translocation of protein phosphatase 1 catalytic subunits ... ·...

ICANCERRESEARCH55,774-750,February15,l@@5l

ABSTRACT

Toelucidatetherolesofproteinphosphatasestype1(PP1)andtype2A(PP2A) in 1,25-dibydroxy-cholecalciferol [1,25(OH)2D3j-hnduceddifferentiation of HL-60cellsInto monocytes,weexaminedtheenzymeactivityand the protein and gene expressions of PP1 and PP2A In these cells.Calyculin-A augmented the 1,25(OH)2D3-lnduced differentiation of thecells.Treatment of thecellswith l,25(OH)2D3led to a decreasein PP1-likeactivity in the cytosol fraction, with a concomitant increase in the membrane and nuclear PP1-llke activity, as determined when protein phosphatase activity was assayed using myosin light chain as substrate In thepresenceof S aM okadaic acid. Western blot analysis with antibodiesspecific for PP1 catalytic subunit Isozymes(PP1a, PP1y, and PP1&)showed that all three PP1 isozymes were expressed but were differentiallydistributed in each cellular fraction. Subcellular redistribution of PP1-likeactivity during 1,25(OH)2D3-induced differentiation was mainly attributed to PPIyand PP1aproteins.In contrast, the localizationsof PP1SandPP2A catalytic and regulatory subunits were not significantly affected by1,25(OH)2D3 treatment. The gene expressions of PP1a and PP1y ap.pearedto be constantduring processesof monocyticdifferentiation. Thecorrelation between phenotypic and functional changes of HL-60 cells onthe onehandand subcellularredistributionof PP1-likeactivity on theother suggestthat the translocationsof PP1aand PP1i isozymesmaycontribute to the l,25(OH)2D3-induced monocytic differentiation of HL-60cells

INTRODUCTION

HL-60 cells differentiatealong the granulocyticor the monocyticpathwaywhen treatedwith the respectiveinducermolecules(1). Thischaracteristicfeatureof HL-60 cells attractedresearchers'interest,and this cell line hasbecome a frequently described model system forcell differentiationin vitro (1).

1,25(OH)2D33induceshumanmyelogenousleukemicHL-60cells,as well as normal human bone marrow cells, to differentiate terminally into maturemonocytes(1, 2). Differentiationwith this agentiscoupledto inhibition of proliferation.The intracellularmechanismsleadingto terminaldifferentiationhaveyet to be clarified(2). Becauseretinoic acid has proved to be effective in differentiationtherapyofacute promyelocytic leukemia, analoguesof 1,25(OH)2D3 also arecurrently being investigated as putative differentiation-inducingagentsto treat patientswith leukemia(3).

Kinases and phosphatases modulate the protein phosphorylationâ€œstatusâ€•of a cell, which in turn directly regulatesnumerousfundamentalbiologicalphenomenonsuchascell divisionanddevelopment

Received9/26/94; accepted1219/94.Thecostsof publicationof thisarticleweredefrayedin partbythepaymentof page

charges.This article mustthereforebe herebymarkedadvertisementin accordancewith18 U.S.C. Section 1734 solely to indicatethis fact.

1 This work was supported in part by grants for research from the Ministry of

Education,Scienceand Culture of Japan.2 To whom requests for reprints should be addressed.

3 The abbreviations used are: 1,25(OH)2D3, 1,25-dihydroxy cholecalciferol; PP1,proteinphosphatasetype 1; PP2A,proteinphosphatasetype 2A PP2A-C,proteinphosphatasetype2A catalyticsubunit;OKA, okadaicacid;NBT, nitrobluetetrazolium;PAGE,polyacrylamidegel electrophoresis;Cal-A, calyculinA; MLC, myosinlight chain;PKC,proteinkinaseC.

(4, 5). Accordingly, protein phosphatases may also be involved in the

process of HL-60 cell differentiation (6). The major serine/threonineprotein phosphatasecatalytic subunitsof mammaliancells comprisefour forms which havebeendesignatedas PP1, PP2A, PP2B (calcineurin), and PP2C, based on a classification system proposed byCohen (7) and Mumby and Walter (8). PP1 and PP2A do not have an

absoluterequirementfor divalentcations,whereasproteinphosphatases PP2B and PP2C are Ca2@/calmodulin- and Mg@4-dependent,respectively.OKA, a recentlydiscoveredtumor promoter,is a polyether fatty acid with IC5@sof 60â€”200nM for PP1 and 1â€”10nM forPP2A (9); therefore, it can distinguish between PP1 and PP 2A. Cal-A

is a spiroketal fatty acid with IC5@,sof 2 flM for PP1 and 1 nt@ifor PP2A(10). While the regulatorysubunitsof PP2A havebeenpurified andcloned,much lessis known of the regulatorysubunitsof PP1.Threedifferentbut highly similar isoformsof PP1catalyticsubunits(PP1a,PP1@y,andPP16)with an85â€”95%aminoacidsequenceidentity havebeenidentifiedby cDNA cloning,andtheir mRNAs are expressedina wide range of mammalian tissues (11). PP1 isozymes have beenreported to be identified in both soluble and particulate cellularfractionsand in variousforms(12). The glycogen-associatedform ofPP1,termedPP1G,consistsof thecatalyticsubunitcomplexedtoaMr124,000 G subunit which targets PP1 to glycogen particles (13).Recently, a myosin-bound PP1 holoenzyme in smooth muscle cellswas isolated and was found to consistof a catalytic subunit, Mr130,000 and Mr 20,000 regulatory subunits (13). In leukemic cells,including HL-60 cells, the regulationof PP1is not well understood.PP1 andPP2A are involvednot only in glycogenmetabolismandmusclecontractionbut also regulateprocessessuchas the cell division cycle (13). We reportedthat OKA and Cal-A augmentedthegranulocytic differentiation of FIL-60 cells induced by retinoic acid(14), andwe alsofound that retinoicacid inducesdown-regulationofthe PP2A catalytic subunit associatedwith granulocyticdifferentiation (15â€”17).To further elucidatethe involvementof protein phosphatases in HL-60 cell differentiation, we investigated changes in

