Aginactin, an Agonist-regulated F-actin Capping Activity Is Associated with an Hsc7O in Dictyostelium*

We have previously isolated an agonist-regulated actin filament capping activity, called aginactin, that is associated with a 70-kDa protein (Sauterer, R. A., Eddy, R. J., Hall, A. L., Coneelis, J. S. J. Biol. Chem. 266,24533-24539). A 2.0-kilobase clone isolated from a Dictyostelium Xgtll cDNA library screened with affinity-purified aginactin antibodies displays an overall sequence identity of 73% to the 70-kDa heat shock cognate protein, Hsc70, from various species. Aginactin capping activity and the 70-kDa protein bind to ATP-agarose columns and are quanti-tatively depleted from the load, indicating that an Hsc70 is associated with aginactin activity. Moderate stringency Southern blots indicate the presence of no fewer than six Hsc70-related sequences. Immunoflu- orescent staining of vegetative Dictyostelium AX3 cells with aginactin antibodies reveals a colocalization of aginactin-associated Hsc70 in F-actin-rich regions of the cell cortex and cell protrusions. Nuclei and or- ganelles lacked positive staining indicating that the aginactin-associated Hsc7O is cytosolic. The levels of cytoskeletal-associated Hsc70 correlate with the loss of barbed end capping activity following cAMP stimulation, suggesting that the uncapping of barbed fila- ment polyclonal as aginactin- associated 70-kDa protein SDS only the purified 70-kDa as described aginactin pool was electrophoresed on a 10% SDS-polyacrylamide gel and Western blotted onto nitrocellulose (Schleicher The location of the 70 kDa protein was determined by staining of the nitrocellulose with 0.1% ponceau S in 1% acetic acid. The 70 kDa band was excised, blocked, and incubated with aginactin polyclonal antisera for 16 h at 4 with Tris-buffered (TBS), purified polypeptide kDa


Chemoattractant-induced increases in actin polymerization have been observed in a variety of cells including neutrophils
macrophages (3), lung carcinoma cells (4,5), and Dictyostelium amoebas (6, 7). In these diverse cell types, the underlying mechanism for increases in actin polymerization following chemoattractant stimulation appears to be conserved. In Dictyostelium, cell lysates show a cytochalasinsensitive actin nucleation activity that exhibits a transient increase within 5 s of stimulation with the chemoattractant, CAMP. Following low speed centrifugation of cell lysates, the nucleation activity is found associated with the F-actin containing cytoskeletal pellet suggesting that the nucleation activity is associated with the barbed ends of actin filaments. The nucleation activity in the cytoskeletal pellet is stable, * This work was supported by research grants from the National Institutes of Health and the New York Lung Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted

L22736.
to the GenBankTM/EMBL Data Bank with accession number(s) $ To whom correspondence should be addressed. Tel.:  however, addition of the lysate supernatant to the pellet causes a rapid loss of nucleation activity, suggesting an inhibitor of actin nucleation is present in the supernatant (7). Upon stimulation with the chemoattractant CAMP, the inhibitor is regulated with kinetics reciprocal to the nucleation activity, suggesting that dissociation of a capping protein from the barbed or preferred end of F-actin is responsible for the increase in nucleation activity following stimulation.
To distinguish the agonist-regulated capping activity from other capping activities in Dictyostelium, the following purification strategy was devised. Since the agonist-regulated capping activity is at a minimum 5 s after stimulation with CAMP, lysate supernatants from unstimulated cells and cells lysed 5 s post-stimulation were run on parallel columns. Only the peak of capping activity that showed a significant decline after cAMP stimulation was pooled and used as the starting material for the next column. Following sequential DE52 anion-exchange, hydrophobic interaction, fast protein liquid chromatography anion-exchange, and hydroxyapatite chromatography, we have purified a 70-kDa protein associated with this cytosolic activity that inhibits actin nucleation and called it aginactin for AGonist-regulated INhibitor of ACTIN polymerization (8).
Aginactin is a barbed-end capping activity based on several criteria and is associated with a 70-kDa protein. Aginactin has an apparent Kd for capping of 2.7 nM, neither nucleates nor severs F-actin and is Ca2+ insensitive for all activities. In addition, the 70-kDa protein associated with aginactin can bind directly to actin filaments in a cosedimentation assay (8). In this paper, we present molecular genetic and biochemical evidence that the 70-kDa protein associated with aginactin is a heat shock cognate protein, Hsc70.

