TI - Brain beta-spectrin phosphorylation : phosphate analysis and identification of threonine-347 as a heparin-sensitive protein kinase phosphorylation site . AB - Phosphorylation of brain spectrin was studied by a combination of in vivo and in vitro approaches . Chemical analysis of phosphate groups on electrophoretically purified mouse brain beta-spectrin yielded a stoichiometry of 3.2 +/- 0.18 mol of PO4/mol of beta-spectrin . The spectrin isolated by chromatographic methods from mouse brain , pig brain , and human erythrocytes yielded 4.1 , 5.6 , and 3.2 mol of PO4/mol of spectrin heterodimer , respectively . The 32P labeling of spectrin in retinal ganglion cell neurons or NB 2a/d1 neuroblastoma cells with [32P] orthophosphate showed phosphorylation of only beta-spectrin in vivo . Two-dimensional phosphopeptide map analyses showed that most of the in vivo sites on beta-spectrin were phosphorylated by either a heparin-sensitive endogenous cytoskeleton-associated protein kinase or protein kinase A . Phosphoamino acid analysis of in vivo and in vitro phosphorylated beta-spectrin showed that [32P] phosphate groups were incorporated into both serine ( &gt ; 90% ) and threonine residues . In vitro , phosphate groups were incorporated into threonine residues by the heparin-sensitive endogenous protein kinase . The amino acid sequence VQQQLQAFNTY of an alpha-chymotryptic 32P-labeled peptide phosphorylated by the heparin-sensitive cytoskeleton-associated endogenous protein kinase corresponded to amino acid residues 338-348 on the beta1 repeat of beta-spectrinG (betaSPIIa) gene . These data suggest that phosphorylation of Thr347 , which is localized on the presumptive synapsin I binding domain of beta-spectrinG , may play a role in synaptic function by regulating the binding of spectrin to synaptic vesicles .