phosphataseactivity and expressionpatternsof PP1 and PP2A proteins and mRNA expressions of PP1 isozymes during the monocyticdifferentiation induced by 1,25(OH)2D3.

MATERIALS AND METHODS

Reagents. 1,25(OH)@D3purchased from Solvay-Duphar B.V. (Weesp, theNetherlands)wasdissolvedin ethanol(10@M)andstoredat â€”20Â°Cwithprotection from light. CAL-A and OKA were purchased from Wako Pure

ChemicalIndustries,Ltd. (Osaka,Japan).Determination of Cell Differentiation. Proceduresusedfor the mainte

nanceof HL-60 cells and determinationof viable cell countswere doneasdescribed elsewhere (18, 19). The extent of differentiation was assessed by

morphology, the ability to produce superoxide as monitored by the reductionof NBT (SigmaChemicalCo.), nonspecificesterasepositivity,andsurfaceantigen analysis. For morphological assessment,cytospin was prepared andstained with May-Giemsa. Differential cell counting was done on 200 to 400stainedcells from at least two preparationsfor each experimentalpoint.Surfaceantigenswereexaminedby cytofluorometryin a FACSCan(BectonDickinson, Mountain View, CA) using monoclonalantibodies,including

774

Translocation of Protein Phosphatase 1 Catalytic Subunits during 1,25-Dihydroxyvitamin D3-induced Monocytic Differentiation of HL-60 Cells1

Serdar B. Omay, Hitoshi Ogasawara, Hideki Toyoda, Keiji Nakai, Hiroshi Shima, Minako Nagao, Marc C. Mumby,Hiroshi Shiku, and Masakatsu Nishikawa2

The 2nd Department ofln:ernal Medicine. Mie UniversftySchoolofMedicine, 2-174 Edobashi. Tsu, Mie 514, Japan (S. B. 0.. H. 0.. H. T.. K. N., H. S., M. NJ; CarcinogenesisDivision, National Cancer ResearchInstitute. Tokyo,Japan [H. S., M. NJ; and Department of Pharmacology, University of Texas, SouthwesternMedkal Center, Dallas, Texas75235-9041 IM. C. M.J

Research. on October 11, 2020. © 1995 American Association for Cancercancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

Table1Protein phosphazaseactivities and quantitativeanalysisof immunoreactivePP1 isozymes and PP2A-C in subcellularfractions ofHL-6OcellsProtein

phosphatase gig/mgprotein'@activityâ€•nmol/min/mg

(PP1-likeactivity) PP1a2 PP1y@ PP1B2PP2A-C2Cytosol27.1

+ 2.2 0.42 0.39 0.14 0.50(16.2 +1.9)Nuclear1

1.4 + 1.8 0.03 0.24 0.06 0.13(5.3 +1.5)Membrane

4.1 + 1.4 0.31 0.17 0.02 0.12(4.0 + 1.6)

PP1 TRANSLOCATION IN l,2S(OH)@D3-tNDUCED DIFFERENTIATION

CD1lb (OKM1;OrthoDiagnosticSystem,Inc.,Raritan,NJ),CD1lc (LeuM5;Becton Dickinson), and CD14 (My4; Coulter Immunology, Hialeah, FL).

Preparation of Cellular Fractions. HL-60 cells (1 X 10' cells for eachsample) were harvested by centrifugation and washed twice with ice-coldTris-HC1-buffered saline, pH 7.4. All preparative procedures were carried outat 4Â°C;thencells werehomogenizedin a buffer containing20 mMTris-HC1(pH 7.4), 2 mM DTF, 2 mM EDTA, 2 mM EGTA, 0.25 M sucrose, and a cocktail

of proteaseinhibitors (18) by a glass-to-glassPotter-Elvehjemhomogenizer.Thehomogenateswereimmediatelycentrifugedat 1,000X g for 10min,andtheresultingpelletwasusedasthecrudenuclearfraction.Thesupernatantwascollected and centrifuged at 100,000 X g for 1 h to separate cytosol andmembranefractions.Themembraneandnuclearfractionswererinsedwith thehomogenizationbuffer and subsequentlyresuspendedin homogenizationbuffer followedby glass-to-glasshomogenization.By furthercentrifugationat100,000 x g for 30 min, the membrane fraction was obtained.

