Brain beta-spectrin phosphorylation: phosphate analysis and identification of threonine-347 as a heparin-sensitive protein kinase phosphorylation site.
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 (>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.
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Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892-4062, USA.
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D001921:Brain / Q000378:metabolism*; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D010449:Peptide Mapping; D010710:Phosphates / Q000032:analysis; D019606:Phosphoamino Acids / Q000032:analysis; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D013049:Spectrin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013552:Swine
D010710:Phosphates; D019606:Phosphoamino Acids; D013049:Spectrin; D006493:Heparin; D011494:Protein Kinases