gCap39 Is Phosphorylated

THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268. No. 6, Issue of February 25, pp. 4106-4112, 1993 Printed in U. S.A.

gCap39 Is Phosphorylated STIMULATION BY OKADAIC ACID AND PREFERENTIAL ASSOCIATION WITH NUCLEI*

(Received for publication, September 9, 1992)

Koji Onoda and Helen L. Ying From the Department of Physiology, The University of Texas, Southwestern Medical Center, Dallas, Texas 77235-9040

gCap39 is an actin filament end-capping protein which is a member of the gelsolin family. Unlike gelsolin, gCap39 does not sever actin filaments and is a cytoplasmic as well as nuclear protein. We report here that gCap39 is phosphorylated, while gelsolin is not. gCap39 is phosphorylated on serines and threonines at multiple sites, and phospho-gCap39 is resolved by isofocusing into multiple isoforms which are more acidic than unphosphorylated gCap39. In vitro dephosphorylation eliminates the acidic isoforms. Okadaic acid, a protein phosphatase inhibitor, stimulates gCap39 phosphorylation in vivo. It preferentially increases labeling of several peptides and enhances labeling of phosphothreonines relative to phosphoserines. The phosphorylation state of gCap39 in cells is therefore regulated by a balance between kinases and okadaic acid-sensitive phosphatases, and phosphorylation sites containing threonines appear to be particularly sensitive to the phosphatases. Subcellular fractionation shows that the nuclear fraction contains 17 f 5% ( n = 3) of total gCap39. Compared with the soluble cytoplasm, nuclear gCap39 has a 1.7 f 0.2 ( n = 3) fold increase in the amount of 32P label incorporation and a higher ratio of acidichasic gCap39. We conclude that phospho-gCap39 is preferentially associated with nuclei and suggest that phosphorylation of gCap39 is functionally significant.

gCap39 is a Ca2+- and polyphosphoinositide-regulated actin filament barbed-end capping protein (1) which is very abundant in macrophages (2-5) and also present in a variety of other cells (I, 5, 6). It is a member of the gelsolin extended family (7, 8) but is unique in several respects: gCap39 is functionally distinct from many other gelsolin-like proteins identified to date in that it does not sever actin filaments (1, 9,lO); gCap39 is present in the nucleus and the cytoplasm (5, 6), while other gelsolin-like proteins are exclusively cytoplasmic ( 5 , 11, 12). The existence of nuclear gCap39 was first reported by Prendergast and Ziff (6) who accidentally cloned a gCap39-like cDNA (called mbhl) with a primer containing a c-myc basic motif. mbhl contains a c-myc-like DNA binding motif and a potential nuclear targeting signal, which would be consistent with a nuclear function and localization, respectively. We confirmed the nuclear localization of gCap39 by cDNA mediated overexpression (5) and found that during

* Supported by National Institutes of Health Grant HL29113. 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.

$ To whom correspondence should be addressed: Dept. of Physi- ology, The University of Texas, Southwestern Medical Ctr., 5323 Harry Hines Blvd., Dallas, TX 77235-9040.

mitosis it is redistributed throughout the cytoplasm following the dissolution of the nuclear envelope.

In this paper, we report that gCap39 is phosphorylated in vivo and phospho-gCap39 is preferentially associated with the nucleus. gCap39 is phosphorylated on serine and threonine residues at multiple sites, and phospho-gCap39 can be resolved into several isoelectric variants. The acidic isoforms disappear after in vitro dephosphorylation, indicating that isoform variabilities are directly attributable to differential phosphorylation. In vivo phosphorylation of gCap39 is enhanced by okadaic acid (13), a specific inhibitor of protein phosphatase type 2A and type 1 (14), which are two of the four major serine and threonine phosphatases in eukaryotic cells (15). Phospho-gCap39 in cells is therefore dephosphorylated by okadaic acid-sensitive phosphatases. Subcellular fractionation studies show that the nuclear fraction contains gCap39 and i s enriched in the acidic phospho-gCap39 isoforms. These results suggest that gCap39 phosphorylation may be important for its function within the nucleus, and that gCap39 is a target for protein phosphatases.

