A Model System for Activation-Induced Alternative Splicing of CD45 Pre-mRNA in T Cells Implicates Protein Kinase C and Ras

Abstract

Multiple isoforms of the protein tyrosine phosphatase CD45 are expressed on the surface of human T cells. Interestingly, the expression of these isoforms has been shown to vary significantly upon T-cell activation. In this report, we describe a novel cell line-based model system in which we can mimic the activation-induced alternative splicing of CD45 observed in primary T cells. Of the many proximal signaling events induced by T-cell stimulation, we show that activation of protein kinase C and activation of Ras are important for the switch toward the exclusion of CD45 variable exons, whereas events related to Ca²⁺ flux are not. In addition, the ability of cycloheximide to block the activation-induced alternative splicing of CD45 suggests a requirement for de novo protein synthesis. We further demonstrate that sequences which have previously been implicated in the tissue-specific regulation of CD45 variable exons are likewise necessary and sufficient for activation-induced splicing. These results provide an initial understanding of the requirements for CD45 alternative splicing upon T-cell activation, and they confirm the importance of this novel cell line in facilitating a more detailed analysis of the activation-induced regulation of CD45 than has been previously possible.

ACKNOWLEDGMENTS

We thank Haruo Saito for the gift of plasmid pSV-MiLCA2 and thank Christine Guthrie and members of the Weiss and Guthrie laboratories for valuable discussions and suggestions. We also thank Ravi Majeti, Xiang-Dong Fu, Brenton Graveley, Amy Kistler, and Jon Staley for critical reading of the manuscript.

This work was supported by NIH grant GM-39553. K.W.L. was supported by a Cancer Research Institute fellowship.