mirk protein kinase is a mitogen-activated protein kinase ...kangmoon lee, xiaobing deng, and eileen...

[CANCER RESEARCH 60, 3631–3637, July 1, 2000]

Mirk Protein Kinase Is a Mitogen-activated Protein Kinase Substrate ThatMediates Survival of Colon Cancer Cells1

Kangmoon Lee, Xiaobing Deng, and Eileen Friedman2

Upstate Medical University, Pathology Department, Syracuse, New York 13210

ABSTRACT

We have cloned a novel genemirk (minibrain-related kinase) encoding aprotein kinase that enables colon carcinoma cells to survive under certainstress conditions. Mirk is a mitogen-activated protein kinase substrate butis down-regulated by activated extracellular signal-regulated kinases(erks) in vivo. Mirk contains a PEST region characteristic of rapidlyturned over proteins and is broken down to aMr 57,000 form only in thenucleus. In each of three colon carcinoma cell lines, mirk levels wereincreased 20-fold when erk activation was blocked by the MEK inhibitorPD98059 in serum-free medium. Addition of IGF-I to activate erksblocked this increase. Mirk was stably overexpressed in two colon carci-noma cell lines to attain levels seen in colon cancers. Each of five mirktransfectants proliferated when switched to serum-free medium and re-gained rapid growth when serum was restored, whereas five vector con-trol transfectants and three kinase-dead mutant mirk transfectants didnot. mirk mRNA levels were elevated in several types of carcinomas, andmirk protein was detected in each of seven colon carcinoma cell lines.mirkwas expressed at a higher protein level in Western blots from three ofeight colon cancers compared with paired normal colon tissue, suggestingthat mirk plays a role in the evolution of a subset of colon cancers.mirkis not mutated in colon carcinomas. Mirk may mediate tumor cell survivalin mitogen-poor environments or early in colon cancer development be-fore many autocrine growth factors have been induced.

INTRODUCTION

Eukaryotic cells are constantly subjected to growth, differentiation,and stress signals and use cascades of protein kinases called MAPKs,3

and their activators to respond to these varied signals. The proteinkinase cascades translate signals from cell surface receptors throughthe cytoplasm to the nucleus, to cytoskeletal elements, and to otherparts of the cell. There are three major MAPK cascades in mammaliancells, consisting of three sequentially activated protein kinases, namedfor the final kinase in the cascade: the mitogen-activated erk1/2cascade, the stress-activated protein kinase/c-Jun NH2-terminal kinasecascade, and the high osmolarity-activated p38 kinase cascade (1–3).In earlier studies, we had identified a protein kinase that was selec-tively activated in colon tumor cells compared with normal colonepithelial cells (4, 5). We have purified and cloned this kinase andnow report that it is a MAPK substrate, is broken down to a smallerform in cells with active MAPKs, and can mediate cell growth andsurvival only under conditions in which the erk1/2 class of MAPKs isnot highly activated.

MATERIALS AND METHODS

Materials. Phosphospecific (T202/Y204) monoclonal antibody detectingdoubly phosphorylated erk1/erk2 and rabbit polyclonal antibody to total erk1/erk2 were purchased from New England Biolabs, and IGF-I from UpstateBiotech. Human tissues were obtained from the Cooperative Human TissueNetwork and Northern blots from Clontech.

Isolation of mirk cDNA Clone. Mirk was purified from diolein-treated(5 min) HP1 colon carcinoma cells by sequential fractionation of S100 cytosolsby DEAE-cellulose and phenyl Sepharose fast protein liquid chromatography,followed by affinity purification on immobilized pan-MAPK polyclonal anti-body directed to the conserved kinase subdomain 11 sequence DRLTAEE-ALSHPYMSIYSFPTDE. Eluted protein was fractionated by SDS-PAGE andelectroblotted onto nitrocellulose, and the Ponceau S-stainedMr 57,000 bandwas excised and processed for internal amino acid analysis. HPLC peakfractions were subjected to chemical sequencing by an Applied Biosystems477A sequenator optimized for femtomol level analysis. One peak corre-sponded to amino acids 379–390 of mirk and was used to identify its cDNAclone. Degenerate oligonucleotide primers from conserved protein kinasesubdomains II and VIb were used to amplify a cDNA fragment made usingHD3 colon carcinoma cell mRNA with the Access RT-PCR system (Promega).Sixteen different cDNA bands were amplified, purified on agarose gel, insertedinto the pGEM-T Easy vector (Promega), and then transfected intoEscherichiacoli JM109. Of 180 colonies isolated, 36 were sequenced, yielding three erk2s,one erk1, one MEKK, and one novel 440-bp fragment that was used as a probeto screen a human SW480 colorectal adenocarcinoma cDNA (Clontech)library. More than 1,000,000 plaques were screened. Seventeen positive cloneswere obtained, subcloned into the pGEM vector, and analyzed by sequencingfor the presence of amino acids 379–390 in purified mirk. pG9R contained a2541-bp longmirk cDNA with a start codon, poly(A) sequence, and coded forthe amino acid sequence found in purified mirk. The DNA sequence wasdeposited in GenBank under accession number AF205861.

