Detection and identification of Vav1 protein in primary cultured murine cerebellar neurons and in neuroblastoma cells (SH-SY5Y and Neuro-2a)

Abstract

Vav1 was detected in neuronal cells during a screening for 1-methylthiodihydroceramide (1-MSDH-Cer) binding proteins. 1-MSDH-Cer is a metabolically stable analogue of dihydroceramide that was reported to strongly interfere with the formation of ceramide and hence the biosynthesis of all sphingolipids in neuronal cells. To identify target proteins that function as putative mediators of this molecule, a 1-MSDH-Cer affinity chromatography was utilised. When the cytosolic fraction of human neuroblastoma SH-SY5Y cells was subjected to 1-MSDH-Cer affinity chromatography, the sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the eluted protein fraction revealed an about 2-fold enrichment of the 98 kD protein band. Tryptic digestion of the excised band in combination with MALDI mass spectrometry strongly suggested that this band contained Vav1 protein. This was surprising since Vav1 in contrast to the other two isoforms Vav2 and Vav3 is believed to be exclusively expressed in hematopoietic cells and has not been detected in neuronal cells until now. The expression of Vav1 was confirmed in human SH-SY5Y neuroblastoma cells and additionally in murine Neuro-2A neuroblastoma cells as well as in primary cultured murine cerebellar neurons by Western blot analysis and reverse transcription polymerase chain reaction.