Brain β-Spectrin Phosphorylation: Phosphate Analysis and Identification of Threonine-347 as a Heparin-Sensitive Protein Kinase Phosphorylation Site |
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Authors: | Ram K Sihag |
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Institution: | Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland;and Department of Psychiatry, McLean Hospital and Harvard Medical School, Belmont, Massachusetts, U.S.A. |
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Abstract: | Abstract: 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 β-spectrin yielded a stoichiometry of 3.2 ± 0.18 mol of PO4/mol of β-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 β-spectrin in vivo. Two-dimensional phosphopeptide map analyses showed that most of the in vivo sites on β-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 β-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 α-chymotryptic 32P-labeled peptide phosphorylated by the heparin-sensitive cytoskeleton-associated endogenous protein kinase corresponded to amino acid residues 338–348 on the β1 repeat of β-spectrinG (βSPIIa) gene. These data suggest that phosphorylation of Thr347, which is localized on the presumptive synapsin I binding domain of β-spectrinG, may play a role in synaptic function by regulating the binding of spectrin to synaptic vesicles. |
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Keywords: | β-Spectrin Phosphorylation Heparin-sensitive protein kinase Neuronal cytoskeleton |
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