Measurement of Phosphatase Activity. 32P-phosphorylated myosin lightchains were used as substrates of the phosphataseassaybecause they are goodsubstrates for both mammalian PP1 and PP2A (14). Phosphatase activity was

determined by the liberation of 32Pi from the substrate (32P-labeled Mr 20,000myosinlight chain)at 30Â°C,accordingto themethodsof PatoandKerc (20).The extentof dephosphorylationwas restrictedto below 10%.Under theseconditions, rates of dephosphorylation were linear with respect to time and

enzyme dilution.Immunoblot Analysis. Antisera against PPI catalytic subunit isoforms,

PP2A-C, A, and B regulatory subunits were obtained by immunizing a rabbitwith the synthesized fragments of PP1 (11) or PP2A (21, 22). HL-60 cells wereincubated with 1,25(OH)2D3 (20 nM), and the cellular fractions were obtainedasdescribedandsubjectedto SDS-PAGEusinga 12.5%runninggel followedby electrophoreticaltransferonto Immobilonpolyvinylidenedifluoride membrane(Millipore, Bedford, MA). The membranewas reactedwith specificantibodiesforPP1andPP2A,andimmunoreactiveproteinswerestainedusingthe avidin-biotin peroxidasecomplexmethod(Vectastain;Vector Laboratoties,Burlingame,CA).PrestainedSDS-PAGEstandards(Bio-Rad,Richmond,CA) were used as molecular weight standards. Quantitative estimation of thelevel of PP1isozymesandPP2A-Cwascarriedout densitometricallywith aMolecularDynamicsscanningdensitometer(Sunnyvale,CA) in conjugationwiththeImageQuantprogramrunona Dell PersonalComputer(Austin,TX)by scanningthe immunoreactiveband after immediatelyphotographingthevisualized band. The area of an individual peak was measuredabove background in densitometric tracings and was expressed as arbitrary unit (count).Thesignalwasthencomparedwith thatof knownamountsof thepurifiedrecombinant PP1a, PP1y, PP16, and PP2A-C. Routinely, at least four differentamountsof each enzyme were included as standardsto quantify the level ofeachenzyme.The immunoreactivesignalsdeterminedby this methodwere

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Fig. 1. Quantification of PP1 isozymes and PP2A-C by immunoblotting. PurifiedrecombinantPP1a,PP1@y,andPP16isozymesandPP2A-Cwereseparatedby SDS-PAGEandelectrophoreticallytransferredto Immobilonpolyvinylidenedifluoridemembranes.EachmembranewasincubatedwithPP1a,PP1y,PP16,andPP2A-C-specificantibodies,andtheimmunoreactivebandswerescannedwith a MolecularDynamicsdensitometerimmediately after photographingthesebands.

a Protein phosphatase activity (n = 3) was measured as described in â€œMaterialsandMethods.â€•PPI-Iike activity is the MLC phosphataseactivity in the presenceof 5 muOKA.

b The quantitative analysis of PP1 isozymes and PP2A-C were performed as described

in â€œMaterialsandMethods.â€•Thevaluesrepresentthemeanof threeexperiments.SDwaslessthan 10%.

proportional to increasing amounts of each enzyme (Fig. 1). If the signal of the

samplewasoff thelimit oflinearity shownin Fig. 1,theloadedquantityof thesample was adjusted until a signal in the linear range was obtained. The levelof PP1isozymesandPP2A-Cwereestimatedaccordingly(Table 1).

RNA Isolation and Northern Blot Analysis. Total cellular RNA wasextractedusingtheguanidiniumisothiocyanatetechnique(23). Twenty g.@goftotalRNA of HL-60cellswaselectrophoresedthrough0.8%agarosewith 18%formaldehydeandtransferredto Nytran membranes(SchleicherandSchuell,Dassel,Germany).After baking at 95Â°Cfor 2 h undervacuum,blots werehybridized at 42Â°Cin 50% formamide, 5X Denhardt's solution, 5X SSPE (1XSSPE= 0.15MNaCI,[email protected],and0.1 mMEDTA), 0.5%SDS,200@ig/mlof denatured salmon sperm DNA, and 32P-labeled cDNA probe. TheprobesusedwerethecDNAprobesof therat PP1aandPP1y(12,22,24).They were labeledwith [a-32P]dCTPusinga multiprime labelingsystemkit(Amersham, Buckinghamshire, England). Hybridized blots were finallywashedwith 0.1X SSC,(1X SSC 0.15MNaCI-l5 mMtrisodiumcitrate)and0.5% SDS at room temperature and autoradiographed.

RESULTS

Effect of Cal-A on 1,25(OH)2D3-induced Cell Differentiation.1,25(OH)2D3suppressedtheproliferationof HL-60 cells andinducedthesecells to differentiate terminally into monocyticcells. Morphological examination of May-Giemsa stained cytospin preparationsshowed that untreated HL-60 cells retained a blast-like phenotypethroughoutall of the experiments,whereasthe 1,25(OH)2D3-treatedcells gradually acquireda differentiatedpattern indicatedby surfacemarkers (CD1 ib, CD1 ic, and CD14), ability to reduce NBT, and

nonspecific esterasepositivity after 4 days of treatment (Fig. 2).Treatment of HL-60 cells with 1,25(OH)2D3 resulted in a timedependentincreasein NBT-positive andnonspecificesterase-positivecells and an increasein reactivitiesof the cells with CD1 ib, CD1 ic,and CD14 (Fig. 2), although the kinetics of each differentiationmarker acquisition was not always identical. The reactivities of HL-60cellswith CD1 lb and CD14 reacheda plateaumore rapidly than thatof CD1lc. The reductionof NBT and the positivity of nonspecificesteraseprovide functional markersfor monocyticdifferentiationofHL-60 cells. Cal-A at concentrations of 1 ni@ior lower had no effecton proliferation (14). As shown in Fig. 3, the addition of Cal-A incombinationwith l,25(OH)2D3led to further maturationwhen corn

2@' paredto findingswith Cal-Aalone.Cal-Aaloneled to no increaseinâ€˜p the numbers of NBT- and nonspecific esterase-positive cells.

