Membrane Raft-Dependent Regulation of Phospholipase Cγ-1 Activation in T Lymphocytes

Abstract

Numerous signaling molecules associate with lipid rafts, either constitutively or after engagement of surface receptors. One such molecule, phospholipase Cγ-1 (PLCγ1), translocates from the cytosol to lipid rafts during T-cell receptor (TCR) signaling. To investigate the role played by lipid rafts in the activation of this molecule in T cells, an influenza virus hemagglutinin A (HA)-tagged PLCγ1 was ectopically expressed in Jurkat T cells and targeted to these microdomains by the addition of a dual-acylation signal. Raft-targeted PLCγ1 was constitutively tyrosine phosphorylated and induced constitutive NF-AT-dependent transcription and interleukin-2 secretion in Jurkat cells. Tyrosine phosphorylation of raft-targeted PLCγ1 did not require Zap-70 or the interaction with the adapters Lat and Slp-76, molecules that are necessary for TCR signaling. In contrast, the Src family kinase Lck was required. Coexpression in HEK 293T cells of PLCγ1-HA with Lck or the Tec family kinase Rlk resulted in preferential phosphorylation of raft-targeted PLCγ1 over wild-type PLCγ1. These data show that localization of PLCγ1 in lipid rafts is sufficient for its activation and demonstrate a role for lipid rafts as microdomains that dynamically segregate and integrate PLCγ1 with other signaling components.

ACKNOWLEDGMENTS

We thank E. W. Shores and S. Kozlowski for discussion, comments, and review of the manuscript.

This research was supported in part by the appointment of R.R. and C.N. to the Research Participation Program at the Center for Biologies Evaluation and Research administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the U.S. Food and Drug Administration.