(CANCER RESEARCH56. 138-144. January I. 19961

ABSTRACT

Contact between lymphokine-activated killer (LAK) cells and naturalkiller-resistant tumor targets SK-Mel-1 (human melanoma) or Raji (human lymphoma) stimulates phosphorylation of two Mr 65'000 LAK proteins (pp65a and pp65b) with nearly identical isoelectric points. Phos.phoamino acid analysis of pp65a and pp6Sb detected phosphorylation

exclusively on senne residues. Phosphotyrosine could not be detected oneither substrate after immunoblotting with an antiphosphotyrosine anti.body, and herblmycm A treatment failed to inhibit p65 phosphorylationinduced by target contact. However, phorbol myristate acetate treatmentalone Induced LAK pp65a and pp65b phosphorylation, suggesting phos

phorylation may be mediated by protein kinase C or a protein kinaseC-regulated kinase. The molecular weight and Isoelectric points of pp65aand pp65b are similar to that reported for the human actin-bundlingprotein, L-plastin (L-flmbrln). On two-dimensional SDS-PAGE gel humunoblots, a peptide specific anti-L-plastin antiserum bound to pp65a andpp65b,suggestingthat the phosphoproteinsare similaror IdenticaltoL-plastin. In addition, two adjacent Mr 65@000LAK proteins were alsodetected by the antiserum and may correspond to unphosphorylatedforms of L-plastin. On the basis of known properties of phosphorylated

L.plastln, It Is hypothesized that p65 phosphorylation in LAKS mayregulate adhesion to tumor targets.

INTRODUCTION

Coincubation between human LAKs4 and the NK-resistant tumortargets SK-Mel-l (human melanoma) or Raji (human lymphoma) waspreviously shown to stimulate phosphorylation of two Mr 65,000 LAKproteins (pp65a and pp65b) with nearly identical isoelectric points on2-D PAGE gels (1). Increased phosphorylation of these substrates inLAKs was found to require target cell contact and did not occur inresponse to soluble factors generated by tumors. Induction of LAKp65 phosphorylation correlated with target recognition because forcedcontact with normal peripheral blood lymphocyte targets did not leadto increased phosphorylation. In addition, cross-linking CDI6, thereceptor that triggers antibody-dependent cellular cytotoxicity (analternate cytolytic pathway), also increased pp65a and pp6sb phosphorylation. Therefore, prior correlative evidence suggested that p65phosphorylation might have a role in the cytolytic function of LAKS.

In the present report, the kinase pathways responsible for p65phosphorylation in LAKs are further characterized. Using a highlyspecific antiserum, pp65a and pp6sb are tentatively identified as

Received 8/10195; accepted 10131/95.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

I Supported by National Cancer Institute Award RO1-CA45225 (E. A. G.) and NIH

Grant CA 33305 (L. V. R.).2 This manuscript is the second in a series. See Frederick, M. J., Rodriguez, LV.,

Johnston, D.A., and Grimm, E.A. Tumor target binding induces phosphorylation of twoMr 65,000 lymphokine-activated killer proteins. Cancer Res., 56: 127—137,1996.

3 To whom requests for reprints should be addressed, at Department ofTumor Biology,

Box 79, M. D. Anderson Cancer Center, 1515 Holcombe Avenue, Houston, TX 77030.4 The abbreviations used are: LAK, lympokine-activated killer; IL-2, interleukin 2;

NK, natural killer; 2-D PAGE, two-dimensional SDS-PAGE; PMA, phorbol myristateacetate; mAb, monoclonal antibody; ECL, enhanced chemiluminescence; p1, isoelectricpoint; PKC, protein kinase C.

phosphorylated forms of the actin-bundling protein L-plastin(L-fimbrin). On the basis of the known biochemical and functionalproperties of L-plastin and its phosphorylated forms, a hypothesis toexplain the role of p65 phosphorylation in LAK-mediated cytotoxicityis proposed.

MATERIALS AND METHODS

Cells and Reagents. Cell cultures were grown in RPMI 1640 supple

mented with 10% fetal bovine serum, penicillin, streptomycin, 2 mM glutamine, and 20 mM HEPES. CD3-CD56+ LAKs were prepared as describedpreviously. Essentially, enriched CD3-CD56+ LAKs were generated fromperipheral blood mononuclear cells according to the method described byPerussia et a!. (2). Contaminating I cells were removed by adherence to

anti-CD3 Micro—Cellectorflasks (Applied Immune Sciences, Santa Clara, CA).Resulting effectors were >90% CD3-CD56+ and >80% CDI6+, as confirmed by FACScan analysis using a panel of mAbs (Becton Dickinson,Mountain View, CA), and exhibited potent lytic activity when tested againstNK-resistant targets. The Burkitt B-cell lymphoma line Raji and the nonad

herent human melanoma cell line SK-Mel-l (Mel-l) were obtained fromAmerican Type Culture Collection. Both tumors are NK resistant but LAKsensitive. The EBV-transformed lymphoma RPMI 8866 used as feeder cells

was kindly provided by Dr. 1. Whiteside (Pittsburgh, PA). All tumor cell lines

were Mycoplasma free.