Preparation of Expression Plasmids.After EcoRI digestion,mirk cDNAwas ligated into pcDNA3.1/HisA (Invitrogen), yielding the hexahistidine fu-sion plasmid pHX9. Hexahistidine fusionmirk cDNA was isolated from pHX9by HindIII, XhoI double digestion and blunt ended. AfterStuI digestion, theblunted mirk cDNA was ligated into the pBacPAK8 (Clontech) plasmid,yielding recombinant baculovirus pBH. Aftermirk was excised from pG9R byStuI digestion, it was ligated into pGEX-4T-1 (Pharmacia), yielding the GSTfusion plasmid pGD. Aftermirk was excised from pG9R byEcoRI digestion,it was ligated into pLXSN (Clontech), yielding pLXSN-D2. pLXSN-D2 wastransfected into the PT67 retrovirus packaging cells, and recombinant retrovi-rus was harvested from the supernatant at 105–106 virions/ml. Stablemirktransfectants were isolated in G418 after retroviral infection of HD3 coloncarcinoma cells.

Mutagenesis/Coupled Transcription Translation/in Vitro Kinase As-says. Site-directed mutagenesis was performed with mutagenic oligonucleo-tides using the pGEM-11Zf(1) vector (Promega) to transform PMH 71–18mutS competent cells, and the products were sequenced to verify the mutation.Hexahistidine fusion mirk was produced by a coupledin vitro transcription andtranslation system (Promega) using 1mg of pHX plasmid/reaction. Producedprotein was immunoprecipitated overnight by using 6x(His) mAb (Clontech)and 10ml of 50% Protein A-Sephadex (Sigma). The immunoprecipitate waswashed three times with RIPA buffer and three times with kinase buffer [10mM Tris (pH 7.4), 150 mM NaCl, 10 mM MgCl2, and 0.5 mM DTT]. Theimmunocomplex was incubated for 15 min at 37°C with 40ml of the kinasebuffer containing 25mM ATP, 2.5 mCi [32Pg]ATP, and 1.0 mg/ml MBP assubstrate and then analyzed by electrophoresis and autoradiography.

Phosphatase Treatment of Phosphorylated MBP.The kinase reactionmixture described above was incubated for 30 min at 30°C with either proteinphosphatase-1 [0.5 unit in 20 mM 4-morpholinepropanesulfonic acid (pH 7.5),

Received 11/10/99; accepted 4/27/00.The costs of publication of this article were defrayed in part by the payment of page

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

1 Supported by National Cancer Institute RO1 CA67405 (to E. F.).2 To whom requests for reprints should be addressed, at Pathology Department,

Upstate Medical University, State University of New York, 2305 Weiskotten Hall, 750East Adams Street, Syracuse, NY 13210. Phone: (315) 464-7148; Fax: (315) 464-8419;E-mail: [email protected].

3 The abbreviations used are: MAPK, mitogen-activated protein kinase; erk, extracel-lular signal-regulated kinase; IGF, insulin-like growth factor; MBP, myelin basic protein;mirk, minibrain-related kinase; MEK, MAPK kinase; RT-PCR, reverse transcription-PCR.

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60 mM 2-mercaptoethanol, 1 mM MgCl2, and 0.1 mg/ml serum albumin],protein tyrosine phosphatase-b [2 units in 25 mM HEPES (pH 7.2), 50 mMNaCl, 5 mM DTT, and 2.5 mM EDTA], or PP-1 (0.5 unit) with okadaic acid(100 nM) and then analyzed by electrophoresis and autoradiography.

Western Blotting. Samples were prepared according to Denget al. (6). Allmirk blots used affinity-purified polyclonal antibody directed to the mirkunique C9terminus unless noted, except blots of the human tissue samples,which used monoclonal antibody. Western blotting for activated and totalMAPKs was performed exactly according to Yanet al. (7). Band density inautoradiograms was measured using a Lacie Silverscanner and silverscannerIII software and analyzed by the IP LabGel program.

