(SPy) as hnRNP K

Identi®cation of the SRC pyrimidine-binding protein(SPy) as hnRNP K: implications in the regulation ofSRC1A transcriptionShawn A. Ritchie, Mohammed K. Pasha1, Danielle J. P. Batten, Rajendra K. Sharma1,

Douglas J. H. Olson2, Andrew R. S. Ross2 and Keith Bonham3,*

Department of Biochemistry and 1Department of Pathology, College of Medicine, University of Saskatchewan,Saskatoon, Saskatchewan S7N 5E5, Canada, 2National Research Council Canada, Plant Biotechnology Institute,110 Gymnasium Place, Saskatoon, Saskatchewan S7N 0W9, Canada and 3Cancer Research Unit, SaskatchewanCancer Agency and the Division of Oncology, College of Medicine, University of Saskatchewan, 20 Campus Drive,Saskatoon, Saskatchewan S7N 4H4, Canada

Received as resubmission November 12, 2002; Revised and Accepted December 12, 2002

ABSTRACT

The human SRC gene encodes pp60c±src, a non-receptor tyrosine kinase involved in numeroussignaling pathways. Activation or overexpression ofc-Src has also been linked to a number of importanthuman cancers. Transcription of the SRC gene iscomplex and regulated by two closely linked buthighly dissimilar promoters, each associated withits own distinct non-coding exon. In many tissuesSRC expression is regulated by the housekeeping-like SRC1A promoter. In addition to other regulatoryelements, three substantial polypurine:polypyrimi-dine (TC) tracts within this promoter are required forfull transcriptional activity. Previously, we describedan unusual factor called SRC pyrimidine-bindingprotein (SPy) that could bind to two of these TCtracts in their double-stranded form, but was alsocapable of interacting with higher af®nity to all threepyrimidine tracts in their single-stranded form.Mutations in the TC tracts, which abolished theability of SPy to interact with its double-strandedDNA target, signi®cantly reduced SRC1A promoteractivity, especially in concert with mutations incritical Sp1 binding sites. Here we expand upon ourcharacterization of this interesting factor anddescribe the puri®cation of SPy from human SW620colon cancer cells using a DNA af®nity-basedapproach. Subsequent in-gel tryptic digestion ofpuri®ed SPy followed by MALDI-TOF massspectrometric analysis identi®ed SPy as hetero-geneous nuclear ribonucleoprotein K (hnRNP K), aknown nucleic-acid binding protein implicated invarious aspects of gene expression including tran-scription. These data provide new insights into the

double- and single-stranded DNA-binding speci®-city, as well as functional properties of hnRNP K,and suggest that hnRNP K is a critical component ofSRC1A transcriptional processes.

INTRODUCTION

The human SRC proto-oncogene encodes the 60 kDa non-receptor tyrosine kinase pp60c±src, which has been implicatedin many diverse cellular processes including differentiation,focal adhesion dynamics, regulation of cell±cell contact, boneremodeling, mitosis and others (1±4). Numerous ®ndingsthroughout the past two decades have also pointed towards arole for SRC in the process of cellular transformation andcancer (2). For example, activation and/or overexpression ofpp60c±src has been observed in many colon (5±8) as well asbreast (9±12) carcinomas, and activating mutations in the SRCgene have been detected in a limited number of advancedcolon tumors (13). Further evidence from our own laboratorysuggests that in certain colon cancer cell lines, increased c-Srcprotein levels can be attributed to the transcriptional activationof the SRC gene (14). Therefore, to elucidate the mechanismsresponsible for regulating SRC transcription we have beenstudying the regulatory motifs within the 5¢ region of the SRCgene and have identi®ed two promoters: SRC1a and SRC1A.The SRC1a promoter, located ~1 kb upstream of the SRC1Apromoter, is regulated by the liver-enriched hepatocytenuclear factor 1 family of transcription factors, and transcriptsoriginating from this promoter show a restricted tissue-speci®cexpression pattern (15). Transcripts arising from the SRC1Apromoter, on the other hand, show a ubiquitous expressionpattern more typical of housekeeping genes. Consistent withthis classi®cation, the SRC1A promoter is GC rich and usesmultiple transcription start sites; however, it also containsthree perfect polypurine:polypyrimidine (TC) tracts, TC1,TC2 and TC3. We have previously shown that SRC1Apromoter activity was dependent upon two critical Sp1 sites,

