©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Reactivation of Phosphorylated Actin Depolymerizing Factor and Identification of the Regulatory Site (*)

(Received for publication, February 21, 1995; and in revised form, April 24, 1995)

Brian J. Agnew , Laurie S. Minamide , James R. Bamburg (§)

From the Department of Biochemistry and Molecular Biology and the Program in Neuronal Growth and Development, Colorado State University, Fort Collins, Colorado 80523

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

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, [P]pADF was purified and digested with endoproteinase Lys-C. The digest contained only one P-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 (Ser 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 or Thr, express and phosphorylate the exogenous ADF. Cells also expressed high levels of mutant ADF when Ser was deleted or converted to either Ala or Glu. However, none of these mutants was phosphorylated, confirming that Ser in the encoded ADF is the single in vivo regulatory site.


INTRODUCTION

Actin depolymerizing factor (ADF),()is an 18.5-kDa protein with a pH-dependent F-actin binding/depolymerizing activity (1, 2) very similar to that of its structural homolog, cofilin(3, 4) . Proteins in the ADF/cofilin family appear to be ubiquitous in eukaryotes (for review, see (5) ). Mutations that inactivate the ADF/cofilin gene in Saccharomyces cerevisiae(6) , Drosophila melanogaster(7) , and Caenorhabditis elegans(8) are lethal.

Phosphorylated forms of both ADF and cofilin have been identified in vertebrate cells(9, 10, 11) . We isolated phosphorylated ADF and showed it to be inactive in depolymerizing F-actin and in inhibiting the assembly of G-actin(11) . Phosphoamino acid analysis identified phosphoserine as the phosphorylated amino acid(11) . 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.


MATERIALS AND METHODS

Purification of Proteins

pADF was isolated free from ADF and cofilin from embryonic day 10 (E10) or E11 chick brain as described previously(11) . 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.(12) . Skeletal muscle actin was purified from rabbit muscle acetone powder(13) .

Dephosphorylation of pADF

The in vitro reactivation of pADF by treatment with alkaline phosphatase (Sigma) is described under ``Results.''

ADF Activity Assay

The amount of G-actin depolymerized from F-actin in the presence of ADF was quantified by using the DNase I inhibition assay(10, 14) .

Sample Preparation for Electrophoresis

Cells in 10-cm culture dishes were scraped in 300 µl 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 µl/ml protease inhibition mixture)(11) , sonicated, and immersed 5 min in a boiling water bath. After cooling the samples on ice, proteins were precipitated with chloroform/methanol(15) . 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 (16) . Protein concentrations were determined by the solid phase dye-binding method(17) .

Gel Electrophoresis and Immunoblotting

SDS-PAGE was performed by the method of Laemmli (18) on 15% acrylamide (2.7% cross-linker) isocratic mini-slab gels. Two-dimensional, nonequilibrium pH gradient electrophoresis (19) 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(10) . 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 MgCl), 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.

Metabolic Labeling of Chick Skin Fibroblasts

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. [P]Orthophosphate (5 Ci/mmol; 100 µCi/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 P-labeled pool of pADF was then purified as described under ``Results.''

Protease Digests

For endopeptidase digestion, approximately 60 µg of precipitated ADF or pADF were solubilized in 20 µl of 8 M urea, 20 mM Tris-Cl buffer, pH 9.0. The volume was adjusted to 100 µl 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 µl of 8 M urea, 50 mM sodium phosphate, pH 8.0. The volume was adjusted to 60 µl 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.

HPLC Separation of Peptides

Peptides from Lys-C digests of ADF or [P]pADF were fractionated by HPLC on a column of Microsorb-MV C18, 5 µm, 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.

Mass Spectrometry

HPLC electrospray mass spectrometry and tandem mass spectrometry were performed at the Beckman Institute, City of Hope, Duarte, CA on a TSQ-700 triple quadrupole mass spectrometer (Finnigan-MAT, San Jose, California), equipped with an electrospray ion source operating at atmospheric pressure. Mass spectra were recorded in the positive ion mode. The solvent delivery system, fused silica capillary columns, and UV detection system have been described in detail(20) .