In Vivo Phosphorylatlon. in vivo phosphorylation experiments were carned out as described previously (1). Briefly, CD3-CD56+ LAKs were prelabeled with 0.75 mCi/mI [32P1P@and 1 mCi total L-[355]methioninefor 2 h at37°Cin phosphate- and methionine-deficient DMEM supplemented with 20mM HEPES, 2% dialyzed fetal bovine serum, and 100 units/mi dialyzed

recombinant IL-2. Postlabeled LAKs were cocentrifuged at room temperature

with an equal number of unlabeled Raji targets or one-half as many Mel-l

targets and incubated for various times at 37°C,before addition of ice-cold stopbuffer containing PBS and phosphatase inhibitors. In experiments examiningthe effect of protein tyrosine kinase inhibition on p65 phosphorylation, herbimycin A was added at a final concentration of 10 @xg/mlfor the full duration

of the labeling reaction. Herbimycin A was prepared in advance by dissolvingin DMSO at 1.4 mg/ml and was stored at —80°C until used. The final

concentration of DMSO was 0.7% in all drug incubations and controls.Cell Lysis and 2.D PAGE. Cell pellets were lysed in “ureamix―(9.0 M

urea-4% NP4O-2% DTT; Ref. 3) containing 2% ampholytes (80% pH range,

3.5—10;10%, 2.5—4.0;and 10%, 5—8)and the following phosphatase inhibitors: 1 mM sodium orthovanadate; 2 mM EDTA; 10 mM sodium PP1; and 10 mM

sodium fluoride. Where applicable, presolubilized tumor targets were added to

control samples containing LAKs alone so that final composition of proteinswas the same for all samples. Complete solubilization was achieved bymagnetic spin vanes stirred overnight at room temperature. Nucleic acids wereremoved by ultracentrifugation at 150,000 X g for 2 h, and samples werestored at —80°C.Proteins were resolved by 2-D PAGE using an ISO-DALTsystem as described previously (I). After fixation (10% HAc-30% methanol),gels were either treated with En3Hance (DuPont) for fluorography or neutral

ized in 30% ethanol before they were dried and exposed to Kodak X-OMAT

XAR-5 film. To detect phosphoproteins only, a sheet of colored paper (black,red, or blue) enclosed in a plastic sheet protector was placed between the geland film to selectively block @Semissions. Intergel comparisons were madeafter equal film exposure times.

ImageAnalysisand ProteinQuantitation.Autoradiographsweredigitized using a Compaq 386/20 running a General Purpose Image Digitizing

138

Characterization of the Mr 65,000 Lymphokine-activated Killer ProteinsPhosphorylated after Tumor Target Binding: Evidence That pp65aand pp65b Are Phosphorylated Forms of as2

Mitchell J. Frederick, Lewis V. Rodriguez, Dennis A. Johnston, Bryant G. Darnay, and Elizabeth A. Grimm3

Departments of Tumor Biology fM. J. F., E. A. G.J, Molecular Pathology (L V. RI, Biomathematics fD. A. ii. and Molecular Oncology lB. G. DI. The University of TexasM. D. AndersonCancer Center, Houston, Texas 77030

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CHARACTERIZATIONOF LAK pp65a AND pp65b

System (4) interfaced to a Megavision 1024 X M Digitizer/Display with a

Texas Instruments 1230 X 1024 by 8-bit CCD black and white camera. Themethod of protein (spot) quantitation has been described previously in detail(I). The net integrated optical density (1. 0. D.) for each spot was calculatedby the formula:

I.O.D., = {T.O.D.@—(Area, X p)]

where T.O.D.@stands for total optical density of a spot, and Area, equals thearea of a spot in pixels and is the average background gray value per pixel in

the vertical neighborhood of the spot. Levels of 35S-labeledf3-.and ‘y-actinwere quantitated with a Betascope (Betagen, Waltham, MA) and used tocompare differences in protein load between samples.

Immunoblotting.AntiphosphotyrosinemAb PY2Owas obtainedfromTransduction Laboratories (Lexington, KY), and isoform specific rabbit anti

human L-plastin antiserum R325.4 (5) was generously provided by Dr. P.Matsudaira (Cambridge, MA). Proteins resolved by l-D or 2-D PAGE were

transferred to nitrocellulose filters using a Trans-Blot Semidry electroblotting

apparatus (Bio-Rad, Richmond, CA). For detection of phosphotyrosine, filterswere blocked for 1.5 h at room temperature with PBS containing I% BSA and0. I% Tween 20. Filters were then incubated with a 1:900 dilution of antiphosphotyrosine mAb PY2O in blocking buffer for 1.5 h, washed 4 times in PBScontaining 0.1% Tween 20, and then incubated with horseradish peroxidaseconjugated goat antimouse secondary antibody (Transduction Laboratories) for1 h at I:4000 in blocking buffer. After additional washes, bound secondaryantibody was detected via the nonradioactive ECL system (Amersham, Ar

lington Heights, IL) using the manufacturer's instructions. For detection of

L-plastin, filters were blocked with PBS containing 5% nonfat dry milk beforeincubation for 1.5 h with a 1:1000 dilution of rabbit anti-L-plastin antiserum

R325.4 in antibody dilution buffer containing PBS, 2.5% dry milk, and 0.05%

Tween 20. Washes were performed with PBS containing 0.05% Tween 20, andbound primary antibody was detected by a I-h incubation with a 1:4000dilution of horseradish peroxidase goat antirabbit serum (Transduction Laboratories) in antibody dilution buffer. Immune complexes were visualized usingthe ECL nonradioactive detection system as above. Radiolabeled proteins were

visualized before and after immunodetection by prolonged exposure of blots to

film.Phosphoamino Acid Analysis. Phosphoaminoacid analysis was carried

out using the method described by Cooper et a!. (6). Essentially, spots ofinterest identified by autoradiography were excised from dried gels, rehydrated, and eluted overnight in 50 mM ammonium bicarbonate (pH 7.6)containing 0. 1% SDS and 5% f3-mercaptoethanol. Eluted proteins were pre

cipitated with trichloroacetic acid, resolubilized, and partially hydrolyzed by

boiling in 5.7 MHC1for 60 mm. Free phosphoamino acids were resolved bytwo-dimensional thin-layer electrophoresis.

