Differential Phosphorylation of Human Thymidine Kinase in ...

THEJOURNALOF BIOLOGICAL CHEMISTRY

Vol. 269,No.33, Issue of August 19,pp. 21249-21254, 1994 Printed in U.S.A.

0 1994 by T h e American Society for Biochemistry and Molecular Biology, Inc.

Differential Phosphorylation of Human Thymidine Kinase in Proliferating andM Phase-arrested Human Cells* (Received for publication, April 26, 1994)

Zee-Fen ChangS, Duen-Yi Huang,and Nai-Che Hsue From the Department of Biochemistry, Chang Gung College of Medicine a n d Technology, 259 Wen-Hwa 1 Road, Tao-Yuan 3333, Taiwan, Republic of China

be distinctly different. It is clearthat The expression of cytosolic human thymidine kinase HeLa cells were found to activity is needed for DNA replication duringthe S phase showthat TK is hyperphosphorylatedduringthe M but not forM phase progression.Yet, the amount of TK protein phase in several human cell lines. Our data from char- always reachesits maximal level at the M phase in HeLa cells. acterizing TK phosphorylation in proliferating and M Therefore, it is intriguing to knowthe relationship between the phase-arrested HeLa cells suggest that the polypeptide catalytic efficiency of TK a n d its hyperphosphorylated form.We of TK is differentially phosphorylated during the pro- found that hyperphosphorylation of TK in mitotically blocked gression of the cell cycle. TK in the M phase-arrested HeLa cells is accompanied by a decrease in its affinity for its HeLa cells was found to have a 10-fold lower affinity forsubstrate, thymidine. Phosphorylationhas been considered to its substrate, thymidine, than in the proliferating cells. play a central rolein regulating and interplaying the biochemiWe propose that phosphorylation of TK by the mitotic cal eventsthat coordinate the progression of the cell cycle. For kinase(s) mayprovide an attenuatingmechanismto example, it is now knownthat t h e cdc2 kinase-cyclin B complex prevent unnecessary synthesis of dTTP at the time of is responsible for the phosphorylation of a number of proteins, mitosis. such as nuclear lamins, vimentin, and caldesmon, that a r e required for their molecular reorganization during the M phase (reviewed by Noburyand Nurse (1992)). Here, we proposethat The eukaryotic cellcycle is composed of an ordered series of the physiological functionof t h e specific TK phosphorylation by events regulated bya network of enzymes and protein factors. the M phase kinase(s) is to decrease its catalytic efficiency to During the transition fromthe G, t o t h eS phase, the activities prevent unnecessary synthesis of dTTP in mitotic cells.

