Reactivation of Phosphorylated Actin Depolymerizing Factor and Identification of the Regulatory Site

Abstract

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, [32P]pADF was purified and digested with endoproteinase Lys-C. The digest contained only one 32P-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 Ser3 (Ser2 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 Ser24 or Thr25, express and phosphorylate the exogenous ADF. Cells also expressed high levels of mutant ADF when Ser3 was deleted or converted to either Ala or Glu. However, none of these mutants was phosphorylated, confirming that Ser3 in the encoded ADF is the single in vivo 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, [32P]pADF was purified and digested with endoproteinase Lys-C. The digest contained only one 32P-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 Ser3 (Ser2 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 Ser24 or Thr25, express and phosphorylate the exogenous ADF. Cells also expressed high levels of mutant ADF when Ser3 was deleted or converted to either Ala or Glu. However, none of these mutants was phosphorylated, confirming that Ser3 in the encoded ADF is the single in vivo regulatory site. Actin depolymerizing factor (ADF),1 1The abbreviations used are: ADFactin depolymerizing factorE10embryonic day 10DTTdithiothreitolPAGEpolyacrylamide gel electrophoresisHPLChigh pressure liquid chromatographyHAPhydroxylapatiteGTPγSguanosine 5’-3-O-(thio)triphosphate. is an 18.5-kDa protein with a pH-dependent F-actin binding/depolymerizing activity (1Hayden S.M. Miller P.S. Brauweiler A. Bamburg J.R. Biochemistry. 1993; 32: 9994-10004Crossref PubMed Scopus (203) Google Scholar, 2Hawkins M. Pope B. Maciver S.K. Weeds A.G. Biochemistry. 1993; 32: 9985-9993Crossref PubMed Scopus (240) Google Scholar) very similar to that of its structural homolog, cofilin(3Nishida E. Maekawa S. Sakai H. Biochemistry. 1984; 23: 5307-5313Crossref PubMed Scopus (253) Google Scholar, 4Yonezawa N. Nishida E. Sakai H. J. Biol. Chem. 1985; 260: 14410-14412Abstract Full Text PDF PubMed Google Scholar). Proteins in the ADF/cofilin family appear to be ubiquitous in eukaryotes (for review, see (5Sun H.-Q. Kwiatkowska K. Yin H.L. Curr. Opin. Cell Biol. 1995; 7: 102-110Crossref PubMed Scopus (171) Google Scholar)). Mutations that inactivate the ADF/cofilin gene in Saccharomyces cerevisiae(6Moon A.L. Janmey P. Louie K.A. Drubin D. J. Cell Biol. 1993; 120: 421-435Crossref PubMed Scopus (201) Google Scholar), Drosophila melanogaster(7Edwards K.A. Montague R.A. Shepard S. Edgar B.A. Erikson R.L. Kiehart D.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4589-4593Crossref PubMed Scopus (59) Google Scholar), and Caenorhabditis elegans(8McKim K.S. Matheson C. Marra M.A. Wakarchuk M.F. Baillie D.L. Mol. & Gen. Genet. 1994; 242: 346-357Crossref PubMed Scopus (100) Google Scholar) are lethal. actin depolymerizing factor embryonic day 10 dithiothreitol polyacrylamide gel electrophoresis high pressure liquid chromatography hydroxylapatite guanosine 5’-3-O-(thio)triphosphate. Phosphorylated forms of both ADF and cofilin have been identified in vertebrate cells(9Ohta Y. Nishida E. Sakai H. Miyamoto E. J. Biol. Chem. 1989; 264: 16143-16148Abstract Full Text PDF PubMed Google Scholar, 10Bamburg J.R. Minamide L.S. Morgan T.E. Hayden S.M. Giuliano K.A. Koffer A. Methods Enzymol. 