EXPERIMENTAL PROCEDURES

Cell Culture and Biosynthetic Labeling-Peritoneal exudate cells were obtained from mice (CSH/NIH) 4 days after they were injected intraperitoneally with thioglycollate broth (16). The cells were centrifuged at 400 X g for 10 min, resuspended in RPMI 1640 (GIBCO) containing 10% fetal bovine serum and 1 X lo7 cells were plated on 60-mm tissue culture dishes. After 2 h. a t 37 “C in 95% air, 5% CO,, the nonadherent cells were removed by washing, and adherent macrophages were cultured overnight in RPMI containing 10% fetal bovine serum. Macrophages were incubated with phosphate-free Krebs-Ringer bicarbonate solution containing 2% bovine serum albumin (17) for 60 min, and then exposed to [32P]orthophosphate (1 mCi/ml, Du Pont-New England Nuclear) for 2-4 h with or without the addition of okadaic acid (1 bM, Calbiochem). Macrophages were incubated in methionine-free Dulbecco’s modified Eagle’s medium for 1 h and then labeled with Tra r~~~S- labe l (0.2 mCi/ml) in methionine-free Dulbecco’s modified Eagle’s medium for 6 h.

Zmmunoblotting-Rabbit antigCap39 was generated by immunizing rabbits with recombinant mouse gCap39 (9) and affinity-purified by absorption against gCap39-Sepharose. Rabbit antimouse gelsolin was obtained by immunizing with natural mouse plasma gelsolin and affinity-purified against nitrocellulose strips containing recombinant mouse gelsolin.

Western blotting was carried out using the Amersham enhanced chemiluminescence system. The affinity-purified polyclonal antig- Cap39 was used a t 0.5 pg/ml, and horseradish peroxidase-conjugated secondary antibody (Bio-Rad) was used at a 1:2500 dilution. The blots were exposed to x-ray film (Kodak Blue) for different intervals (30 s to 2 min), and exposures within the linear range of resolution of the x-ray film were chosen for quantitation. The intensities of the stained bands were determined by scanning with a Molecular Dynam- ics 300A computing densitometer.

Immunoprecipitation-The cell monolayers (in 60-mm dish) were washed with cold phosphate-buffered saline, and scraped in 1 ml of RIPA lysis buffer (50 mM Tris-HCI, pH 7.5, 150 mM NaCI, 2.5 mM EGTA, 2.5 mM EDTA, 1% Nonidet P-40,0.1% SDS, 0.5% deoxycho- late, 50 mM NaF, 1 rnM Na3V04, 30 mM sodium pyrophosphate, 1

4106

gCap39 Gelsolin Phosphorylation Okadaic Acid Nuclei 4107

mM phenylmethylsulfonyl fluoride, 2 pg/ml each of leupeptin, pepstatin, and antipain). Following a 30-min incubation on ice, the lysates were centrifuged a t maximum speed in an Eppendorf micro- centrifuge for 10 min a t 4 "C. The supernatants were incubated with 30 pg of affinity-purified rabbit anti-gCap39 for 1 h on ice, and then with excess formalin-fixed Staphylococcus aureus (Cowan strain 2) bearing protein A (IgGsorb, New England Enzyme Center). The immune complexes were pelleted by centrifugation and washed exten- sively. Under these conditions, gCap39 was quantitatively immunoprecipitated, as determined by Western blotting of the remaining supernatant.

Radioactivity incorporated into gCap39 was detected by autoradiography (at -70 "C with two intensifying screens, on Kodak Blue film) after gel electrophoresis or by scintillation counting of the excised gel band.

Subcellular Fractionation-Cells cultured on 150-mm plates were washed three times with phosphate-buffered saline, scraped in 2 ml of hypotonic buffer (10 mM Tris, pH 7.5, 10 mM NaF, 1 mM MgC12, 1 mM NanV04) and incubated on ice for 10 min to allow the cells to swell (18). The swollen cells were disrupted in a Dounce homogenizer (B pestle) with 30 strokes. Cell lysis was monitored by microscopy, and was greater than 98%. Protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 2 pg/ml each of leupeptin, antipain, and pepstatin) and 10 mM sodium pyrophosphate were added immediately after the first stroke. The lysate was centrifuged a t 400 X g for 10 min, and the pellet (crude nuclear fraction) was washed once with a buffer containing 50 mM Tris, pH 7.5, 0.25 M sucrose, 5 mM MgCI2, 1 mM CaCI2, 25 mM KCI, 1 mM Na3V04, 10 mM sodium pyrophosphate, and 60 mM NaF. The 400 X g supernatant was clarified by centrifuging at 100,000 X g. The 100,000 X g pellet contained less than 5% of total gCap39 and was not studied further. The 100,000 X g supernatant was defined as the soluble cytoplasm.