ImmuneComplexKinaseAssays. AfterincubationwithherbimycinA orsolvent, LAKs were washed three times in PBS and resuspended in ice-coldlysis buffer [25 mM Tris-140 m@.t NaCl-l% NP4O (pH 7.5)] containing 5

mM EDTA, 0.2 mM orthovanadate, 10 mM NaF, leupeptin, aprotinin, andphenylmethylsulfonyl fluoride. Lysates were then stored at —80°C.Forkinase assays, lysates were thawed on ice and clarified by centrifugation atmaximum speed for 30 mm in a microfuge, and protein concentrations weredetermined with a BCA protein assay kit (Pierce Chemical Co., Rockford,IL). Immunoprecipitations were performed in duplicate by preclearingequal amounts of LAK protein (—100 jig) with normal rabbit serum

preabsorbed to protein A-Sepharose, incubating it with 20 @grabbit polyclonal anti-p56'@ (Upstate Biotechnology, Lake Placid, NY) for 1 h at 4°C,and recovering immune complexes with protein A-Sepharose. Immunecomplexes were washed four times with lysis buffer supplemented with 0.2mM orthovanadate and once with Tris-buffered saline (pH 7.5), and wereresuspended in kinase reaction buffer containing 25 mM HEPES, 100 mMNaCI, 10 mM MnCI2, and 5 m@iMgCl2 (pH 7.5). The kinase reaction wasperformed for I 5 mm at room temperature in 60 p1 of kinase buffer

containing 1 uM ATP, 10 @xCiof [32PJATP, and 6 @xgof enolase. Reaction

products were eluted from immune complexes by boiling in 3X Laemmli

buffer (I X SSC = 6% SDS, 0. 18 M Tris, 30% glycerol, 6% @-mercaptoethanol) and electrophoresed on 9% SDS-polyacrylamide gels. Gels werefixed (30% methanol-lO% HAc) overnight, dried, and exposed to X-rayfilm for autoradiography. Phosphorylation levels of reaction products werequantitated directly from gels with a Betascope.

139

RESULTS

Herbimycin A Does not Block p65 phosphorylation. The ability ofthe tyrosine kinase-specific inhibitor herbimycin A to block LAK p65phosphorylation induced by tumor target contact was examined. Pretreatment of LAKs with 10 p.g/ml herbimycin A for 2.5 h did notinhibit the phosphorylation of pp6sa or pp6sb induced by contact withRaji tumor cells (Fig. 1). Although the area of spot pp6sb appearedslightly larger on the gel from control LAKS exposed to Raji (Fig. 1A,lower panel) when compared to pp65b from LAKs pretreated withherbimycin A, no quantitative difference existed between these spotswhen the vertical backgrounds were subtracted after computer-assisted image analysis (Fig. 1C). To confirm that the kinase inhibitorwas working properly, in vitro kinase assays were performed withanti-p56 Ick immune complexes derived from LAKs pretreated withherbimycin A. Three reaction products corresponding to p60ld@,p56―@―,and enolase (Fig. 1D) were visible after in vitro kinasereactions performed with immunoprecipitates isolated from LAKstreated with DMSO alone. When normal rabbit serum was substitutedfor anti-p56'@@in the immunoprecipitation, no bands were visible afterthe in vitro kinase assay, confirming the specificity of the reaction.Pretreatment of LAKs with 10 @g/mlherbimycin A before immunoprecipitation with anti-p561'@ decreased the phosphorylation of allthree in vitro reaction products by greater than 50%, as measured witha Betascope.

Phosphotyrosine Is Not Detected on pp65a or pp65b. Althoughthe experiment performed with herbimycin A suggested that de novop65 phosphorylation did not involve a tyrosine kinase pathway, it wasstill possible that pp6sa or pp6sb contained constitutively phosphorylated tyrosine residues. Immunoblotting was used to examinewhether pp65a and pp6sb contained phosphotyrosine. After stimulating 32P-labeled LAKs for 5 mm with Raji, proteins resolved by 2-DPAGE were transferred to nitrocellulose, and phosphoproteins weredetected by autoradiography (Fig. 2A). Proteins on the same blotreacting with an antiphosphotyrosine mAb PY2O (Fig. 2B) were thenvisualized using the ECL nonradioactive detection system and filmexposure times less than 1 h. Before immunodetection, no phosphoproteins were visible by autoradiography with film exposure timesunder 1 h; thus, proteins detected by PY2O were easily distinguishedfrom radiolabeled proteins. Both pp65a and pp6sb were clearly visibleby autoradiography (Fig. 24), but phosphotyrosine was not detectedon either substrate after a 10-mm ECL exposure (Fig. 2B), or evenupon longer ECL exposure times. The adjacent marker phosphoproteins “X―,“Y―,and “Z―were not apparent by autoradiography in Fig.2A and were occasionally absent in other experiments as well, possibly because constitutive phosphorylation of these proteins during thelabeling reaction was insufficient. Nevertheless, pp65a and pp6sbwere easily identified on the basis of molecular weight, p1, andinduction by tumor contact. A number of proteins, including thosevisible in Fig. 2B, reacted with PY2O mAb.