*To whom correspondence should be addressed: Tel: +1 306 655 2313; Fax +1 306 655 2910; Email: [email protected] address:Shawn A. Ritchie, Phenomenome Discoveries, Inc., 204±407 Downey Road, Saskatoon, Saskatchewan S7N 4L8, Canada

1502±1513 Nucleic Acids Research, 2003, Vol. 31, No. 5DOI: 10.1093/nar/gkg246

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GC1 and GA2, as well as the binding of a factor called SRCpyrimidine-binding protein (SPy) (16). SPy was originallydescribed as a factor capable of binding speci®cally to TC1and TC2 (but not TC3) in their double-stranded form, and toall of the TC tract pyrimidine sequences in single-strandedform. TC1 and TC2 were found to share a common motif(CTTCC), which was absent from TC3. Mutation of thissequence to CTTTC within either TC tract abolished SPydouble-stranded binding and compromised SRC1A promoteractivity; however, this mutation failed to inhibit SPy single-stranded pyrimidine binding (16). Therefore, it appeared thatSPy had differential binding speci®cities for both double- andsingle-stranded DNA. In addition, the apparent af®nity of SPyfor single-stranded DNA was observed to be greater than forthe double-stranded counterparts (16). Lastly, SPy was shownto regulate the SRC1A promoter cooperatively with thetranscription factor Sp1 (16).

Given the highly unusual DNA-binding characteristics ofSPy and its requirement for maximum SRC1A transcription,we decided to study this factor in greater detail and describehere its puri®cation using a DNA-af®nity based approach.Subsequent matches between experimental tryptic peptidemasses determined by matrix-assisted laser desorption/ioniza-tion-time of ¯ight (MALDI-TOF) mass spectrometry and insilico tryptic digests derived from protein sequence databasesidenti®ed SPy as heterogeneous nuclear ribonucleoprotein K(hnRNP K). This highly modular protein interacts with manybiomolecules including RNA, single-, double- and triple-stranded DNA (17±19), as well as a number of importanttranscriptional regulators and signaling molecules (17,20).Interestingly, these include pp60c±src itself as well as otherc-Src family members (21). The functions of hnRNP K, whilegenerally poorly de®ned, appear to be numerous, and theprotein has been implicated in multiple levels of geneexpression including transcription, translation and the pro-cessing of RNA (22±25). Our data show that hnRNP K alsoplays an important role in the regulation of the SRC1Apromoter and provides new insights into the DNA-bindingspeci®city and biological activity of hnRNP K.

MATERIALS AND METHODS

Cells and media

SW480 and SW620 human colon cancer cells were obtainedfrom the American Type Culture Collection and grown at37°C, 5% CO2 in DMEM containing 10% FCS (Cansera) and1% PenStrep (Life Technologies).

Oligonucleotides

All oligonucleotides were purchased from Life Technologies.Double-stranded TC1 and mutant TC1 oligonucleotides weregenerated by annealing with their complementary respectivestrands (Life Technologies). The TC1 and TC2 wild-type anddouble-stranded mutant sequences have been previouslyreported (16). Sequences of the TC1 and TC2 single-strandedmutant oligonucleotides used for electrophoretic mobility shiftassays (EMSAs), site-directed mutagenesis and constructionof the SPy DNA af®nity column are as follows.

EMSA oligonucleotides. TC1ss mut: 5¢CTTGCTGCTTG-CTGCTCCTCCC; TC2ss mut: 5¢CTCCCTCCCTCGTGTTG-CCGTCCCC.

Site-directed mutagenesis oligonucleotides. TC1ss mutCT:5¢GTCCCCGCGCGCTTGCTGCTTGCTGCTCCTCCCGG-CTGGCCTGCC; TC1ss mutGA: 5¢GGCAGGCAGCCGGG-AGGAGGAGCAAGCAGCAAGCGCGCGGGGAC; TC2ssmutCT: GGCCCGGGGACGGCAACACGAGGGAGGGA-GCG; TC2ss mutGA: CGCTCCCTCCCTCGTGTTGCCG-TCCCCGGGCC.

DNA af®nity oligonucleotides. TC1 af®nity CT: 5¢GAT-CCTTCCTCCTTCCTCCTCCTCCCGATCCTTCCTCCTT-CCTCCTCCTCCC; TC1 af®nity GA: 5¢GATCGGGAGGA-GGAGGAAGGAGGAAGGATCGGGAGGAGGAGGAAG-GAGGAAG.