Plasmids and Site-directed Mutagenesis

A 656-base pair EcoRI/AvrII fragment containing the full ADF cDNA was excised from pUC 19 (21) and cloned into the EcoRI-XbaI sites of M13mp18. Oligonucleotide-directed point mutagenesis (22) was used to mutate the Ser and Thr codons to Ala using the mutagenic primers 5`-CCTCAGGCGCTGCGCATTTCCG-3`, 5`-CCTCAGGCGTTGCGCCATTT-3`, 5`-CCTCAGGCGCAGAGCATTTCCG-3` giving the ADF(A24,25), ADF(A24), and ADF(A25) mutant cDNAs, respectively. NcoI/HindIII fragments encoding ADF(A24,25), ADF(A24), or ADF(A25) were subcloned into the modified pBR 322 bacterial expression vector (pET) as described previously(21, 23) . BamHI/AvrII fragments (560 base pairs) from pET containing the full ADF or ADF(A24,25) cDNAs were cloned into the multi-cloning site of the eukaryotic expression vector pcDNA3 (Invitrogen, San Diego, CA), giving the vectors pcDNA3ADF and pcDNA3ADF(A24,25). Site-directed mutagenesis was performed on pcDNA3ADF using the Transformer Mutagenesis Kit (Clontech, Palo Alto, CA)(24) . Three mutagenic primers were used, encoding mutations in the Ser codon of the ADF cDNA (Ser Ala, 5`-CTTGTACTCCAGCTGCCATGGATC-3`; Ser Glu, 5`-CGGCAACTTGTACTCCTTCTGCCATGGATCCGAGC-3`; Ser deletion, 5`-CGGCAACTTGTACTCCTGCCATGGATCCGAGC-3`). A selection primer, eliminating a unique pcDNA3 HindIII restriction site, was also synthesized (5`-GGTACCAAGCAAGGGTCTCCC-3`). All mutant cDNAs were confirmed by DNA sequencing (Sequenase Version 2.0 DNA Sequencing Kit, U. S. Biochemical Corp., Cleveland, OH). All oligonucleotides were synthesized by Macromolecular Resources, Colorado State University, Fort Collins, CO.

Bacterial Expression and Isolation of Recombinant Proteins

Bacterial expression of the ADF(A24,25) and ADF(E3) mutants was performed as described previously for the expression of ADF (21) . The postinduction bacterial pellets were resuspended in 50 mM Tris, pH 8.0, 2.0 mM EGTA, 2.4 mM phenylmethylsulfonyl fluoride, 20 µl/ml protease inhibition mixture(11) , and then lysed in 2.0 mg/ml lysozyme on ice for 15 min, followed by addition of 15 mM MgCl, 0.2 mM MnCl, and 8 µg/ml DNase I. After incubation at room temperature for 10 min, 20 mM DTT and 10 µl/ml protease inhibition mixture were added. After centrifugation at 60,000 g for 30 min at 4 °C, the supernatant was passed through a column of DEAE-cellulose (DE-52; Whatman), and the flow-through was applied to a column of Green A-agarose (Amicon Corp., Danvers, MA). The ADF was eluted with 175 mM NaCl in 10 mM Tris, pH 7.6, 5 mM DTT, concentrated on a Centricon-10 (Amicon Corp.), frozen in liquid nitrogen, and stored at -80 °C.

Cell Transfections

HeLa cells were cultured in minimal essential medium (Sigma) containing 10% fetal bovine serum, 50 units/ml penicillin, and 50 µg/ml streptomycin. Cells at 30-50% confluency on 10-cm dishes were transfected by the calcium phosphate method (25) with 10 µg of pcDNA3 vector containing ADF cDNA or one of the site-directed mutants of ADF cDNA. The precipitate was removed after 12 h by washing 3 times with phosphate-buffered saline, and extracts of cells were prepared for SDS-PAGE 48 h after transfection as described above.


RESULTS

pADF Is Reactivated by Alkaline Phosphatase

We previously demonstrated that the phosphorylated form of ADF, partially purified from chick embryo brain, had no actin depolymerizing activity(11) . Partially purified preparations of pADF contain proteases that degrade ADF when incubated at 37 °C(11) . While the SDS-denatured pADF could be dephosphorylated by alkaline phosphatase, we did not demonstrate reactivation. We found that brain ADF, denatured by boiling in 1% SDS, could be completely reactivated after removal of SDS and renaturation. We used these conditions to renature ADF produced from denatured pADF following alkaline phosphatase treatment. Complete ADF activity was regained after dephosphorylation, whereas no ADF activity was found in the pADF control (Fig. 1). Electrophoresis of both samples confirmed that dephosphorylation occurred in the alkaline phosphatase-treated sample, as previously reported(11) .


