Phosphorylation of ankyrin down-regulates its cooperative interaction with spectrin and protein 3.
Ankyrin mediates the primary attachment between beta spectrin and protein 3. Ankyrin and spectrin interact in a positively cooperative fashion such that ankyrin binding increases the extent of spectrin tetramer and oligomer formation (Giorgi and Morrow: submitted, 1988). This cooperative interaction is enhanced by the cytoplasmic domain of protein 3, which is prepared as a 45-41-kDa fragment generated by chymotryptic digestion of erythrocyte membranes. Using sensitive isotope-ratio methods and nondenaturing PAGE, we now demonstrate directly (1) the enhanced affinity of ankyrin for spectrin oligomers compared to spectrin dimers; (2) a selective stimulation of the affinity of ankyrin for spectrin oligomer by the 43-kDa cytoplasmic domain of protein 3; and (3) a selective reduction in the affinity of ankyrin for spectrin tetramer and oligomer after its phosphorylation by the erythrocyte cAMP-independent membrane kinase. The phosphorylation of ankyrin does not affect its binding to spectrin dimer. Ankyrin also enhances the rate of interconversion between dimer-tetramer-oligomer by 2-3-fold at 30 degrees C, and in the presence of the 43-kDa fragment, ankyrin stimulates the rate of oligomer interconversions by nearly 40-fold at this temperature. These results demonstrate a long-range cooperative interaction between an integral membrane protein and the peripheral cytoskeleton and indicate that this linkage may be regulated by covalent protein phosphorylation. Such interactions may be of general importance in nonerythroid cells.
Cianci
C D
CD
Department of Pathology, Yale University School of Medicine, New Haven, CT 06510.
Giorgi
M
M
Morrow
J S
JS
D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism*; D017487:Ankyrins; D001667:Binding, Competitive; D001798:Blood Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism; D010766:Phosphorylation; D013049:Spectrin / Q000302:isolation & purification / Q000378:metabolism*
D001457:Anion Exchange Protein 1, Erythrocyte; D017487:Ankyrins; D001798:Blood Proteins; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D009842:Oligopeptides; D013049:Spectrin