IntroductionThe lymphocyte antigen 6 (Ly‐6) supergene family encodes proteins of 12–14 kda in molecular mass that are either secreted or anchored to the plasma membrane through a glycosyl‐phosphatidylinisotol (GPI) lipid anchor at the carboxy‐terminus. The lipidated GPI‐anchor allows localization of Ly‐6 proteins to the 10–100 nm cholesterol‐rich nano‐domains on the membrane, also known as lipid rafts. Ly‐6A/Sca‐1, a member of Ly‐6 gene family is known to transduce signals despite the absence of transmembrane and cytoplasmic domains. It is hypothesized that the localization of Ly‐6A/Sca‐1 with in lipid rafts allows this protein to transduce signals to the cell interior.Methods and ResultsIn this study, we found that cross‐linking mouse Ly‐6A/Sca‐1 protein with a monoclonal antibody results in functionally distinct responses that occur simultaneously. Ly‐6A/Sca‐1 triggered a cell stimulatory response as gauged by cytokine production with a concurrent inhibitory response as indicated by growth inhibition and apoptosis. While production of interleukin 2 (IL‐2) cytokine by CD4+ T cell line in response to cross‐linking Ly‐6A/Sca‐1 was dependent on the integrity of lipid rafts, the observed cell death occurred independently of it. Growth inhibited CD4+ T cells showed up‐regulated expression of the inhibitory cell cycle protein p27kip but not of p53. In addition, Ly‐6A/Sca‐1 induced translocation of cytochrome C to the cytoplasm along with activated caspase 3 and caspase 9, thereby suggesting an intrinsic apoptotic cell death mechanism.ConclusionsWe conclude that opposing responses with differential dependence on the integrity of lipid rafts are triggered by engaging Ly‐6A/Sca‐1 protein on the membrane of transformed CD4+ T cells.

The Ly-6 family of glycosylphosphatidylinisotol proteins is excellent differentiation markers of immune cells during their development and they regulate signaling responses. Specifically, Ly-6A is up-regulated on a variety of transformed cells therefore making it a potential biomarker for cancer detection and therapy. Previous work in our laboratory revealed that a transformed CD4+ T lymphocyte cell line shows growth inhibition and apoptotic cell death when Ly-6A proteins are engaged. We have investigated whether this mechanism involves p53 protein, which is known to regulate the cell cycle, trigger growth inhibition and apoptotic cell death in several normal cell types. In cancer cells, p53 is often mutated, resulting in uncontrollable cell growth. The cell cycle is regulated by several proteins known as cyclin dependent kinases that bind cyclin proteins and usher the cell past checkpoints in the cell cycle. Therefore, we have examined the expression of relevant cell cycle and apoptotic proteins. We find that while growth inhibition and cell death in the T cell line was p53 independent, the cell cycle inhibitor gene p27 becomes significantly up-regulated. Furthermore, we extended this study beyond the analysis of one Ly-6A antibody and one T-cell line. We find that the responses were tied primarily to the extent of cross-linking with anti-Ly-6A antibody. Other transformed T-cell lines also exhibited growth inhibition and cellular death when the Ly-6A protein was engaged.