1,25(OH)2D3-induceddifferentiationwas dosedependentat concentrationsof l,25(OH)2D3 from 2 to 100 flM.Exposureto Cal-A shiftedthe dose-responsecurveby l,25(OH)2D3 of monocyticdifferentiationof HL-60 cells to the left. However, Cal-A did not facilitate thedifferentiation induced by 100 nM l,25(OH)2D3. The addition of OKAin combinationwith l,25(OH)2D3did not augmentthe l,25(OH)2D3-

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induceddifferentiation,andhigherconcentrationsof OKA could notbe appliedin culturesbecauseof toxicity (datanot shown).

Phosphatase Activity during 1,25(OH)2D3-lnduced MonocyticDifferentiation. The activity of serine/threonineproteinphosphatasein HL-60 cells was assayedin the absenceof divalentcations,using32P-phosphorylated MLC of smooth muscle as substrate for bothmammalian PP1 and PP2A (15). Alteration of phosphatase activitywas examined in cytosolic, nuclear, and membrane fractions following treatmentof 1,25(OH)2D3(Fig. 4). Table 1 showsproteinphosphatase activities of cytosol, nuclear and membrane fractions, inuntreatedHL-60 cells.In HL-60 cells,approximately60â€”65%of totalactivity of MLC phosphatasewas presentin the cytosolfraction.Theexposureof exponentiallygrowingcells to l,25(OH)2D3resultedin agradual decreasein cytosolicphosphataseactivity, and the activityreachedthe lowest level (approx.40% decreaserelative to that inuntreatedHL-60 cells)4 dayslater.Concomitantwith thedecreaseinthecytosolicMLC phosphataseactivity in these1,25(OH)2D3-treatedHL-60 cells, there was a significant increasein this activity in both thenuclear and membrane fractions (Fig. 4). Treatment with 20 n@i1,25(OH)2D3 for 4 days resulted in 1.8- and 1.9-fold increasesin thenuclearandmembranefractions,respectively.Thesedatasuggestthatmostof the lost activity canbe accountedfor by associationwith the

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Time ( hr)

PP1 TRANSLOCAI1ON IN 1,25(0H)@D3-INDUCED DIFFERENTIATION

nuclearandmembraneactivity during the 1,25(OH)@D3-induceddifferentiation.A decreasein phosphataseactivity appearedto coincidewith increasesin thepositivity of the surfacemarkerCD11candthenonspecific esterasepositivity.

To elucidatewhich type of phosphataseis associatedwith thedecrease of MLC phosphatase activity in the cytosol of HL-60 cells

after treatment of 1,25(OH)2D3, inhibition assaywith 5 n@iOKA wasalso done (Fig. 4). While PP2A is inhibited mostly by 5 n.i OKA, PP1is almostnot affectedat this5 flMconcentration(9). The influenceofPP2B and PP2C which absolutely require divalent cations (8) waslittle, since a sufficient amount of EGTA was present in the MLCphosphataseassaymixture. MLC phosphataseactivity in the presenceof 5 n@iOKA is thought to be mostly PP1-like activity. The phosphatase activity, subtracting the PP1-like activity from total MLC phosphataseactivity is thought to be PP2A-like activity. Fig. 4 shows that60% of the phosphataseactivity in the cytosol fraction, 46% in the

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Fig. 2. Time courseof surfacemarkers,NBT positivity, and nonspecificesterasepositivity ofHL-6Ocells induced by 1,25(OH)@D3.HL-60 cells were incubated with 20 mu1,25(OH)@D3,andsurfacemarkers(A)usingCD11b(0), CD11c(@),andCD14(Ci)wereanalyzedby flow cytometry.B, the percentagesof NBT-positive(â€¢)and nonspecificesterase-positiveJ) cells after treatmentof 20 [email protected],25(OH)@D3.Points,meansfromthree separateexperiments; bars. SD (less than 10%).

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Fig. 3. Effect ofCal-A on 1,25(OH)@D3-induceddifferentiationofKL.60 cells.HL-60cells (4 X 10'/nil) were culturedwith variousconcentrationsof 1,25(OH)@D3in theabsence(U, â€¢)andpresence(0, 0) of 1 muCal-A.CelldifferentiationwasevaluatedbyNBT positivity (â€¢,0) andnonspecificesterasepositivity (U, 0) after72 h. , P < 0.05;â€¢@,P<0.02.

776



24 48 72 96

â€˜ â€˜ â€˜ . â€” differentiation, protein expressions of PP1 isozymes and PP2A in

cellular fractions were investigatedby Western blot analysis usingpolyclonal antibodies specific against PP1 isozyme catalytic subunits (PP1a, PP1y, and PP1&) and PP2A-C, A-, and B-regulatorysubunitsof PP2A. As shown in Fig. 5, the antibodiesagainstPPla,PP1y, and PP1& isozymes detected major immunoreactive bandswith Mr 36,000, Mr 36,000, and Mr 37,000, respectively, in eachcellular fraction of HL-60 cells. PP2A-C was detectedas a majorsingle band of Mr 35,000, the A-regulatory subunit as a major bandof Mr 65,000,and the Ba-regulatory subunit asa major bandof Mr55,000 (Fig. 6). Bf3- and B'-regulatory subunits of PP2A proteinswere not detectable in HL-60 cells. These results obtained wereeven better illustrated by densitometer scanning as shown in Fig.5B and Fig. 6B. We determined the protein amounts of PP1catalytic subunits and PP2A-C in the cytosol, nuclear and rnernbranefractions of HL-60 cells, using purified recombinantproteins