Immunofluorescence Staining-MDCK' cells were fixed and stained as described in (5). Isolated nuclei were cytospun a t 850 rpm for 10 min onto glass slides. The nuclei were fixed and processed as for intact cells. Fluorescence microscopy was carried out with a Zeiss fluorescence microscope, using 63 X and 100 X oil immersion objec- tives.

Gel Electrophoresis-One-dimensional analysis of samples was carried out in the presence of SDS with the discontinuous pH, polyacrylamide gradient slab gel system of Laemmli (19).

Isoelectric focusing was performed according to the method of O'Farrell(20) on a minigel apparatus (Hoeffer Scientific Instruments) with some modifications. The samples were resuspended in a buffer containing 9.5 M urea, 2% Nonidet P-40, 5% 6-mercaptoethanol, 1.6% and 0.4% of Bio-lyte 5/7 and 3/10, respectively (Bio-Rad). Isoelectric focusing gels for the first dimension were cast in 1.5-mm diameter glass tubes and contained 8.5 M urea, 4% acrylamide, 2% Nonidet P-40, 2% Bio-lyte 3/10 ampholyte. They were prerun a t 150 and 200 V for 30 min sequentially. Samples were loaded and focused at 200 V for 14 h, and then 300 V for 2 h. Proteins were separated in the second dimension by SDS-polyacrylamide gel electrophoresis.

Phosphatase Treatment-gCap39 immunoprecipitates were washed to remove phosphatase inhibitors and treated with 1 mg/ml potato acid phosphatase (Sigma) in 40 mM PIPES (pH 6) or 20 pg/ ml of the catalytic subunit of protein phosphatase type 2A (21) (PP2Ac, gift of Dr. Marc Mumby, University of Texas Southwestern Medical Center) in 10 mM MOPS (pH 7.4, 0.5 mg/ml bovine serum albumin, 1 mM dithiothreitol) for 30 min a t 30 "C. The solutions also contained 2 pg/ml each of leupeptin and aprotinin. The immunoprecipitates were washed three times with phosphate-buffered saline (containing phosphatase inhibitors), and analyzed by one- or two- dimensional gel electrophoresis. Control samples were processed in parallel, without addition of phosphatases.

Phosphoamino Acid Analysis-The gCap39 immunoprecipitate was electrophoresed on a 10% SDS-polyacrylamide gel, and the 3pP- labeled gCap39 band was detected by autoradiography, excised from the gel, and hydrolyzed with 6 N HCI for 2 h a t 110 "C. The hydrol- ysate was dried in a Speed-Vac, dissolved in water, mixed with phosphoserine, phosphothreonine, and phosphotyrosine standards (5 pg each), and subjected to thin-layer electrophoresis on cellulose plates (Kodak). Phosphoamino acid standards were also spotted separately on adjoining lanes. Electrophoresis was performed a t 1200 V a t 4 "C in pyridine:acetic acid:H20 (1:10:189). Radioactive phospho-

' The abbreviations used are: MDCK, Madin Darby canine kidney; MOPS, 4-morpholinepropanesulfonic acid; PIPES, 1,4-piperazinedi- ethanesulfonic acid; 2D, two-dimensional.

amino acids were detected by autoradiography and the unlabeled phosphoamino acid standards detected by ninhydrin staining (22).

Two-dimensional Phosphopeptide Mapping-The procedures used were similar to that described in Ref. 23. The immunoprecipitated gCap39 band excised from a wet gel was eluted electrophoretically (Schelicher & Schuell, Elutrap), lyophilized, and dissolved in 475 p1 of 50 mM NH4HCOn (pH 8.4). 25 pl of a 1 mg/ml L-1-tosylamido-2- phenylethyl chloromethyl ketone-treated trypsin (sequencing grade, Boehringer Mannheim Biochemical) was added. After digestion for 20 h a t 30 "C, the sample was boiled for 5 min, freeze-dried, and subjected to high voltage thin-layer electrophoresis on thin-layer chromatography plates (Kodak) in 2% ammonium carbonate. The plates were then chromatographed in the second dimension in n- butano1:pyridine:acetic acidHz0 (37.5:25:7.5:30). Dinitrophenyl ly- sine and phosphoserine were included as standards. The plate was dried and the phosphopeptides were located by autoradiography.

