Erythrocyte adducin

Two major substrates for human erythrocyte protein kinase C (PK‐C) of M r 120 000 and 110 000, previously named PKC‐1 and PKC‐2 [Palfrey, H. C. & Waseem, A. (1985) J. Biol. Chem. 260 , 16021–16029] have been found to be identical to CaM‐BP 103/97 or ‘adducin’, recently described by K. Gardner and V. Bennett [(1986) J. Biol. Chem. 261 , 1339–1348; (1987) Nature (Lond.) 328 , 359–362]. These proteins have been purified from the membrane skeleton by high‐salt extraction, ion‐exchange and gel filtration chromatography. The two proteins co‐fractionate in a ratio of ∼ 1:1 under a number of conditions suggesting that they exist as a complex. Physicochemical data indicate that the native adducin complex is probably an asymmetric heterodimer of α and β subunits. Adducin binds to a calmodulin (CaM) affinity matrix in a Ca 2+ ‐dependent manner and is specifically eluted with EGTA. Fingerprinting of the iodinated peptides derived from the α and β subunits using three different proteases yields 16–37% overlapping peptides, indicating limited similarity between the two polypeptides. Affinity‐purified polyclonal antibodies against each protein show little or no cross‐reactivity with the other, indicating that the β subunit is not derived from the α subunit or vice versa. Proteins reactive with both anti‐(α‐adducin) and anti‐(β‐adducin) antibodies are found in erythrocytes from rat, rabbit, pig, ferret and duck. Immunoblots of adducin after non‐ionic detergent extraction of ghosts reveal that a significant fraction of the protein may associate with non‐skeleton membrane components. The phosphorylation of adducin is stimulated by both phorbol esters and cAMP analogues in intact erythrocytes. Fingerprinting suggests that protein kinase C preferentially phosphorylates four distinct sites on the two proteins. Phosphopeptide maps of α‐adducin are virtually identical to those of β‐adducin after phorbol ester stimulation of intact cells, or after PK‐C‐catalyzed phosphorylation of the purified protein, indicating strong local similarities in the two proteins. Such maps also suggest that cAMP‐dependent protein kinase (cAMP‐PK) modifies adducin at some similar and some distinct sites as those modified by PK‐C. In vitro phosphorylation of isolated adducin by purified PK‐C results in rapid incorporation of phosphate to a final level of ∼ 1.5 mol/mol in both α and β subunits. Phosphorylation by the catalytic subunit of cAMP‐PK is equally rapid, with α‐ adducin being preferentially phosphorylated to a level of ∼ 1.8 mol/mol. Increased phosphorylation enhances the extraction of adducin from the membrane by non‐ionic detergent. The multivalent properties of this protein complex suggest that it may play a role in mediating the interactions of components of the membrane skeleton with the inner surface of the membrane and that CaM and/or phosphorylation may modulate this function.