1991; 196: 125-140Crossref PubMed Scopus (21) Google Scholar, 11Morgan T.E. Lockerbie R.O. Minamide L.S. Browning M.D. Bamburg J.R. J. Cell Biol. 1993; 122: 623-633Crossref PubMed Scopus (143) Google Scholar). We isolated phosphorylated ADF and showed it to be inactive in depolymerizing F-actin and in inhibiting the assembly of G-actin(11Morgan T.E. Lockerbie R.O. Minamide L.S. Browning M.D. Bamburg J.R. J. Cell Biol. 1993; 122: 623-633Crossref PubMed Scopus (143) Google Scholar). Phosphoamino acid analysis identified phosphoserine as the phosphorylated amino acid(11Morgan T.E. Lockerbie R.O. Minamide L.S. Browning M.D. Bamburg J.R. J. Cell Biol. 1993; 122: 623-633Crossref PubMed Scopus (143) Google Scholar). Here, we demonstrate that pADF can be completely reactivated following phosphatase treatment. We also determine the location of the phosphoserine and show by site-directed mutagenesis and expression in vertebrate cells that this is the site for regulation by phosphorylation in vivo. pADF was isolated free from ADF and cofilin from embryonic day 10 (E10) or E11 chick brain as described previously(11Morgan T.E. Lockerbie R.O. Minamide L.S. Browning M.D. Bamburg J.R. J. Cell Biol. 1993; 122: 623-633Crossref PubMed Scopus (143) Google Scholar). It was further purified for digestion and sequence analysis as described under “Results.” Chick brain ADF was purified according to the method of Giuliano et al.(12Giuliano K.A. Khatib F.A. Hayden S.M. Daoud E.W.R. Adams M.E. Amorese D.A. Bernstein B.W. Bamburg J.R. Biochemistry. 1988; 27: 8931-8938Crossref PubMed Scopus (43) Google Scholar). Skeletal muscle actin was purified from rabbit muscle acetone powder(13Pardee J.D. Spudich J.A. Methods Cell Biol. 1982; 24: 271-289Crossref PubMed Scopus (340) Google Scholar). The in vitro reactivation of pADF by treatment with alkaline phosphatase (Sigma) is described under “Results.” The amount of G-actin depolymerized from F-actin in the presence of ADF was quantified by using the DNase I inhibition assay(10Bamburg J.R. Minamide L.S. Morgan T.E. Hayden S.M. Giuliano K.A. Koffer A. Methods Enzymol. 1991; 196: 125-140Crossref PubMed Scopus (21) Google Scholar, 14Harris H.E. Bamburg J.R. Bernstein B.W. Weeds A.G. Anal. Biochem. 1982; 119: 102-114Crossref PubMed Scopus (24) Google Scholar). Cells in 10-cm culture dishes were scraped in 300 εl of ice-cold extraction buffer (10 mM Tris, pH 7.6, 1% SDS, 15 mM NaF, 10 mM dithiothreitol (DTT), 2 mM EGTA, 0.3 mM sodium orthovanadate, 10 εl/ml protease inhibition mixture)(11Morgan T.E. Lockerbie R.O. Minamide L.S. Browning M.D. Bamburg J.R. J. Cell Biol. 1993; 122: 623-633Crossref PubMed Scopus (143) Google Scholar), sonicated, and immersed 5 min in a boiling water bath. After cooling the samples on ice, proteins were precipitated with chloroform/methanol(15Wessel D. Flügge U.I. Anal. Biochem. 1984; 138: 141-143Crossref PubMed Scopus (3185) Google Scholar). The pellets were dissolved in sample preparation buffer (0.125 M Tris, pH 6.8, 1% SDS, 5% glycerol, 10% 2-mercaptoethanol, 0.01% bromphenol blue) for SDS-polyacrylamide gel electrophoresis (PAGE) or in lysis buffer (9.5 M urea, 2% Nonidet P-40, 2% ampholytes, pH 3-10, 10% 2-mercaptoethanol) for two-dimensional gels (16O'Farrell P.H. J. Biol. Chem. 1975; 250: 4007-4021Abstract Full Text PDF PubMed Google Scholar). Protein concentrations were determined by the solid phase dye-binding method(17Minamide L.S. Bamburg J.R. Anal. Biochem. 1990; 190: 66-70Crossref PubMed Scopus (237) Google Scholar). SDS-PAGE was performed by the method of Laemmli (18Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207478) Google Scholar) on 15% acrylamide (2.7% cross-linker) isocratic mini-slab gels. Two-dimensional, nonequilibrium pH gradient electrophoresis (19O'Farrell P.Z. Goodman H.M. O'Farrell P.H. Cell. 1977; 12: 1133-1141Abstract Full Text PDF PubMed Scopus (2583) Google Scholar) on mini-gels, immunoblotting onto polyvinylidene difluoride membrane (Immobilon P; Millipore Corp., Bedford, MA) for 1 h at 0.3 A, and immunostaining for ADF were performed as described previously(10Bamburg J.R. Minamide L.S. Morgan T.E. Hayden S.M. Giuliano K.A. Koffer A. Methods Enzymol. 