Eur. J. Biochem. 234, 1-7 (1995) 0 FEBS 1995

Identification of two sites in gelsolin with different sensitivities to adenine nucleotides Lorraine E. LAHAM I , Michael WAY ’, Helen L. YIN ’ and Paul A. JANMEY ’ ’ Division of Experimental Medicine,

Department of Medicine, Brigham and Women’s Hospital and the Program i n Cell and Developmental Biology, Harvard Medical School, Boston, USA

Department of Physiology, The University of Texas Southwestern Medical Center, Dallas, USA * European Molecular Biology Laboratory, Heidelberg, Germany

(Received 13 July 1995) - EJB 95 1149/1

The affinity of monomeric actin for several actin-binding proteins, including gelsolin, depends on adenine nucleotides. Gelsolin binds faster and with higher affinity to ADP-actin than to ATP-actin. Here, we show that the C-terminal actin-binding domain of gelsolin, which is required for filament nucleating activity but not for filament severing activity, contains the site that distinguishes between ATP-actin and ADP-actin monomers.

In contrast, actin binding to the N-terminal half of gelsolin depends on solution ATP concentrations, but not on the nucleotide (ATP or ADP) tightly bound in the cleft of the actin monomer. Binding is stronger in the absence of free nucleotide or i n the presence of 0.5 mM ADP than in solutions containing 0.5 mM ATP. Complexes formed using different nucleotide concentrations differ in their filament-severing activities as well as in their abilities to increase the fluorescence of 4-chloro-7-nitrobenzeno-2-oxa-l,3- diazole-labeled actin monomers. These results suggest that, at physiologic concentrations of nucleotides, both free and actin-bound ATP may affect the binding of actin to its accessory proteins and that gelsolin, actin, or the gelsolin-actin complex, contains a low-affinity nucleotide-binding site.

Keywords: gelsolin ; actin ; nucleotides ; ATP.

Actin monomers bind ATP with nanomolar affinity and ex- change ATP at a low rate (for reviews, see [I -41). Bound ATP is hydrolyzed to ADP and P, after actin monomers assemble into filaments, a reaction which generates both ATP-actin and ADP- actin in cells [5 , 61. One possible consequence of the ATPase activity of actin is that the actin-bound ATP or actin-bound ADP regulates the binding of proteins to actin monomers (G-actin) and polymers (F-actin). For example, thymosin /74 171 has a higher affinity for ATP-actin than for ADP-actin in vitro, while gelsolin [8], actophorin [91 and actin-depolymerizing factor [ 1 O] bind ADP-actin tighter than ATP-actin. Under some conditions, profilin is reported to bind ATP-actin more tightly [ I l l , while under other conditions, profilin binds ADP-actin more tightly than ATP-actin [12]. The ability of profilin to accelerate nucleo- tide exchange from actin may also be part of its physiologic function 112-141.

Gelsolin belongs to a family of actin-binding proteins that sever and cap actin filaments, and have characteristic conserved repeating domain structures [I 5 - 191. In addition to reactions with actin filaments, gelsolin can nucleate new filament poly-

Correspondence to L. E. Laham, Dept. of Cellular and Molecular Biology, M-815, Dana-Farbcr Cancer Institute and Harvard Medical School, 55 Binney Street, Boston MA 021 IS , USA

Abbreviations. tS1-3,45-kDa thermolysin-generated N-terminal half of gelsolin; 64-6, 47-kDa thermolysin-generated C-terminal half of gel- solin ; F-actin, filamentous actin; G-actin, monomeric actin; NBD: 4- chloro-7-nitrobenzeno-2-oxa-1,3-diazole; rS1-3, recombinant plasma gelsolin containing amino acids 1-406; S1-3, gelsolin N-terminal half fragment containing homologous repeats 1-3 ; S4-6:gelsolin C-terminal half fragment containing homologous repeats 4-6.

merization by binding to two actin monomers [15, 20, 211. The different functions of gelsolin depend on how actin subunits in- teract with one or more of at least three distinct actin-binding sites [22-241 contained within the six homologous segments (termed S1 -S6) of gelsolin [24]. Actin binding to these sites is regulated in vitro by pH [25], Ca2+ 126-291 and phosphoinosi- tides [30-321.

