Identification of two cAMP-dependent phosphorylation sites on erythrocyte protein 4.1.
In human erythrocytes, dibutyryl cyclic AMP induces the phosphorylation of protein 4.1 on sites within the adjacent 16 kDa and 10 kDa chymotryptic domains (Horne, W.C., Leto, T.L. and Marchesi, V.T. (1985) J. Biol. Chem. 260, 9073-9076). The 10 kDa domain also contains the spectrin/actin-binding site (Correas, I., Leto, T.L., Speicher, D.W. and Marchesi, V.T. (1986) J. Biol. Chem. 261, 3310-3315) and it has been shown that phosphorylation of protein 4.1 by cyclic AMP-dependent protein kinase inhibits the binding of protein 4.1 to spectrin and actin (Ling, E., Danilov, Y.N. and Cohen, C.M. (1988) J. Biol. Chem. 263, 2209-2216). In this study, we have identified two sites on protein 4.1 which account for 80% of the phosphate incorporated into protein 4.1 during metabolic labelling of erythrocytes in the presence of dibutyryl cyclic AMP. More than 95% of the 32P incorporated into protein 4.1 was in the form of phosphoserine. Reverse-phase HPLC of the peptides generated by digestion of the isolated protein with trypsin or endoproteinase lysine C produced two major radioactive peaks. The phosphorylation sites, identified by gas phase sequencing of the purified phosphopeptides and confirmed by determining the residues converted to S-ethylcysteine by reacting the phosphopeptides with ethanethiol under alkaline conditions, were Ser-331, in the 16 kDa domain and Ser-467, in the 10 kDa domain.
Horne
W C
WC
Department of Pathology, Yale University School of Medicine, New Haven, CT 06510.
Prinz
W C
WC
Tang
E K
EK
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001798:Blood Proteins / Q000737:chemistry* / Q000378:metabolism*; D003994:Bucladesine / Q000494:pharmacology; D000242:Cyclic AMP / Q000502:physiology*; D003598:Cytoskeletal Proteins; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000097:blood* / Q000737:chemistry*; D008969:Molecular Sequence Data; D009479:Neuropeptides; D010446:Peptide Fragments / Q000302:isolation & purification; D010750:Phosphoproteins / Q000737:chemistry; D010766:Phosphorylation
D000596:Amino Acids; D001798:Blood Proteins; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D009479:Neuropeptides; D010446:Peptide Fragments; D010750:Phosphoproteins; C028841:erythrocyte membrane band 4.1 protein; C120557:erythrocyte membrane protein band 4.1-like 1; D003994:Bucladesine; D000242:Cyclic AMP