RESULTS

Separation of Phospho-gCap39 by 2D-Gel Electrophoresis- gCap39 which was immunoprecipitated from 3sS-labeled macrophages was resolved into three spots by ZD-gel electrophoresis (labeled a-c); a was a major spot and had a PI of 6.7; b and c were less intensely labeled and had more acidic PIS (PI of c was 6.5). The appearance of multiple isoelectric variants raises the possibility that gCap39 has several isoforms which may have different primary sequences and/or are modified post-translationally to different extents. The two murine gCap39 cDNA clones isolated thus far from a fibroblast (6) and a kidney (1) library have some differences in their deduced amino acid sequence. However, comparison of these sequences with that of a human gCap39 cDNA (3) (also called macrophage capping protein) suggested that some of these differences, especially in the most variable region between the two murine clones, may have been due to small errors in sequencing. When our kidney gCap39 cDNA was expressed in bacteria (9), the recombinant gCap39 was also resolved into multiple isoforms (data not shown), suggesting that the isoform differences were most likely due to post-translational modifications, and not to differences in primary sequence.

["P]Orthophosphate metabolic labeling experiments showed that gCap39 was phosphorylated. PhosphogCap39 was resolved by 2D-gel electrophoresis into at least four major spots, three of which coincided with the "S label spots (Fig. 1). To determine precisely which of the gCap39 isoform was phosphorylated, the immunoprecipitated gCap39 was trans- - I E F

32P

3ss

PI 617 6:5 FIG. 1. 2D gel electrophoresis of gCap39. gCap39 was immu-

noprecipitated from macrophages metabolically labeled with ["PI orthophosphate or Tran:'2S-label. The monolayers were washed and lysed in RIPA buffer containing protease and phosphatase inhibitors. The lysates were immunoprecipitated with affinity-purified rabbit anti-mouse gCap39. The immunoprecipitated proteins were analyzed by isoelectric focusing followed by SDS-polyacrylamide gel electrophoresis. The PI values of the most basic and acidic "S-labeled isoforms were 6.7 and 6.5, respectively.

4 108 gCap39 Gelsolin Phosphorylation Okadaic Acid Nuclei

ferred to a nitrocellulose filter. Phospho-gCap39 was detected by autoradiography and gCap39 isoforms by Western blotting of the same filter (Fig. 2, A and A'). The Western blotting pattern was essentially similar to that obtained by "S labeling, except that a fourth minor spot (d in Fig. 2 A " ) was also detected (Fig. 2A). Careful alignment showed that the pre- dominant gCap39 isoform (a) was not phosphorylated (Fig. 2, A and A', diagramed in A") except for a small amount of "P label at its acidic boundary. This was probably due to overlap of spot a with a minor phosphorylated species. Isoforms b and c were phosphorylated. Spot g was "P-labeled but not detected by Western blotting or '"S labeling, presumably because it was a minor isoform which was highly labeled. Densitometry scanning of the 2D Western blot shown in Fig. 2.4 revealed that the acidic gCap39 isoforms accounted for 19% of total gCap39. This value varied between cell preparations, with a mean of 25 k 7% (n = 5 ) . The variability was not due to isofocusing artefacts, because the isoform distribution of a single sample was reproducible on multiple analysis. It may reflect instead the different activation states of the macrophages, which were not controlled due to variability in animals and tissue culture conditions.

It has been shown previously that gelsolin, which is a related actin modulatory protein, was not phosphorylated (24). This was confirmed in the macrophage system. No "P-labeled gelsolin band was detected (data not shown), even though the antibody was capable of immunoprecipitating gelsolin ( 5 ) . In Vitro Dephosphorylation by Phosphatases-Since the

acidic variants were more highly phosphorylated, it is likely that the isoelectric point differences between the gCap39 variants are due to phosphorylation. This was established by treating immunoprecipitated gCap39 with phosphatases. Fig. 3A showed that potato acid phosphatase decreased the amount of "P associated with gCap39 (left panel). Western blotting (right panel) showed that phosphatase-treated gCap39 had considerably less acidic isoforms compared with the untreated sample. Since dephosphorylation was accom- panied with the loss of acidic isoforms, we conclude that the isoelectric differences between most of the gCap39 isoforms are due to phosphorylation.

