ISOLATION AND CHARACTERIZATION OF A REGULATED FORM OF ACTIN DEPOLYMERIZING FACTOR

Actin depolymerizing factor (ADF) is an 18.5-kD protein with pH-depend ent reciprocal F-actin binding and severing/depolymerizing activities. We previously showed developing muscle down-regulates ADF (J. R. Bamb urg and D. Bray. 1987. J Cell Biol. 105: 2817-2825). To further study this process, we examined ADF expression in chick myocytes cultured in vitro. Surprisingly, ADF immunoreactivity increases during the first 7-10 d in culture. This increase is due to the presence of a new ADF s pecies with higher relative molecular weight which reacts identically to brain ADF with antisera raised against either brain ADF or recombin ant ADF. We have purified both ADF isoforms from myocytes and have sho wn by peptide mapping and partial sequence analysis that the new isofo rm is structurally related to ADF. Immunoprecipitation of both isoform s from extracts of cells prelabeled with [P-32]orthophosphate showed t hat the new isoform is radiolabeled, predominantly on a serine residue , and hence is called pADF. pADF can be converted into a form which co migrates with ADF on I-D and 2-D gels by treatment with alkaline phosp hatase. pADF has been quantified in a number of cells and tissues wher e it is present from approximately 18% to 150% of the amount of unphos phorylated ADF. pADF, unlike ADF, does not bind to G-actin, or affect the rate or extent of actin assembly. Four ubiquitous protein kinases failed to phosphorylate ADF in vitro suggesting that ADF phosphorylati on in vivo is catalyzed by a more specific kinase. We conclude that th e ability to regulate ADF activity is important to muscle development since myocytes have both pre- and posttranslational mechanisms for reg ulating ADF activity. The latter mechanism is apparently a general one for cell regulation of ADF activity.