The DNA af®nity sequences were then ligated to formconcatenated double-stranded multimers and coupled directlyto CNBr-activated Sepharose, as previously described (26).

Plasmids and reporter gene constructs

The human SRC promoter-CAT constructs p0.38SRC-CATand the TC1, TC2 and TC1-TC2 double-stranded mutatedforms of p0.38SRC-CAT have been previously described (16).Construction of p0.38SRC-CAT harboring single-strandedSPy-binding mutations in either TC1 or TC2 were generatedby the QuickChange site-directed mutagenesis protocol(Stratagene) using either the TC1 or TC2 mutagenic sequenceslisted in the oligonucleotides section. To generate theconstruct harboring single-stranded mutations in both TC1and TC2, p0.38SRC-CAT containing the TC1 single-strandedmutation was used as the template in a QuickChange reactionwith the mutant TC2 oligonucleotides. All mutant plasmidsgenerated in the PCR stage were then digested with NarI andSacII to isolate the promoter region, and the fragment ligatedback into a wild-type p0.38SRC-CAT construct to ensure theabsence of possible mutations elsewhere in the plasmid thatcould have occurred during the mutagenesis step.

Transient transfections and CAT assays

Transfection-grade supercoiled plasmids were prepared usingan EndoFree Plasmid Maxi Kit (Qiagen). SW480 cells (3.75 3105) in six-well plates were transfected with CAT expressionvector (3.0 mg) and pCH110 b-galactosidase expression vector(1.0 mg) using Superfect (Qiagen) according to the manufac-turer's instructions. In the case where single-strandedoligonucleotides were added to titrate out SPy/hnRNP Kprotein, the oligonucleotides were added at various concen-trations (0.5, 1.0, 1.5 and 2.0 mg) to the transfection reagent atthe same time as the plasmid. Cells were harvested 48 h aftertransfection with lysis buffer provided with the CAT ELISAkit (Roche), and protein levels determined using BradfordProtein Reagent (Bio-Rad). b-Galactosidase levels weredetermined as previously described (27), and CAT proteinlevels quanti®ed using the CAT ELISA kit (Roche). ReportedCAT values were standardized to total protein and b-galactosidase expression and are representative of at leastthree duplicate experiments.

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Preparation of nuclear extracts and EMSA

Nuclear extracts were prepared from human cells according tothe method of Andrews and Faller (28). Typically, three150 mm plates of con¯uent cells were scraped, washed threetimes in PBS, and suspended in 2 ml ice cold buffer A (10 mMHEPES±KOH pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mMDTT and 0.2 mM PMSF) on ice for 10 min. An aliquot of 25 ml10% NP-40 was added and the cells vortexed. Lysis of theplasma membrane was monitored by nuclear staining with0.4% trypan blue. The nuclei were pelleted by centrifugation(in a Sorvall 6000D at 1100 r.p.m.), the supernatants discardedand the pellets resuspended in 100±200 ml ice cold buffer C(20 mM HEPES±KOH pH 7.9, 25% glycerol, 420 mM NaCl,1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT and 0.2 mMPMSF) on ice for 20 min. The samples were transferred tomicrofuge tubes, centrifuged at 13 000 r.p.m. in a microfugeat 4°C for 5 min and the supernatants removed and stored at±80°C. For EMSAs, double-stranded probes were generatedby annealing complementary single-stranded oligonucleotidesand labeling with in-®ll reactions using Klenow fragment and[a32P]dCTP. Single-stranded probes were end-labeled with[g32P]ATP and T4 polynucleotide kinase. Typically, nuclearextracts were incubated with 50 000±100 000 c.p.m. of probein binding buffer (25 mM HEPES pH 7.0, 4 mM Tris±HCl pH8.0, 5 mM MgCl2, 1 mM CaCl2 and 1 mM DTT) and 3 mgpoly(dI-dC). When competitor oligonucleotides or antibodieswere included, they were added to the binding reaction andincubated on ice for 15 min before the addition of probe,followed by incubation at room temperature for 30 min. Theanti-hnRNP K monoclonal was the kind gift of Dr GideonDreyfuss, University of Pennsylvania School of Medicine(29), while the anti-hnRNP K polyclonal was kindly providedby Dr Miau, Institute of Molecular Medicine, Dr H.L. TsaiMemorial Laboratory, College of Medicine, National TaiwanUniversity (30). The Ets-1 antibody was purchased from SantaCruz, Inc. The reactions were then resolved on 6%polyacrylamide gels in 13 Tris±glycine running buffer [50mM Tris base, 380 mM glycine and 2 mM EDTA] at 150 V for~3 h at 4°C. Gels were dried and visualized either byautoradiography or by phosphorimage analysis using a Bio-Rad Molecular FX Imager.