To better define the phosphatases which may act upon gCap39 in the cell, the catalytic subunit of protein phosphatase 2A (21), which dephosphorylates serine and threonine (15), was used. Fig 3B showed that it dephosphorylated con- stitutively phosphorylated gCap39 as well as gCap39 phos-

FIG. 2. Alignment of phospho- gCap39 with gCap39 isoforms. Mac- rophages were metabolically labeled with [:'2P]orthophosphate (for 4 h in A and 2 WESTERN h in R ) . 1 PM okadaic acid or vehicle alone was added during the last hour of labeling to R and A , respectively. gCap39 was immunoprecipitated and analyzed by 2D-gel electrophoresis. Isofocusing was in the horizontal direction, and SDS-polyacrylamide gel electrophoresis was in the vertical direction. Proteins were transferred electrophoretically to nitrocellulose filters. Phospho-gCap39 was detected by autoradiography ( A and H ) , and gCap39 isoforms were identified by Western blotting (A' and R') . A" and b"', diagrams identifying the isoforms of pCap.79. The open circle denotes isoform p which was phosphorylated but not detected by Western blotting.

32P

phorylated in the presence of okadaic acid (see below). Okadaic Acid Increased Recovery of Phospho-gCap39-The

effect of okadaic acid on gCap39 phosphorylation was exam- ined by incubating "P-labeled macrophages with okadaic acid, Fig. 5A showed that okadaic acid increased the incorporation of radioactivity into gCap39 in a time-dependent manner. Besides gCap39, the immunoprecipitate of the 60-min sample contained several additional phosphorylated bands. These proteins were nonspecifically adsorbed because similar bands were observed with nonimmune IgG.

Scintillation counting of the gCap39 bands (excised from the gel shown in Fig. 4A) showed that there was a 5- and 17- fold enrichment after a 15- and 60-min treatment, respectively. Similar increases were observed in two other experiments. 2D-gel electrophoresis showed that 32P label incorporation into each gCap39 isoform was increased (Fig. 4B). Short exposure of another autoradiogram revealed that two new (not detected in untreated gCap39) phosphorylated species, e and f, were more highly phosphorylated than the others (compare Fig. 2A' and E ) , and they also appeared as novel spots in the Western blot (Fig. 2B). Densitometry scanning of the Western blot showed that the acidic phospho-gCap39 accounted for 29% of total gCap39 in these okadaic acid- treated cells, compared with 19% in the parallel untreated sample. Results from multiple experiments showed that after okadaic acid treatment, the acidic gCap39 accounted for 45 k 15% (n = 4) of total gCap39 and the mean increase, based on comparison of three paired experiments, was 1.5 ?z 0.2 fold ( n = 3).

Our results showed that phosphorylated gCap39 is a target of okadaic acid-sensitive phosphatases, and some sites were preferentially dephosphorylated. Okadaic acid inhibited the phosphatase, increasing the recovery of all phosphorylated gCap39, allowing detection of the most labile isoforms.

Characterization of the Phosphorylation Sites-Phosphoa- mino acid analyses showed that in vivo labeled gCap39 from untreated macrophages was phosphorylated predominantly on serines and to a lesser extent on threonines (Fig. 5). After okadaic acid treatment, there was increased label incorporation, but unlike the untreated gCap39, phosphothreonines predominated. This switch was observed in three separate experiments. We conclude that phosphothreonines are preferentially dephosphorylated by the okadaic acid-sensitive protein phosphatases.

Two-dimensional tryptic peptide analyses of gCap39 from

w/o OKADAIC ACID WITH OKADAIC ACID

A" d

0 .. 0 0

a b c g

0 "03

a b e c 9

a b c g a a e c

gCap39 Gelsolin Phosphorylation Okadaic Acid Nuclei 4109

A. PAP 32P WESTERN

0 15' 60' - ---- - cCAP39

+

B. PP2Ac 32P

W f O OKA WITH OKA - + - +

- - -

- - -

FIG. 3. I n vitro dephosphorylation of gCap39. The gCap39 immunoprecipitates were incubated with potato acid phosphatase (PPA, 1 mg/ml) or the catalytic subunit of protein phosphatase type 2A (PP2Ac, 20 pg/ml) for 30 min a t 30 "C and analyzed by SDS- polyacrylamide gel electrophoresis (left panels) or 2D-gel electrophoresis (right panel). Control samples were processed in parallel, without added phosphatase. Phospho-gCap39 was detected by autoradiography, and gCap39 isoforms in 2D gels were detected by Western blotting. A, gCap39 without (-) or with (+) PPA treatment; R, gCap39 without (-) or with (+) PP2Ac. The Western blot in A had several nonspecific spots, which were not consistently observed (see Figs. 2 and 9).

