Erythrocyte adducin

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Abstract
Two major substrates for human erythrocyte protein kinase C (PK-C) of Mr 120 000 and 110 000, previously named PKC-1 and PKC-2 [Palfrey, H. C. and 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 skelton by high-salt extraction, ion-exchange and gel filtration chromatography. The two proteins co-fractionate in a ratio of .apprxeq. 1:1 under a number of conditions suggest that they exist as a complex. Physicochemical data indicate that the native adducin complex is probably an asymmetric heterodimer of .alpha. and .beta. subunits. Adducin binds to a calmodulin (CaM) affinity matrix in a Ca2+-dependent manner and is specifically eluted with EGTA. Fingerprinting of the iodinated peptides derived from the .alpha. and .beta. 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 .beta. subunit is not derived from the .alpha. subunit or vice versa. Proteins reactive with both anti-(.alpha.-adducin) and anti-(.beta.-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 significant fraction of the protein may associate with non-skeleton membrane component. The phosphorylation of adducin is stimulated by both phorbol esters and cAMP analogues in tact erythrocytes. Fingerprinting suggest that protein kinase C preferentially phophorylates four distinct sites on the two proteins. Phosphopeptide maps of .alpha.-adducin are virtually identical to those of .alpha.-adduction are virtually identical to those of .beta.-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 .ALPHA.PP 1.5 mol/mol in both .alpha. and .beta. subunits. Phosphorylation by the catalytic subunit of cAMP-PK is equally rapid, with .alpha. adducin beng preferentially phosphorylated to a level of .apprxeq. 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.