MATERIALS AND METHODS
In Vitro Actin Capping Assay-All polymerization assays were performed as described (7). Actin polymerization was monitored by the increase in fluorescence of pyrene-labeled actin in a mixture containing approximately 30% pyrene-labeled G-actin. The pyrenelabeled G-actin mixture was added to a final concentration of 2 p~ in APAB (10 mM PIPES,' pH 7.0, 50 mM KCI, 0.1 mM MgCl,, 2.5 mM EGTA, 0.5 mM ATP, 1 mM dithiothreitol). For capping assays, fluorescence was monitored and actin polymerization was initiated 2-5 min later by the addition of 0.5 p~ sheared F-actin seeds.
Protein Purification-Aginactin was isolated from Dictyostelium discoideum strain AX3 as described (8). ATP-agarose chromatography was performed as described (9) with slight modifications. The hydroxapatite pool containing active, purified aginactin was dialyzed against buffer D (20 mM Tris, pH 7.5, 20 mM NaCI, 0.1 mM EDTA, 3 mM MgC12,l mM dithiothreitol) and mixed with ATP-agarose resin equilibrated with buffer D. The mixture was then incubated at 4 "C for 1 h with gentle agitation, poured into a 1 X 4-cm Dispo column. The column was sequentially washed with 2 column volumes each of buffer D, 0.75 M NaCI, buffer D and 1 mM GTP to elute any GTP-' The abbreviations used are: PIPES, 1,4-piperazinediethanesulfonic acid kb, kilobase(s); bp, base pair(s). binding proteins. Bound Hsc70 was eluted with 3 mM ATP in buffer D, and fractions were analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting with aginactin antibodies.
Affinity Purification of Antibodies-A polyclonal antibody was raised against purified aginactin contained in the hydroxyapatite pool as described (8). The hydroxyapatite pool contained the aginactinassociated 70-kDa protein which was judged to be approximately 80% pure by Coomassie Blue staining of SDS gels and was the only major protein observed in the gels. Aginactin antibodies were affinity purified against the 70-kDa protein as described (10) with modifications. The hydroxyapatite aginactin pool was electrophoresed on a 10% SDS-polyacrylamide gel and Western blotted onto nitrocellulose (Schleicher & Schuell). The location of the 70 kDa protein was determined by staining of the nitrocellulose with 0.1% ponceau S in 1% acetic acid. The 70 kDa band was excised, blocked, and incubated with aginactin polyclonal antisera for 16 h at 4 "C with agitation. The pieces were washed with Tris-buffered saline (TBS), pH 7.5 (2 X 5 min), 1 M NaCl in TBS, pH 7.5 (1 X 5 min), and TBS, pH 7.5. Antibodies were eluted with 0.1 M glycine-HC1, pH 2.8, 1 mM EGTA for 2 min, immediately neutralized with 1 M Tris, pH 8.5, dialyzed into TBS, pH 7.5, and used for incubaticns. Affinity purified aginactin antibodies recognize a single polypeptide band at 70 kDa in Western blots of Dictyostelium AX3 whole cells, ATP-agarose purified Dictyostelium Hsc70, and bovine brain Hsc7O.
Protein An.alysis-SDS-polyacrylamide gel electrophoresis was performed as described (11). For Western blots, proteins were transferred to 0.45 p~ nitrocellulose membrane (Schleicher & Schuell) (12), incubated with affinity purified aginactin antibodies, and processed and labeled with lZ61-protein A as described (13). All autoradiographic images were scanned with an Ektron 1412 high resolution CCD camera, processed by Adobe Photoshop 2.5, and printed with a Sony Mavigraph Color Video Printer VP-500.