Due to its multifunctional nature, activated gelsolin can act as a potent modulator of microfilament restructuring. Monomer- binding sites have been localized to S1 and S4-6, each site being required for either the strong filament severing (Sl) or the filament nucleating (S4-6) activities of gelsolin [23, 24, 33-35]. A third actin-binding site within repeats 2-3 (S2-3) binds to the side of actin filaments 1321 and promotes filament nucleation [24]. How the actin-binding functions of gelsolin are controlled in vivo is unclear. Studies in which gelsolin and gelsolin frag- ments were microinjected into cells yielded conflicting results 136-381 which, in some cases, depended on the source of the purified gelsolin. Chemical crosslinking [39-421 and binding inhibition studies [33, 43, 441 have suggested that multiple con- formations of gelsolin-actin complexes can be formed under dif- ferent experimental conditions.

We recently reported that gelsolin binds ADP-actin preferen- tially to ATP-actin [8]. In this report, we localize the site on gelsolin that distinguishes between ADP-actin and ATP-actin monomers to its C-terminal half. In contrast, the actin-binding domains located at the N-terminal half of gelsolin do not distin- guish between the nucleotides bound to the tight binding cleft of actin, but the rates and affinity of binding depend strongly on sub-millimolar concentrations of nucleoside triphosphates in

2 Laharn et al. (ELK J . Biochem. 234)

solution. In addition to the nucleotide bound within the tight binding pocket of actin, an additional low-affinity nucleotide- binding site on gelsolin, actin, or gelsolin-actin complexes may also regulate the rate of actin binding to gelsolin and the result- ing activity of the protein complexes.

MATERIALS AND METHODS

Materials. All nucleotides were purchased from either Sigma or Boehringer Mannheim. MagFURA-5, pyrene iodo- acetamide, and 4-chloro-7-nitrobenzeno-2-oxa-l,3 diazole (NBD) were purchased from Molecular Probes. Coomassie blue R-250 was purchased from BioRad. All other chemicals and ma- terials were purchased from Sigma or Fisher Scientific. The anti- gelsolin 2C4 monoclonal antibody was either purchased from Sigma or obtained as a generous gift from Christine Chaponnier (University of Geneva, Switzerland) and immobilized on CNBr- activated Sepharose (Pharmacia) as described [45].

Protein preparations. Rabbit skeletal muscle actin was purified according to published methods 1461. Monomeric actin was prepared as described [S] after extensive dialysis in 2 mM Tris/HCI, pH 7.8, 0.5 mM ATP or ADP, 0.2 mM CaCI,, 0.2 mM dithiothreitol (buffer A) and stored in frozen aliquots at -80°C. Actin concentrations were determined by the absorbance at 290 nm using the absorption coefficient 0.62 mg I ml cm-’ 1471, and polymerized by the addition of 2 niM MgCl, and 150 mM KCI (to form buffer B). Actin was labeled with pyrene iodoacetamide [48] or NBD (NBD-actin) [49] as described.

Generation of gelsolin fragments. Recombinant human gelsolin fragment containing S1-3 (amino acids 1-406; rS1-3) was generated according to published methods [24]. Human or bovine plasma gelsolin was cleaved with thermolysin to generate a 45-kDa N-terminal (tS1-3) fragment and a 47-kDa C-terminal (tS4-6) fragment, as previously described [45 I with the following modifications. Gelsolin (0.38 mg/ml) was dialyzed against 20 mM TrisHCl, pH 7.4, 30 mM NaCI, 0.2 mM CaC1, (buffer C), then digested with thermolysin at a 1 :2000 (by mass) ratio for 30 min at room temperature. 1 mM EGTA was added to stop the digestion prior to separation of the fragments by FPLC (Pharmacia LKB LCC 500 plus). The digest was loaded onto a I-ml Mono Q column, equilibrated with buffer C/1 mM EGTA, and 0.5-ml fractions were collected. The basic amino-terminal fragment (SI-3) eluted in the flow through while the acidic C- terminal fragment (S4-6) eluted from the column at 0.20- 0.25 M NaCl using a 0- 1 .0 M NaCl gradient in buffer C/EGTA. Prior to use in immunoprecipitation assays, the C-terminal S4-6 fragment was dialyzed against buffer C to remove excess EGTA.

