Spatiotemporal Regulation of C-Kit Signaling through Lipid Rafts.

Abstract

Recently, the understanding of membrane receptors has been transformed by studies characterizing the topology of the plasma membrane. The T-cell receptor (TCR) has been most studied for interactions of receptors with the lipid bilayer. TCR signaling has been shown to be dependent on the localization of the TCR complex to specific cholesterol- and sphingolipid-rich membrane subdomains, also called microdomains or lipid rafts. The definition of lipid rafts as assembly platforms to initiate membrane receptor signaling has induced a novel view of the plasma membrane as a compartmentalized structure. To investigate the role of lipid rafts in the signal transduction by the prototype receptor tyrosine kinase (RTK) c-kit, lipid raft, plasma membrane and cytosol fractions were obtained by subcellular fractionation of Mo7e cells widely used to study c-kit signaling. The purity of fractions was verified by the exclusive presence of marker proteins in their respective fraction. Time course experiments using non-stimulated and kit ligand (KL)-stimulated cells harvested after 5 and 20 minutes (′) revealed that non-activated c-kit was mainly localized within the membrane and that KL-induced activation of c-kit resulted in the redistribution of c-kit protein from the membrane fraction into lipid rafts. Activated c-kit was seen exclusively in lipid rafts at 5′ of KL-stimulation and was redistributed to the membrane after 20′.Analysis of downstream targets of c-kit revealed that various src-family kinases previously shown to be crucially involved in c-kit activation were predominantly present within the lipid raft fraction independently of c-kit activation. Investigating the main survival/proliferation pathway activated by c-kit we found that the p85 subunit of PI3-K was recruited to lipid rafts at 5′ of c-kit stimulation and was redistributed to the membrane fraction after 20′. Accordingly, PTEN, the central negative regulator of PI3-K, was present in lipid rafts in non-activated cells and was withdrawn from lipid rafts upon c-kit stimulation. PKB/Akt was not detected within lipid rafts but accumulated within the membrane fraction after 20′ of c-kit activation.Like PKB/Akt, PKC, Plcγ as well as PDK and adaptor molecules like Grb2, Grb4/Nckβ and Grb10 were predominantly localized in the cytosol and accumulated in the membrane fraction at 20′ of c-kit activation.To determine the biological role of lipid rafts in c-kit signaling we analyzed the effect of non-toxic concentrations of methyl-beta-cyclodextrin (MBCD) on c-kit dependent proliferation. MBCD has been shown to disrupt lipid rafts by removal of cholesterol from the plasma membrane. MBCD treatment of Mo7e cells resulted in complete inhibition of KL-mediated growth of Mo7e cells without inhibiting tyrosine phosphorylation of c-kit.We conclude that c-kit signaling is initiated in lipid rafts and that c-kit mediated proliferation is dependent on the integrity of lipid rafts. The predominant presence of src-family kinases in lipid rafts prior to activation of c-kit supports a crucial role for these signaling molecules in the initiation and amplification of c-kit signaling. The recruitment of p85 to lipid rafts and the synchronous withdrawal of PTEN from lipid rafts suggests that lipid rafts are the location of c-kit mediated activation of PI3-K.We propose a significant role for lipid rafts in the spatiotemporal regulation of c-kit signaling and hypothesize, that cell type- and cell state-specific compositional and topological variations of lipid rafts significantly influence the signaling outcome of c-kit and other RTKs.