(TK)occurs in a cell cycle-dependent manner. Here, weTK

of several enzymes related to DNA replication are promoted (reviewed by Hofbauer and Denhardt (1991)). One of these MATERIALSANDMETHODS enzymes is cytosolic thymidine kinase (TK).’ T K is an enzyme Cell Cultures and M Phase Arrest-HeLa and LMTk- cells were that catalyzes the transfer of the terminal phosphateof ATP to maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. K562 and HL-60 cells were maintained at a the 5’-hydroxyl groupof thymidine to form dTMP, which the is salvage pathwayfor dTTP synthesis. It is well documentedthat density of 2 x lo5 celldml in RPMI 1640 medium supplemented with the transcriptional, post-transcriptional, and translational ac- 10%heat-inactivated fetal bovine serum in ahumidified atmosphere of tivation of TK expression lead to the elevation of its activity at 5% CO, in air. For M phase arrest, NOC, at a final concentration of 0.5 pg/ml, was added to HL-60 and K562 cells at a density of 5 x 105/mland the Gl/S phase (Stuart etal., 1985; Coppock and Pardee, 1987; to subconfluent HeLa monolayer cultures. After 17 h, cells were harStewart, et al., 1987; Lieberman et al., 1988; Ito and Conrad, vested and washed three times. Byflow cytometric analysis, using 1990; Sherley and Kelly, 1988).The level of T K in HeLa cells propidium iodideDNA staining, cells treated under the above condition reaches its maximum at the M phase, and a subsequent deg- were shown to be arrested at theG@l phase. Production and Characterization ofAntiserum against Human Thyradation of TK polypeptide occurs during the mitosis (Kauffmidine Kinase-An ScaI-BamHI restriction fragment containing the man a n d Kelly, 1991). Studies on TK regulation during the cell cycle have provided a good model for understandingthe molec- nucleotides of the human thymidine kinase codingsequence was blunted with the Klenow fragment of DNApolymerase I,fused in frame ular events regulatingthe progression of t h e cell cycle. to the unique SmaI site of a glutathione S-transferase gene in an exOur laboratoryhas previously shown that TK was phospho- pression vector (Smith and Johnson, 1988), and introduced into rylatedinhumanpromyeloleukemiacells in responseto Escherichia colicells. The bacterial extracts were fractionated on a growth stimulation (Chang, and Huang, 1993). In this study, glutathione-Sepharose 4B column (Pharmacia Biotech Inc.). After exTK phosphorylation was investigated in proliferating and mi- tensive washing with phosphate-buffered saline, the protein-bound Sepharose beads were boiled in SDS-PAGE loading buffer. The 52-kDa totically blocked cells. When cells were M phase-arrested by fusion protein was resolved by 10% SDS-PAGE.The gel band containing treatment with t h e microtubule-depolymerizing drug, nocoda- the glutathione S-transferase-TK fusion protein was mincedand eluted zole (NOC), TKbecameheavilyphosphorylated in HL-60, for immunization. Rabbits received an intravenous injection of 100 pgof K562, and HeLa cells. Furthermore, the phosphorylated sites purified glutathione S-transferase-TK followed by booster injections at 3-week intervals with 100 pg of fusion protein. Animals were bled 1 in TKpolypeptide in proliferating and mitoticallyblocked week after each boost,and antisera were tested for the ability to detect TK by immunoblotting and immunoprecipitation. * This research was supported by Grant CMRP366 from the Chang human Construction ofCMV-TK Plasmid and DNADansfection-The Gung College of Medicine and Technology and Grant NSC-83-0412-B182-020-MO2from the National Science Councilin Taiwan. The costs of CMV-TK plasmid was constructed by cloning the ScaI-BamHI restricpublication of this article were defrayed in part by the payment of page tion fragment containing cDNA nucleotides of the human TK into downcharges. This article must therefore be hereby marked “advertisement” stream of cytomegalovirus (CMV)immediately early promoter of a vector pCDM8 (Seed, 1987). LMTk cells (5 x lo5)plated on 60-mm dishes in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $To whom correspondence should be addressed: Fax: 886-03-328- were transfected by incubation with 3 pg of CMV-TKplasmid or control vector plus 18 pg of lipofectin (Life Technologies,Inc.) mixture in 2 ml 3031. The abbreviations used are: TK, cytosolic thymidine kinase; NOC, of medium for 6 h. The medium was then changed with Dulbecco’s nocodazole; PAGE, polyacrylamide gel electrophoresis;CMV, cytomega- modified Eagle’s medium containing 10% fetal bovine serum. Twentylovirus. four hours after transfection, cells werelabeled with 1ml of Dulbecco’s

21249

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Phosphorylation of Human ThymidineKinase