Filament-severing assays. The rate of depolymerization when filaments are diluted below the critical concentration of polymerization can be measured as the loss of tluorescence from pyrene-labeled filaments and is proportional to the number of filament ends [35]. Gelsolin and its homologues increase the rate of F-actin depolymerization by severing filaments and capping the newly formed barbed ends, a process that generates new pointed ends from which depolymerization occurs. The rate of complex formation between filament-severing proteins and actin monomers can be measured by the decrease in the ability of the severing protein to sever filaments and accelerate filament depolymerization.

Gelsolin N-terminal half fragments (0.02-0.04 pM, depend- ing on intrinsic severing activity) were incubated with a 4 : 1 molar excess of G-actin. At various times after adding unlabeled G-actin to the severing protein, an aliquot of pyrene-labeled F- actin from a 15 pM stock was added to a final concentration of 0.25-0.30 pM. Severing activity was measured as the initial

rate of fluorescence decrease as the diluted filaments depolymer- ized. The calcium-insensitivity of the N-terminal half fragments (tS1-3 and rS1-3) of gelsolin was verified by repeating the sever- ing assays in buffer B containing either 1 mM EGTA or 1 mM EGTA and 2 mM EDTA. To examine the effects of nucleotides on gelsolin-actin complexes, ATP-actin was incubated with the N-terminal half fragments in buffer A containing 0.5 mM vari- ous nucleotides (ATP, CTP, GTP, ATP[yS], UTP, ITP, ADP and AMP) as indicated. All fluorescence measurements were made with a Perkin-Elmer luminescence spectrophotometer model LSSOB. The fluorescence intensity of pyrene-labeled actin was measured using excitation and emission wavelengths of 365 nm and 386 nm, respectively, while that of NBD-labeled actin was measured at 478 nm and 520 nm, respectively.

Immunoprecipitation and nucleotide analysis of gelsolin- actin complexes. gel solin-actin complexes were immunopreci- pitated using a monoclonal anti-gelsolin from solutions of dif- ferent ionic strengths and nucleotide compositions which con- tained either monomeric ADP-actin or ATP-actin, according to previously described methods [S]. For immunoprecipitations of gelsolin C-terminal half fragment and actin complexes, 400 p1 solutions of 1 pM tS4-6 were incubated with 1 pM ADP-actin or ATP-actin for 15 min in ADP/buffer A or B or ATPhuffer A or B containing 20 p1 1 : 1 suspension of anti-gelsolin monoclo- nal antibody coupled to Sepharose. Precipitations were washed three times with their respective buffer solutions, then analyzed by standard SDSlPAGE techniques. Coornassie-blue-stained protein bands were analyzed by densitometer scanning using a Lightning Scan 256 instrument and the integrated density func- tion of Image 1.29 software.

NBD-actin fluorescence assays. The binding of the N-ter- minal half fragment, rS1-3 (0.045 pM) of gelsolin, to NBD-la- beled ATP-actin monomers (0.092 pM) was measured following the fluorescence increases of actin in buffer A solutions that varied in nucleotide (0.5 mM ATP, CTP, GTP, UTP, ITP, ATP[;S], ADP, GDP, or no nucleotide) and salt (0-10mM KCI), as indicated. Gelsolin was added with continuous stirring to solutions containing NBD-actin 25-30 s after the start of fluorescence measurements. To rule out the possibility that any differences in fluorescence between the gelsolin-actin complexes formed in the ATPhuffer A and ADPhuffer A was due to differ- ences i n calcium concentrations, additional assays were per- formed using ADP/buffer A solutions containing 0.1 mM EGTA or ADP at a final concentration of 1 mM. The relative fluores- cence is calculated by the following expression :

