HETEROGENEOUS PHOSPHORYLATION OF ERYTHROCYTE SPECTRIN BETA-CHAIN IN INTACT-CELLS

Human erythrocyte spectrin is an alphabeta heterodimer which forms tet ramers by self-association. This association involves the N-terminal r egion of the a chain and the C-terminal region of the beta chain. The latter contains a cluster of four phosphorylation sites (one phosphoth reonine and three phosphoserine residues). The role of this phosphoryl ation 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 : P-32 peptide maps obtained by 2-nitro-5-thiocyanobenzoic acid (NTCB) cleavage revealed the presen ce of six components with apparent molecular masses of 17.5 kDa, diffe ring in their isoelectric points; this is most simply interpreted as r eflecting the presence of six exchangeable phosphorylation sites in th e spectrin beta chain, rather than four as had been supposed. When the alphabeta dimers were partly dissociated by urea, the most highly pho sphorylated fraction of the beta chain was found in the undissociated dimers. This high specific activity in the undissociated dimer reflect ed multiple phosphorylated sites, as revealed by NTCB cleavage. The de phosphorylation or the hyperphosphorylation of spectrin beta chains di d not modify the equilibrium between dissociated and undissociated spe ctrin dimers in the presence of urea. However, the data revealed the e xistence of two spectrin dimer populations in respect to phosphate tur nover and spectrin dimer dissociation.