______________________________________ as standards (Table 1). We could not estimate the amounts of A-and Ba-regulatory subunits becauseof not having the purifiedsubunits. We found that each isozyme of PP1 catalytic subunitswas distributed differentially in subcellular fractions of HL-60cells, although all isoforms were present in each cellular fraction.The nuclearfractionmainlycontainedPP1y, whereasthe membrane fraction contained mostly PP1a. PP16 was a minor isotypein all cellular fractions. Most of PP2A-C was detected in thecytosol fraction. The addition of l,25(OH)2D3 led to a dramaticdecreasein the levels of PP1y and PP1a in cytosol fraction, witha concomitant increase of PP1'y and PP1a in nuclear and mern

______________________________________ branefractions.After 4 days of incubationwith l,25(OH)2D3,approximately 90% of cytosol PP1y hadbeenredistributed to boththe nuclear and membrane fractions. The doublet in nuclearfractions for PP1-yand PP1a might be proteolytic degradation. Incomparison, the decreasein cytosol PP1a protein was slower thanthat of PPl@y,and PP1a appeared to be redistributed mostly tothe membrane fraction. Thus, treatment of HL-60 cells withl,25(OH)2D3 led to a redistribution of PP1 isozymes from cytosolto nuclear and membrane fractions, and each PP1 isozymeappearedto be differentially translocatedduring the l,25(OH)2D3-induced differentiation. The distribution of PP2A-C, A-, andBa-regulatory subunits of PP2A among the subcellular fractionswere unchanged, consistent with the finding that PP2A-like activ

ity is unchanged during 1,25(OH)2D3-induced differentiation ofHL-60 cells (Fig. 4). In the membrane fraction, A-regulatorysubunit could not be detected until we applied a triple proteinamount for immunoblot analysis. It may explain why PP2A-likeactivity was very low in the membrane fraction. PP2A-C andB-regulatory subunits were detected,but PP2A-C/A complex formation was probably little for PP2A activity, because of therelatively small amount of A-regulatory subunit.

To determine whether the 1,25(OH)2D3-inducedreduction ofPP1a and PP1'y proteins in the cytosol fraction is reflected at thereducedlevel of RNA transcripts,Northern blot analysiswas doneon total cellular RNA preparedfrom HL-60 cells, before and aftertreatmentwith 1,25(OH)2D3(Fig. 7). The PP1-yand PP1a probesdetected major bands of 1.8 and 1.6 kilobases, respectively, inHL-60 cells. Expressions of both PP1'y and PP1a transcripts inHL-60 cells were relatively unalteredfollowing treatmentwith1,25(OH)2D3. Rehybridization of these blots with the glyceraldehyde-3-phosphatedehydrogenasecDNA probe confirmed that relatively equal amounts of RNA were loaded in each lane. Theseresults provide further support that cytosolic PP1y and PPlccproteins were translocatedto the membrane and nuclear fractionsduring l,25(OH)2D3-induced differentiation.

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PP1 TRANSLOCATION IN 1,25(OH)@D3-INDUCED DIFFERENTIATION

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Fig. 4. Time courseof proteinphosphataseactivity in cellularfractionsof HL-60 cellsduring 1,25(OH)2D3-induceddifferentiation.HL-60 cells (1 X 10@cells) were treatedwith 20 flM 1,25(OH)@D3,andcytosolic(A), nuclear(B), andmembrane(C) fractionsofHL-60 cells were prepared at indicated times. Protein phosphataseactivity was measuredin the absence(â€¢)or presence(A) of 5 muOKA, as describedin â€œMaterialsandMethods.â€•Points,meansfrom threeseparateexperiments;bars,SD (lessthan 10%).

nuclear fraction, and 98% in the membranefraction, respectively,show PP1-likeactivity in untreatedHL-60 cells.Thereappearsto bevery little PP2A-like activity in the membrane fraction of HL-60 cells.Fig. 4 indicatesthat the PP1-likeactivity wasgraduallydecreasedinthe cytosol fraction and increasedin the nuclear and membranefractionsduring 1,25(OH)2D3-induceddifferentiationof HL-60 cells.On the other hand,PP2A-like activity was relatively constantin thecytosol and nuclear fractions during 4 days after addition of1,25(OH)2D3. These data suggest that PP1-like activity can translocate from the cytosol to the nuclear and membranefractions aftertreatmentof HL-60 cells with 1,25(OH)2D3.

