REACTIVATION OF PHOSPHORYLATED ACTIN DEPOLYMERIZING FACTOR AND IDENTIFICATION OF THE REGULATORY SITE

Actin depolymerizing factor (ADF) occurs naturally in two forms, one of which contains a phosphorylated Ser and does not bind G-actin or depolymerize F-actin. Removal of this phosphate in vitro by alkaline phosphatase restores full F-actin depolymerizing activity. To identify the phosphorylation site, [P-32]pADF was purified and digested with endoproteinase Lys-C. The digest contained only one P-32-labeled peptide. Further digestion with endoproteinase Asp-N and mass spectrometric analysis showed that this peptide came from the N terminus of ADF. Alkaline phosphatase treatment of one Asp-N peptide (mass 753) converted it to a peptide of mass 673, demonstrating that this peptide contains the phosphate group. Tandem mass spectrometric sequence analysis of this peptide identified the phosphorylated Ser as the encoded Ser(3) (Ser(2) in the processed protein). HeLa cells, transfected with either chick wild-type ADF cDNA or a cDNA mutated to code for Ala in place of Ser(24) or Thr(25), express and phosphorylate the exogenous ADF. Cells also expressed high levels of mutant ADF when Ser(3) was deleted or converted to either Ala or Glu. However, none of these mutants was phosphorylated, confirming that Ser(3) in the encoded ADF is the single in vivo regulatory site.