Relative fluorescence = (F, - b)/(F, - b ) ,

where F, represents the fluorescence of the sample at time t , F, represents the initial fluorescence of NBD-actin in the absence of gelsolin, and h represents the background fluorescence of the buffer alone in the absence of actin. To determine the number of actin monomers bound to S1-3 in ADP/buffer A and nucleo- tide-free buffer A, increasing concentrations of rS1-3 (0- 0.094 pM) were added to a constant concentration of NBD-ATP- actin (0.090 pM). Mixtures were incubated at room temperature for at least 1 hour prior to final fluorescence readings. The final fluorescence of the samples is plotted against the S1-3/actin con- cen tration.

Measurement of free CaZ+ concentrations. The concentra- tion of free Ca” was determined by a method similar to that described in Lamb et al. [25] with the exception that the indica- tor MagFURA-5 [SO] was substituted for FURA-2. MagFURA- 5 has 400-times higher affinity for Ca*+ (Kd = 25 pM) than for ME’ ’ (Kd = 14.7 mM) and can be used for measuring free Ca2+ in the 1-100-pM range 1.511. The fluorescence intensity of 1.6 pM MagFURA-5 was determined for ATPhuffer B and

Laham et al. (ELK J . Biochern. 234) 3

ATP-actin ADP-actin

G- A-

Buffer A ADP ATP ATP ADP ATP condition: + S

Fig. 1. The actin-binding site in the C-terminal half of gelsolin binds preferentially to ADP-actin monomers. The C-terminal half fragment of gelsolin, generated by thermolysin cleavage, 64-6 (1 pM), was im- munoprecipitated using monoclonal anti-gelsolin coupled to Sepharose beads from either ATP-actin or ADP-actin (1 pM) solutions that varied in their nucleotide (0.5 mM ADP or 0.5 mM ATP) and salt contents following a 15-min incubation at room temperature. The complexes were washed three times with their respective incubation solutions, denatured by heating i n SDS/PAGE sample buffer, then analyzed by standard SDS/ PAGE followed by Coomassie blue staining. The positions of tS4-6 (G) and actin (A) are labeled to the left of the gel, the type of actin used in the precipitations is labeled above the gel, and the buffer conditions used for the precipitations are labeled below the gel with S representing the presence of 2 mM MgCI, and 150 mM KCI.

ADP/buffer B or buffer A containing 150 mM KCl at 340 nni and 380 nm excitation with emission at 509 nm. The minimum fluorescence intensity ratio of 1.6 pM MagFURA-5 (R,,,,,,) was determined by addition of 2 mM EGTA to each solution. The maximum fluorescence intensity ratio of 1.6 pM MagFURA-5 (R,,,,) was determined after addition of 10 mM CaCI,.

RESULTS

The C-terminal gelsolin domain distinguishes between ADP- actin and ATP-actin. When the C-terminal half of gelsolin (S4-6) is precipitated by a monoclonal antibody from solutions that contain G-actin, the amount of actin co-precipitated depends on the nucleotide tightly bound to the actin, but not on the nucle- otide free in solution. The 47-kDa thermolysin-generated C-ter- minal half of gelsolin (tS4-6) [45] precipitates as a 1 : 1 complex with ADP-actin in either ADP-containing or ATP-containing buffers, but precipitates alone when mixed with ATP-actin under the same conditions (Fig. 1). Although addition of 2 mM MgC1, and 150 mM KCI increases the amount of ATP-actin co-precipi- tating with full-length gelsolin (data not shown), addition of these salts to the tS4-6-actin mixture fails to increase the amount of precipitable actin. Since the calcium-sensitive C-terminal ac- tin-binding domain of gelsolin precipitates ADP-actin from both ATP-containing and ADP-containing solutions, the levels of free calcium are sufficient for the activation of its actin-binding ac- tivity and these concentrations were confirmed to be greater than 50 LIM by direct measurements (Table 1).