Isolation and Sequencing of cDNA Clones-A Xgtll cDNA library prepared against AX3 Dictyostelium discoideum amoebas ( t = 4) was kindly provided by P. Devreotes, Johns Hopkins University, and screened with affinity purified polyclonal aginactin antibodies labeled with '"I-protein A (ICN). Library screening and bacteriophage X isolation were performed as described by Maniatis et al. (14). X-DNA was digested with EcoRI and electrophoresed in 1% agarose in TBE, pH 8.0. Inserts were then subcloned into pBLUESCRIPT I1 SK+ phagemid vector (Stratagene) for sequencing. Sequencing reactions were performed using dideoxy chain termination method (Sequenase, Version 2.0, United States Biochemical Cor.) using T7, T3, and sequence-specific primers and confirmed by automated DNA sequencing (Applied Biosystems). For DNA and protein sequence analysis, FASTA and GAP programs included in the GCG-Wisconsin were used (15). To prepare for peptide microsequencing, purified aginactin associated 70-kDa protein was dissolved in 8 M urea, 0.4 M NHIHC03, reduced with with 45 mM dithiothreitol, cysteine alkylated with 100 mM iodoacetic acid, and digested with trypsin for 16 h as described (16). Peptides were resolved on a Vydac C18 reverse-phase high performance liquid chromatography column using a trifluoroacetic acid-acetonitrile gradient. Selected tryptic peptides were subjected to Edman degradation and microsequenced at Harvard Microchemistry Facility, Harvard University.
Immunofluorescence Microscopy-Dictyostelium AX3 cells were harvested and washed in PB (14.8 mM NaHzP04, 5.2 mM KZHP04, pH 6.6) and allowed to settle on ethanol-cleaned 12-mm circular coverslips (Fisher) for 30 min at 1 X lo6 cells/ml. The cells were then overlayed with a 1.5% agarose sheet as described (18), but not allowed to flatten, fixed for 10 min in 2.5% formaldehyde (Baxter) in PB, and then extracted in -20 "C acetone for 5 min. Coverslips were then washed briefly in PB, blocked for 15 min, and stained with 30 pg/ml affinity purified aginactin antibodies and 0.17 p~ rhodamine phal-loidin (Cappel) as described (19). Control antibody was prepared by incubating aginactin antibodies with 10-fold excess of purified 70-kDa protein immobilized on nitrocellulose for 16 h at 4 "C with gentle agitation. Fluorescein-labeled goat anti-rabbit IgG (Cappel) was prepared by preabsorption against formaldehyde fixed, acetone-extracted Dictyostelium AX3 cells (19), and used at 10 pg/ml. Cells were examined in a Bio-Rad MRCGOO scanning confocal microscope equipped with a Kr/Ar laser to ensure complete separation of fluorescein and rhodamine channels. 1.5-pm optical sections of stained cells were imaged with a Nikon 60 X 1.4 numerical aperture flat field objective on a Nikon Diaphot. Prints were made using a Sony Mavigraph Color Video Printer VP-500.
Preparation of Whole Cell Cytoskekton/Membrane Fraction-1 X lo7 Dictyostelium AX3 cells/ml were starved for 6 h in PB, treated with caffeine as described (7), and diluted to 3 X IO6 cells/ml with nanopure HzO to facilitate lysis. At various times following stimulation with 2' deoxy-CAMP (Sigma), cells were lysed by forced passage through a 3-pm Nucleopore filter (Millipore) (20). Lysis efficiency was >95% as determined by phase contrast microscopy. Lysates were spun in a microfuge at 8700 X g at 4 "C for 10 min. The supernatant was removed and the pellets resuspended in PB, Western blotted, and probed with affinity purified aginactin antibodies. Levels of aginactin-associated Hsc70 was quantitated by scanning laser densitometry of autoradiograms and normalized for total cell protein and cytoskeletal F-actin content. Pellets contained the cellular F-actin and unsealed plasma membrane sheets (data not shown).

RESULTS
Primary Structure of the 70-kDa Protein Associated with Aginuctin-To obtain the primary sequence of the 70-kDa protein, we cloned the respective cDNA from a Xgtll library (t = 4) prepared from the AX3 strain of D. discoideum. The library was screened with affinity purified antibodies raised against a highly purified hydroxylapatite pool of aginactin. A single clone of 2.0 kb, pAG-2.0, was isolated and sequenced. The pAG-2.0 cDNA has an overall length of 2,021 bp, contains an open reading frame of 1,908 bp coding for 636 amino acids, and terminates in a poly(A) tail (Figs. 1 and 2). Based on sequence comparison with other Hsc7Os, pAG-2.0 lacks the first 24 nucleotides (8 amino acids) from the 5' end, including the start AUG codon. Four additional positive clones were analyzed by EcoRI digestion and contained larger 5' truncations than pAG-2.0 and were not characterized further. pAG-2.0 also terminates on a rare TGA codon. An analysis of codon usage in 56 Dictyostelium genes has shown that the TGA codon occurred with a frequency of 1/17,921 total codons (21).