Results and Discussion

We have cloned a novel gene using sequence data from a proteinpurified from colon carcinoma cells (see “Materials and Methods”).The deduced protein structure is shown in Fig. 1A. BLAST searchconfirmed that this gene was a homologue of aDrosophilagene calledminibrain (Mnb; Ref. 8) ordyrk (9, 10); therefore, it was calledmirk.A initial report of this work has been presented (11). MutantMnb fliesare characterized by a marked reduction in size of the optic lobes andcentral brain hemispheres (8).Dyrk1/minibrain is a strong candidatefor the gene duplicated in Down Syndrome (12), but no function has

been established for this gene aside from its kinase activity. Morerecently, several other members of theMnb/dyrk family have beencloned, but no function for any of these genes has been elucidated(13). OneMnb/dyrkgene,dyrk1B,has three splice variants, encodingputative proteins of 629, 601, and 589 amino acids (14), the longestbeing identical to mirk. Amirk splice variant of 601 amino acids hadbeen isolated in our initial screen but was not studied further becauseit did not encode the peptide found in mirk protein purified from coloncarcinoma cells (see “Methods and Materials”). This splice variantwas generated by alternative splicing within exon 9 ofmirk (Fig. 1B),which encodes amino acids found in the conserved kinase domainbetween subdomains X and XI.

mirk encodes a 629-amino acid protein with a bipartite nuclearlocalization signal in the N9terminal nonconserved region, the 11canonical kinase subdomains, a PEST sequence common to rapidlydegraded proteins following the kinase domain, and a consensusMAPK phosphorylation sequence in the nonconserved C9 terminus(Fig. 1A). The amino acid sequence of mirk is 56% identical withinthe conserved kinase domain to the human forms of dyrk1 (mini-brain), dyrk2, and dyrk3, with much less identity in the N9terminusand C9 terminus. All dyrk family members are serine/threonine ki-

Fig. 1. A, deduced amino acid sequence encoded by themirkgene of a 629-amino acid protein, with an initiating methioninedefined by a consensus Kozak sequence and ended by an in-framestop codon. The position of the bipartite nuclear localization signalis italicized. The conserved kinase domain is inboldfacewith thekinase subdomains indicated by roman numerals. The PEST re-gion often seen in rapidly degraded proteins isunderlined. TheDNA sequence was deposited in GenBank with accession numberAF205861.B, genomic structure ofmirk drawn to scale with 11exons (heavy lines) and introns (light lines). Exon 1 is 108 bp,whereas intron 1–2 is 2054 bp. The cDNA sequence ofmirk wascompared by BLAST search to sequences deposited in GenBankfrom the Human Genome Project. The genome sequence of themirk gene was found in chromosome 19q13.1, and intron/exonjunctures were determined by comparison of the two sequences.C,in vitro activity of mirk. Left, recombinant mirk with an NH2-terminal hexahistidine tag (rMirk lanes) was expressed in sf21insect cells and then purified on nickel-charged agarose resin(Invitrogen) and either assayed for protein kinase activity onimmobilized MBP in anin-gel kinase assay or Western blottedwith anti-phosphotyrosine antibody. Parallel experiments wereperformed with an empty vector (C lanes).Center, MBP waslabeled with [32P]ATP in anin vitro kinase reaction with recom-binant mirk from sf21 insect cells and then subjected to phospho-amino acid analysis by TLC.M, marker controls;D, digest.Lowerleft, MBP was phosphorylated by mirk in anin vitro kinasereaction, subjected to phosphatase treatment, and then electro-phoresed and autoradiographed.CT, control; PTP-b, tyrosinephosphorylaseb; PP1,protein phosphatase-1;OA,okadaic acid, aPP1 inhibitor.Right, point mutants. Mutants were analyzed bycoupled transcription translation, followed byin vitro kinase as-says. The amount of mirk generated in each reaction mixture wasverified by Western blot using affinity-purified NH2-terminal an-tibody. Autophosphorylation lanes show autoradiograms of la-beled mirk on SDS gel.Vec,vector control.