Since the epitope for the monoclonal antibody used to pre- cipitate the C-terminal half fragment of gelsolin lies in gelsolin segment 4, and there are no available antibodies to the N-termi- nal half of human gelsolin, a functional assay that measures complex formation by loss in severing activity was used to de- termine if the actin-binding sites within S1-3 discriminate be- tween ATP-actin and ADP-actin monomers. In contrast to S4- 6, the N-terminal half (Sl-3) of gelsolin is insensitive to the nucleotide contained within the nucleotide-binding cleft of actin

Table 1. Concentration of free Ca" in buffer A and buffer B. The concentration of free calcium in buffer A and buffer B used to form the gelsolin-actin complexes was determined using the Mg' ' and CaZ indicator MagFura-5 [SO, 511. The fluorescence intensity of MagFura-5 (1 .h mM) was measured at 340 nm and 380 nm excitation with emission at SO9 nm using a Perkin Elmer LS-50 instrument.

Buffer conditions Measured free [Ca"']

ATPIhuffer A ADP/buffer ATPhuffer B ADP/buffer B

PM 51

162 168 129

0 ' ' " " ' I ' I " ' 0 60 120 180 240 300

Time (s)

Fig. 2. N-terminal actin-binding sites of gelsolin are insensitive to the nucleotide bound to actin monomers, The N-terminal half fragment of gelsolin, generated by thermolysin cleavage tS1-3 (0.03 pM), was incubated with either ATP-actin (0.1 2 pM; hollow symbols, dashed lines) or ADP-actin (0.12 pM; solid symbols, solid lines) in solutions that varied in their nucleotide contents (0.5 mM ATP or 0.5 mM ADP) for the times indicated prior to determination of filament-severing activ- ity. The severing activity of tS1-3 and actin complexes is expressed rela- tive to the activity of the gelsolin fragment in the absence of actin incu- bation. The ionic contents of solutions are represented as follows: ATP/ buffer A (0, O), ATP/buffer B (A, A), ADPhuffer A (0, m), and ADP/ buffer B (0, +). Buffer A contained 2 mM TrdHCl, pH 8.0, 0.2 mM CaCI2, 0.2 mM dithiothreitol and 0.5 mM ADP or ATP, while buffer B contained an additional 2 mM MgCI, and 1.50 mM KCI.

(Fig. 2 ; compare open and closed symbols). The calcium-insen- sitive 45-kDa N-terminal fragment generated by thermolysin cleavage (tS1-3) rapidly forms non-severing complexes with both ADP-actin and ATP-actin. The reaction rate is, however, dependent on the ions present in solution. Both 0.5 mM ATP and 150 mM KCI retard the reaction between S1 -3 and either ATP-bound or ADP-bound actin monomers. Similar results were obtained using human and bovine gelsolin N-terminal half frag- ments generated by either bacterial expression or thermolysin cleavage, respectively (data not shown).

Actin binding to the N-terminal half of gelsolin is sensitive to solution ATP. Adenine nucleotides also influence the rate of actin-gelsolin binding as measured by changes i n the fluores- cence of NBD-labeled G-actin. Addition of gelsolin to ATP-ac- tin in the absence of free ATP (in 0.5 mM ADP or nucleotide- free solutions, data not shown) causes a rapid increase in fluo- rescence (Fig. 3A). The fluorescence increase is much slower

4 Laham et al. (Euv. J . Biochenz. 234)

A

1.7

a, 1.6 0 c 8 1.5 u) a, z 1.4 3 - 1.3 a, > 'L 1.2 m a,

-

- a 1.1

1

0 30 60 90 120 150 180 Time (s)

