Heterogeneous phosphorylation of erythrocyte spectrin beta chain in intact cells.
Human erythrocyte spectrin is an alpha beta heterodimer which forms tetramers by self-association. This association involves the N-terminal region of the alpha chain and the C-terminal region of the beta chain. The latter contains a cluster of four phosphorylation sites (one phosphothreonine and three phosphoserine residues). The role of this phosphorylation is as yet unknown. We show in this paper that the spectrin beta chain occurs in the cell in subpopulations differing in the degree of occupancy of their phosphorylation sites: 32P peptide maps obtained by 2-nitro-5-thiocyanobenzoic acid (NTCB) cleavage revealed the presence of six components with apparent molecular masses of 17.5 kDa, differing in their isoelectric points; this is most simply interpreted as reflecting the presence of six exchangeable phosphorylation sites in the spectrin beta chain, rather than four as had been supposed. When the alpha beta dimers were partly dissociated by urea, the most highly phosphorylated fraction of the beta chain was found in the undissociated dimers. This high specific activity in the undissociated dimer reflected multiple phosphorylated sites, as revealed by NTCB cleavage. The dephosphorylation or the hyperphosphorylation of spectrin beta chains did not modify the equilibrium between dissociated and undissociated spectrin dimers in the presence of urea. However, the data revealed the existence of two spectrin dimer populations in respect to phosphate turnover and spectrin dimer dissociation.
Pedroni
S
S
INSERM U.160, Hôpital Beaujon, Clichy, France.
Lecomte
M C
MC
Gautero
H
H
Dhermy
D
D
D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification; D010766:Phosphorylation; D013049:Spectrin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D010750:Phosphoproteins; D013049:Spectrin