Phosphoamino Acid Analysis. Because herbimycin A did notprevent p65 phosphorylation, it seemed likely that the de novo phosphorylation was occurring either on Ser or Thr. Spots correspondingto pp65a and pp65b were excised from gels and subjected to phosphoamino acid analysis. Spot pp65b contained only phosphoserine(Fig. 3). Similarly, phosphorylation was detected exclusively on 5crinc residues for pp6sa as well (data not shown). Although we cannotcompletely exclude the possibility that other residues were phosphorylated and not detected due to limiting amounts of material, serineappears to be the major residue phosphorylated on pp65a and pp65b.

Phorbol Ester Induces p65 Phosphorylation. Data from phosphoamino acid analysis, immunodetection, and pharmacological studies using herbimycin A all suggested that a 5cr kinase was responsible

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CHARACTERIZATIONOF LAK pp65a AND pp65b

- + kD

XI... XI...Rajisup

Az

5.2

Az

5.2

Fig. 1. Herbimycin A does not inhibit p65 phosphorylation. LAKs were prelabeled for 2.5 h with[35S]methionine and 132P1P in the presence ofDMSO solvent (A ) or 10 @g/mlherbimycin A (B)before a 5-mm coincubation with either Raji supernatant (top panels) or Raji cells (bottom panels).Only phosphoproteins are shown. Three phosphoproteins labeled X, Y, and Z were chosen as landmark spots to facilitate localization of pp65a andpp65b. C, computer-assisted image analysis wasused to quantitate levels of pp65a and pp65b fromautoradiographs in the bottom panels. I.O.D. (integrated optical density) values were adjusted to account for protein load differences. D, LAKs werepretreated with the indicated concentrations of herbimycin A or with DMSO for 2.5 h and washedthree times in PBS. and proteins were immunoprecipitated with polyclonal anti-p56@' or normal rabbit sera (NRS) before immune complex kinase assays were performed and reaction products resovedby 1-D PAGE. kD, molecular weight in thousands.

C. D.

for p65 phosphorylation in LAKs. Consequently, the ability of PMA,a direct activator of PKC, to stimulate LAK p65 phosphorylation inthe absence of tumor targets was examined. A 10-mn treatment ofLAKs with 100 ng/ml of PMA dramatically increased pp65a andpp65b phosphorylation when compared to treatment with solventalone (Fig. 4). Levels of p65 phosphorylation after PMA treatmentwere determined to be at least 100-fold higher when compared toLAKs treated with solvent alone (Fig. 4C).

Both pp65a and pp65b React with an Antiserum Specific forL-Plastin. The apparent molecular weight, isoelectric point, andphosphorylation characteristics of pp6sa and pp6sb suggested thesesubstrates might be identical to L-plastin, a Mr 65,00068,000 actinbundling protein that becomes phosphorylated in leukocytes after avariety of stimuli (7—10).Matsudaira et al. (5) described an antiserum

highly specific for L-plastin that was generated against a synthetic

peptide containing unique NH2-terminal sequences. The immunoreactivity of this antiserum is demonstrated in Fig. 5. Total cellularlysates from HeLa cells, heterogeneous 5-D LAK cultures, or enriched CD3-CD56+ LAKs were resolved by SDS PAGE on l-D gels,transferred to nitrocellulose, and detected with anti-L-plastin antiserum. A single band with an apparent molecular weight of Mr 65,000was detected in lysates derived from all three cell types. No bands

were visible when control blots were reacted with normal rabbit serumalone (data not shown). To determine whether pp6sa and pp65breacted with anti-L-plastin antiserum, LAKs prelabeled with [355]methionine and [32P]P1were stimulated with Raji targets for 5 mm, andsolubilized proteins resolved by 2-D PAGE were transferred to nitrocellulose. Phosphoproteins from Raji-stimulated LAKs were detected

140

A. 0.7%DMSO B. lOpg/mlHerbA

Stimulus

;a1 -@‘b@ :.@

@*

p1 5.12, ..Raji xJp,s*@

cells “a1 .ii@•@ z@

p1 5.2

U)Ca,

(Jo4.',-0.xOU).Dll)

-69

kD

-69

.@

Q@

_@ —++ + +

p60 lckp56lck. ••@

Enolase-@

10 Ab@ ick NRS

0.7% lOpg/mIDMSO HerbA

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CHARACTERIZATIONOF LAK pp65a AND pp65b

B.ECLdetection

10-mm exposurekD

+ — -69

a4 “b

. . -46

S

p1 5.2

Fig. 2. Phosphotyrosine is not detected on ppb5aor ppfl5b. LAKs prelabeled with [32P]P were coin- kDcubated for 5 mm with Raji cells, and proteinsresolved by 2-D PAGE were transferred to nitro- 69cellulose. LAK phosphoproteins from the same blotwere visualized first by autoradiography (A) andthen by ECL after immunodetection with antiphosphotyrosine mAb PY2O (B). Spots a and b correspond to ppfl5a and pp65b, respectively. 0. positions corresponding to pp65a and pp65b on theimmunoblot. Marker spots X. Y, and Z were not 46visible in the experiment shown. kD, molecularweight in thousands.

by autoradiography after exposing the blot to film for 24 h, and bothpp65a and pp65b were clearly visible (Fig. 6A). Although marker

phosphoproteins X, Y, and Z do not appear in Fig. 6, spots pp6sa andpp65b were easily identified on the basis of their molecular weight, p1,induction by tumor contact, and relative position to adjacent@labeled proteins. Direct exposure of the blot to film for 24 h in theabsence of a barrier allowed visualization of LAK proteins labeledwith @Sor 32P (Fig. 6B). Because the blot was not processed forfluorography, however, only some 35S-labeled proteins were visible.Nevertheless, pp65a and pp65b were detected, along with the two

adjacent unphosphorylated Mr 65,000 proteins designated “p65a―and“p6Sb―(a and b). The same blot was then probed with anti-L-plastinantiserum (Fig. 6C), and four spots were visible that correspondexactly in position to pp65a, p65a, pp6sb, and p65b, respectively,when aligned with the autoradiograph depicted in Fig. 6B. To confirmalignment of spots reacting with antiserum, ECL exposure time was

extended to 24 h, thus allowing simultaneous detection of both radio

labeled proteins and proteins detected by immunoblotting. Spots detected with anti-L-plastin antiserum were easily distinguished fromradiolabeled proteins because the former appeared considerablydarker after extended ECL exposure (Fig. 6D) when compared toautoradiography performed before immunodetection (Fig. 6B).