Comparison of the pAG-2.0 cDNA sequence with the GenEMBL data base using the FASTA program indicated the highest overall amino acid sequence identity (71-74%) was found between Hsc70 proteins from various eukaryotic species including human, rat, cow, and Drosophila. Fig. 3 shows a direct amino acid comparison between pAG-2.0 and Hsc70 from bovine brain and Drosophila using GAP program. Allowing for maximum alignment, aginactin displays an overall 74% identity, 84% similarity with bovine Hsc70 and 71% identity, 82% similarity with Drosophila Hsc7O.
All Hsc70s yet described are composed of two distinct domains. The ATPase domain which is contained in the NH2terminal 450 amino acids is the most highly conserved domain among all Hsc70s (22). As expected, the percent identity of pAG-2.0 within the ATPase domain of bovine and Drosophila Hsc70 was higher at 80 and 79%, respectively. The more variable COOH-terminal200 amino acids which contains the proposed substrate-binding domain was less conserved at 57.5 and 50%, respectively.
The deduced amino acid sequence of pAG-2.0 was confirmed by direct sequencing of peptides generated by tryptic cleavage of a purified pool of aginactin. Three separate internal tryptic peptides spanning a total of 30 amino acid residues

GCTGATCTTTTCCGTGGTTGTTTAGATCCAGTTG~GTATT~GATAGTAAATTGGATAAGAAATCAATTCATGAAATTGT~TA -G C L D P V E K V L K D S K L D K K S I H E I V L
GTTGGTGGTTCAACTCGTCTTCCAAAGGTACAACAATTATTACAAG~~TC~CAAT~TAAAGAATTGAATAAATCAATTAATCCAGAT Nucleotide sequence and deduced amino acid sequence derived from Dictyostelium Hsc70 derived from the cDNA clone pAG-2.0. The translated amino acid is shown in single letter code below the nucleotide sequence. Amino acid sequence is numbered on the left, and the nucleotide sequence is on the right. Tryptic peptides derived from purified Hac70 associated with aginactin was subjected to microsequencing. Exact matches between selected tyrptic peptides and the deduced amino acid sequence are underlined. matched precisely the deduced amino acid sequence of pAG-2.0 (Fig. 2).

TAAAGCTTCTCATATATAGTACACATT-
In order to determine the number of Hsc70-related sequences present in Dictyostelium AX3 cells, Southern blots were performed (Fig. 4). The probe fragment consisted of a 222-bp EcoRI/RsaI restriction fragment derived from nucleotide 90-312 of 5' end of pAG-2.0 and shared a >80% sequence identity to the highly conserved ATPase domain of various Hsc7O proteins. Of the eight restriction digests performed, EcoRI yielded the greatest number of bands (Fig. 4, lane 6 ) with three strongly hybridizing bands at 6.4, 6.2, and 3.3 kb and three less intense bands at ~1 5 , 8.5, and 4.7 kb under moderate stringency conditions, suggesting that Dictyostelium contains no fewer than six Hsc70-related sequences. Aginactin Binds to ATP-Agarose Affinity Columns-One unique property of all Hsc7O proteins is the binding to ATPagarose affinity columns and their elution with ATP (9). Since the deduced amino acid sequence of the pAG-2.0 and direct sequencing of tryptic peptides predicts that 70-kDa component of aginactin is an Hsc70, a hydroxyapatite pool of purified aginactin was analyzed by ATP-agarose chromatography. A Western blot of a representative experiment (Fig.   5B) shows binding of the 70-kDa immunoreactive band to the ATP-agarose column (lanes 1 and 2) and specific elution with  6 and 7). Capping assays performed on the ATP-agarose load and flow-through demonstrate that in the absence of ATP, approximately 63% of the aginactin capping activity is depleted from the load (Fig. 5A, shaded bar). However, in the presence of 0.4 mM ATP (open bur), only 2% of the capping activity is depleted from the load. Recovery of capping activity depleted from the ATP-agarose load was nonquantitative, with approximately 10-20% of the depleted capping activity recovered in the ATP eluent.