b

66 -

4 5-

-29

29-

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C (kDa) 1

2

3

4

I I mm

97-

66-

T

-

45-

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29 24

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I

0

5

24 kDa

modified Eagle's medium with 0.05 m\c methionine plus 10'; dialyzed fetal bovine serum containing 500 pCi of [""Slmrthionine IArnrrsham After washing with cold phosphatr-huffrred s:jlinr. crlls Corp.) for 12 h. were lysed with a lysis buffer containing50 m u Tris-HC'I buffrr ( p H 7.51. 18 Nonidet P-40, 150 msc NaCI. 50 m\c SaF, 0.2'; sodium drrrxyrholatr. 0.2% sodiumdodecylsulfate, 1 m\c \3-mrrcaptorthanol. 1 m\c phrnylmethylsulfonyl fluoride, 10 p g h l each of Ieuprptin and nprotinin fnr immunoprecipitation analysis. Immr~noblotting and Immunoprrripitnlionn-Rnhhit a n t i s r n l m against glutathione S-transferase-TK prrparedas descrihrti ahovr was used for t h e immunological procrdurrs. Thr cytoplasmic rxtracts(80 pg of protein) were separated by 1 0 7 SDS-PAGE fnllowrd hy electrophoretic transfer onto a polyvinylidene difluoridr memhranr 1 Millipore Corp.) (Towbin et a/..19791,After hlocking with 57 powdrrrd m~lk. thr membrane was incubated with antiserum against glutathionr S-transferase-TK (1:1500) for 4 h and treated for 2 h with alkalinr phosphatase-conjugated goat anti-rabbit I d ; a n t i b d y (Promegat. Thr alknline phosphatase color drvelopmrnt was prrformrd according to t h e vender's instructions i Promega I . For immunoprecipitation. crll lysates were preclenrrd hy thr addition of 30 pl of protein A-Sepharosr1 Pharmacia I and crntrifugation. TK antiserum or preimmune serum 15 p11 were added to thr clanfird Iysates and incubatedfor 2 h. Immunncomplrxes were then adsorbed onto protein A-Sepharose and washed five times with lysis huffrr prior to SDS-PAGE. Metabolic h h e l i n g with f'PIOrthopho.qphntr-Suhconflurnt mnnolayer of HeLa, K562, and HL-60 crlls at a density of 5 x 10."ml m c h were pulse-labeled for 2 h at 37 -,C with '?PO; 1500 pCi~ml,Arnrrsham Corp.) in phosphate-free medium. Cells wrrr washrd with phosphatebuffered saline and lysed in the lysisbuffer. Equal trichlorrrncrtic acidinsoluble counts of lysates were used for immunoprrcipitation. fnllowrd by SDS-PAGE and autoradiography. Phosphrmmino Acid Analysis and Prptirfr Mapping-Crlls wrrr Iabeled with ' T O : f 1 mCi/ml, Amersham Corp.1 in phosphatr-frrr medium as described above. The immunoprecipitated '.'P-lahrlrd TK was separated on SDS-PAGE. transferred onto a polyvinylidenr difluoride membrane, and digested with 6 s HCI (Cooper r t 01.. 198.31.T h r hydrolysates were mixed with unlaheled phosphoserinr. phosphothrroninr. hy thin-layrr rlrrtroandphosphot-yrosincstandardsandanalyzed phoresis followed by thin-layerchromatography.Thin-layer rlrctrnphoresis was performedin X 8 7 formicacld'glacialaceticacitl'water (25:78:897, vlviv) a t 1350 V for 20 min. and nscrnding chromatography was performed in isobutyric acid. 0.5 \I NH,OH 1.53. v ' v ~ . Cyanogen Bromide Clral~ageofTK-The '"I'-labrled TK immunoprrcipitates from proliferating andNOC-treated HeLn cells wrrr srparatrd by SDS-PAGE and transferred onto nitrncellulosr memhranrs for CNRr cleavage analysis. as descrihrd hv Luo. rt al. ( 1991 1. Thr memhranrs were incubated with 100 m g h l CNRr in 707 formic acid for 1.5 h a t ronm temperature. ARer the supernatants wrre dried. the prptides were separated by electrophoresis on a 2.19 i w / v ) acrylnmidr. 0.054'; (w/v)bisacrylamide gel. ATricinecathodebuffercontaining 0.01 \c Tricine, 0.13SDS, 0.1 SI Tris base ( p H X.25, was usrd for rrsolving low molecular weight peptides. (Schagger and von .Jagow. 19H71.The gel was dried and exposrd to an intenrifving screen for autoradiography, Thymidine Kinasr Assay-Cells werr Iysrd in 50 m\c Tris-II('I ( p H 7.9). 50 mhf NaF, and 1 m\c @-mercaptoethanolhv sonlcation. T h r cytoplasmic extracts were obtained by centrifuging thr lvsatcs at 1f)).000x g for 10 min. C-ytosolic extract (10 pg of protrlnt was incuhatrd fnr 10 min a t 37 "C in a final volumr of 100 p1 of reaction mixturr containing 50 mhc Tris-HCI ( p H 7.9). 3 mh! /3-mercaptwthanol. 2.5 m\r M&l,. 5 m\c ATP, 0.l"r bovine serum albumin, 90 p \ ~thymidinr. 5 m\c SaFqand 2 pCi of ["Clthymidine (50mCi/mmol. Amersham ('trrp. 1. I"C:IThymid~ne nucleotide formation was determined as drscribrd r l s r w h r r r ( I r r and Cheng, 1976,. ~