1991; 196: 125-140Crossref PubMed Scopus (21) Google Scholar). Alkaline phosphatase-conjugated secondary antibody (Amersham Corp.) was used, and blots were developed after a quick rinse in high pH buffer (100 mM Tris, pH 9.5, 100 mM NaCl, 50 mM MgCl2), first with Lumiphos substrate (Boehringer Mannheim, Indianapolis, IN) and then in 0.165 mg/ml of 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt and 0.33 mg/ml of nitro blue tetrazolium chloride (Life Technologies, Inc.) diluted in the pH 9.5 buffer. Hyperfilm ECL (Amersham Corp.) exposures of the Lumiphos images and the stained blots were scanned using a Microscan 2000 image analysis system (Technology Resources, Inc., Knoxville, TN). Internal standards of chick brain ADF were included on all one-dimensional immunoblots. Chick skin fibroblasts, prepared by trypsin dissociation of skin from the dorsal side of 8-day embryos, were grown in 10-cm culture dishes in Dulbecco's modified Eagle's medium (Sigma) containing 10% fetal bovine serum. When 70% confluent, cells were transferred to a low phosphate medium (Dulbecco's modified Eagle's medium without phosphate) (Sigma) containing 10% fetal bovine serum that had been exhaustively dialyzed against 0.15 M NaCl. [32P]Orthophosphate (5 Ci/mmol; 100 εCi/ml culture medium) was added for 12 h. The cells were washed with 3 × 10 ml of 4°C phosphate-buffered saline, and then harvested in extraction buffer as described above. The cell lysate was added to a homogenate prepared from 6 g of 10-11-day chick embryo brain in 10 ml of the same buffer, and the mixture was further homogenized with five to six more strokes of a Teflon-glass homogenizer. The 32P-labeled pool of pADF was then purified as described under “Results.” For endopeptidase digestion, approximately 60 εg of precipitated ADF or pADF were solubilized in 20 εl of 8 M urea, 20 mM Tris-Cl buffer, pH 9.0. The volume was adjusted to 100 εl with 20 mM Tris-Cl, pH 9.0. Endopeptidase Lys-C (Wako Bioproducts, Richmond, VA) was added at an enzyme/substrate molar ratio of 1:50, and the mixture was incubated at 30°C for 8 h. Aliquots were removed periodically to evaluate the progress of digestion by SDS-PAGE. The digested samples were either immediately fractionated or frozen in liquid nitrogen and stored at −80°C for later use. For endopeptidase Asp-N digestion of the N-terminal fragment from Lys-C digestion, the appropriate fractions were lyophilized and resolubilized in 10 εl of 8 M urea, 50 mM sodium phosphate, pH 8.0. The volume was adjusted to 60 εl with 50 mM sodium phosphate, pH 8.0. Endopeptidase Asp-N (Boehringer Mannheim) was added at an enzyme/substrate weight ratio of 1:20. The mixture was incubated for 8 h at 36°C and then lyophilized. Peptides from Lys-C digests of ADF or [32P]pADF were fractionated by HPLC on a column of Microsorb-MV C18, 5 εm, 300-Å pore (5.6 mm × 25 cm) (Rainin Inst., Woburn, MA) eluted with a linear step gradient (2.0-37.5% B, 60 min; 37.5-75% B, 30 min; 75-90% B, 15 min; solvent A, 0.1% trifluoroacetic acid; solvent B 80% acetonitrile, 0.1% TFA) (Millipore, Waters Chromatography, Milford, MA). The flow rate was 0.5 ml/min, and the column effluent was monitored at 220 nm. Fractions (0.25 ml) were collected every 30 s, and Cerenkov radiation was measured. HPLC electrospray mass and mass were performed at the of on a mass with an electrospray at were in the The solvent and system have been described in Methods Enzymol. 1995; Scholar). fragment containing the full ADF cDNA was from M.E. Minamide L.S. Bamburg J.R. Biochemistry. 