These results demonstrate that 1) antibodies prepared against aginactin recognize an Hsc7O and 2) the 70 kDa band and associated capping activity can bind to ATP-agarose and are specifically eluted with ATP suggesting that aginactin, an agonist-regulated, barbed-end capping activity, is associated with an Hsc70 in Dictyostelium. Based on these data and the high sequence identity with various Hsc70s, we conclude that the 70-kDa protein associated with aginactin is an Hsc7O of Dictyostelium.
Cytoskeletul Levels of Aginuctin-associated Hsc70 Correlate with Decreases in Capping Activity-To quantitate changes in the level of cytoskeletal associated Hsc70, AX3 cells were starved for 6 h and lysed through a Nucleopore filter at various times following CAMP stimulation. A membrane-cytoskeletal pellet fraction was obtained from lysates by low speed centrifugation, Western blotted, and probed with aginactin an- tibodies (Fig. 7). During the first 5-9 post-stimulation, the level of Hsc70 associated with the cytoskeleton decreases, mimicking the decrease in the level of capping activity. This suggests that the uncapping of barbed filament ends upon stimulation leads to a decrease in cytoskeletal-associated Hsc70 and a corresponding increase in actin nucleation sites in the actin cytoskeleton as reported elsewhere (7). pAG-2.0. Lanes 1, XbaI; 2, BglII; 3, ClaI; 4, NsiI; 5, BamHI; 6, EcoRI; DISCUSSION When cells are exposed to an external stress such as heat shock, they induce the synthesis of several classes of chaperone proteins that are thought to facilitate the stabilization and refolding of denatured proteins, assist in the proper assembly or disassembly of certain oligomeric protein complexes, and participate in maintaining precursor proteins destined for translocation across organellar membranes in an open, translocation-competent conformation (23). In recent years, the role of the 70-kDa heat shock protein class of chaperones has been intensively studied. This class includes forms that are strongly inducible by heat shock or other conditions of cellular stress (Hsp70) as well as forms which are constitutively expressed (Hsc7O). Hsc7O (heat shock cognate 70) is a protein chaperone that assists in preventing premature or inappropriate folding of nacent polypeptides. Several Hsc70 proteins have been described that possess essential functions in the absence of stress. These include the yeast cytosolic Ssa proteins and the mammalian Hsc70, both which can participate in the ATP-dependent disassembly of clathrin-coated vesicles. Organellar Hsp7Os can also be found within the mitochondria (Ssclp of yeast) and the endoplasmic reticulum (Kar2p in yeast and BiP in mammalian cells) (23).
In most eukaryotic organisms, Hsc70/Hsp70 proteins are members of multigene families. This complexity most likely reflects the involvement of certain members in stress response as well as constitutive and organellar functions. In Saccharomyces cereuisiae, eight distinct Hsp70-related genes have been  6 and 7). characterized which function in a variety of cellular compartments including endoplasmic reticulum, mitochondria, nuclei, and cytosol (22,23). Sequence analysis of the pAG-2.0 clone showed highest identity to the cytosolic Ssalp and Ssa2p (72%) and less identity to Ssblp, Kar2p, and Ssclp (56.5, 58.6, and 48.695, respectively) suggesting that the Hsc7O associated with aginactin is a cytosolic isoform. This is confirmed by the nonvesicular, cytosolic localization of Hsc7O by immunofluorescence in Dictyostelium. Southern blots of Dictyostelium DNA probed with sequence derived from the highly conserved NH2-terminal domain of pAG-2.0 clone suggest the presence of six Hsc7O-related sequences, three of which show a strong hybridization with this probe under moderate stringency hybridization conditions. A unique feature of Hsc70/Hsp70s is their ability to bind Time after Stimulation (s) FIG. 7. Correlation between cytoskeletal Hsc7O and levels of capping activity following chemotactic stimulation. Closed circles represent the levels of aginactin-associated Hsc70 present in the membrane-cytoskeletal pellet fraction of cell lysates following cAMP stimulation as measured by Western blotting with aginactin antibodies. Data shown were normalized for total cell protein. Normalization to cytoskeletal F-actin content gave similar results. The prestimulation level of Hsc7O is set at 100%. Closed squares represent the levels of capping activity (% inhibition) in cell lysate supernatants following cAMP stimulation as measured by pyrene-actin polymerization assay.
to ATP-agarose affinity resin and be eluted with ATP (9).