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MIRK PROTEIN KINASE AND SURVIVAL OF COLON CANCER CELLS



nases that also can autophosphorylate on tyrosine; therefore, they aredual function kinases (8, 9, 10, 13). Their activation sequence is YQYor YTY, with both tyrosines phosphorylated. This YxY sequencealigns with the TxY activation sequence in MAPKs. The cDNAsequence ofmirk was compared by BLAST search to sequencesdeposited in GenBank from the Human Genome Project. The genomesequence of themirk gene was found in chromosome 19q13.1, andintron/exon junctures were determined by comparison of the twosequences.mirk has 11 exons and 10 introns encompassing 8.8 kb ofgenomic DNA (Fig. 1B).mirk was not mutated in the SW480 coloncarcinoma cells from which it was cloned, because its sequence didnot differ from the genomic DNA sequence.

Recombinant mirk expressed in sf21 insect cells was purified andthen either assayed for protein kinase activity on immobilized MBP inan in gel kinase assay or Western blotted with anti-phosphotyrosineantibody. Recombinant mirk had MBP kinase activity atMr 70,000,the molecular weight of mirk deduced from its amino acid sequence,and was phosphorylated on tyrosine (Fig. 1C). Phosphotyrosine wasalso detected in mirk expressed inE. coli, indicating that mirk hastyrosine autophosphorylation capacity (data not shown). Mirk activityis dependent on phosphorylation of tyrosines 271 and 273 (Fig. 1C)located between subdomains VII and VIII in the sequence YQY,which aligns with the erk1 activation sequence TEY (15). A mirkmutant at the ATP-binding site (K140R) and a double mutant at theputative activation sequence YQY, to FQF, were generated. Bothmutations greatly decreased the kinase activity of mirk on MBP, aswell as the capacity of mirk for autophosphorylation (Fig. 1C) and theautophosphorylation of mirk on tyrosine (data not shown). Westernblotting confirmed that equal levels of the wild-type and mutant

proteins were present in each reaction (Fig. 1C). Analysis of thephosphorylated amino acids in MBP by TLC and of labeled MBP forphosphatase sensitivity (Fig. 1C) demonstrated that mirk phosphoryl-ates only serine and threonine residuesin vitro. Mirk-phosphorylatedMBP was a substrate for the serine-threonine phosphatase PP1 but notfor the phosphotyrosine-specific phosphatase, PTP-b (Fig. 1C). Inaddition, mirk had no detectablein vitro activity on two syntheticphosphotyrosine substrates while phosphorylating certain substratesof the Ser/Thr kinase MAPKAP kinase-2 (Ref. 16; data not shown).Therefore, although mirk kinase is activated by autophosphorylationon tyrosine at the Y271/Y273 site, mirk itself is a serine-threoninekinase on exogenous substrates.

mirk exhibits restricted mRNA expression in normal tissue (Fig. 2,A andB), with highest expression in skeletal muscle, testes, heart, andbrain, and with little expression in normal colon (Fig. 2A, Lane c) andnormal lung (Fig. 2B, Lane lu).mirk is highly expressed in SW480colon carcinoma cells and A549 lung carcinoma cells (Fig. 2C) andother tumor cell lines including HeLa ovarian carcinoma cells, K562chronic myelogenous leukemia cells, molt4 lymphoblastic leukemiacells, and G361 melanoma cells.mirk is not detectably expressed inHL60 promyelocytic leukemia cells or Raji lymphoma cells (Fig. 2C).Thus,mirk is expressed in few normal tissues but in many types ofcancer.

Two polyclonal antibodies to nonconserved regions of mirk wereraised and affinity purified, one to amino acids 1–19 of mirk and thesecond, to amino acids 595–609 in the C9 terminus of mirk. Bothsequences were unique by BLAST search. A monoclonal antibody torecombinant mirk was also raised. In Western blots with C9-terminal

Fig. 2. A–C, Northern analysis ofmirk expression in poly(A)1RNA extracted fromvarious normal human tissues and carcinoma cell lines withb-actin as loading control andeach blot probed under identical conditions. Tissues:sp,spleen;th, thymus;pr, prostate;ts, testis;ov,ovary;sb,small bowel;c, colon; leu,peripheral blood leukocytes;hrt, heart;br, brain;pl, placenta;lu, lung; li, liver; skms,skeletal muscle;kd,kidney;pan,pancreas.Carcinoma cell lines:HL60, promyelocytic leukemia;HeLa, ovarian carcinoma;K562,chronic myelogenous leukemia;Molt4, lymphoblastic leukemia;Raji, Burkitts’s lym-phoma;SW480,colon carcinoma;A549, lung carcinoma;G361,melanoma.