DISCUSSION

Previously, we reported that tumor-target binding induces phosphorylation of two Mr 65,000 proteins in LAKs (1), pp65a and pp6sb. Theapparent molecular weight, pis, and phosphorylation properties ofthese two LAKS proteins suggested that they might be related to theactin-bundling protein L-plastin. In the present report, we testedwhether pp65a or pp65b resolved by 2-D PAGE cross-reacted with apolyclonal rabbit antiserum specific for L-plastin. The antiserumreacted with both Mr 65,000 phosphoproteins, suggesting that they aresimilar or identical to L-plastin. In addition, two 35S-labeled proteinsthat migrated in the immediate vicinity of pp6sa and pp65b were alsodetected by the antiserum and most likely represent unphosphorylatedforms (or apoproteins) of L-plastin.

Because an allelic charge variant of L-plastin occurs frequently inhumans (7, 8), pp65a and pp65b could represent distinct electro

. . . phoretic phosphoisoforms encoded by these alleles. The two unphos

phorylated proteins that also reacted with anti-L-plastin antiserumwould then be their corresponding apoproteins. In heterozygous mdividuals, the different electrophoretic isoforms of L-plastin focus asfour closely spaced spots with slightly different pis and nearly identical molecular weights when resolved by 2-D PAGE (8). The mostacidic of these spots is the phosphorylated form of normal L-plastin,which is followed in order of increasing p1 by the normal apoprotemn,the phosphorylated variant protein, and the variant apoprotein. Anidentical pattern of alternating phosphorylation was also observed forthe Mr 65,000 LAK proteins detected by the anti-L-plastin antiserum.Moreover, all four Mr 65,000 LAK proteins focus at the same positions relative to /3- and ‘y-actinthat have been observed for the fourelectrophoretic isoforms of L-plastin from heterozygous individualson 2-D gels.

Although originally characterized as a Mr 66'000 molecule (9),molecular weights ranging from Mr 64'000 to 68,000 have beenreported for normal L-plastin (8—12).The mobility of variant Lplastin appears slightly increased relative to normal L-plastin whenresolved by 2-D PAGE (8). Consistent with this finding, pp65b andp65b also had increased mobility relative to pp65a and p65a in someexperiments (i.e., Fig. 6). Thus, in addition to cross-reacting with aspecific antiserum, pp6sa and pp65b had the same appearance and

...... S...

@j @‘(@TfI

k@ . . . . .. .. . . . . . ..

pH 1.9

:@

Fig. 3. Spot ppb5b contains phosphoserine. 32P-labeled LAKs were coincubated withRaji cells for 5 mm, and proteins were resolved by 2-D PAGE. Spots corresponding topp65b were excised, eluted, and TCA precipitated. Subsequently, p65 was hydrolyzed,and phosphoamino acids resolved by two-dimensional thin layer electrophoresis weredetected by a Phosphorlmager. Positions of phosphoamino acid standards were visualizedwith ninhydrin. S. phosphoserine; T, phosphothreonine; Y, phosphotyrosine.

141

A.Autoradiography24-h exposure

+

a

p1 5.2

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CHARACTERIZATION OF LAK pp65a AND pp65b

Phosphorylation of L-plastin occurs exclusively on 5cr residues(1 1, 13, 14). In agreement with these findings, serine was the onlyresidue found to be phosphorylated on pp65a and pp65b by phosphoamino acid analysis. Furthermore, tyrosine phosphorylation couldnot be detected on either pp6sa or pp65b by immunoblotting. PMAalone was a potent stimulator of p65 phosphorylation in LAKS,suggesting that PKC or a PKC-regulated kinase might be responsiblefor the phosphorylation induced by tumor-target binding. A concomitant decrease in the relative amount of 35S-labeled p65a or pp65b ongels also occurred after PMA treatment, consistent with the hypothesisthat these proteins are unphosphorylated forms of pp65a and pp65b,respectively. PMA is also known to stimulate L-plastin phosphorylation in leukocytes (13, 15), further strengthening the argument thatpp6sa and pp6sb are identical to L-plastin. Moreover, the PTKinhibitor herbimycin A does not block phosphorylation of L-plastininduced by chemotactic factors in polymorphonuclear leukocytes(15). Similarly, herbimycin A did not inhibit LAK p65 phosphorylation induced by tumor targets. Thus, both L-plastin and p65 phosphorylation are similar in that protein tyrosmne kinases are unlikely to beinvolved. Presently, the kinase responsible for plastin phosphorylationis unknown, but it is unlikely to be directly mediated by PKC because

L-plastin is not a substrate for this kinase in vitro (15).Increased phosphorylation of pp6sa and pp65b in LAKs was shown

previously to correlate with tumor-target binding and to occur aftercross-linking FcyIIIa receptors that trigger an alternate antibodydependent cytotoxic pathway expressed by LAKs (1). Thus, circumstantial evidence suggests that p65 phosphorylation may have a role inLAK-mediated cytotoxicity. Assuming that p65 is similar or identicalto L-plastin, a possible role for pp65a and pp6sb in LAK-mediatedcytotoxicity can be postulated based on the known properties ofplastins.