~

~~

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ase and glutathione S-transferase-TK fusion proteins arr indicatrd hy

arrows. h, LMTk- cells were transfected with control vrctor plasmid (CDMFI; lane 1 J or with the construct encoding human TK p~C\lV-Ttir (lane 2 ) . I"'S1Methionine-Iaheled protein rxtracts wrrr suhjrctrrl to Immunoprecipitation with TK antiserum. r , HrLa cells mrt:~lnrlically Iabeled with I~"'Slmethionine (500 p C i / m l ~wrrr immunopreclpitated hy Flc. 1. Expression of glutathione S-transferase-TKfusion pro- preimmune ( l a w I ) and TK antiserum ( l a m 2 I. TK mtiserum prradtein and generation of antibody against cytosolic TK.a,glutathisorhed on glutathione S-transferase glutathione-Srpharosr ( h n r .7 I , and glutathione S-transferase-TK fusion pro- preadsorbedonglutathioneS-transferasr-Ttiglutathionr-Srpharose one S-transfrrase (GST) tein (GST-TK) precipitatedfrombacteriallysateswithglutathione(lane 4 ), individually. Cytosolic extract(100 pg o f protrin t of 1lrI.a crlls Sepharose. Proteins wereresolved by 10"r SDS-PAGE and visualized by was separated on the same gel for IVrstern hlot analysisdrtectrd hy T t i staining with Coomassie Brilliant Blue R-250. Glutathione S-transfer- antiserum (lane 5 ). Std, molecular weight standards.


21251

L5

29 21 kDa

20

FIG.2. TK is hyperphosphorylated in mitotically blocked HL-60, HeLa, and K562 cells. a , logarithmically growing HIAX) cells or NO(:-treated cells were laheled hy I"'Plorthophosphate for 2 h. Cells were lysed and immunoprecipitated by antiserum against glutathione S-transferase-TK ( T ) and preimmune serum( C ) . Sampleswereseparated by 10% SDSPAGE. transblotted to polyvinylidene difluoride membrane, and visualizedby autoradiography. The region corresponding to molecular size of 21 kDa of the same blot was immunodetected by antiserum agaistglutathioneS-transferase-TK. h, two-dimensional analysis of phosphoamino acids of "P-labeled TK in NOCtreated HL-60 cells; c and d. TK phosphorylation in HeLa and K562 cells with or without NOC arrest was performed as in a . The same sets of cultures were harvested and lysed for immunoblot analysis. TK, as indicated by douhle arrows, was detected by antiserum against glutathione S-transferase-TK.e , TK phosphorylation in HeLa cells treated with aphidicolin ( M H , 5 pghnl) for 16 h was compared with that in proliferating( P r o )and NOCtreated cells.

RESULTS

Tk ImmuneSerumProductionandCharacterization-We expressed human thymidine kinase in E. coli as the fusion protein glutathione S-transferase-TK containing the enzyme glutathione S-transferase fused to the N terminus of thymidine kinase. The resulting fusion protein (glutathione S-transferase-TK) migrated on SDS-PAGE with an apparent molecular

-

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4

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size of 52 kDa (Fig. la j. This gel-purified glutathione S-transferase-TKfusionproteinwasusedtogenerateantiserum against human TK. To verify the ability of this antiserum to recognize the human TK polypeptide, humanTK cDNA under the controlof C M V immediately early gene promoter was transientlyexpressedinTK-deficientcells (LMTk-). Cellswere ["S1methionine-labeled forimmunoprecipitation by this TK