B

2 a, 0 6 1.8 0 v) al z 1.6 3 - c

Q, 1.4 > a .- - - 2 1.2

:'ir w

1 0 1 , 1 1 1 1 1 , 1 1 1 , / , 1 1 1 1 1 1

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 S1-3:actin ratio

Fig.3. The binding of S l -3 to actin is affected by nucleotides in solu- tion. (A) Complex formations between rS1-3 (0.045 pM) and ATP-actin (0.092 pM) were measured by the increases in NBD-actin fluorescence in solutions that varied in their nucleotide contents (ADPlbuffer A, 0; ATPhuffer B, 0). To control for differences in the calcium concentration and the ionic strength between buffer A solutions, additional fluores- cence assays were performed using ADPlbuffer A solutions that con- tained an additional 0.1 mM EGTA (0). 10 mM KCI (B), or containing ADP to a final concentration of 1 mM (W). (B) The number of mono- mers bound to rS1-3 in ADPlbuffer A (0) was determined from the final fluorescence of gelsolin-actin complexes after 1 hour incubations at room temperature. The concentration of NBD-labeled ATP-actin mo- nomers was held constant at 0.090 pM while the concentration of rS 1-3 was varied at 0-0.096 pM. Each point represents the average tluores- cence obtained from two separate mixtures. The fluorescence plateau near 0.5 suggests the formation of 1 : 2 gelsolinhctin complexes. Similar results were obtained using nucleotide-free buffer A (data not shown).

when free ATP is present. To control for differences in Ca'+ concentrations and ionic strength between ATP-containing and ADP-containing solutions (Table l ) , fluorescence assays were repeated using ADP solutions containing 0.1 mM EGTA, 10 mM KCI, or ADP to a final concentration of 1 mM. The in- creases in NBD-actin fluorescence appear to be independent of differences in the calcium concentration and ionic strength. The number of actin monomers bound to gelsolin in ADP-containing solutions was determined by the S1-3/actin ratio required to sat- urate the fluorescence increase 124. 26, 521. These measure-

6

a, 0 c a, 0 v)

0 2

2 - .c

a, > a a,

.- c - a

l l l l l l l l l l l l l l l l l l l l l .L

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 [Nucleotide or EGTA] (mM)

ADP 1.6 IT P

UTP 1.5 y S-ATP

1.4

1.3

-

-

-

-

GDP GTP

l I I I l 1 l I I ~ l 1 1 1

0 30 60 90 120 150 180 210 Time (s)

Fig.4. ATP is the most potent inhibitor of complex formation be- tween actin and the N-terminal half of gelsolin. (A) rS1-3 (0.025 pM) was incubated with ATP-actin (0.075 pM) for 180 s in buffer A solutions that varied in their ATP concentrations over 0 - 2 m M prior to use in filament depolymerization assays (0). To control for artifactual results due to calcium chelation, incubation assays were repeated with the sub- stitution of EGTA (0-2 mM) for the ATP present in the solutions (X). To examine the effects of other nucleotides on gelsolin and actin com- plex formation, 0.5 mM concentrations of CTP, GTP, ATP[yS], UTP, ITP, ADP, AMP, or no nucleotides (blank) were substituted for ATP in the incubation solution. The resulting severing activities of gelsolin-actin complexes from these incubations are shown in the panel inset. All points represent the average value obtained from 3-5 incubations. (B) Complex formations between rS1-3 (0.045 pM) and NBD-labeled ATP- actin (0.092 pM) were measured by following the fluorescence increase in buffer A that varied in nucleotide contents. 0.5 mM concentrations of different nucleotides were added as indicated to nucleotide-free buffer A prior to adding gelsolin and actin.

ments confirm the formation of 1 :2 gelsoldactin complexes in ADP-containing solutions (Fig. 3 B). Similar resultc were ob- tained using nucleotide-free solutions (data not shown).

To determine the concentration of ATP required to affect the interaction of the N-terminal half fragment of gelsolin with ac- tin, S1-3 was mixed with unlabeled monomeric actin in solu- tions that varied in nucleotide concentrations prior to use in fila- ment-severing assays. Increasing the concentration of ATP from 0 mM to 2 mM reduces the extent by which actin monomers inhibit the S1-3 severing (Fig. 4A). One non-specific effect of nucleoside triphosphates is to chelate the divalent cations that are needed to maintain native G-actin structure. Therefore, the