Fig. 3.A, upper panel, Western blot showing elevated mirk expression within three ofeight colon cancer cases (C) compared with paired normal (N) colon tissue from the samepatient. Monoclonal antibody 11 raised to recombinant mirk detected full-length mirk atMr 70,000 (lower panel) Blots were stripped and reblotted;b-actin in region of blotMr

z44,000 is shown.B, lysates of mirk stable transfectant T24 and vector control trans-fectant V1, both transfectants of HD3 colon carcinoma cells, were run in triplicate in SDSPAGE and transferred to membranes. The parallel blots were analyzed for mirk expressionlevels by ECL incubation with preimmune mouse serum, monoclonal antibody 11 raisedto recombinant mirk, and monoclonal antibody 11 preabsorbed with GST-mirk beforeimmunoblotting.Arrows, positions of full-length mirk and another mirk species atMr

57,000.Left, molecular weight markers of 194,000, 99,000, 67,000, 43,000, and 29,000.A Mr 57,000 mirk species were also detected by polyclonal antibody raised to a sequencein the N9 terminus of mirk but not detected by an antibody directed to the C9terminus ofmirk, suggesting theMr 57,000 species is generated by C9-terminal deletionin vivo (seeC). Blots were stripped and reblotted;b-actin in region of blotMr z44,000 is shown.C,Western blotting demonstrates that lengthMr 70,000 mirk (arrow) is expressed in each ofseven colon carcinoma cell lines, using mirk C9terminal directed affinity-purified poly-clonal antibody.Right, molecular weight markers of 103,000, 68,000, 44,000 and 28,000.Two mirk forms atMr 70,000 andMr 67,000 clearly seen in HD3 cells and present in otherlines may have been caused by alternative splicing. A splice variant within exon 9 wascloned from HD3 cells encoded a shorter form of mirk.Lower panel, Western blottingusing mirk NH2-terminal-directed, affinity-purified polyclonal antibody.Arrows, the threemirk species detected, full-lengthMr 70,000 mirk, atMr 67,000 form, and aMr 57,000mirk species not detected with the C9-terminal antibody, which may be caused byC9-terminal deletion of mirk in each line.

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antibody (Fig. 3C, upper panel), with N9-terminal antibody (Fig. 3C,lower panel, upper arrow), or with monoclonal antibody 11 (Fig. 3B),mirk was detected at the expected translational size ofMr 70,000. AMr 67,000 mirk form was detected in several lines by both N9-terminaland C9-terminal antibodies. Therefore, theMr 67,000 mirk must becaused by an internal deletion. A splice variant caused by internaldeletion within exon 9 was cloned in our initial screen (see “Materialsand Methods”). This splice variant encoded aMr 67,000 mirk proteinafter in vitro coupled transcription/translation (data not shown) andtherefore is very likely to be the source of the smaller mirk form. Inaddition to the splice variants, a mirk breakdown form ofMr 57,000was detected by N9-terminal antibody and monoclonal antibody butwas not detected by C9-terminal-directed antibody, indicating that itwas generated by C9-terminal deletion.

Monoclonal antibody raised to recombinant mirk was used to detectmirk in the stable mirk transfectant T24 and a vector control trans-fectant V1. Mirk was more abundant in the T24 overexpressing linethan the control cells, and aMr 57,000 degradation product of mirkwas also detected in the overexpressing line (Fig. 3B). Monoclonalantibody activity was removed by preabsorption with recombinantmirk protein, and preimmune mouse serum did not detect mirk inparallel blots (Fig. 3B). The blots were stripped and reblotted forb-actin, demonstrating equal loading and transfer (Fig. 3B, lowerpanels). When eight cases of colon cancer tissue with paired normalcolon were examined by Western blotting with this monoclonal anti-body, mirk was found to be much more abundant in the cancer tissuein three of eight cases (nos. 1, 3, and 6) when normalized to actinlevels (Fig. 3A, upper panel). Lack of degradation of protein withinthe clinical samples was demonstrated by reblotting forb-actin pro-tein (Fig. 3A, lower panel). Mirk was localized predominately withinthe cytoplasm of the colon carcinoma cells within sectioned coloncancer tissue from three additional cases, not within the stroma (datanot shown), as shown by immunocytochemistry with monoclonalantibody to mirk. Therefore, the greater abundance of mirk protein byWestern blotting in a subset of colon cancer tissues was attributable toincreased mirk expression within the colon cancer cells themselves,not another cell type.

