title: Characterization of an ecto-phosphorylated protein of cultured cerebellar granule neurons.
abstract: Previous work identified the phosphorylation by extracellular ATP of an endogenous 45-kDa protein substrate and established the presence of ecto-protein kinase activity associated with cultured cerebellar granule neurons (Volonté et al.: J Neurochem 63:2028-2037, 1994). In this work, we characterize such ecto-phosphorylated 45-kDa protein substrate and its association with the cellular membrane. The total radioactive content of the 45-kDa protein is stable for the first 15 min after phosphorylation, and decreases about 70% in 30 min and 90% in approximately 2 hr. Rinsing the cells after the phosphorylating reaction causes a 50% removal of the incorporated radioactivity. Glycosidic residues are present on the 45-kDa ecto-protein, which is held in position on the cellular membrane through a specific glycosyl-phosphatidylinositol anchor. The extracellular incorporation of phosphate on the 45-kDa protein is not modulated by agents interfering with cytoskeleton stability, such as colchicine and taxol, or by gangliosides. The extracellular phosphorylation occurs mostly on serine residues, since the phosphate ester linkage is unstable at high pH and only antibodies raised against phosphoserine are capable of recognizing the 45-kDa ecto-protein.
authors.affiliation: Institute of Neurobiology, Consiglio Nazionale Ricerche, Rome, Italy.
authors.forename: D
authors.initials: D
authors.lastname: Merlo
authors.forename: R
authors.initials: R
authors.lastname: Anelli
authors.forename: P
authors.initials: P
authors.lastname: Calissano
authors.forename: M T
authors.initials: MT
authors.lastname: Ciotti
authors.forename: C
authors.initials: C
authors.lastname: Volonté
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002531:Cerebellum / Q000378:metabolism*; D007150:Immunohistochemistry; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar
chemical_list: D010768:Phosphoserine; D011494:Protein Kinases