Ultraviolet crosslinking analysis

An EMSA gel containing a shifted SPy-TC1 double-strandedradiolabeled oligonucleotide complex was exposed to UVradiation in a Stratagene Stratalinker (auto cross-link mode).The SPy-oligonucleotide complex was then excised from theEMSA gel and either physically inserted into the well of anSDS±PAGE gel (12%) and the complex resolved at 150 V for1 h, or eluted from the gel fragment by overnight incubation at37°C in elution buffer (20 mM Tris±HCl pH 8.5, 1 mMEDTA) followed by SDS±PAGE analysis as described above.When complete, the gel was dried and visualized byautoradiography using Kodak X-Omat ®lm at ±80°C over-night.

Western blotting

For western blot analysis, polypeptides from af®nity fractionswere separated by 12% SDS±PAGE and transferred tosupported nitrocellulose using a wet transfer apparatus

running at 50 V for 2 h. The membrane was probed withmonoclonal anti-hnRNP K antibody 3C2 (29) and detectedusing PicoWest Enhanced Chemiluminescent substrate(Pierce) and Kodak X-Omat XB-1 ®lm at room temperature.

Puri®cation of SPy

Crude SW620 nuclear extracts were dialyzed in buffer Z(10 mM Tris±HCl pH 7.7, 1 mM EDTA, 0.01% NP-40, 10%glycerol, 1 mM DTT) and applied to an SP-Sepharose column(1.5 3 4.0 cm). After loading, the column was washedextensively with buffer Z, and SPy activity subsequentlyeluted with a step gradient of 0.4 and 0.8 M NaCl in buffer Z.Fractions of 2 ml were collected and assayed for SPy bindingactivity by EMSA. Active fractions were pooled, dialyzed inbuffer Z and applied to a second SP-Sepharose column (1.5 34.0 cm). The column was washed with buffer Z and elutedusing a linear gradient of 0±0.4 M NaCl in buffer Z. Fractionscontaining SPy activity (determined by EMSA) were pooled,dialyzed and applied to a DNA af®nity column (1.5 3 4.0 cm)constructed by coupling concatenated double-stranded oligo-nucleotides containing SPy binding sites to CNBr activatedSepharose according to the method of Kadonaga and Tjian(26). The af®nity column was washed and eluted using a lineargradient of 0±1 M KCl in buffer Z. Fractions were assayed forSPy activity by EMSA and the active SPy fraction concen-trated by lyophilization, analyzed by SDS±PAGE, and stainedwith SYPRO Ruby-Red protein gel stain (Bio-Rad). The gelwas visualized on a Bio-Rad Molecular FX Imager.

Trypsin digestion of gel-separated polypeptides

The 60 kDa polypeptide present in the SPy-active af®nityfraction, along with background spots from the gel, wereexcised from SYPRO Ruby-Red stained SDS±PAGE gel, cutinto 1 mm squares and placed into a 1.5 ml tube. Gelmanipulation was performed in a laminar ¯ow hood tominimize keratin contamination. A 50 ml aliquot of 25 mMammonium bicarbonate in 1:1 (v/v) water:acetonitrile wasadded and the samples vortexed for 10 min. The solution wasdiscarded and the previous step repeated twice. Samples werethen dried for 30 min in a vacuum centrifuge followed byincubation with 50 ml freshly prepared 10 mM DTT in H2O for60 min at 56°C in the dark. The DTT was removed and 50 mlof 55 mM iodoacetamide (IAA) in H2O was added followedby incubation for 45 min at 22°C in the dark with occasionalvortexing. The IAA solution was removed and samplesvortexed in 50 ml of 25 mM ammonium bicarbonate in H2Ofor 10 min. The IAA solution was removed and the previoustwo steps repeated twice. Gel pieces were dried for 30 min in avacuum centrifuge and resuspended in 100 ml of 25 mMammonium bicarbonate/5 mM calcium chloride containing25 ng/ml modi®ed porcine trypsin (sequencing grade;Promega). Samples were incubated at 4°C for 30 min, thetrypsin solution removed and 100 ml of 25 mM ammoniumbicarbonate added. The tubes were sealed with para®lm andincubated overnight at 37°C. The supernatant was removedand placed into a new tube (tube B). The gel pieces wereresuspended in 100 ml of H2O and vortexed for 5 min,followed by sonication for 10 min. The supernatant wastransferred to tube B and the gel fragments re-suspended in50 ml of 5% tri¯uoroacetic acid (TFA) in 1:1 (v/v) water:acetonitrile for 15 min. The supernatant was transferred to

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tube B and the previous step repeated once more. The pooledsupernatants in tube B were evaporated in a vacuum centrifugeand the tryptic peptides re-suspended in 10 ml of 0.1% TFA inH2O.