Recombinant mirk is phosphorylated by all three classes ofMAPKs, the erks, the SAP/c-Jun NH2-terminal kinases and p38 (Fig.4A). Western blotting then showed that mirk protein levels are down-

Fig. 4. A, recombinant mirk was phosphorylatedin vitro by purified erk2, stress-activated protein kinasea, and p38 kinase. GST-p38 kinase is also phosphorylated underthe reaction conditions.B–E,Western blots show that mirk protein levels are controlledby the erk class of MAPK, and that inhibition of erk activity by removal of exogenousserum mitogens and by addition of a MEK inhibitor increases mirk protein levels18–20-fold.B, upper panel,mirk protein levels increase approximately 5-, 6-, and 7-fold,respectively, when U9 colon carcinoma cells are cultured in serum-free medium for 1, 2,and 3 days. Similar data were seen for two other colon carcinoma cell lines (not shown).B, lower panel,treatment of U9 cells for 24 h in serum-free medium with the MEKinhibitor PD98059 induces a dose-dependent increase in mirk levels, above that caused byserum-free culture. The 5-fold increase in mirk levels induced by serum-free culture (Lane1) is further increased 4-fold by addition of the MEK inhibitor (Lane 4). The increase inmirk caused by both serum-free culture and the MEK inhibitor is blocked by concurrenttreatment of cells with the serum mitogen IGF-1 (Lanes 5–8).Lane 5 with no MEKinhibitor and 10 ng/ml IGF-I treatment shows a mirk level 18-fold less thanLane 4. Tenng/ml IGF-1 causes an activation of erk1 and erk2, which persisted for at least 3 h (datanot shown).C, a time course demonstrates that mirk levels increase as the fraction ofactivated erk1 and erk2 decreases. The mirk double band is better resolved than inA orB because the gel fractionation was extended for a longer time. No variation in totalerk1/erk2 abundance was seen (lowest panel). U9 colon carcinoma cells were placed inserum-free media with or without 10 ng/ml IGF-I. Parallel immunoblots for mirk proteinusing affinity-purified mirk antibody (upper blot), phosphorylated, activated erk1 anderk2 using monoclonal antibody directed to the erk1 and 2 phosphorylated activationsequence TEY (middle blot), and antibody to total erk1/erk2 (lower blot). Similar datawere obtained with SW480 and HD3 colon carcinoma cells (not shown).D, the stablemirk transfectants of U9 colon carcinoma cells, T3 and T7, express elevated levels ofmirk protein compared with vector controls, 9V1 and 9V2 (Lanes 1–4), and culture in

serum-free media for 24 h (Lanes 5–8) increases the abundance of both endogenous andtransfected mirk protein.E, the stable mirk transfectant T3 expresses about 4-fold theamount of mirk as vector control V1 cells after cells are grown in serum-free medium, andan increase in both endogenous mirk in V1 vector control transfectants and transfectedmirk in T3 cells caused by serum-free growth is blocked by concurrent treatment withIGF-I. F, analysis of mirk mRNA levels by RT-PCR and by transient transfections of mirkpromoter constructs (not shown) confirmed that much of the modulation of mirk proteinlevels seen inA–E was caused by posttranscriptional mechanisms.mirk mRNA levelswere analyzed by RT-PCR in 9V2 vector control and T7 mirk transfectant cells culturedin control (CT) serum-containing medium or in serum-free media (SF) for 24 h, alone orwith 10 mM PD98059 (MEK inhibitor) or 10 ng/ml IGF-I. Primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were included in every reaction mixture as aninternal control. Added PD98059 had no effect and IGF-I had a minor effect on endog-enous mirk mRNA levels in 9V2 vector control transfectants, whereas IGF-I had nodiscernible effect on the combination of exogenous and endogenous mirk mRNA levels inthe T7 transfectant.G, upper panel,mirk expression in V1 and V2 vector control HD3transfectants and mirk stable overexpressors, T19 and T24, was analyzed by Westernblotting using affinity-purified antibody directed to the N9terminus of mirk.Lower panel,mirk expression in V1 and V3 vector control U9 transfectants and mirk stable overex-pressors, T7 and T22, was analyzed by Western blotting using affinity-purified antibodydirected to the N9terminus of mirk. This antibody also detectsMr 57,000 mirk cleaved atthe C9 terminus (Fig. 3C; also data not shown). Cells were cultured in serum-containinggrowth media. Fractionation of cells into nuclear (N) and cytoplasmic (C) fractionsdemonstrated that aMr 57,000 mirk breakdown product was found solely in the nucleusof each cell type, whereas higher levels of full-length,Mr 70,000 mirk are found in eachmirk transfectant. TheMr 57,000 mirk breakdown product could not be detected withC9-terminal-directed antibody (not shown), indicating that the C9terminus of mirk wasdeleted in the nuclear species.