21252


Prollferaltng antiserum. A specifically precipitated polypeptide of 24 kDa NOC-treated was detected in CMV-TK-transformed LMTk- cells but not in (rnln) 0 30 60 120 the cells transfected with the controlvector. (Fig. lh). This TK ( m i n ) o 30 6 0 120 antiserum was also specifically reactive with I"S1methionine1 labeled TK (24 kDa) synthesized in HeLa cells (Fig. IC, lanes and 2 ) . When the TK antibodies were depleted by preadsorption of TK antiserum on glutathione S-transferase-TK-Sepharose, no 24-kDa polypeptide could be detected(Fig. IC, lane3 ) . However, its detection was not affected when TK-antiserum S-transferase-Sepharose. A waspreadsorbedonglutathione band of the same molecular mass could be observed in the cytosolic extract from HeLa cells by immunoblotting (Fig. IC, lane 5).These experimental results confirmed that the specific 24-kDa polypeptide recognizedby our antiserum was authentic thymidine kinase. Mitotic Phosphorylationof TKon Serine in Cytosolic Extracts of HL-60, HeLn, and K562 Cells-We have previously shown thatTKisprogressivelyphosphorylatedinserum-starved HL-60 cells after growth stimulation (Chang and Huang, 1993). In order to examine whether TK polypeptide is phosphorylated in a cell cycle-specific manner, the extent of TK phosphorylation was compared in proliferating HL-60 cells without and with M phase arrest by NOC overnight treatment. The R2P-labeled TK FIG.3. Comparison of the stability of TK phosphorylation in proliferating and mitotically hlocked HeLa cells. SO('-trr.ated in proliferating and M phase-arrested HL-60 cells was immu- and Proliferating IIeLa cells were I a l ~ c . l ~in d :I phosphate-frrc. mpdium noprecipitated, analyzed on SDS-PAGE, transferred toa mem- containing 250 pCi!ml [:"Plnrthnphnsphate for 2 h. A t the end of I a h l brane, and autoradiographed. An approximately 6-fold increase ing. complete medium was added for chasing. At the indicated timw. 24-kI)a TI< polypeptldr. in TK phosphorylation was observed in NOC-treated HL-60 cytoplasmicextractswerepreparedandthe hy immunoprecipitation f o l l o w e d (indicated by thenrrowsj was detected cells as compared with proliferating cells (Fig. 2a). The same by SDS-PAGE. membrane was then immunodetected by TK antibody, which indicated that similar levels of TK immunoprecipitated in proliferating and NOC-treated cells (Fig. 2a). Phosphoamino acid difference in the extentof TK phosphorylation. Since phosphoanalysis revealed that TK was phosphorylated on serine in rylation of the TK polypeptide remained quite stable in mitotiNOC-treated HL-60 cells (Fig. 2h). Thus, an unknown fraction cally blocked cells, it is likely that the heavily phosphorylated of the total TK polypeptide becomes hyperphosphorylated on TK its observed in these cells results from the specific M phase serine residue(s) duringM phase arrest in HL-60 cells. To con- phosphorylation of newly synthesized TK. a general phenomenon, phosphorylationof firm whether this is TK Phosphorylation Can Re Inhihited hy Staurosporinr in TK was also investigated in other human cell lines, HeLa andMitotically Blocked Cells-The effect of inhihitors of protein K562 (Fig. 2,c and d ). Mitotic phosphorylation of TK was even kinases on TK phosphorylation was examined in mitotically stronger in these two cell lines. Analysis by immunoblotting blocked HeLa cells. The inhibitors, H7, sphingosine, and staushowed that the amount ofTKpolypeptide was clearly increased 2 h of rosporine, were each added to the cells separately during in M phase-arrested HeLa cells relative to proliferating cells. "P-labeling (Fig. 4). Staurosporine treatment completely abolNormalizationbydensitometricscanning of theautoradioished TK phosphorylation, whereas H7 and sphingosine treatgraphs and immunoblottings indicated that TK phosphorylaments did not inhibit the extent of phosphorylation. Therefore, tion in M phase-arrested cells was for HL-60 as well as HeLa we can exclude the possible involvement of protein kinase C, cells approximately 6-8-fold and for K562 cells 12-fold higher CAMP-dependent kinase, cGMP-dependent kinase, and Ca"than in proliferating cells.To further confirm that the observeddependentcalmodulinkinaseinTKphosphorylation in M heavy phosphorylation of TK is M phase specific, HeLa cells phase-arrested HeLa cells since H7 and sphingosine, at the were G,/S phase arrested by aphidicolin treatment overnight concentration used, are known inhibitors of these protein kia low level of nases (Hidaka and Kobayashi. 1992: Hannun and and "P-labeled for TK immunoprecipitation. Only Bell, 1987; TK phosphorylation was seen in proliferating and aphidicolin- Jefferson and Schulman, 1988). It still remains to he deterarrested HeLa cells (Fig. 2 e ) . is remined which mitotic kinase, sensitive to staurosporine, Stahi1it.y of TK Phosphorylation in Proliferatinga n d Mitoti- sponsible for the mitotic phosphorylationof TK. cally Blocked HeLa Cells-To understand the mechanism conCyanogen Bromide Cleavageof TK-To know whether differtributory to the significant increaseof TK phosphorylation in e n t sites in TK are being phosphorylated in proliferating and mitotically blocked cells, the stability of TK phosphorylation in NOC-treated HeLa cells, we analyzed the phosphopeptide fragboth proliferating and mitotically blocked HeLa cells was dements (derived from CNRr treatment) of TK labeled in L'IL'O. termined to address the question of whether TK is being con- Cleavage of TK of NOC-arrested HeLa cells resulted in the stantly phosphorylated and dephosphorylated in a cell cycle- resolution of at least 2 "2P-labeled bands around 12 and9 kDa, dependentmanner.Theintensity of TKphosphorylation respectively (Fig. 5). while cleavage of phosphorylated TK in remainedunchangedduringthe 2-h chaseinthecomplete proliferating cells gavea major phosphopeptide at 3 kDa, indiphosphate containing medium in the NOC-treated HeLa cells cating that the phosphorylation sites in TK in proliferating and (Fig. 3). For proliferating cells,an approximate20% decrement mitotically blocked HeLa cells are not identical. of TK phosphorylation occurred during the initial 30-min chaseKinetic Analysis of TK in Prolifrrating and NOC-twated period, after which TK phosphorylation remained unaltered for HeLa Cells-To examine the physiological roleof mitotic phos3). The overall dephosphorylation of TK in phorylation of TK during the M phase,we performed a kinetic another 90 min (Fig. proliferating cells appeared to be negligible, suggesting that analysis of TK in the crude extracts from NOC-treated and in the dephosphorylation rate does not contribute to the dramaticproliferating HeLa cells. TK activity was measured with ATP