untreated macrophages showed that there were two major (Fig. 6, labeled 2 and 4 ) and three minor ( I , 3, and 5 ) phosphopeptides. This is consistent with the data from isofocusing which also indicate that gCap39 is phosphorylated. After okadaic acid treatment, these phosphopeptides and several new peptides were detected. The intensities of phosphopeptides 1, 3, and 5 were increased, suggesting that they were highly phosphorylated but were particularly susceptible to the endogenous phosphatases. Although peptides 2 and 4 did not appear as highly labeled in the okadaic acid-treated sample as the untreated sample, the extent of phosphorylation was probably comparable, because the former was labeled for half as long and exposed to x-ray film for shorter intervals (see Fig. 6 legend). Our results show that some phosphorylation sites are more readily dephosphorylated, and this may account for a change in the gCap39 isoform profile and phosphoserine/threonine ratio after okadaic acid treatment.

Preferential Association of Phospho-gCap39 with Nuclei- gCap39 is a nuclear as well as cytoplasmic protein. The

A

IEF + V I

v) n o

15 '

60'

B FIG. 4. Effect of okadaic acid on gCap39 phosphorylation.

Macrophages were labeled with ["'Plorthophosphate for 2 h, and 1 p~ okadaic acid was added at the final 15 or 60 min of labeling. gCap39 was immunoprecipitated, analyzed by 1D- or 2D-gel electrophoresis and the phosphorylated proteins were detected by autoradiography. A, autoradiogram of SDS-polyacrylamide gel: R, autoradi- ograms of immunoprecipitated gCap39 after 2D-gel electrophoresis. Films were exposed for 14 h.

nuclear localization of gCap39 was demonstrated by immunofluorescence staining (5, 6) and subcellular fractionation (6). There was however no information on the amount of gCap39 in nuclei and the phosphorylation state of nuclear gCap39. We used hypotonic lysis and dounce homogenization, followed by low speed centrifugation to obtain a fraction enriched in nuclei. The presence of gCap39 in isolated nuclei was demonstrated by immunofluorescence staining (Fig. 7 ) . MDCK cells were used because they have large nuclei and are particularly rich in nuclear gCap39 (Fig. 7 A ) . The nuclear envelope was fluorescent, and the nucleoplasm had diffuse staining. The brightly stained spots were probably nonspecific because they were observed with nonimmune IgG (Fig. 7 0 ) . Otherwise, labeling was specific for gCap39, because antigel- solin (Fig. 7 C ) and nonimmune IgG did not stain (Fig. 7 0 ) .

Macrophages also contained nuclear gCap39. The relative

a b c 9 a a e c

'1 110

W/O OKA WITH OW

PI

PI,

PS. PT PY

0

PS PT

PY

0

1

2 -

3

5

* a

- + +

WITH OKA

l ? 2 . 5

3 e 4

-+ +

amounts o f gCap:K) in the macrophage nuclear (N) and 100,000 X g supernatant (soluhle cytoplasms) fractions were quant,itated hv densitometry scanning of the gCap39 immunoprecipitates (Fig. 8). The results of this experiment, and two additional experiments were shown in Tahle I. The nuclear fraction contained 12-22% of total gCap39 (mean of 17 ? 4 % ( n = 3 ) ) . Contamination with rytoplasmic proteins, as monitored hy the recovery of gelsolin, was less than 4'5 (data not shown; also see Fig. 7c'). Although the okadaic acid- treated cells in Experiments 2 and :1 had less nuclear gCap39 than the untreated cells shown in Experiment 1, these differences were not drle to okadaic acid, hecause a similar range was ohserved with and without okadaic acid in additional experiments not included in the tahle. The reasnns for the variable recovery were not identified.