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regulated by activated erksin vivo. Inhibition of erk activity byremoval of serum mitogens and by addition of a MEK inhibitorincreased endogenous mirk protein levels 18–20-fold. When U9 co-lon carcinoma cells were placed in serum-free medium for 1, 2, and 3days, levels of endogenous mirk were increased 5-, 6-, and 7-fold,

respectively (Fig. 4B). These data suggested that serum mitogensactivated some signaling path that either down-regulated mirk mRNA,mirk protein levels, or both. U9 cells exhibit autocrine regulation byepidermal growth factor, fibroblast growth factor, and transforminggrowth factorb family members (17–19); therefore, U9 cells exhibit

Fig. 5. When mirk is present above a threshold level, itcan mediate short-term survival of colon cancer cells in theabsence of serum mitogens. Stable mirk transfectants inHD3 colon carcinoma cells (E–G,T10, T11, and T19) andin U9 colon carcinoma cells (A–D,T7 and T3) exhibited3–9-fold increases in cell number compared with controlsafter 5 days of growth in serum-free medium. Little growthin serum-free conditions was seen in stable transfectants ofmirk mutated at the ATP binding site (KR mutants), mirkmutated at the activation site of YQY to FQF, three vectorcontrol U9 lines (9V1, 9V2, and 9V3), and two vectorcontrol HD3 transfectants (V1 and V2). One representativeexperiment of three is shown (M) with cell number assayedby MTT analysis (A, C, D, E,andG; bars,SE) or by directcell counting (Band F). Bars are not shown in the linegraphs if the SE measurement is,5% of the mean value,which is the default level for the graphing program. How-ever, all data are the mean of triplicate measurements.Similar experiments using [3H]thymidine incorporation as aproliferation assay had been performed with similar out-comes with each point a mean of triplicate measurements(not shown). InH, U9 mirk transfectants were cultured 7days serum free and then switched to serum-containingmedia and showed rapid resumption of growth, whereas twovector control transfectants showed no sustained growth.

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some activation of erks in the absence of serum mitogens (Fig. 4C).Treatment of U9 cells for 24 h in serum-free medium with the MEKinhibitor PD98059 induced a 4-fold, dose-dependent increase in mirklevels, above that caused by serum-free culture alone (Fig. 4B, Lanes1–4), probably because the MEK inhibitor blocked the activation oferks by autocrine growth factors. This total 20-fold increase in mirkabove background levels was reversed by concurrent treatment ofcells with the serum mitogen IGF-I (Fig. 4B, Lanes 5–8), whichactivated erk1 and erk2 for at least 3 h (data not shown). Therefore,blocking erk activation by removal of serum mitogens and blockingany residual erk activation from autocrine growth factors by the MEKinhibitor elevated mirk levels 20-fold, whereas activation of erks withIGF-I reduced mirk levels to those seen in cells cultured in serum-containing medium. Similar data were seen with SW480 and HD3colon carcinoma cell lines (data not shown).

A time course then demonstrated that mirk levels increase as thefraction of activated erk1 and erk2 decreases (Fig. 4C). The mirkdouble band is better resolved than inA or B because the gel frac-tionation was extended for a longer period. No variation in totalerk1/erk2 abundance was seen (lowest panel). U9 colon carcinomacells were placed in serum-free media with or without 10 ng/ml IGF-I.Parallel immunoblots were probed for mirk protein (upper blot), forphosphorylated, activated erk1 and erk2 using monoclonal antibodydirected to the erk1 and 2 phosphorylated activation sequence TEY(middle blot), and for total erk1/erk2 using an antibody which did notdistinguish between activated and inactivated erk forms (lower blot).Mirk levels began to increase by 3 h of serum-free culture andcontinued to rise for 24 h, coincident with the decrease in activated erkforms. The slow decrease in activated erks may be attributable toautocrine growth factors. However, when IGF-I was added to main-tain activation of erk1 and erk2 (Fig. 4C, Lanes 7–12), no increase inmirk levels was observed.

