθ (PKCθ) turning on the Erk MAP kinase pathway, but the biochemical mechanism responsible is unclear. We generated T-cell clones deficient in phorbol myristate acetate (a surrogate for DAG)-induced Ras activation. Analysis of a RasGRP1-deficient Jurkat T-cell clone and RasGRP1 RNA interference in wild-type cells revealed that RasGRP1 is required for optimal, antigen receptor-triggered Ras-Erk activation. RasGRP1 relies on its DAG-binding domain to selectively activate Erk kinases. Activation of Erk correlates with the phosphorylation of threonine residue 184 in RasGRP1. This phosphorylation event requires the activities of novel PKC kinases. Conversely, active PKCθ depends on RasGRP1 sufficiency to effectively trigger downstream events. Last, DAG-PKC-RasGRP1-driven Ras-Erk activation in T cells is a unique signaling event, not simply compensated for by SOS activity.

ACKNOWLEDGMENTS

We express our gratitude to Warner Greene for the PKCθA148E and -K409R constructs, Stacey Stang for preparation of anti-RasGRP1 antibodies, and Yong Zheng for preparation of the P-T184 antibody. We are grateful to Hanne Ostergaard and Nicholas Gascoigne for communicating unpublished results. We thank the members of the Weiss lab for their suggestions and comments and Rene Bernards and Hans Bos for continuous support.

J.R. is grateful for grants from The Netherlands Organization for Scientific Research (NWO) and the Dutch Cancer Society (KWF). This work was supported in part by a grant from the NCI (CA 72531).