U373-MG cells were transfected with 50?nM control (luciferase, siLuc) and ASM siRNAs (siASM) for 48?h and the cells were harvested in the Brij58-containing lysis buffer and fractionated and analyzed as in B. can regulate the trafficking of SNAP23 from the XL765 Golgi to the plasma membrane. Our studies suggest that ASM, acting at the plasma membrane to produce ceramides, regulates the localization and trafficking of the palmitoylated proteins. gene (also called and identified the worm homolog of ASM, homolog of ASM is a positive regulator of the conserved IGF-1R-like signaling pathway (Kim and Sun, 2007, 2012). We wondered if ASM might regulate the localization of proteins in the ceramide-rich lipid rafts, and whether these proteins might be positively involved in receptor tyrosine kinase signaling under physiological conditions. To identify such proteins, we took a biochemical approach to isolate lipid rafts and analyze the associated proteins by mass-spectrometry. By comparing the lipid raft proteomes identified in cells with ASM or without ASM, we aim to identify the lipid raft-associated proteins that are regulated by ASM. The sphingomyelin-enriched XL765 lipid microdomains are known to be relatively resistant to nonionic detergents, such as Triton X-100, and can be isolated as the detergent-resistant membrane (DRM) fractions, which can be separated from the detergent-soluble fractions using a sucrose gradient and ultracentrifugation (Harder et al., 1998; Schuck et al., 2003). Since Rabbit polyclonal to CDC25C lipid microdomains are heterogeneous with varying lipid composition and protein content, their resistances to various detergents are known to be different (Giurisato et al., 2003; Radeva and Sharom, 2004; Schuck et al., 2003). The detergent XL765 Brij has been shown to preserve the lipid raft localization of transmembrane receptors (e.g. T cell receptor) better than Triton X-100 (Giurisato et al., 2003; Montixi et al., 1998; R?per et al., 2000). Human IGF-1R can also be fractionated in the detergent Brij-resistant membrane (DRM) fractions (Remacle-Bonnet et al., 2005). Since our genetic studies have established that the worm homolog of ASM regulates the IGF-1R-like signaling pathway in (Kim and Sun, 2012), it is likely that human IGF-1R is also regulated by ASM. Indeed, in human glioblastoma U373-MG cells, which are highly sensitive to ASM inhibition (Zhu et al., 2016), we found there is a small fraction of IGF-1R localized in the DRM fractions (fraction #1C4). However, most of the IGF-1R protein was localized in the Brij-soluble fractions (fractions #13C16) (Fig.?1A,B). We also found that the detergent Brij58, rather than Triton X-100, was more efficient in preserving the lipid raft localization of IGF-1R (data not shown). In cells treated with desipramine, the localization of IGF-1R in the DRM fractions was reduced (Fig.?1B). Desipramine is a tricyclic amine anti-depression drug that acts as a functional inhibitor of ASM, and the drug blocks the interaction of ASM with membrane inside the lysosomes and causes ASM degradation (Albouz et al., 1981; Jaffrzou et al., 1995; Jenkins et al., 2011; Zhu et al., 2016). Indeed, the ASM activity was potently suppressed in cells treated with desipramine, confirmed by assaying the ASM activities using 14C-sphingomyelin as a substrate (Fig.?1E). Open in a separate window Fig. 1. Fractionation of the ASM-regulated membrane-associated proteins by discontinuous sucrose gradient ultracentrifugation. (A) A schematic workflow XL765 of the discontinuous sucrose gradient fractionation procedure. (B) The distribution of tyrosine kinases IGF-1R and Yes in the discontinuous sucrose gradient in control (DMSO) and desipramine (Desi, 25?M, 12?h) treated U373-MG cells by anti-IGF-1R and anti-Yes antibody immunoblotting. Flotillin was used as a lipid raft marker. Fractions were collected from the top (fraction #1) to the bottom of the gradient (fraction #16). The distribution of IGF-1R or Yes was reduced in the DRM fractions (#1C4) after ASM inhibition. (C) Loss of ASM reduced the levels of Yes in the detergent resistant membrane fraction..