1990; PubMed Scopus Google Scholar) and the of mutagenesis J.D. R.A. Methods Enzymol. PubMed Scopus Google Scholar) was used to the Ser24 and to Ala using the the and mutant or were the modified expression as described M.E. Minamide L.S. Bamburg J.R. Biochemistry. 1990; PubMed Scopus Google Scholar, Methods Enzymol. 1990; PubMed Scopus Google Scholar). from containing the full ADF or were the site of the expression the and mutagenesis was performed on using the J.A. Anal. Biochem. PubMed Scopus Google Scholar). were used, in the Ser3 of the ADF cDNA Ser Ser a site, was also mutant were by U. S. Corp., were by Resources, expression of the and mutants was performed as described for the expression of ADF M.E. Minamide L.S. Bamburg J.R. Biochemistry. 1990; PubMed Scopus Google Scholar). The pellets were in 50 mM Tris, pH mM EGTA, mM 20 εl/ml protease inhibition T.E. Lockerbie R.O. Minamide L.S. Browning M.D. Bamburg J.R. J. Cell Biol. 1993; 122: 623-633Crossref PubMed Scopus (143) Google Scholar), and then in mg/ml on for 15 by of 15 mM mM and 8 DNase After at for 10 20 mM and 10 εl/ml protease inhibition mixture were After at × for 30 min at the was a column of and the was to a column of Corp., MA). The ADF was eluted with mM in 10 mM Tris, pH 7.6, 5 mM on a frozen in liquid and stored at HeLa cells were in medium (Sigma) containing 10% fetal bovine 50 and 50 Cells at on 10-cm dishes were transfected by the phosphate method J. Scholar) with 10 εg of containing ADF cDNA or one of the site-directed mutants of ADF The was removed after 12 h by 3 with phosphate-buffered saline, and of cells were prepared for SDS-PAGE h after as described above. We that the phosphorylated of purified from chick embryo had actin depolymerizing T.E. Lockerbie R.O. Minamide L.S. Browning M.D. Bamburg J.R. J. Cell Biol. 1993; 122: 623-633Crossref PubMed Scopus (143) Google Scholar). purified of pADF that ADF when incubated at T.E. Lockerbie R.O. Minamide L.S. Browning M.D. Bamburg J.R. J. Cell Biol. 1993; 122: 623-633Crossref PubMed Scopus (143) Google Scholar). the pADF be by alkaline we not demonstrate We that brain by boiling in 1% SDS, be completely reactivated after of and We used these to ADF from pADF following alkaline phosphatase treatment. ADF activity was after ADF activity was in the pADF of both samples that in the alkaline as T.E. Lockerbie R.O. Minamide L.S. Browning M.D. Bamburg J.R. J. Cell Biol. 1993; 122: 623-633Crossref PubMed Scopus (143) Google Scholar). The homogenate from the 32P-labeled chick skin and chick embryo brain was at × pADF was purified from the by chromatography on and hydroxylapatite SDS-PAGE of fractions and the demonstrate that [32P]pADF eluted in the for as identified by T.E. Lockerbie R.O. Minamide L.S. Browning M.D. Bamburg J.R. J. Cell Biol. 1993; 122: 623-633Crossref PubMed Scopus (143) Google Scholar). The fractions were in 0.1% SDS, and to 100 εl in a The volume was adjusted to 1 ml with M Tris-Cl, pH 7.6, and the protein was and with J. Methods Enzymol. 1990; PubMed Scopus Google Scholar). Proteins were precipitated D. Flügge U.I. Anal. Biochem. 1984; 138: 141-143Crossref PubMed Scopus (3185) Google Scholar) and in sample preparation buffer for on a SDS-PAGE mini-slab gel After proteins were by Bamburg J.R. Anal. Biochem. PubMed Scopus Google Scholar), and the pADF was from the pADF was a in 0.5 × SDS-PAGE buffer (18Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207478) Google Scholar) and to ml by of this was on SDS-PAGE to for ADF and pADF were D. Flügge U.I. Anal. Biochem. 