In vivo phosphorylation of human erythrocyte spectrin occurs in a sequential manner.
Spectrin is the major component of the erythrocyte membrane skeleton and exists as a 526 kDa alphabeta heterodimer. The 246 kDa beta-chain of human spectrin is phosphorylated near the C-terminus, but the exact phosphorylation sites are unknown and the role of this phosphorylation is not fully characterized. In this study, we produced a monoclonal antibody, Sp316, capable of recognizing the C-terminal region of beta-spectrin regardless of its phosphorylation state and used it to purify the phosphorylated region after 2-nitro-5-thiocyanobenzoic acid cleavage of spectrin. Two-dimensional gels, mass spectrometry, and reversed-phase high-performance liquid chromatography were used to characterize these phosphorylation states. Only about 1.5% of spectrin isolated from fresh blood is unphosphorylated, about 9% has more than four phosphates per molecule, and the majority of the protein has one to four phosphates per molecule. A total of six phosphorylation sites were identified by tandem mass spectrometry. Quantitative analysis of the phosphorylation states by reversed-phase high-performance liquid chromatography revealed that phosphorylation of beta-spectrin occurs in a sequential manner where each specific site is completely phosphorylated before the next site is modified. The first phosphorylation event occurs on Ser-2114, followed by Ser-2125, Ser-2123, Ser-2128, Ser-2117, and Thr-2110. The identification of the specific phosphorylated beta-spectrin residues and the ordered sequence of phosphorylation events in vivo should provide an invaluable basis for further studies of the role of these posttranslational modifications in spectrin function in situ.
Tang
Hsin-Yao
HY
The Wistar Institute, 3601 Spruce Street, Philadelphia, Pennsylvania 19104, USA.
Speicher
David W
DW
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D004910:Erythrocyte Membrane / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D010449:Peptide Mapping; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013049:Spectrin / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013861:Thiocyanates / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D013861:Thiocyanates; D013049:Spectrin; D014357:Trypsin; C008658:2-nitro-5-thiocyanobenzoic acid