- kD

@69

kD

-69

Az

5.2

5.2

DMSO PMA

N-J a. I@ = It) 0-

kD

69-@@ -@“@-L-plastin

46-

30-

Fig. 5. Detection of L-plastin in human LAKs by immunoblotting. Total cellularprotein (0.5 X 106 cell equivalents) from HeLa cells, 5-day-old heterogeneous LAKcultures,and highlypurifiedCD3-CD56+LAKswere resolvedby l-D SDS-PAGE,transferred to nitrocellulose, and detected with rabbit anti-L-plastin R325.2 antiserum(1:1000) as described in “Materialsand Methods.―kD, molecular weight in thousands.

142

A. DMSOControl

+ - +

A A

p1

XII..

:@ 5@2

C.

B. PMA(100 ng/mI)

x@

y1P@• b

.*

.@% 250 Dpp65a

.@@ 200 ‘ • pp65b

:@;i150

o@@

.5..-4..

C

Fig. 4. PMA stimulates LAK p65 phosphorylation. Prelabeled LAKS were stimulatedfor 10 mm at 37°Cwith either 0.1% DMSO solvent (A) or 100 ng/ml PMA (B), andproteins resolved by 2-D PAGE were detected as in Fig. 1. The same gel pair was exposedto film by two methods, producing autoradiographs of proteins labeled with 35Sand/or 32P(top panels) or 32Palone (bottom panels). Spots labeled a and b correspond to pp65a andpp65b, respectively. Spots labeled a and@ correspond to unphosphorylated proteinsdesignated p65a and pp65b, respectively, which decreased in intensity after PMA treatment. kD, molecular weight in thousands.

relative position on 2-D gels expected for L-plastin. Because L-plastinis known to be expressed in NK cells (8), and the CD3-CD56+ LAKs

in these studies were derived from NK cells, it seems likely that pp6saand pp65b are phosphorylated forms of L-plastin. Unequivocal proofof this identification will require obtaining enough material formicrosequencing.

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CHARACTERIZATIONOF LAK pp65a AND pp65b

69-

46-

.:., ••

69-A Aa b

4.@

a@69-46-46-

@, \

pI 512 5@2

Fig. 6. Both ppfl5a and ppfl5b are recognized by anti-L-plastin R325.2 antiserum. LAKs prelabeled with [35Slmethionine and [32PIP were coincubated for 5 mm with Raji cells,and proteins resolved by 2-D PAGE were transferred to nitrocellulose. The same blot was exposed to film in the absence or presence of a barrier to detect proteins labeled by 32P alone(A ) or proteins labeled by 355and/or 32P(B). The blot was then reacted with anti-L-plastin antiserum (1:1000) as described in “Materialsand Methods,―and reactive proteins werevisualized after a 3-s (C) or an extended (D) ECL exposure to simultaneously detect radiolabeled proteins. Spots labeled a and b correspond to pp65a and pp65b, respectively. Spotslabeled a and /@correspond to unphosphorylated proteins designated p65a and pp65b. respectively, which also reacted with anti-L-plastin antiserum. kD, molecular weight in thousands.

35 32B. SI P-image:1-dayexposure

.

A. 32P-image:1-dayexposure

5.2

kD+

69-

46-

kD+

p1

.

5!2

D. Antl-L-plastln(1:1000)ECLand autoradiography:1-day exposure

phosphorylated form of L-plastin is found in the insoluble cytoskeleton fraction (13), which consists primarily of proteins found inpodosomes (points of tight contact between the plasma membrane andthe substratum). On the basis of the macrophage data, Matsudaira (16)has proposed that L-plastin may have a role in cell adhesion. Celladhesion is an important step in the killing process mediated byLAKs, as well as other cytotoxic lymphocytes. When viewed byscanning electron microscopy, LAKs appear attached to tumor cellsthrough pseudopod-like extensions (22, 23). By transmission electronmicroscopy, LAKs appear bound to tumor targets via tight plasmamembrane bonds, including numerous plasma membrane interdigitations (22). In addition, broad LAK cell processes are observed protruding into the cytoplasm of targets (23). Possibly, phosphorylationof L-plastin could regulate adhesion of LAKs to tumor targets.

Because staurosporine inhibits p65 phosphorylation without affecting the number of LAK-tumor target conjugates formed (I), it isunlikely that p65 phosphorylation is involved in initial adhesion ofLAKs to tumor cells. However, binding of lymphocytes to target cellsis probably a multistep process that involves signal transduction and

several states of adhesion (24, 25). Models proposed for T-cell bind

143

C. Anti-L-plastin(1:1000)ECL:3-sec exposure

Plastins (fimbrins) are a family of homologous genes that encodeactin-bundling proteins (16). Although L-plastin was first identified asa protein unique to human cancer cells based on comparative 2-D gel

analysis, it was latter determined to be an abundant protein in freshlyisolated T cells, NK cells, and other normal leukocytes (8). Currently,three genes encoding distinct isoforms of human plastin have beenidentified and are expressed in a somewhat tissue-specific manner (5,

17). L-plastin is expressed exclusively in leukocytes and is the only

form known to become phosphorylated. The other two isoforms, Tand 1-plastin, are not detected in leukocytes. All plastins have threefunctional domains consisting of two putative calcium-binding sites, aputative calmodulin-binding site, and two tandem actin-binding domains that probably permit actin cross-linking (5, 18, 19). In addition,

all three plastin isoforms are known to bundle actin filaments in vitro(16).