1984; 138: 141-143Crossref PubMed Scopus (3185) Google Scholar), dissolved in buffer, and digested with endopeptidase Lys-C. The were by HPLC and Cerenkov in determined single to the pADF was identified single peptide contained of the onto the the for in the and a in sequence be by sequence analysis of the single from the same digest were that the peptide contained a N terminus and from the N terminus of a peptide containing only a single Ser The N-terminal Lys-C peptide from pADF contains only a single Ser and the sequence B is and is sequence that the N-terminal is removed and that the Ala is sequence was by mass spectrometric analysis as The isolated Lys-C of the was digested with Asp-N and on The peptide and two peptide of the were The of the two Asp-N are and as determined by electrospray mass spectrometric to the for from the sequence A, (mass B, (mass the peptide was identified by its mass of of the N-terminal peptide by mass the in The at the for that of the N-terminal peptide. The fragment and are in and are in the sequence confirming the of the peptide. The to the N-terminal fragment in the are the of phosphate as acid mass to The N-terminal peptide in was isolated by HPLC and with alkaline of the peptide by mass the in The at is that of the peptide mass an sequence are by mass to the of are the of acid to The fragment contains only one and the phosphorylation site on pADF is in the encoded of the phosphorylation site by mass A, N-terminal Asp-N peptide from B, same peptide after alkaline phosphatase treatment. peptide from the N-terminal and of the peptide. that are identified in the mass are in and B. acid B is phosphoserine in the image from HeLa cells, transfected with wild-type or expressed the transfected forms of ADF as on two-dimensional HeLa cell ADF only with the chick ADF the to the expressed of from cells wild-type ADF or the mutant show two the more one from the in vivo phosphorylation of ADF. of cells transfected with or not the phosphorylated forms of these confirming that Ser3 of the encoded protein is the regulatory site for phosphorylation in vivo. expressed forms of wild-type and were purified from of Proteins were on SDS-PAGE and were to brain ADF in depolymerizing F-actin. The a structural of the phosphorylated had F-actin depolymerizing activity that was 10% of the wild-type of proteins by phosphorylation is a for activity (for review, see D.A. P. S.M. 1991; PubMed Scopus Google (for review, see Cell. 1991; Full Text PDF PubMed Scopus Google and protein M. K. N. N. N. J. PubMed Scopus Google Scholar). ADF and cofilin H. K. Biochemistry. 1990; PubMed Scopus Google Scholar, H. Cell 1993; PubMed Scopus Google Scholar), and both can be to the of cells under E. K. N. S. Sakai H. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, S. H. J. Cell 1993; PubMed Scopus Google Scholar), phosphorylation in cells be in one or more of the to demonstrate that the phosphorylation of ADF its we to show that it can be reactivated by of the purified of pADF which ADF when incubated at T.E. Lockerbie R.O. Minamide L.S. Browning M.D. Bamburg J.R. J. Cell Biol. 1993; 122: 623-633Crossref PubMed Scopus (143) Google Scholar). However, by boiling the proteins with SDS, these are ADF can be reactivated after and by the activity of ADF in ADF is in chick muscle in vivo not in of in in vitro for to ADF expression by the ADF to the inactive which the of in vitro T.E. Lockerbie R.O. Minamide L.S. Browning M.D. Bamburg J.R. J. Cell Biol. 1993; 122: 623-633Crossref PubMed Scopus (143) Google Scholar). ADF with actin in the membrane of cells in containing actin J.R. D. J. Cell Biol. PubMed Scopus Google Scholar). of actin be by ADF show phosphorylation of both ADF and cofilin are by that are by and of with or the and of the actin and of both the regulatory of and J. Cell Biol. PubMed Scopus Google Scholar). treatment of of cells with and a of the actin as as of both ADF and J. Cell 1994; PubMed Scopus Google Scholar). the be by it that the of ADF is in both and cells by a cofilin in be by or by free not by of protein