Characterization of an ecto-phosphorylated protein of cultured cerebellar granule neurons

Daniela Merlo, Roberta Anelli, Pietro Calissano, MariaTeresa Ciotti, Cinzia Volonté

Research output: Contribution to journalArticle

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 (Volonte 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.

Original languageEnglish
Pages (from-to)500-508
Number of pages9
JournalJournal of Neuroscience Research
Volume47
Issue number5
DOIs
Publication statusPublished - Mar 1 1997

Keywords

  • ecto-kinase
  • ecto-protein substrate
  • extracellular phosphorylation
  • glycosyl-phosphatidylinositol anchor
  • signal transduction

ASJC Scopus subject areas

  • Neuroscience(all)

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