Figure 1: Reactivation of pADF by alkaline phosphatase. Partially purified pADF, which was eluted from an HAP column, contained no cofilin or ADF as determined by two-dimensional gel electroblotting. It was boiled in 1% SDS to inactivate protease and phosphatase activity. The protein was precipitated, dissolved in SDS, and treated with 10 units of alkaline phosphatase () in the presence of a 5-fold excess of Triton X-100 to bind excess SDS. After 60 min at 37 °C, the protein was reprecipitated, solubilized in 8 M urea, and dialyzed against 10 mM Tris, pH 7.5 containing 1 mM DTT. A control sample of pADF () was treated identically, except no alkaline phosphatase was added. Activity was assayed by the DNase I inhibition assay(10, 14) . Actual ADF content of the partially purified sample was determined by quantitative immunoblotting. The theoretical curve for 100% activity (dashedline) and points obtained with renatured recombinant ADF () are shown for comparison.



Purification of pADF

The combined homogenate from the P-labeled chick skin fibroblasts and chick embryo brain was centrifuged at 100,000 g. pADF was partially purified from the supernatant by chromatography on DEAE-cellulose, Green A-agarose, and hydroxylapatite (HAP). SDS-PAGE of HAP fractions (Fig. 2A) and the corresponding autoradiograph (Fig. 2B) demonstrate that [P]pADF eluted in the position expected for pADF, as previously identified by immunoblotting(11) . The pADF-containing fractions were boiled in 0.1% SDS, combined, and concentrated to less than 100 µl in a Centricon-10 microconcentrator. The volume was adjusted to 1 ml with 0.1 M Tris-Cl, pH 7.6, and the protein was reduced, and alkylated with 4-vinylpyridine(26) . Proteins were precipitated (15) and redissolved in sample preparation buffer for separation on a preparative SDS-PAGE mini-slab gel (0.5 mm). After electrophoresis, proteins were visualized by KCl precipitation(27) , and the pADF band was excised from the gel. pADF was electroeluted into a Centricon-10 chamber in 0.5 SDS-PAGE running buffer (18) and concentrated to about 0.1 ml by centrifugation. An aliquot of this material was rerun on SDS-PAGE to check for homogeneity (Fig. 2C).


Figure 2: Purification of pADF. A, fractions from the HAP column were subjected to SDS-PAGE and silver stained. The pADF ran just slightly above the ADF standard (S) with the major amounts eluting in fractions 4-6. B, autoradiograph of the gel in A showing that [P]orthophosphate was incorporated into the pADF. C, Coomassie Blue-stained gel of the pooled HAP fractions (4, 5, 6) before (lane1) and after (lane2) reduction and alkylation. The pADF has a slightly slower mobility than the ADF standard (compare lanesS and 1), which is amplified following alkylation with 4-vinylpyridine (lane2). The purified electroeluted pADF is shown in lane3.



pADF Contains Only a Single Phosphopeptide

Electroeluted ADF and pADF were precipitated(15) , dissolved in buffer, and digested with endopeptidase Lys-C. The resulting peptides were separated by HPLC and Cerenkov radioactivity in each fraction determined (Fig. 3). A single phosphopeptide, unique to the pADF digest, was identified (Fig. 3, arrow). This single peptide contained about 60% of the total radioactivity loaded onto the column, the remainder being accounted for in the unbound fraction and a few small peaks visible in Fig. 3. No sequence could be obtained by direct sequence analysis of the single phosphopeptide, whereas neighboring peptides from the same digest were readily sequenced. This suggested that the labeled peptide contained a blocked N terminus and might arise from the N terminus of ADF, a peptide containing only a single Ser residue.


Figure 3: HPLC peptide maps comparing endopeptidase Lys-C digests of purified brain ADF and pADF. The position of the single P-labeled phosphorylated peptide is shown by the arrowhead with the CPM in each fraction shown below.