The (list rihution of:'"I'-laheled gCap39 into the nuclear and

COOMASSIE BLUE 32P

S N S N

L &CAP39

TAF3I.I.: I Suhrdlulnr distrih~rtirm o f p h o . s p h r ) - g f ' n p : ~ ~

Experiment I was from untreated marrophages laheled with ""1' for 4 h; experirnrnts 2 and 3 were from okadair acid-treated macrophages laheled for 2 h. S, 1 0 0 , 0 0 0 X g supernatant (soluble cytoplasm); N, 400 X g crr~de nuclear pellet. After autoradiographv. thr gCapX1 hand was excised, soluhilizetl, and the 9' was quantitatrd t y scintillation counting. I'rotein determined hy densitomrter scanning of Coomassie 13lue-stained gel of immunoprecipitated gCap:19, expressed in nrhitrary units of specific activity, expressed as cpm/protein units and -fold enrichment o f sprrific activity of nurlear frnrtion relative to that o f the soluhle rvtoplasm. 'The Coomassie Hlue-stained gel and autoradiogram for Experiment 2 is shown in Fig. 8. The percent o f total gCap:19 and phosphorylated g(hp39 recovered in the nurlear fractions were I 7 f 5 and 22 * 6 ( n = 3 ) . respectivelv.

:II 1, 7 Total

I'mtc.in "I' I'rotein Spwific nctivity

~ _ _ _ _ _ ..~ ". "" .~ -

47 X:\ 1 3 1 . 6 17

W I O OKA S

N

S+N

WITH OKA S

4

N

relative to total gCap:19 (defined as S A . ) in the nuclear fraction was higher than the supernatant fraction (1.7 5 0.2- fold, n = 3) , indicating that there was a preferential association of phospho-gCap39 with the nucleus.

An enrichment. of phosphogCap39 in the nuclear fraction was corroborated hy an increase in acidic gCap.39. This was demonstrated bv western blotting of the 2D gels of the nuclear a n d cytoplasmic fractions (Fig. 9). At the exposure shown, t h e soluhle cytoplasm from cells not treated with okadaic acid

4112 gCap39 Gelsolin Phosphorylation Okadaic Acid Nuclei

tases. gCap39 has multiple phosphorylation sites and those containing phosphothreonines are particularly sensitive. In- hibition of the phosphatase results in an apparent “activation” of the gCap39 kinase. The final level of gCap39 phosphorylation in a cell reflects a balance between the kinase and phosphatase activities. We have not yet identified the gCap39 kinase(s) and phosphatase(s) except to the extent that protein phosphatase type 2A is a candidate phosphatase. Computer analysis using the IntelliGenetics PC/Gene Prosite program shows that gCap39 has many potential phosphorylation sites for a variety of kinases. Significantly, many of these sites are present on gCap39 but not on gelsolin. For example, gCap39 has three unique potential protein kinase C phosphorylation sites (2 serines, 1 threonine) and four casein kinase I1 phosphorylation sites (2 serines, 2 threonines). Besides phosphorylation, gCap39 is also unique among the gelsolin family of proteins in that it is present both in the nucleus and cytoplasm (5,6). We have begun to investigate the possibility that the two properties are related. In our subcellular fractionation studies we recovered 12-22% (n = 3) of total gCap39 in the nuclear fraction. Because gCap39 is extremely abundant in macrophages, accounting for 0.6-1% of total macrophage protein (3), and the nucleus occupies much less volume than the cytoplasm, gCap39 may be a major nuclear protein.

Our results clearly showed that the gCap39 recovered in the nuclear fractions contained a higher percent of acidic phosphorylated gCap39 than the soluble cytoplasm (46 us. 7%, respectively, in one experiment), as well as higher level of 32P label incorporation. The 1.7 f 0.1-fold ( n = 3) increase in specific activity of labeling was consistent with the increased recovery of the acidic phosphorylated isoforms. The enrichment of phospho-gCap39 in the nuclear fraction suggests that phosphorylation may be related to its nuclear function and/ or localization. One possibility is that gCap39 is preferentially phosphorylated by a nuclear kinase, accounting for the phosphorylation of a subset of gCap39. However, since a substan- tial proportion of phospho-gCap39 was also recovered in the cytoplasm, it will be necessary to hypothesize that the same or a different kinase is present in the cytoplasm, but is less active, either inherently or is counteracted more effectively by cytoplasmic phosphatases. Phosphorylation can modulate the nuclear import of a number of proteins (28), so another possibility is that gCap39 entry is facilitated by its phosphorylation. However, since we did not observe an increase in the proportion of phospho-gCap39 in the nucleus after

okadaic acid treatment even though the amount of total phospho-gCap39 was increased, this possibility cannot be substantiated at present. It is possible that, because gCap39 is such an abundant protein, the nuclear import machinery is already saturated by the phospho-gCap39, so increased phosphorylation will not produce additional import.