Expressions of PP1 Isozymes and PP2A during 1,25(OH)2D3-inducedDifferentiationof HL.60 Cells. To furtherclarifythe selective decrease in PP1-like activity during 1,25 (OH)2D3-induced

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subunits of H.L-60 cells during 1,25(OH)@D3-induceddifferentiation. HL-60 cellsTime( hr) (1 x io@cells)weretreatedwith20mul,25(OH)@D3,andeachcellularfractionwas

obtainedasdescribedin â€œMaterialsandMethods.â€•A, 10 @gproteinof cytosol,nuclear,andmembranefractionswereanalyzedbyiminunoblotanalysisusingantiseraspecificfor

Fig. 5. Time courseof changesin immunoreactivePPm, PP1-y,and PP18 catalytic@ catatytic(PP2A-C) andA- andBa- regulatorysubunits,asdescribedin â€œMaterialssubunits of HL-60 cells during 1,25(OH)@D3-induceddifferentiation. HL-6O cells and Methods.â€•Twenty g.@gwere applied from nuclear and membranefractions for(1 X 10@@cells)weretreatedwith 20 mu1,25(OH)@D3,andeachcellularfractionwas@ and30 pg proteinof membranefractionwereappliedfor the A-regulatoryobtainedasdescribedin â€œMaterialsandMethods.â€•A, theproteinsof cytosol,nuclear,and subunit.B, graphicdisplayof PP2A catalyticand regulatorysubunits(A), as quantitaledmembranefractionswere analyzedby immunoblotanalysisusing antiseraspecific for by densitometry(C, PP2A-C;0, A-regulatorysubunit;0, Ba-regulatorysubunit).ResultsPP1y,PP1a,andPPIBisozymesasdescribedin â€œMaterialsandMethods.â€•Ten @gofproteinsamplewereappliedtoeachlane,while20 g.@gwereappliedfromnuclearfraction a@eexpreSSedasthepercentageof themeancontrolvalueinuntreatedHL-60cells.These

data are from one representativeexperiment, but similar results were obtained in two otherfor PPm andmembranefractionfor PP18.B, graphicdisplayof PP1isozymes(A), asquantitated by densitometry (@, PP1'y',A, PP1a; A, PP18). Results are expressed as the exPeriments.percentageof the meancontrolvaluein untreatedHL-60 cells.Thesedataarefrom onerepresentativeexperiment,but similar resultswereobtainedin two otherexperiments.

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pP1 TRANSLOCATION IN 1,25(OH)@D3-INDUCED DIFFERENTIATION

ulocytic inducers share PKC as a target molecule and that the activation and/or inductionof PKC is a common mechanismfor bringingabout terminal differentiation of HL-60 cells, irrespective of theterminal cellular phenotype. The biochemical activation of PKC aloneis apparently insufficient to account for the differentiation responsesto monocytic or granulocytic phenotypes,and additional elementsprobablycontributeto eachdifferentiation pathway.

We reported that Cal-A, a potent inhibitor of PP1 and PP2A,

augmentedthe retinoic acid inducedgranulocyticdifferentiationbutnot the phorbol ester induced macrophage-like differentiation ofHL-60cells(14).Moreover,PP2Aisdown-regulatedduringretinoicacid-induced granulocytic differentiation, while PP1 is unchanged(15â€”17).The finding that Cal-A also augments l,25(OH)2D3-inducedmonocytic differentiation of HL-60 cells is evidence for the involvement of protein phosphatases.Treatment of HL-60 cells with1,25(OH)2D3 led to a decrease in cytosolic PP1-like activity with aconcomitantincreasein PP1-like activity of membraneand nuclearfractions.Reductionof cytosolicPP1-like activity correlatedwith theincreased expression of certain differentiation markers related to

monocytes. Subcellular redistribution of PP1-like activity induced byl,25(OH)2D3wasmainlydueto theselectivetranslocationof cytosolic PP1'y and PP1a proteins to membrane and nuclear fractions,whereasthe distributionof PP18 and PP2A was relatively constant.Interestingly, translocation of PPFy was more rapid than that of PP1a,henceindividual PP1 isozymesmight be differentially regulated.PP1was shown to be colocalized with chromosomes in mammalian cellsat mitosis (28). Intracellular distributionof PP1 activity may be animportant aspect of regulation related to the differentiation of HL-60cells. Induction of c-fos mature transcriptsand an increasein AP-lactivity havebeennotedin casesof 1,25 (OH)2D3-induced inhibitionof proliferation andmonocyticdifferentiation (29). l,25(OH)2D3wasfoundto causea decreasein c-myc mRNA expressionin HL-60 cells(30). These nuclearevents may be relatedto the increasedPP1'yactivity in the nucleus, facilitating the activated state of definite

transcriptionfactors, including 1,25(OH)2D3 receptor,and affectingprocessesof differentiation. It has been shown that PP1 activity isassociatedwith glycogenparticlesandSR membranesand is enrichedin other particulate fractions and organellessuch as microsomes,ribosomes,nuclei,andmyofibrils (5). The increaseof PP1y andPP1aprotein in the membranefraction translocatingfrom cytosol fractionmay be indicativeof the changingtargetingspecificity.The decreasein immunoreactive PP1y and PP1a isoforms of cytosol fraction during 1,25(OH)2D3-induced differentiation was not associatedwith amodified expression of PP1'y and PP1a genes. Chronic 1,25(OH)2D3treatment has no significant effect on the expressions.Thus, themodulation of PP1 isozymesmay be involved in the regulation of1,25(OH)2D3-induceddifferentiation.Although PKC is up-regulatedby retinoic acid and 1,25(OH)2D3 (6), protein phosphatasedynamicsdiffer possiblyin associationwith different pathwaysof differentiation. Cal-A augmentedbothretinoicacid-inducedgranulocyticdifferentiation (14) and 1,25(OH)2D3-induced monocytic differentiation;however, during retinoic acid-induced granulocytic differentiation,the down-regulation of PP2A-C occurred (15, 16), whereas during thel,25(OH)2D3-induced monocytic differentiation, translocation and redistribution of PP1 isozymes occurred. On the other hand, Cal-A didnot augmentthe phorbolester-inducedmonocyticdifferentiation,andthere was neither down-regulation nor translocation of PP1 and/orPP2A (14â€”16).When these results of our studies are considered alltogether, it becomesclear that each differentiation pathway has itsspecific phosphatase enzyme dynamics, and the translocation-redistribution of PP1 isozymes during l,25(OH)2D3-induced monocyticdifferentiation that we have reported in this study is specific for thispathway and not a generalphenomenon.