Identification of the Phosphorylation Site by Mass Spectrometry

The N-terminal Lys-C peptide from pADF contains only a single Ser and has the sequence Ac-ABGVQVADEVJRIFYDMK, where B is phosphoserine, and J is pyridylethylcysteine. This sequence suggests that the N-terminal Met is removed and that the penultimate Ala is acetylated. This sequence was confirmed by mass spectrometric analysis as follows. The isolated Lys-C phosphopeptide (containing some of the contaminating trailing peak) was digested with Asp-N and rerun on HPLC. The undigested contaminating peptide and two major peptide fragments of the phosphopeptide were obtained (Fig. 4). The parent ion masses of the two major Asp-N peptides are 753.6 and 1150.3, as determined by electrospray mass spectrometric analysis. These correspond to the masses expected for peptides derived from the sequence shown above: A, Ac-ABGVQVA (mass 753); B, DEVJRIFY (mass 1150). In addition, the C-terminal peptide (C) DMK was identified by its mass of 393. Analysis of the N-terminal peptide by tandem mass spectrometry produced the spectrum in Fig. 5A. The parent ion (P) at m/z 753 matches the value predicted for that of the N-terminal peptide. The predicted fragment ions, type b and y are shown in Fig. 5C, and those ions observed are labeled in Fig. 5A. Fragment ions b through b yield the C-terminal sequence -VQVA, confirming the origin of the peptide. The b ion m/z corresponds to the N-terminal fragment Ac-ABG-. Also apparent in the spectrum are ion species representing the loss of phosphate as phosphoric acid (98 mass units) due to collision. The N-terminal peptide (A) in Fig. 4was isolated by HPLC and treated with alkaline phosphatase. Analysis of the resulting peptide by tandem mass spectrometry yielded the spectrum in Fig. 5B. The parent ion at m/z 673 is that of the dephosphorylated peptide (-80 mass units). Fragment ions b through b yield an identical C-terminal sequence but are decreased by 80 mass units due to the loss of phosphate. Additionally, there are no peaks representing the loss of phosphoric acid due to collision. The b fragment (AcASG-) contains only one phosphorylatable residue, Ser, and therefore, the phosphorylation site on pADF is Ser (Ser in the encoded protein).


Figure 4: HPLC-electrospray mass spectrometry of the Lys-C phosphopeptide following digestion with the endopeptidase Asp-N. The sequence of the N-terminal Lys-C peptide from pADF is shown above with the expected Asp-N peptides underlined. Peptides A, B, and C all arose from the Lys-C peptide. Peptide D was a contaminating peak arising from the large Lys-C peptide, which eluted directly after the phosphopeptide. This peptide contained no Asp and was identified by direct sequencing of the peak from the Lys-C peptide fractionation shown in Fig. 3.




Figure 5: Identification of the phosphorylation site by tandem mass spectrometry. A, N-terminal Asp-N peptide from pADF. B, same peptide after alkaline phosphatase treatment. C, peptide fragments expected from the N-terminal end (b) and C-terminal end (y) of the peptide. Those that are readily identified in the mass spectra are labeled in A and B. p, parent ion. Amino acid B is phosphoserine (serine in the dephosphorylated peptide).



Confirmation of Seras the in Vivo Regulatory Site by Site-directed Mutagenesis

Extracts from HeLa cells, transiently transfected with pcDNA3 carrying wild-type ADF, ADF(A24,25), ADF(A3), ADF(E3), or ADF(3) expressed the transfected forms of ADF as shown on two-dimensional immunoblots (Fig. 6). Endogenous HeLa cell ADF only weakly cross-reacts with the chick ADF antiserum (Fig. 6, 1a), eliminating the need to epitope tag the exogenously expressed protein. Two-dimensional immunoblots of extracts from cells expressing wild-type ADF (Fig. 6, 2a) or the ADF(A24,25) mutant (Fig. 6, 1b) typically show two species, the more acidic one arising from the in vivo phosphorylation of ADF. Extracts of cells transfected with ADF(A3) (Fig. 6, 2b), ADF(E3) (Fig. 6, 3a), or ADF(3) (Fig. 6, 3b) do not contain the phosphorylated forms of these proteins, confirming that Ser of the encoded protein is the regulatory site for phosphorylation in vivo.


Figure 6: Two-dimensional immunoblot analysis of ADF species in extracts of HeLa cells transfected as follows. 1a, nothing; 2a, wild-type chick ADF; 3a, ADF(E3) mutant; 4a and 4b, mixture of samples in 2a and 3a; 1b, ADF(A24,25) mutant; 2b, ADF(A3) mutant; 3b, ADF(3) mutant.



Bacterial Expression and Characterization of Mutants of ADF

Bacterially expressed forms of wild-type ADF, ADF(A24), ADF(A25), and ADF(E3) were purified from supernatants of bacterial lysates. Proteins were homogeneous on SDS-PAGE (not shown). Wild-type ADF, ADF(A24) and ADF(A25) were identical to brain ADF in depolymerizing F-actin. The ADF(E3) mutant, a structural homolog of the phosphorylated ADF, had F-actin depolymerizing activity that was about 10% of the wild-type (Fig. 7).