Aginactin antibodies stain the 70-kDa protein that binds to ATP-agarose columns and specifically elutes with ATP. The 70 kDa band and aginactin capping activity are both depleted by ATP-agarose columns indicating a strong association between aginactin capping activity and Hsc70.
All members of the 70-kDa class of heat shock proteins appear to contain certain structural and functional features. The first 450 NH2-terminal amino acids consist of a highly conserved ATPase domain. The more variable COOH-terminal 200 amino acids is thought to contain the substratebinding domain, however, this remains to be confirmed (22).
The atomic structures of both the 44-kDa NH2-terminal ATPase domain fragment from bovine Hsc70 (24) and rabbit skeletal actin (25) have been solved. Although there is little sequence identity between the two proteins, the structures are remarkably similar, particularly in regions involving ATP binding and hydrolysis (26). This would suggest that the properties of Stimulation of ATP hydrolysis upon ligand binding and dissociation of ligand following release of inorganic phosphate may be similar in both proteins. The role of ATP in actin polymerization appears to be the regulation of filament stability (27). As an ATP-actin monomer polymerizes, nucleotide hydrolysis is stimulated. The release of inorganic phosphate increases the dissociation rate of actin monomers from the barbed or preferred end of the actin filament (27). In addition, Hsc7O can form oligomers which are dissociated upon addition of ATP (28,29). Therefore, in a general sense, Hsc7Os are able to regulate their oligomeric state by a ATPase cycle analogous to that observed for actin. Based on this evidence, it is tempting to speculate that Hsc70s may function as actin-binding proteins through their actin-like domains. Acting as a pseudo-actin, the NH2-terminal ATPase domain of Hsc7O may bind to actin filaments and block the barbed end and/or to actin-binding proteins that recognize the barbed end, thereby regulating actin polymerization. However, this model is complicated by the COOH-terminal substrate recognition domain of Hsc70, which is proposed to mediate the interaction of Hsc70 with a diversity of target proteins (22) and may play a role in actin binding.
Several studies indicate that members of the Hsp7O family are actin-binding proteins in cells other than Dictyostelium. Immunofluorescence with a monoclonal antibody reactive against Hsp7O from rat embryo fibroblasts demonstrates that Hsp70 is concentrated at the leading edge of motile fibroblasts and codistributes with the F-actin-rich lamellopodia, cell cortex, and stress fibers in addition to a prominent nuclear localization (30). Colocalization with F-actin persists in Triton X-100-extracted cytoskeletons suggesting a direct binding to actin containing structures. Furthermore, actin in soluble extracts from nonmuscle (31) and muscle cells (32) binds specifically to Hsp7O affinity columns. A complex of actin and Hsp7O can also bind to Hsp7O antibody affinity columns. Actin is specifically eluted from these affinity columns with ATP (31).
The identification of an Hsc70 associated with an agonistregulated barbed-end F-actin capping activity provides important clues to the understanding of actin dynamics in chemotactic cells in addition to the function of Hsc7O proteins in cellular processes. The levels of Hsc70 associated with the cytoskeleton correlate with the loss of actin capping activity and increase in actin nucleation in cells 5 s following cAMP stimulation. During this peak of nucleation activity, Dictyostelium amoebas undergo a morphological change referred to as the freeze response that corresponds to a global polymerization of actin in the cell cortex (33,34). Antibodies generated against aginactin react with an Hsc7O and Hsc7O can be localized to F-actin-rich regions of Dictyostelium including the cell cortex and cell protrusions. These data lend support to the model where chemotactic stimulation leads to an increase in nucleation activity by uncapping of barbed filament ends, subsequent monomer addition, and filament elongation, followed by recapping of barbed ends. The mechanism by which Hsc7O can regulate polymerization at the barbed end in an agonist-regulated fashion is currently under investigation.