In summary, we have demonstrated that gCap39 is phosphorylated and phospho-gCap39 is enriched in the nucleus. gCap39 phosphorylation is enhanced by okadaic acid treatment, suggesting the regulation of gCap39 phosphorylation is dependent on a balance between kinases and phosphatases. The significance of gCap39 phosphorylation for various gCap39 functions, including nuclear localization and interac- tions with actin, will be explored in the future.

REFERENCES 1. Yu, F.-X., Johnston, P. A,, Sudhof, T. C., and Yin, H. L. (1990) Science

2. Johnston, P. A., Yu, F. X., Reynolds, G. A., Yin, H. L., Moomaw C. R. 250 , 1413-1415

Slaughter, C. A,, and Sudhof, T. C. (1990) J. Biol. Chem. 265 , ’17946- 1 mm

3. Dabiri, G. A,, Young, C. L., Rosenhloom, J., and Southwick, F. S. (1992) J.

4. Southwick, F. S., and DiNubile, M. J. (1986) J. Biol. Chem. 261 , 14191-

* I ““I

Biol. Chem. 2 6 7 , 16545-16552

14195 5. Onoda, K., and Yin, H. L. (1992) J. Cell Biol. 115 , 327 (abstr.) 6. Prendergast, G. C., and Ziff, E. B. (1991) EMBO J. 10,757-766 7. Yin, H. L. (1987) BioEssavs 7. 176-178 8. Matsudaira, P., and Janmey, P. (1988) Cell 5 4 , 139-140 9. Yu, F.-X., Zhou, D., and Yin, H. L. (1991) J. Biol. Chem. 266, 19269-

19975 10. YoGig,-C. L., Southwick, F. S., and Weber, A. (1990) Biochemistry 2 9 ,

11. Yin, H. L., Albrecht, J., and Fattoum, A. (1981) J. Cell Bid. 91,901-906 12. Cooper, J. A., Loftus, D. J., Frieden, C., Bryan, J., and Elson, E. L. (1988)

13. Cohen, P., Holmes, C. F. B., and Tsukitani, Y. (1990) Trends Biochem. Sci.

14. Bialojan, C., and Takai, A. (1988) Biochem. J. 256,283-290 15. Ingebritsen, T. S., and Cohen, P. (1983) Eur. J. Biochem. 132,255-261 16. Yin, H. L., Alley, S., Bianco, C., and Cohn, Z. A. (1980) Proc. Natl. Acad.

17. Smith, C. J., Rubin, C. S., and Rosen, 0. M. (1980) Proc. Natl. Acad. Sci.

18. Thomas, T. P., Talwar, H. S., and Anderson, W. B. (1988) Cancer Res. 4 8 ,

20. O’Farrell, P. H. (1975) J. Bid. Chem. 250,4007-4021 19. Laemmli, U. K. (1970) Nature 227,680-685

21. Mumby, M. C., Green, D. D., and Russell, K. L. (1985) J. Biol. Chem. 260 ,

22. Cooper, J. A,, Sefton, B. M., and Hunter, T. (1983) Methods Enzymol. 9 9 ,

2232-2240

J. Cell Biol. 106, 1229-1240

15,98-112

Sci. U. S. A. 77,2188-2191

U. S. A. 77 , 2641-2645

1910-1919

13763-13770

7Q7-An9 23.

24.

25.

26. 27.

28.

Boyle, W. J., van der Geer, P., and Hunter, T. (1991) Methods Enzymol.

Wang, E., Yin, H. L., Krueger, J. G., Caliguiri, L. A., and Tamm, I. (1984)

Hasegawa, T., Takahashi, S., Hayashi, H., and Hatano, S. (1980) Biochem-

Furuhashi, K., and Hatano, S. (1990) J. Cell Biol. 111 , 1081-1087 Furuhashi, K., Hatano, S., Ando, S., Nishizawa, K., and Inagaki, M. (1992) J. Bid. Chem. 267,9326-9330

Hunt, T. (1989) Cell 59,949-951

”. 2 0 1 , 110-152

J. Cell Biol. 9 8 , 761-771

istry 19 , 2677-2683

Documents

gCap39 Is Phosphorylated