Phosphorylation of Human Thymidine Kinase NOC-treated

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FIG.4. Effects of inhibitors of protein kinase on TK phosphorylation in HeLa cell. Proliferating HeLa cells were treated withNOC (0.5 pg/m\J for 16 h. These cells were then treated with sphingosine ( S p h ; 1 p ) , staurosporine (Sfnuro;50 nMJ, and H7 (100~ M during J 2h of:"P-laheling. T h e control I-J was performed in cells treated with0.1'7 dimethyl sulfoxide. Cell lysates were immunoprecipitated by antiserum againstElutathioneS-transferase-TK,followed by SDS-PAGEand autoradingraphy.

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Fir;. 6. Kinetic analysin of TK in HeLa celln with or without M Phase arrest. TK of thr crude extracts from t h e proliferatlnfi 1lrLa cells with or without NOC overnight treatment was assaycd at : C (' a t various concentrations of th-ymidlne (4.5 22.5 p v In ~ thf. presence of 5 m y ATP ( A J or at various concentrationsofATP 10.313 -- 5 mv I In t h r presence of 22.5 p~ thymidine cnr. T h er e s u l k am displayed as Lineweaver-Burk plots. Points are the averagr of duplicate ;~.;.;ays.

cally blocked cells than in proliferating cells, confirming that t h e level of TK is higher in NOC-treated HeLa cells, whichis in good afleement with the data derived from immunohlotting analysis. When TK kinetics were assayed with ATP varying fixed at from 5 to 0.313 m>f with the concentration ofthymidine 22.5 p , the apparent K,,, was 0.22 m>! for NOC-treated HeLa and 0.17 mw for proliferating cells (Fig.6R 1. Clearly. the affinity ofTKfor its substrate, ATP. was not significantly affectedhy the status of TK phosphorylation. DISCUSSION

5. Analysis of CNBr-generated phosphopeptides of TK.In "P-labeled TK proteins from proliferating ( I ' ) and NOC-treated (NI HeLa cells were cleaved by CNRr and separatedby SDS-PAGE. T h e sizes of all phosphopeptide handsare given according to the mobilityof t h e myoglohin fragments (Sigma)run on the same gel. FIG.

