Linker for Activation of T-cell Family Member2 (LAT2) a Lipid Raft Adaptor Protein for AKT Signaling,Is an Early Mediator of Alkylphospholipid Anti-leukemic Activity

Lipid rafts are highly ordered membrane domains rich in cholesterol and sphingolipids that provide a scaffold for signal transduction proteins; altered raft structure has also been implicated in cancer progression. We have shown that 25 μm 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC), an alkylphospholipid, targets high cholesterol domains in model membranes and induces apoptosis in leukemia cells but spares normal hematopoietic and epithelial cells under the same conditions. We performed a quantitative (SILAC) proteomic screening of ODPC targets in a lipid-raft-enriched fraction of leukemic cells to identify early events prior to the initiation of apoptosis. Six proteins, three with demonstrated palmitoylation sites, were reduced in abundance. One, the linker for activation of T-cell family member 2 (LAT2), is an adaptor protein associated with lipid rafts in its palmitoylated form and is specifically expressed in B lymphocytes and myeloid cells. Interestingly, LAT2 is not expressed in K562, a cell line more resistant to ODPC-induced apoptosis. There was an early loss of LAT2 in the lipid-raft-enriched fraction of NB4 cells within 3 h following treatment with 25 μm ODPC. Subsequent degradation of LAT2 by proteasomes was observed. Twenty-five μm ODPC inhibited AKT activation via myeloid growth factors, and LAT2 knockdown in NB4 cells by shRNA reproduced this effect. LAT2 knockdown in NB4 cells also decreased cell proliferation and increased cell sensitivity to ODPC (7.5×), perifosine (3×), and arsenic trioxide (8.5×). Taken together, these data indicate that LAT2 is an early mediator of the anti-leukemic activity of alkylphospholipids and arsenic trioxide. Thus, LAT2 may be used as a target for the design of drugs for cancer therapy.The development of resistance to drugs that inhibit signaling pathways in cancer cells has emerged as a major limitation of targeted therapy. While the major mechanism of acquired resistance is the emergence of additional mutations or growth factor receptor overexpression (1), recent studies have shown an interesting mechanism of constitutional resistance to epidermal growth factor receptor inhibitors in breast cancer cells, which involves structural alterations in lipid rafts and is independent of the kinase itself (2).Lipid rafts or membrane rafts are highly ordered membrane domains that are rich in cholesterol and sphingolipids which function by compartmentalizing diverse cellular processes (3, 4), including signal transduction (5–7). Emerging evidence associates altered raft structure with cancer progression (8–10). Therefore, the development of therapeutic strategies for disrupting raft-based cell signaling in cancer represents a potentially useful approach. We and others have presented evidence that alkylphospholipid (APL)¹ drugs target raft structure in leukemia (11) and lymphoma cells (12). One such APL, perifosine, is currently in clinical trials as an anti-cancer therapeutic agent (13).We demonstrated that 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC) targets high cholesterol raft-like domains in model membranes and induces apoptosis in leukemia cells, with an effective dose of 25 μm after 24 h in NB4 cells, but has no effect on normal hematopoietic and epithelial cells under the same conditions (11).Here we present evidence based on quantitative proteomics (14) that the APL ODPC targets proteins recovered in a lipid raft-enriched fraction of leukemic cells. Proteins with predicted palmitoylation sites located in lipid rafts are reduced in abundance after treatment with ODPC. We provide evidence that an adaptor protein for cell signaling, linker for activation of T-cells-2 (LAT2)/non-T-cell activation linker (NTAL)/linker for activation of B-cells (LAB) (15), is involved in early events of ODPC anti-leukemic activity. Additionally, we show that LAT2 knockdown cells obtained with shRNA have suppressed AKT activation, decreased cell proliferation, and increased cell sensitivity to drugs such as ODPC, perifosine, and arsenic trioxide (ATO), indicating that LAT2 is a potential target for the design of drugs for cancer therapy.