6

GAPOH'@

0 12 24 48 72Qw)

PPIa @-S@@I4@@m16kb

I

GAPOH @-@ â€”@@

0 12 2448 72(1w)Fig.7. Time courseof PP1yandPP1aisozymesmRNA expressionsin HL-60 cells

stimulatedwith 1,25(OH)2D3.HL-60 cells(2 X iO@cells)weretreatedwith 1,25(OH}@D3.Total RNA (20 @g)was isolated from HL-60 cells at the indicated periods of time belowthecorrespondinglanes.Northernblotsoftotal RNAwere hybridizedto 32P-labeledPP1'y(first lane) and PPm (third lane) cDNA probes.The same RNA blots were hybridizedto glyceraldehyde-3-phosphatedehydrogenaseeDNA probe for assessmentof RNAamounts in each lane (second and fourth lanes). Similar results were obtained in twoindependentexperiments.

DISCUSSION

Although a number of phosphatases have been isolated and char

acterized and some functions clarified, data regarding their role incellular differentiation, especially related to leukemia, are scanty. Thepresentstudieswere designedto define PP1 andPP2A expressionsinHL-60 cells, to examine the effects of l,25(OH)2D3 treatment on the

phenotypeand functionof thesecells, and to correlatethem with theeffects on enzymatic activity of PP1 and PP2A and on expression ofimmunoreactiveproteinsof PP1andPP2A.Using antibodiesspecificfor eachof three PP1 catalyticsubunitisozymes,a, y, and 6 and forPP2A-C and A- and B-regulatory subunits of PP2A, we followedchangesin individual PP1 speciesat the protein level.

l,25(OH)2D3 @5reported to exert its effects on the cells in twoways, by rapidly stimulatingmembranephosphoinositidebreakdown,thereby activating PKC (25), and by binding to its nuclear hormonereceptor (26). The 1,25(OH)2D3 receptor is classified as a member ofnuclearreceptorfamilywithsequencesincommonwiththosefortheretinoic acid receptor (26). 1,25(OH)2D3 receptor regulation as atranscription factor is considered to be governed by phosphorylation

of the serine51 residueby PKC (27). 1,25(OH)2D3-inducedmonocytic differentiationof HL-60 cells is associatedwith an increaseinboth PKC activity and expression of PKCx and PKCj3, as well as thesteady-statelevels of PKCa and PKC@ mRNA expressions(6). Onthe other hand, the levels of both PKC activity and the expressionPKCE, PKC@3,and PKCy isoforms have also been shown to increaseduring granulocytic differentiation of HL-60 cells induced by DMSOandretinoic acid (6). Thesestudiessuggestthat monocyticandgran

779

PPI'y@.@ .@18kbI-I.



PPI TRANSLOCATIONIN 1,25(OH)@D@-INDUCEDDIFFERENTIATION

rat tissues. Biochem. Biophys. Res. Commun., 192: 1289â€”1296,1993.12. Zhang, Z., Bai, G., Shima, M., Thuo, S., Nagao, M., and Lee, E. Y. C. Expression and

characterization of rat protein phosphatases-la, -lyl, -1y2, and -18. Arch. Biochem.Biophys., 303: 402â€”406,1993.

13. Alessi, D., Macdougall,L. K., SoIL M. M., Ikebe,M., andCohen,P. The controlofprotein phosphatase-1by targetingsubunits. Eur. J. Biochem., 210: 1023-4035,l9fl

14. Morita, K., Nishikawa, M., Kobayashi, K., Deguchi, K., Ito, M., Nakano, T., Shims,H., Nagao, M., Kuno, T., Tanaka,C., and Shirakawa,S. Augmentationof retinoicacid-induced granulocytic differentiation in HL-60leukemia cells by serine/threonineprotein phosphatase inhibitoN FEBS Left., 314: 340-344, 1992.

15. Tawara, I., Nishikawa, S., Morita, K., Kobayashi, K., Toyoda, H., Omay, S. B.,Shims, H., Nagao, M., Kuno, T., Tanaka, C., and Shirakawa, S. Down-regulation byretinoic acid of the catalytic subunit of protein phosphatase type 2A during granulocytic differentiation of HL-60 cells. FEBS Lell., 321: 224â€”228,1995.

16. Nishikawa, M., Omay, S. B., Toyoda, H., Tawara, I., Shima, H., Nagao, M.,Hemmings, B. A., Mumby, M. C., and Deguchi, K. Expression of the catalytic andregulatory subunits of protein phosphatase type 2A may be differentially modulatedduringretinoicacid-inducedgranulocyticdifferentiationofHL-60cells.CancerRes.,54: 4879-4885, 1994.

17. Omay, S. B., Nishikawa, M., Morita, K., Toyoda, H., Shims, H., Nagao, M.,Kitagawa, S., Saito, M., and Shiku, H. Decreased expression of protein phOsphatasetype 2A in }II@-60variant (HL-6ORA')cells resistant to induction of cell differentiation by all-transretinoicacid. Exp. Haematol.,in press, 1995.