A variety of agents are capable of stimulating L-plastin phosphorylation in leukocytes, including IL-I, IL-2, tumor necrosis factor,lipopolysaccharide, IL-8, chemotactic factors, and PMA (10—12,20,21). All of these factors are also capable of altering the adhesiveproperties of cells. Moreover, in adherent macrophages, most of the

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CHARACI@ERIZAT1ONOF LAK pp65a AND pp65b

ing (24, 25) may explain the way LAKs interact with tumor cells. Insuch models, initial binding occurs through low-affinity interactionsbetween relatively large integrins and their ligands, establishing pointsof contact that still permit lateral diffusion of smaller membraneproteins such as CD2 (or other signaling receptors) so that they findtheir perspective ligands. Once triggering molecules are engaged,second messenger generation can lead to phosphorylation of proteinssuch as LFA-l, which strengthen binding. Possibly, establishment oftight plasma membrane bonds, plasma membrane interdigitations, orprotrusion of LAK cell processes into the cytoplasm of targets coulddepend on signal transduction and p65 phosphorylation. Examinationof the ultrastructure of LAKs bound to tumor cells under conditionsthat inhibit p65 phosphorylation would help to clarify this possibility.

Phosphorylation of other actin-cross-linking proteins has been

shown to inhibit their actin-bundling properties (18). A mechanismhas been proposed (19) that might explain how phosphorylation ofL-plastin could change its association with actin. It has been demonstrated in vitro that the ability of L-plastin to bundle actin filaments isinhibited by increasing concentrations of Ca2@ (26). Because theserine residue phosphorylated on L-plastin resides in the first Ca2tbinding domain (19), phosphorylation could affect affinity of Lplastin for Ca2@ and influence the actin-bundling properties of plastin.

Currently, it is not known which LAK receptors are involved in p65phosphorylation induced by tumor target binding. Although no anti

gen-specific receptors have been identified for LAKs, considerableevidence suggests that LAKs utilize some of the same general celladhesion molecules used by NK and T cells when binding targets. Thecell adhesion molecules LFA-l and CD2 are present on the majorityof LAKs, and antibodies to these molecules inhibit LAK killing (27,28). Some of these adhesion molecules probably contribute to signal

transduction as well (14, 29). In NK and T-cells, antibodies to CD2reportedly induce phosphorylation of a Mr 67,000 protein that has alsobeen tentatively identified as L-plastin (14), based on cross-reactivitywith antiserum and p1. Our data that p65 phosphorylation occursduring tumor-target binding suggests that L-plastin phosphorylationalso occurs when receptors on LAKs are stimulated by their natural orphysiological ligands. On the basis of identification of pp6sa andpp65b as L-plastin, CD2 is a likely candidate for the receptor onLAKs that triggers p65 phosphorylation. Because both cytotoxic Tcells and freshly isolated NK cells also utilize CD2 to bind susceptibletargets, it will be of interest to examine whether phosphorylation ofL-plastin occurs in these cytotoxic lymphocytes as well, after targetcell binding.

ACKNOWLEDGMENTS

We thank Dr. Theresa Whiteside and members of her laboratory for excellent advice regarding cultivation of CD3 negative LAKS,and Kuang Tang andPeggy Daniels for assistance with the image analysis software.

REFERENCES

1. Frederick, M. J., Rodriguez, L. V., Johnston, D. A., and Grimm, E. A. Tumor-targetbinding induces phosphorylation of two 65-kD lymphokine-activated killer proteins.Cancer Res., 56: 127—137,1996.

2. Perussia, B., Ramoni, C., Anegon, I.. Cuturi, M. C., Faust, J., and Trinchieri, G.Preferential proliferation of natural killer cells among peripheral blood mononuclearcells cocultured with B lymphoblastoid cell lines. Nat. Immun. Cell Growth Regul.,6: 171—188,1987.

3. Ramagli, L. S., Womack, J. E., and Rodriguez, L. V. Genetic analysis of nonhistonechromosomal protein inheritance in recombinant inbred mouse strains using twodimensional electrophoresis. Biochem. Genet., 28: 123—136, 1990.

4. Gersten, D. M., Ramagli, L. S., Johnston, D. A., and Rodriguez, L. V. The use of

radioactive bacteriophage proteins as X-Y markers for silver stained two-dimensional electrophoresis gels and quantification of the patterns. Electrophoresis, 13:87—92.1992.

5. Lin, C-S., Shen, W., Chen, Z. P., Tu, Y-H., and Matsudaira, P. Identification of1-plastin.a human fimbrin isoform expressed in intestine and kidney. Mol. Cell. Biol.,14: 2457—2467,1994.

6. Cooper, J. A., Sefton, B. M., and Hunter, T. Detection and quantification of phosphotyrosine in proteins. Methods Enzymol., 99: 387—402, 1983.

7. Goldman, D., and Mcml, C. R. Human lymphocyte polymorphisms detected byquantitative two-dimensional electrophoresis. Am. J. Hum. Genet., 35: 827—837,1983.

8. Goldstein, D., Djeu, J., Latter, G., Burbeck, S., and Leavin, J. Abundant synthesis ofthe transformation-induced protein of neoplastic human fibroblasts, plastin, in normallymphocytes. Cancer Res., 45: 5643—5M7,1985.

9. Leavin, J., Goldman, D., Mcml, C., and Kakunaga, T. Changes in gene expressionaccompanying chemically induced malignant transformation of human fibroblasts.Carcinogenesis (Lond.), 3: 61—70,1982.

