Tyrosine-phosphorylation and activation of glucosylceramide synthase by v-Src: Its role in survival of HeLa cells against ceramide.
Biochim Biophys Acta Mol Cell Biol Lipids
; 1866(1): 158817, 2021 01.
Article
en En
| MEDLINE
| ID: mdl-32980536
ABSTRACT
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.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Fenilalanina
/
Tirosina
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Proteína Oncogénica pp60(v-src)
/
Glucosilceramidas
/
Glucosiltransferasas
Límite:
Humans
Idioma:
En
Revista:
Biochim Biophys Acta Mol Cell Biol Lipids
Año:
2021
Tipo del documento:
Article