Serum-free culture increased the level of transfected mirk (Fig. 4D)in the T3 and T7 stable, clonal mirk transfectants of U9 coloncarcinoma cells, as well as endogenous mirk in the vector controls,9V1 and 9V2. Addition of IGF-I to maintain activated erks caused adecrease in both transfected and endogenous mirk levels (Fig. 4E).The stable mirk transfectant T3 expressed about 4-fold the amount ofmirk as vector control V1 cells after growth in serum-free medium(Fig. 4E).

mirk mRNA levels were analyzed by RT-PCR in 9V2 vectorcontrol and T7 mirk transfectant cells cultured in control serum-containing medium or in serum-free media for 24 h with or without 10mM PD98059 (MEK inhibitor) or 10 ng/ml IGF-I. Primers for glyc-eraldehyde-3-phosphate dehydrogenase were included in every reac-tion mixture as an internal control. Added PD98059 had little effect onendogenous mirk mRNA levels in 9V2 vector control transfectants(Fig. 4F), while dramatically up-regulating mirk protein levels (Fig.4B). IGF-I had no discernible effect on the combination of exogenousand endogenousmirk mRNA levels in the T7 transfectant (Fig. 4F)but slightly decreased mRNA levels of endogenousmirk (seen in threeexperiments). Thus, mirk protein levels are primarily modulated at theposttranscriptional level by the MAPKs erk1 and erk2.

MAPKs dimerize after their activation, and dimers translocate tothe nucleus, where they modify transcription factors (20). Phospho-rylation of proteins by MAPKs has been shown to be a trigger for theirdegradation (21, 22). Western blotting with a mirk N9-terminal anti-body demonstrated that a mirkMr 57,000 breakdown product wasfound solely in nuclear fractions of both mirk HD3 transfectants (T19,T24) and vector control HD3 transfectants (V1, V2; Fig. 4G, upperpanel), placing the mirk breakdown product in the nucleus withactivated MAPKs. Similar cell fractionation data were observed withU9 cells (Fig. 4G, lower panel). We hypothesize that after activation

by serum factors and translocation to the nucleus, erks signal the rapidturnover of mirks by phosphorylation of mirk at the canonical MAPKS557 site in its nonconserved C9terminus and possibly at a second sitewithin the PEST region.4

Mirk protein levels were generally higher in resected colon cancersthan in established cell lines by Western blotting (data not shown),which is not surprising because carcinoma cell lines are established inserum-containing medium that keeps mirk levels low. Cell lines mayup-regulate expression of autocrine growth factors to adapt to passage,and in this way also contribute to the down-regulation of mirk. Wehypothesized that because mirk abundance is rather low in cell lines,we would have to overexpress mirk in established cell lines to studyits function. Clonal stable transfectants expressing elevated endoge-nous levels of mirk were isolated using two human colon carcinomacell lines, HD3 and U9 (23). Mirk levels in these transfectants re-mained elevated 3–4-fold over vector controls in serum-free condi-tions (Fig. 4,E andG). Each of the three mirk HD3 cell transfectantsand both of the mirk U9 cell transfectants proliferated when switchedto serum-free media, whereas none of the five vector control trans-fectants was able to sustain growth under these conditions (Fig. 5).Mutation of mirk at the putative ATP binding site (KR) and at theYQY activation site (FQF) completely inhibited the kinase activity ofmirk on MBP (Fig. 1). Stable transfectants of these mirk mutantslacked the ability to proliferate when switched to serum-free condi-tions (Fig. 5). Similar data were obtained by direct cell counting andby MTT assay. Overexpression of mirk by stable transfection in-creased cell numbers 3–5-fold in HD3 cells and 5–9-fold in U9 cellsafter 5 days of serum-free growth, whereas mutations that blocked thekinase activity of mirk also blocked this biological activity (Fig. 5,A,B, E, andF). After 7 days in serum-free medium, mirk transfectantsremained viable and readily resumed rapid growth when serum wasadded to the cultures (Fig. 5H), whereas vector control transfectantcultures did not re-establish rapid growth, indicating that some cellshad lost viability.

Mirk may mediate tumor cell growth and survival under conditionsin which MAPKs are not highly activated by extracellular signals,such as early in colon cancer evolution when tumor vascularization issuboptimal, or at the tubular adenoma stage when few autocrinegrowth factors have been up-regulated. Mirk is expressed in a widerange of tumor types (Fig. 2) and in a subset of colon carcinomas (Fig.3), indicating that selection for mirk-expressing cells is maintainedafter cancers evolve from premalignant stages. Mirk may mediatetumor cell survival, especially in solid tumors, during periods whencarcinoma cells temporarily outgrow their nutrient support.

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2000;60:3631-3637. Cancer Res Kangmoon Lee, Xiaobing Deng and Eileen Friedman Substrate That Mediates Survival of Colon Cancer CellsMirk Protein Kinase Is a Mitogen-activated Protein Kinase

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