18. Nishikawa,M., Komada,F., Uemura,Y., Hidaka,H., and Shirakawa,S. Decreasedexpression of type II protein kinase C in HL-60 variant cells resistant to induction ofcell differentiation by phorbol theater. Cancer Rca., 50: 621â€”626, 1990.

19. Uemura, Y., Nishikawa, M., Komada, F., and Shirakawa, S. Alteration of intracellularactin levels induced by phorbol diester in human HL-60leukemia cells susceptible orresistant to differentiation, and the effectsofprotein kinase inhibitors. Leukemia Res,13: 545â€”552,1989.

20. Pato, M. D., and Kerc, E. Purification and characterization of a smooth musclemyosin phosphatase from turkey gizzards. J. Biol. Chem., 260: 12359-12366, 1985.

21. Kamibayashi, C., Lickteig@R. L, Extra, R., Walter. G., and Mumby, M. C. Expressionof the A subunitof proteinphosphatase2A andcharacterizationof its interactionswith the catalytic and regulatory subunits. J. Biol. Chem., 267: 21864â€”21872, 1992.

22. Shims, H., Haneji, T., Hatano, Y., and Nagao, M. Expression ofprotein phosphatases1 and 2A in rattestis. Adv. ProteinPhosphatascs,7:489-499,1993.

23. Maniatis, T., Fritsch, E. T., and Sambrook, J. Analysis of RNA. In: C. Nolan (ed.),Molecular Cloning: A Laboratory Manual, Book 1, pp. 7.37-7.84. Cold SpringHarbor, NY: Cold Spring Harbor Laboratory. 1989.

24. Hatano, Y., Shims, H., Haneji, T., Miura, A. B., Sugimura, T., and Nagao, M.Expressionof PP2AB regulatorysubunit@ isotypein rat testis.FEBSLeft.,324:71â€”75,1993.

25. Khare, S., Wilson, D. M., Tien, X., Dudeja, P. K., Wali, R. K., Sitrin, M. D., andBrasitus, T. A. l,25-Dihydroxycholecalciferol rapidly activates rat colonic particulateguanylate cyclase via a protein kinase C-dependent mechanism. Endocrinology, 133:2213â€”2219,1993.

26. Hsieh, J., Jurutka, P. W., Galligan, M. A., Terpening. C. M., Haussler, C. A., Samuels,D. S., Shimizu,Y., Shimizu,N., and Haussler,M. R. HumanvitaminD receptorisselectively phosphorylated by protein kinase C on serine 51, a residue crucial to itstrans-activation action. Proc. Nati. Acad. Sci. USA, 88: 9315â€”9319,1991.

27. Hsieh, J., Jurutka, P. W., Nakajima, S., Galligan, M. A., Haussler, C. A., Shimizu, Y.,Shimizu, N., Whitfield, 0. K., and Haussler, M. R. Phosphorylation of the humanvitamin D receptor by protein kinase C. J. Biol. Chem., 268: 15118â€”15126, 1993.

28. Fernandez, A., Brautigan, D. L, and Lamb, J. C. Protein phosphatase type 1 inmammalian cell mitosis: chromosomal localization and involvement in mitotic exit. J.Cell Biol., 116: 1421â€”1430,1992.

29. Mollinedo, F., Gajate, C., Tugores, A., Flores, I., and Naranjo, J. R. Differences inexpression of transcription factor AP-1 in human promyelocytic HL-60 cells duringdifferentiation towards macrophages versus granulocytes Biochem. J., 294: 137-144,1993.

30. Mangasarian, K., and Mellon, W. S. 1,25-Dthydroxyvitamin D3 destabilizes c-mycmRNA in HL-60 leukemic cells. Biochim. Biophys. Acts, 1172: 55-63, 1993.

780

The mechanisms underlying the different redistribution of PP1isozymes in response to 1,25(OH)2D3 treatment are not clear.PP1 consists of a multimeric structure composed of a catalytic subunitcomplexed to a number of regulatory components that play importantroles in regulating phosphatase activity or targeting it to specificsubcellular locations (8). The free catalytic subunit has not beendetected in cells. The amino acid sequences of amino-terminal andcentral regions of all PP1-catalytic subunit isozymes are highly conserved, and differences are mainly in the carboxy-terminal regions (8).The PP1y, PPTh, and PP1a proteins expressed in Escherichia colihave similar enzymatic properties, with respect to substrate specificities and sensitivities to inhibitor-2 and OKA (8). Different PP1catalytic subunit isoforms may differentially associate with regulatorycomponents through interactions dependent on their unique carboxyterminal regions. The regulatory components of each PP1 isozyme, asyet undefined in HL-60 cells, may modulate the different translocationpatterns of individual PP1 isozymes during monocytic differentiation.However, further studies of regulatory subunits should be performedin HL-60 cells, although physiological importance of this redistribution is unknown at present.

ACKNOWLEDGMENTS

We are thankfulto M. Oharafor criticalreadingof the manuscript.

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11. Shims, H., Hatano, Y., Chun, Y., Sugimura, T., Zhang, Z., Lee, E. Y. C., and Nagao,M. Identificationof PP1catalyticsubunitisotypes PPl@y1,PP18andPP1a in various



1995;55:774-780. Cancer Res Serdar B. Omay, Hitoshi Ogasawara, Hideki Toyoda, et al. Differentiation of HL-60 Cells

-induced Monocytic3during 1,25-Dihydroxyvitamin DTranslocation of Protein Phosphatase 1 Catalytic Subunits

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