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

Actin depolymerizing factor (ADF) occurs naturally in two forms, one o f which contains a phosphorylated Ser and does not bind G-actin or dep olymerize F-actin. Removal of this phosphate in vitro by alkaline phos phatase restores full F-actin depolymerizing activity. To identify the phosphorylation site, [P-32]pADF was purified and digested with endop roteinase Lys-C. The digest contained only one P-32-labeled peptide. F urther digestion with endoproteinase Asp-N and mass spectrometric anal ysis showed that this peptide came from the N terminus of ADF. Alkalin e phosphatase treatment of one Asp-N peptide (mass 753) converted it t o a peptide of mass 673, demonstrating that this peptide contains the phosphate group. Tandem mass spectrometric sequence analysis of this p eptide 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(2 4) or Thr(25), express and phosphorylate the exogenous ADF. Cells also expressed high levels of mutant ADF when Ser(3) was deleted or conver ted to either Ala or Glu. However, none of these mutants was phosphory lated, confirming that Ser(3) in the encoded ADF is the single in vivo regulatory site.