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

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.

Identification of ceramide binding proteins in neuronal cells: a critical point of view.

Much discussion has centered on the biochemical mechanism by which ceramide is produced and functions as a signalling molecule in cells. To identify proteins involved in ceramide signalling, we synthesized a radioactively labelled ceramide analogue equipped with a photosensitive group: N-(p-trifluoromethyl-diazirinyl)phenyl-ethyl-2-[35S]-2-thioacetyl-D-erythro-C18-sphingosine ([35S]-TDS-ceramide). This compound was then employed in photo-affinity labelling experiments in primary cultured cerebellar neurons. Due to the hydrophobic nature of the compound, most of the cell-associated radioactivity was recovered in the lipid fraction while only about 0.1% of radioactivity was photocoupled to proteins. In order to improve protein labelling the cytosolic fraction of rapidly growing human neuroblastoma cells (SH-SY5Y) was isolated and subjected to ceramide affinity chromatography prior to photo-affinity labelling. Following electrophoresis proteins photocoupled to ceramide were identified by MALDI mass spectrometry in combination with tryptic digestion and turned out to be either cytoskeletal or stress proteins that are highly abundant in cytosol and contain at least one hydrophobic domain.