Ldllillll Ct dl. ( C U I

effect of EGTA, which is a much stronger Caz+ chelator, was also examined. Since EGTA has significantly less effect than ATP, Ca2' chelation alone does not account for the inhibitory effect of ATP. To examine the specificity of the nucleotide ef- fects, 0.5 mM concentrations of other nucleotides were substi- tuted for ATP in solution (Fig. 4A, panel inset). CTP also inhib- its complex formation, while GTP, ATP[yS], UTP, ITP, ADP and AMP are less effective. The different nucleotides also vary in their effects on the binding of S1-3 to monomeric actin, as judged by changes in NBD-actin fluorescence (Fig. 4B). Similar to the effects of solution nucleotides on the severing activity of gelsolin-actin complexes, 0.5 mM concentrations of ATP and CTP prevent the rapid increase in NBD-actin fluorescence, whereas GTP, UTP, ITP, ATP[yS], ADP and AMP are less effec- tive.

DISCUSSION

The multi-domain structure of gelsolin combined with its multiple modes of interaction with actin suggest a complicated mechanism for regulation. The two most thoroughly analyzed regulatory mechanisms for gelsolin are activation by Ca'+ [26- 291 and inactivation by polyphosphoinositides [ 30-32, 531, but these two features alone are insufficient to account for changes in actin-gelsolin binding observed in vivo or in cell extracts [36-381. The present results identify the actin-binding sites of gelsolin that depend on adenine nucleotides and show that the ATP dependence is fundamentally different at the C-terminal and N-terminal actin binding domains. The C-terminal actin- binding site of gelsolin binds strongly to ADP-actin monomers but weakly or not at all to ATP-actin monomers, regardless of the ionic and nucleotide composition of the solution. In contrast, the N-terminal actin-binding site of gelsolin is insensitive to the nucleotide tightly bound to the actin cleft, but the binding is inhibited by sub-millimolar concentrations of ATP in solution. These findings have several implications for gelsolin function.

One implication is that the C-terminal half of gelsolin has a primary role in regulating activity. The nucleating, severing and capping activities of gelsolin result from an interplay among at least three actin-binding sites. Occupation of the C-terminal ac- tin-binding site on gelsolin is the rate-limiting step for gelsolin- actin complex formation 145, 54, 551 and is required for strong filament nucleating activity [24]. Since the C-terminal domain does not bind ATP-G-actin well, nucleation might depend on the presence or formation of ADP-actin monomers, even at Ca2+ levels where the binding site is activated. This hypothesis is con- sistent with the recent report that much of the apparent nucle- ation activity of gelsolin in vitro comes from efficient severing of actin filaments rather than from binding of G-actin to gelsolin [56]. If the nucleotide preference also applies to subunits within filaments, then intact gelsolin may selectively sever older fila- ments that have already hydrolyzed their ATP to ADP but be unable to sever newly formed filaments rich in ATP or ADP Pi. Severing by the isolated N-terminal domain would be free of that constraint, consistent with the greater ability of this frag- ment to disrupt F-actin when microinjected in cells as compared to intact gelsolin [36, 381.

The slow spontaneous exchange of ATP from actin mono- mers and filaments makes it possible to examine the effects of nucleotides independent of those bound tightly to actin [57]. Somewhat surprisingly, the actin-binding site in S1-3, which binds to actin monomers regardless of the nucleotide in the tight- binding cleft, is sensitive to ATP in solution with a half-maximal inhibition of binding at 500 pM ATP. There are numerous pos- sible origins for this effect. First, ATP may act as a non-specific

polyanion that screens the electrostatic contribution of binding the basic N-terminal domain of gelsolin to the acidic actin sub- unit. Such electrostatic screening is not negligible, because monovalent salts also somewhat inhibit these interactions (La- ham, L., unpublished observations) and the severing activity of the poly-basic villin N-terminus is known to be inhibited by KCI [SS-60]. However, we present evidence in this report that the effects of ATP are greater than other anions and, in particular, greater than other nucleoside triphosphates. Alternatively, ATP at millimolar concentrations also chelates Ca2+. Direct measure- ments of free Ca" concentrations (Table 1) confirm that the concentrations are still above the levels needed to bind G-actin and to activate the actin-binding capabilities of gelsolin. In addi- tion, actin binding to the site on S4-6 is sensitive to surround- ing Ca?+ concentrations yet, in contrast to intact gelsolin, the thermolysin-generated C-terminal half fragment forms com- plexes with actin in both ATP-containing and ADP-containing solutions. These results suggest that the above mechanisms are unlikely to explain our effects of ATP on gelsolin and actin in- teraction s.

