Ilangumaran S, Arni S, van Echten-Deckert G, Borisch B, Hoessli D C
Department of Pathology, Centre Médical Universitaire, CH-1211 Geneva 4, Switzerland.
Mol Biol Cell. 1999 Apr;10(4):891-905. doi: 10.1091/mbc.10.4.891.
Src family protein-tyrosine kinases are implicated in signaling via glycosylphosphatidylinositol (GPI)-anchored receptors. Both kinds of molecules reside in opposite leaflets of the same sphingolipid-enriched microdomains in the lymphocyte plasma membrane without making direct contact. Under detergent-free conditions, we isolated a GPI-enriched plasma membrane fraction, also containing transmembrane proteins, selectively associated with sphingolipid microdomains. Nonionic detergents released the transmembrane proteins, yielding core sphingolipid microdomains, limited amounts of which could also be obtained by detergent-free subcellular fractionation. Protein-tyrosine kinase activity in membranes containing both GPI-anchored and transmembrane proteins was much lower than in core sphingolipid microdomains but was strongly reactivated by nonionic detergents. The inhibitory mechanism acting on Lck and Fyn kinases in these membranes was independent of the protein-tyrosine phosphatase CD45 and was characterized as a mixed, noncompetitive one. We propose that in lymphocyte plasma membranes, Lck and Fyn kinases exhibit optimal activity when juxtaposed to the GPI- and sphingolipid-enriched core microdomains but encounter inhibitory conditions in surrounding membrane areas that are rich in glycerophospholipids and contain additional transmembrane proteins.
Src家族蛋白酪氨酸激酶参与通过糖基磷脂酰肌醇（GPI）锚定受体的信号传导。这两种分子位于淋巴细胞质膜中富含鞘脂的同一微区的相对小叶中，不直接接触。在无去污剂的条件下，我们分离出了富含GPI的质膜组分，其中也含有跨膜蛋白，该组分选择性地与鞘脂微区相关联。非离子去污剂释放出跨膜蛋白，产生核心鞘脂微区，通过无去污剂的亚细胞分级分离也可获得少量的核心鞘脂微区。含有GPI锚定蛋白和跨膜蛋白的膜中的蛋白酪氨酸激酶活性远低于核心鞘脂微区中的活性，但可被非离子去污剂强烈激活。这些膜中作用于Lck和Fyn激酶的抑制机制独立于蛋白酪氨酸磷酸酶CD45，其特征为混合型非竞争性抑制机制。我们提出，在淋巴细胞质膜中，Lck和Fyn激酶与富含GPI和鞘脂的核心微区并列时表现出最佳活性，但在富含甘油磷脂且含有额外跨膜蛋白的周围膜区域中会遇到抑制条件。