Figure 7: Activity of the recombinant ADF(E3) is reduced to about 10% of that of wild-type ADF. Bacterially expressed wild-type ADF and the ADF(E3) mutant were purified and assayed for their ability to depolymerize F-actin in the DNase I inhibition assay. The equivalent inhibition of DNase I was achieved with 0.4 µg of ADF and 4 µg of ADF(E3). Each point is the average of triplicate assays.




DISCUSSION

Regulation of proteins by phosphorylation is a well established mechanism for controlling biological activity (for review, see (28) ), nuclear translocation (for review, see (29) ), and protein degradation(30) . Since ADF and cofilin contain identical nuclear translocation signal sequences(31, 32) , and both can be transported to the nucleus of cells under stress(33, 34) , their phosphorylation in cells may be involved in regulating one or more of the above events. In order to demonstrate that the reversible phosphorylation of ADF controls its cellular activity, we needed to show that it can be reactivated by removal of the phosphate. Partially purified preparations of pADF contain proteases, which degrade ADF when incubated at 37 °C(11) . However, by boiling the proteins with SDS, these proteases are permanently inactivated. ADF can be reactivated after denaturation and dephosphorylation.

Regulation by phosphorylation/dephosphorylation may control the activity of ADF in cells. ADF is normally down-regulated in developing chick skeletal muscle in vivo but not in primary myocyte cultures of equivalent age in vitro. Interestingly, myotubes developing in vitro compensate for their inability to down-regulate ADF expression by converting the abundant ADF to the inactive form, pADF, which accumulates during the process of in vitro myogenesis(11) . ADF colocalizes with actin in the membrane ruffles of motile cells and, in developing neuronal growth cones, areas containing highly dynamic actin filaments(35) . Rapid cycling of actin assembly/disassembly could be regulated by ADF phosphorylation.

Recent studies show phosphorylation of both ADF and cofilin are altered by various signal transduction pathways that are activated by growth factors and pharmacological agents. Treatment of cultured rat primary astrocytes with dibutyryl-cAMP or the myosin light chain kinase inhibitor, ML-9, causes dramatic shape changes and reorganization of the actin filament network and increased dephosphorylation of both the regulatory light chain of myosin and ADF(36) . Additionally, treatment of primary cultures of dog thyroid cells with thyrotropin causes distinct morphological changes and a redistribution of the actin stress fiber network, as well as rapid dephosphorylation of both ADF and cofilin(37) . Since the thyrotropin effects could be mimicked by forskolin, it seems likely that the dephosphorylation of ADF is regulated in both astrocytes and thyroid cells by a cAMP-dependent process. Additionally, cofilin dephosphorylation in human platelets could be stimulated by GTPS or by free calcium but not by activation of protein kinase C(38) . Again, the GTPS effects could very well be mediated through increases in cAMP via the protein G. Together, this evidence suggests that ADF is a major regulator of actin filament dynamics in vivo and that the regulation of ADF by phosphorylation may provide a rapid, localized, and reversible control mechanism for regulating actin filament dynamics in areas of high filament turnover.

Dephosphorylation of ADF (37) and cofilin (9, 37) accompanies nuclear transport in many cells placed under stress. In addition, the costimulatory signals for human T-cell activation induce the dephosphorylation of cofilin and its translocation to the nucleus of human T-cells(39) . Cofilin dephosphorylation correlates with the induction of interleukin-6 responsiveness and interleukin-2 secretion. Interestingly, cofilin dephosphorylation and translocation to the nucleus occur spontaneously in autonomously proliferating T-lymphoma cells. However, dephosphorylation of ADF and cofilin, which occurs in thyroid cells in response to thyrotropin(37) , is not accompanied by nuclear translocation, suggesting that dephosphorylation is not sufficient for nuclear uptake.

Ser in both ADF and cofilin exists within a conserved CaM kinase II consensus sequence. This seemed a likely site for phosphorylation as it is situated next to a nuclear translocation sequence, conserved between ADF and cofilin, both of which have been shown to translocate to the nucleus. It was suggested that the dephosphorylation of cofilin at this site may be involved in its nuclear translocation(9) . However, ADF was not phosphorylated in vitro to any significant extent with several common kinases including CaM kinase II, protein kinase C, protein kinase A, and myosin light chain kinase(11) . The studies reported here in which the ADF(A24,25) mutant is expressed and phosphorylated in HeLa cells demonstrates convincingly that Ser is not the regulatory site.

