CHARACTERIZATION OF AN ECTO-PHOSPHORYLATED PROTEIN OF CULTURED CEREBELLAR GRANULE NEURONS

Previous work identified the phosphorylation by extracellular ATP of a n endogenous 45-kDa protein substrate and established the presence of ecto-protein kinase activity associated with cultured cerebellar granu le neurons (Volonte et al.: J Neurochem 63:2028-2037, 1994), In this w ork, 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 ph osphorylation, and decreases about 70% in 30 min and 90% in approximat ely 2 hr, Rinsing the cells after the phosphorylating reaction causes a 50% removal of the incorporated radioactivity, Glycosidic residues a re present on the 45-kDa ecto-protein, which is held in position on th e cellular membrane through a specific glycosyl-phosphatidylinositol a nchor, The extracellular incorporation of phosphate on the 45-kDa prot ein 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 agains t phosphoserine are capable of recognizing the 45-kDa ecto-protein. (C ) 1997 Wiley-Liss, Inc.