V~CJO

k e p t a t 5 mM, while thymidine concentration was varied from . apparent K,,, values for thymidine in pro22.5 to 4.5 p ~ The liferating and NOC-treated extracts of HeLa cells were 1.49 and 15.5 p ~ respectively , (Fig. 6A ). Thus, TK in proliferating cells expressed a 10-fold higher affinity for thymidine than in was 4-fold higher in mitotimitotically blocked cells. The VmnX

In this report, we establish that human c.ytosolic thymidine kinase is differentially phosphorylated on its serine rrsiduejsl in proliferating andM phase-arrested HeLa cells. TK phosphorylation drastically increasedby more than R-fold in mitotically blocked HeLa, HL-60, and K562 cells as compared with proliferating cells. Furthermore, phosphorylated TK in proliferating andmitotically blocked HeLacellsyieldeddifferentphosphopeptide patterns upon CNRr treatment, indicating that different sites are phosphorylated in asynchronized flowing and M phase-arrested HeLa cells. The mitotic kinase responsihle for phosphorylation ofTK was shown to be sensitive to staurosporine. Although, protein kinase C, CAMP-dependent kinase, cGMP-dependent kinase, and

21254

Phosphorylation ofThymidine Human Kinase

calmodulin-dependent kinase are known to be the biological target of staurosporine (Abe et al., 1991), the lack of inhibitory effect by H7 andsphingosine excludes the possible involvement of these protein kinases. Several facts have led us to speculate that cdc2 or a related kinase may be involved in TK phosphorylation. First, staurosporine hasbeen shown to be a very potent inhibitorof cdc2 kinase (Gadbois, et al., 1992), which is an M phase kinase. Second, cyanogen bromide cleavage of phosphorylated TK isolated from mitotically blocked HeLa cells yielded a major phosphopeptide of 12 kDa. According to the amino acid sequence of TK (Flemington et al., 1987), this peptide is derived from the C terminus, which contains one consensus site(Ser-Pro) for cdc2 kinase. However, our attempts to phosphorylate TK in vitro by cdc2 kinase were not reproducible. It is, therefore, not clear which mitotic kinase is responsible for TK phosphorylation during the M phase. The apparentK,,, of TK for thymidine was 15.5 PMin mitotically blocked HeLa and 1.49 p~ in proliferating cells, indicating a lower affinity of TK for thymidine in M phase-arrested cells. The apparent K , obtained from the crude extractsof proliferating HeLa cells was very close t o the reported K, values, clustered around 3 p ~ of, purified TK from human breast cancer tissue (Bronzertet al., 1981) and placenta (Eng Gan et al., 1983). Given that TK is heavily phosphorylated in mitotically blocked HeLa cells, it is possible that this phosphorylation event is responsible for the increase in the apparent K,,, of TK in M phase-arrested HeLa cells. Recently, it has been shown that purified cytosolic human thymidine kinase exists in two , forms, with K, values for thymdine of 15 and 0.7 p ~ respectively (Munch-Peterson et al., 1993). Incubation of TK with ATP induced its transition to a more active form witha higher substrate affinity. Accordingly, it is possible that hypophosphorylated TK in proliferating cells represents a more active form, whereas hyperphosphorylated TK in mitotically blocked cells exists as the less active form. Thus, phosphorylation of TK by an M phase-specific kinase, which is sensitive to staurosporine, may decrease its catalyticefficiency. At present, we do not known the precise fluctuation of the thymidine pool in HeLa cells during the cell cycle. If the thymidine pool is being depleted at the endof the S phase, a lower catalytic efficiency of TK during theM phase may avoid unnecM phase essary synthesisof dTTP.Thus, a specific kinase in the of the cell cycle may play a role in attenuating the signalleft from the S phase to provide some biological economy. Here we propose a model that one form of TK with a higher catalytic effkiency is expressed during the earlyG, to the S phase, and another form of TK with a lower catalytic efficiency is expressed during theM phase. Thus, thelevel of TK protein and its catalytic efficiency interact periodically during theprogression of the cell cycle, as depicted in Fig. 7. For examplein HeLa cells, the level of TK protein increases at the G,/S phase and reaches a maximum during the M phase. At this stage, TK activity is no longer needed, since DNA replication is com-

-High

TK catalytic

efficiency

-

-

LOW

FIG.7. Model of the fluctuation of TK protein level and its catalytic efficiency in the HeLa cell cycle. The abundance ofTK protein and the alternation of TK catalytic efficiency interplay during the progression of the cell cycle. The cell cycleproceeds fromleft to rzght.

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