We have identified the single regulatory phosphorylation site within the chick brain ADF primary sequence as Ser of the encoded protein. This is a conserved residue within the ADF/cofilin family, present in mammals, birds, amphibians, and insects (Fig. 8). Echinoderms (40) and yeast (6) have an extra amino acid between this Ser and the initiating Met, while plants (41) have four extra amino acids, two of which are Ser. In the amoeba protein, actophorin(42) , this Ser is adjacent to the initiating Met. In mammals, birds, and amphibians, the only classes so far in which phosphorylation of ADF/cofilin proteins have been demonstrated, a consensus sequence for the phosphorylation site appears to be N-ASGVXVXD. Such a phosphorylation consensus sequence has not been reported(43, 44) . Although autophosphorylation of the N-terminal penultimate Ser residue in c-mos has been characterized, this Ser is surrounded by two Pro residues, which are likely to be involved in determining the phosphorylation specificity of this meiotic cell cycle regulatory protein(30) .


Figure 8: N-Terminal sequences for proteins in the ADF/cofilin family from different sources. Sequences are compared with human ADF and are aligned for maximum homology around Ser. -, gap in sequence for alignment; :, identical amino acid with the human ADF. Reference numbers refer to references at end of manuscript. XenopusADF was as described in Footnote 3.



The small GTP-binding protein Rac1 is a key regulator of the rapid actin reorganization accompanying membrane ruffling during the early phases of growth factor-induced signal transduction (for review, see (45) ). ADF is co-localized with actin in regions where Rac1 induces dramatic alterations in actin dynamics. Overexpression of a constitutively active mutant of Rac1 in Drosophila neurons inhibits nerve growth and increases the rhodamine-phalloidin staining of growth cones(46) . This finding suggests that growth inhibition may arise from a decrease in F-actin turnover. Conversely, overexpression of a dominant negative form of Rac1 also led to inhibition of nerve growth, but in this case growth cones did not stain with rhodamine phalloidin, suggesting that they contained little or no F-actin(46) . One downstream effector of Rac1 is the serine/threonine kinase, p65(47) , which we believe could be the kinase that is directly or indirectly responsible for the phosphorylation of ADF. Interestingly, Drosophila ADF has the phosphorylation consensus sequence found in ADF/cofilin from mammals, birds, and amphibians (Fig. 8). These results are at least consistent with a model in which ADF plays a prominent role in growth cone actin dynamics.

Although the major actin binding regions of proteins in the ADF/cofilin family have been identified as domains in the C-terminal half of the protein(48, 49) , recent evidence suggests that amino acids near the N terminus also play a role.()Introduction of a phosphate group nearby an actin binding domain could directly affect the interactions, but until the structure of the entire molecule is resolved, we can only speculate on how the phosphorylation of the penultimate serine brings about the changes in the actin binding properties of ADF. It is interesting to note, however, that extensions to the N terminus (three amino acids in recombinant ADF (21) or the addition of a 26-kDa glutathione S-transferase fusion protein)()do not inhibit the actin depolymerizing or pH-sensitive F-actin binding of ADF.

The identification of the regulatory phosphorylation site on ADF and the development of site-directed mutants will enable further investigation into the cellular role of this regulation. We are currently isolating stable, clonal populations of various cell lines, which have been transfected with vectors expressing the ADF(A3) and ADF(E3) mutants and will examine the effects of these mutations on actin-based functions including cell motility, neurite outgrowth, and nuclear rod formation.


FOOTNOTES

*
This work was supported in part by National Institutes of Health (NIH) Grants GM35126 and TW01856 (to J. R. B.) and by NIH Grants RR06217 and CA33572. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 970-491-0425; Fax: 970-491-0494; jbamburg{at}vines.colostate.edu

The abbreviations used are: ADF, actin depolymerizing factor; E10, embryonic day 10; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography; HAP, hydroxylapatite; GTPS, guanosine 5`-3-O-(thio)triphosphate.

T. Obinata, personal communication. Presented as abstract (Kusano, K., Abe, H., and Obinata, T. (1993) Cell Struct. Funct.18, 639).

H. Abe, T. Obinata, L. S. Minamide, and J. R. Bamburg, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Dr. K. M. Swiderik, Department of Immunology, Beckman Research Institute of the City of Hope, Duarte, CA for expert technical assistance in obtaining the mass spectrometry results presented here and Craig Miles, Macromolecular Resources, Colorado State University, for protein sequencing. We also thank Judith Sneider for technical assistance and Drs. Norman Curthoys, Barbara Bernstein, and A.-Young Woody for critical reading of the manuscript.


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