Biochem. J. 1994; PubMed Scopus Google Scholar). the very be in the protein this that ADF is a of actin in vivo and that the regulation of ADF by phosphorylation a and for actin in of high of ADF J. Cell 1994; PubMed Scopus Google Scholar) and cofilin Y. Nishida E. Sakai H. Miyamoto E. J. Biol. Chem. 1989; 264: 16143-16148Abstract Full Text PDF PubMed Google Scholar, J. Cell 1994; PubMed Scopus Google Scholar) in cells under the for the of cofilin and its to the of Y. C. S. S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google Scholar). with the of and cofilin and to the in However, of ADF and which occurs in cells in to J. Cell 1994; PubMed Scopus Google Scholar), is not by that is not for Ser24 in both ADF and cofilin a a site for phosphorylation as it is to a ADF and both of which have been to to the It was that the of cofilin at this site be in its Y. Nishida E. Sakai H. Miyamoto E. J. Biol. Chem. 1989; 264: 16143-16148Abstract Full Text PDF PubMed Google Scholar). However, ADF was not phosphorylated in vitro to with protein protein A, and T.E. Lockerbie R.O. Minamide L.S. Browning M.D. Bamburg J.R. J. Cell Biol. 1993; 122: 623-633Crossref PubMed Scopus (143) Google Scholar). The in which the mutant is expressed and phosphorylated in HeLa cells that Ser24 is not the regulatory site. We have identified the single regulatory phosphorylation site the chick brain ADF sequence as Ser3 of the encoded is a the ADF/cofilin in and K. J. Biol. Chem. 1988; Full Text PDF PubMed Google Scholar) and A.L. Janmey P. Louie K.A. Drubin D. J. Cell Biol. 1993; 120: 421-435Crossref PubMed Scopus (201) Google Scholar) have an amino acid this Ser and the Y. Mol. Biol. 1993; PubMed Scopus Google Scholar) have amino two of which are the S. Maciver S.K. C. S.K. D.A. J. Biochemistry. 1993; 32: PubMed Scopus Google Scholar), this Ser is to the and the only in which phosphorylation of ADF/cofilin proteins have been a sequence for the phosphorylation site to be a phosphorylation sequence not been Methods Enzymol. 1991; PubMed Scopus Google Scholar, J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). of the N-terminal Ser in been this Ser is by two which are to be in the phosphorylation of this cell regulatory M. K. N. N. N. J. PubMed Scopus Google Scholar). The protein is a of the actin membrane the of (for review, see A. Cell Biol. 1994; PubMed Scopus Google Scholar)). ADF is with actin in in actin of a mutant of in Drosophila and the of & 1994; PubMed Scopus Google Scholar). that inhibition from a in F-actin of a of also to inhibition of in this not with that contained or & 1994; PubMed Scopus Google Scholar). of is the E. H. Nature. 1994; PubMed Scopus Google Scholar), which we be the that is or for the phosphorylation of ADF. Drosophila ADF the phosphorylation sequence in ADF/cofilin from and are at with a in which ADF a in actin the actin of proteins in the ADF/cofilin family have been identified as in the of the N. Nishida E. M. M. H. Sakai H. J. Biochem. 1989; PubMed Scopus Google Scholar, N. Y. Sakai H. Nishida E. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar), that amino the N terminus also a as and Cell of a phosphate an actin the the of the is we can only on the phosphorylation of the the in the actin of ADF. It is to that to the N terminus amino in ADF M.E. Minamide L.S. Bamburg J.R. Biochemistry. 1990; PubMed Scopus Google Scholar) or the of a S. and J. in not the actin depolymerizing or F-actin of ADF. The of the regulatory phosphorylation site on ADF and the of site-directed mutants further the of this We are of cell which have been transfected with the and mutants and the of these on cell and We K. M. of of the of for in the mass and Resources, for protein We also for and and for of the