10. Matsushima, K., Shiroo, M.. Kung, H. F., and Copeland, T. D. Purification andcharacterization of a cytosolic 65-kilodalton phosphoprotein in human leukocyteswhose phosphorylation is augmented by stimulation with interleukin-l. Biochemistry,27: 3765-3770, 1988.

I 1. Zu, Y., Kohno, M., Kubota, 1., Nishida, E., Hanaoka. M., and Namba, Y. Characterization of interleukin-2 stimulated 65-kilodalton phosphoprotein in human T cells.Biochemistry, 29: 1055—1062,1990.

12. Shibata, M., Yamakawa, Y., Ohoka. T., Mizuno, S., and Suzuki, K. Characterizationof a 64-kD protein phosphorylated during chemotactic activation with IL-8 and fMLPof human polymorphonuclear leukocytes. 11.Purification and amino acid analysis ofphosphorylated 64-kD protein. J. Leukocyte Biol., 54: 10—16,1993.

13. Messier, J. M., Shaw, L. M., Chafel, M., Matsudaira, P., and Mercurio, A., M.Fimbrin localized to an insoluble cytoskeletal fraction is constitutively phosphorylated on its headpiece domain in adherent macrophages. Cell Motil. Cytoskeleton, 25:223—233,1993.

14. Henning, S. W., Meuer, S. c., and Samstag, Y. Serine phosphorylation of a 67-kDaprotein in human T lymphocytes represents an accessory receptor-mediated signalingevent. J. Immunol., 152: 4808—4815,1994.

15. Shibata, M., Ohoka, T., Mizuno, S., and Suzuki, K. Characterization of a 64-kdprotein phosphorylated during chemotactic activation with IL-8 and fMLP of humanpolymorphonuclear leukocytes. I. Phosphorylation of a 64-kd protein and otherproteins. J. Leukocyte Biol., 54: 1—9,1993.

16. Matsudaira, P. Actin crosslinking proteins at the leading edge. Semin. Cell Biol., 5:165—174,1994.

17. Lin, C-S., Aebersold, R. H., Kent, S. B., Varma, M., and Leavitt, J. Molecular cloningand characterization of plastin, a human leukocyte protein expressed in transformedhuman fibroblasts. Mol. Cell. Biol., 8: 4659—4668,1988.

18. Arruda, M. V., Watson, S., Lin, C-S., Leavitt, J., and Matsudaira, P. Fimbrin is ahomologue of the cytoplasmic phosphoprotein plastin and has domains homologouswith calmodulin and actin gelation proteins. J. Cell Biol., 111: 1069—1079, 1990.

19. Zu, Y., Shigesada, K., Nishida, E., Kubota, I., Kohno, M., Hanaoka, M., and Namba,Y. 65-Kilodalton protein phosphorylated by interleukin 2 stimulation bears twoputative actin-binding sites and two calcium-binding sites. Biochemistry, 29:8319—8324,1990.

20. Shiroo, M., and Matsushima, K. Enhanced phosphorylation of65 and 74 kDa proteinsby tumor necrosis factor and interleukin-1 in human peripheral blood mononuclearcells. Cytokine, 2: 13—20,1990.

21. Shinomiya, H., Hirata, H., Saito, S., Yagisawa, H., and Nakano, M. Identification ofthe 65-Wa phosphoprotein in murine macrophages as a novel protein: homology withhuman L-plastin. Biochem. Biophys. Res. Commun., 202: 1631—1638,1994.

22. Hook, G. R., Greenwood, M. A., Barbs, D., Ikejiri, B., Chen, S. N., Oldfield, E. H.,Weber, R. J., and MuuI, L. M. Morphology of interleukin-2-stimulated humanperipheral blood mononuclear effector cells killing glioma-derived tumor cells invitro. J. Natl. Cancer Inst., 80: 171—176,1988.

23. Groscurth, P., Diener, S., Stahel, R., Jost, L., KAgi, D., and Hengartner. H. Morphologic analysis of human lymphokine-activated killer (LAK) cells. Int. J. Cancer, 45:694—704, 1990.

24. Figdor. C. G., van Kooyk, Y., and Keizer. G. D. On the mode of action of LFA-l.Immunol. Today. I 1: 277—280,1990.

25. Springer, T. Adhesion receptors of the immune system. Nature (Lond.), 346:425—434, 1990.

26. Namba, Y., Ito, M., Zu, Y., Shigesada, K., and Maruyama, K. Human T cell L-plastinbundles actin filaments in a calcium-dependent manner. J. Biochem., 112: 503—507,1992.

27. Robertson, M. J., Caligiuri, M. A., Manley, T. J., Levine, H., and Ritz, J. Humannatural killer cell adhesion molecules: differential expression after activation andparticipation in cytolysis. J. Immunol., 145: 3194—3201, 1990.

28. Patarroyo, M., Prieto, J., Rincon, J., Timonen, 1., and Lundberg, C.. Leukocyte-celladhesion: a molecular process fundamental in leukocyte physiology. Immunol. Rev.,114: 67—108,1990.

29. Umehara, H., Takashima, A., Minami, Y., and Bloom, E. T. Signal transduction viaphosphorylated adhesion molecule. LFA-l(CD18): is increased by culture of naturalkiller cells with IL-2 in the generation of lymphokine-activated killer cells. list.Immunol., 5: 19—27,1992.

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1996;56:138-144. Cancer Res   Mitchell J. Frederick, Lewis V. Rodriguez, Dennis A. Johnston, et al.   That pp65a and pp65b Are Phosphorylated Forms of L-Plastin Proteins Phosphorylated after Tumor Target Binding: Evidence

65,000 Lymphokine-activated KillerrMCharacterization of the

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