A third possibility is that a specific ATP-binding site with relatively low affinity (K, = 0.5 mM) for adenine nucleotides exists on gelsolin, actin, or the gelsolin-actin complex. Gelsolin has been reported to bind to adenine nucleotides, but only in the absence of CaZ ' 1611, whereas the differences in gelsolin-actin binding presented here persist in Ca2+-containing solutions. Our data are consistent with the hypothesis raised by several earlier studies 162-651 that actin contains a second nucleotide-binding site distinct from the tight-binding cleft defined in the actin- DNase crystal structure [66]. The location of a potential second nucleotide-binding site on actin was implicated at W356 by pho- toaffinity labeling using azido-ATP [671. This tryptophan residue is very close to the interface with domain 1 of gelsolin, as deter- mined by both the recent gelsolinS1-actin crystal structure [681 and gelsolin-actin interaction domain mapping 1691. The exis- tence of a relatively weak interaction of actin with nucleotides may also relate to the protective effect of ATP on filament dena- turation in vitro [70, 711 and to the apparent association of at least some RNA with the actin cytoskeleton in vivo 1721. Addi- tional evidence for the presence of a second lower-affinity nucle- otide-binding site on actin results from the findings that submil- limolar concentrations of ATP appear to directly affect the tlexi- bility of rhodamine-phalloidin-stabilized actin filaments visual- ized by video microscopy (1731; Kas, J., Janmey, P. and Laham, L., unpublished results). If the binding affinity at the hypotheti- cal second site is in the millimolar range, assays using radio- labeled nucleotides or the fluorescent ATP analog etheno-ATP would not detect this weak binding beyond that of the much tighter binding cleft of actin with a nanomolar affinity.

A relationship between the adenine nucleotide concentration and the actin-binding protein function of gelsolin is not surpris- ing. At least four actin-binding proteins, including gelsolin [ S ] , actophorin [9], actin-depolymerizing factor 1101 and Abl [ 741, bind ADP-actin tighter than ATP-actin. In contrast, thymosin p4 binds ATP-actin more tightly than ADP-actin [71. Under some conditions, profilin binds ATP-actin more tightly than ADP-ac- tin [ll], while under other conditions, profilin binds with higher affinity to ADP-actin 1751. Since the procedure used to make either ATP-actin or ADP-actin generally alters both the nucleo- tide tightly bound to actin and the nucleotide in solution, the nucleotide specificity reported for these interactions may not al- ways depend on the nucleotide bound to the cleft of the actin subunit. Local changes in ADP/ATP ratios near the inner leaflet of the plasma membrane have been suggested to contribute to actin dynamics in motile responses 17, 761. Cellular ATP deple- tion has also been reported to cause major changes in the actin

6 Laham et al. (Eur: J . Biochem. 234)

cytoskeleton, which include the rapid sub-cellular redistribution a n d the side-to-side bundl ing o f actin f i laments [77-791. S i n c e the level of ATP in resting cel ls is in t h e millimoiar range [SO, 81 1, variations o f ATP concentrat ions as well as in the nature of the nucleot ide bound to actin m a y al ter the activity of several key actin-binding proteins. T h e regulation of such interactions b y local changes in cellular ATP levels m a y provide a n e w mechanism to f i n e tune t h e control of cytoskeletal rearrange- ments in vivo.

We thank Maura Shutt for her excellent technical assistance during the completion of this work. We are also grateful to Philip Allen, Josef Kas and Thomas Stossel for their many helpful discussions and sugges- tions. This work was supported by The National Institutes of Health grants AR38910 (PAJ) and the Whitaker Foundation for Biomedical Engineering (PAJ).

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