Erythrocyte adducin. Comparison of the alpha- and beta-subunits and multiple-site phosphorylation by protein kinase C and cAMP-dependent protein kinase.
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)
Waseem
A
A
Department of Pharmacological and Physiological Sciences, University of Chicago, Illinois 60637.
Palfrey
H C
HC
D000818:Animals; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D004372:Ducks; D004910:Erythrocyte Membrane / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism*; D005289:Ferrets; D006868:Hydrolysis; D010446:Peptide Fragments / Q000378:metabolism; D010449:Peptide Mapping; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D013552:Swine; D000099291:Adducins
D002148:Calmodulin-Binding Proteins; D010446:Peptide Fragments; D011493:Protein Kinase C; D011494:Protein Kinases; D000099291:Adducins