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  • Title: Erythrocyte adducin. Comparison of the alpha- and beta-subunits and multiple-site phosphorylation by protein kinase C and cAMP-dependent protein kinase.
    Author: Waseem A, Palfrey HC.
    Journal: Eur J Biochem; 1988 Dec 15; 178(2):563-73. PubMed ID: 3208770.
    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. & 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 approximately 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 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 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 alpha-adducin 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 approximately 1.5 mol/mol in both alpha and beta subunits.(ABSTRACT TRUNCATED AT 400 WORDS)
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