Sphingolipids represent a family of cellular lipid-molecules that regulate physiological and pathophysiological processes. Glucosylceramide (GlcCer), the simplest glycosphingolipid (GSL), is synthesized from ceramide and UDP-glucose by GlcCer synthase (GCS). Both GlcCer (and resulting GSLs) and ceramide regulate various cellular functions including cell death and multiple drug resistance. Src family tyrosine kinases are up-regulated in various human cancer cells. We examined the effect of v-Src expression on GCS activity, the formation of 4-nitrobenzo-2-oxa-1,3-diazole (NBD)-labeled GlcCer from NBD-ceramide, and the effect of tyrosine¹³² mutation in GCS on ceramide-induced cytotoxicity in HeLa cells. Expression of v-Src increased the formation of NBD-GlcCer in both intact cells without marked changes in other sphingolipid metabolites and cell homogenates without changing affinities of NBD-ceramide and UDP-glucose. Expression of v-Src also increased tyrosine-phosphorylated levels in GCS proteins in HeLa and HEK293T cells. In HEK293T cells transiently expressing the GCS mutant, GCS-Y132F-HA, showing replacement of the tyrosine¹³² residue with phenylalanine, tyrosine-phosphorylated levels in GCS proteins were significantly lower than those in control cells expressing the GCS-wild-type-HA. The formation of NBD-GlcCer in HeLa cells stably expressing GCS-Y132F-HA was significantly lower than that in the control. Ceramide-induced cytotoxicity in HeLa-GCS-Y132F-HA cells was significantly greater than in the control. In this study, we showed for the first time that expression of v-Src up-regulated GCS activity via tyrosine phosphorylation of the enzyme in a post-translational manner. Mechanisms of Src-induced resistance to ceramide-induced cytotoxicity are discussed in relation to the Src-induced up-regulation of GCS activity.

鞘脂代表调节生理和病理生理过程的细胞脂质分子家族。葡萄糖基神经酰胺（GlcCer）是最简单的鞘糖脂（GSL），是由神经酰胺和UDP葡萄糖通过GlcCer合酶（GCS）合成的。GlcCer（以及由此产生的GSL）和神经酰胺均调节各种细胞功能，包括细胞死亡和多重耐药性。Src家族的酪氨酸激酶在各种人类癌细胞中上调。我们检查了v-Src表达对GCS活性的影响，NBD-神经酰胺形成4-硝基苯并-2-氧杂-1,3-二唑（NBD）标记的GlcCer的作用以及酪氨酸¹³²的作用GCS突变对神经酰胺诱导的HeLa细胞细胞毒性的影响。v-Src的表达增加了两个完整细胞中NBD-GlcCer的形成，而其他鞘脂代谢产物和细胞匀浆则没有明显变化，而NBD-神经酰胺和UDP-葡萄糖的亲和力却没有改变。v-Src的表达还增加了HeLa和HEK293T细胞中GCS蛋白中的酪氨酸磷酸化水平。在HEK293T细胞中瞬时表达GCS突变体GCS-Y132F-HA，显示酪氨酸^132的替换与苯丙氨酸残基相比，GCS蛋白中酪氨酸磷酸化水平显着低于表达GCS野生型HA的对照细胞。稳定表达GCS-Y132F-HA的HeLa细胞中NBD-GlcCer的形成明显低于对照组。神经酰胺对HeLa-GCS-Y132F-HA细胞的细胞毒性显着大于对照。在这项研究中，我们首次证明了v-Src的表达通过翻译后的酶的酪氨酸磷酸化来上调GCS活性。关于Src诱导的GCS活性上调，讨论了Src诱导的对神经酰胺诱导的细胞毒性的抗性机制。