MALDI-TOF mass spectrometry

Tryptic peptides re-suspended in 0.1% TFA were desalted bysolid-phase extraction using disposable pipette tips(ZipTipC18, Millipore) according to the manufacturer'sinstructions. Peptides were eluted in 1±2 ml of 0.1% TFA/75% acetonitrile directly onto a MALDI target plate. An equalvolume of matrix solution, consisting of 5 mg/ml a-cyano-4-hydroxycinnamic acid (Aldrich) in 0.1% TFA/50% aceto-nitrile, was applied to the sample and allowed to dry. A 1:1mixture (31) of matrix and mass calibrant containing des-ArgBradykinin and ACTH clip 18-39 was also placed beside thesample for close external calibration. Mass spectra of trypticpeptides were acquired on a Voyager-DE STR MALDI-TOFmass spectrometer (Applied Biosystems, Framingham, MA)operating in the positive ion, delayed extraction and re¯ectronmodes. Experimental conditions were as follows: acceleratingvoltage 20 000 V; grid voltage 72.5%; guide wire voltage0.001%; delay 100 ns; laser power 2386. Spectra wereobtained by combining 400 scans and applying mass calibra-tion, baseline correction, noise ®ltering and de-isotopingprocedures using the spectrometer software (Data Explorerv. 4.0, Applied Biosystems). Monoisotopic peptide masseswere submitted to Protein Prospector (http://prospector.ucsf.edu/) for comparison with entries in the National Center forBiotechnology Information (NCBI) sequence database, usingthe MS Fit program.

RESULTS

UV cross-linking analysis suggests that SPy is a singlepolypeptide of ~50±58 kDa

SRC1A expression is dependent upon several cis elementsincluding the GC1 and GA2 sites, which interact with the Sp1family; and three polypurine:polypyrimidine sites, TC1-3,which interact with SPy (Fig. 1A). SPy had been previouslyshown to produce a characteristically broad and fairly rapidlymigrating complex with radiolabeled double-stranded TC1and TC2 oligonucleotides in EMSAs (16). To furthercharacterize the nature of this SPy-DNA complex we carriedout ultra-violet cross-linking of an EMSA gel containing aSPy-double-stranded 32P-labeled TC1 complex. A gel frag-ment encompassing the radioactive cross-linked complex wasthen excised from the EMSA gel and either physically insertedinto the well of an SDS±PAGE gel, or eluted out of the gelfragment and resolved by SDS±PAGE. As shown in Figure 1B,both elution (lane A) and excision (lane B) of the cross-linkedcomplex from the EMSA gel generated a single polypeptidespecies migrating between 50 and 58 kDa on SDS±PAGE.

Puri®cation of SPy by ion-exchange and DNA af®nitychromatography

The puri®cation of SPy comprised a combination of ion-exchange and DNA af®nity chromatography. Initially, SW620nuclear extracts were prepared and puri®ed by ion-exchangeon an SP-Sepharose column (see Materials and Methods),

which was eluted in a step-wise manner with 0.4 and 0.8 MNaCl. Fractions containing SPy protein were identi®ed byEMSAs using radiolabeled double-stranded TC1 oligonucle-otides. The purpose of the Sepharose chromatography stagewas to obtain SPy protein devoid of factors such as nucleasesthat could interfere later with the DNA af®nity column. Asshown in Figure 2A, SPy-binding activity was present withinfractions of the 0.4 M eluate. Fractions 1±5 were pooled,dialyzed and reapplied to another SP-Sepharose column thatwas eluted with a linear NaCl gradient from 0±0.4 M NaCl.`Active' SPy fractions (Fig. 2B) were identi®ed by EMSAs asabove, pooled and dialyzed in preparation for the DNA af®nitycolumn. Fractions from the second ion-exchange column werealso analyzed by EMSA using a radiolabeled TC1-baseddouble-stranded mutant oligonucleotide (see Fig. 6 forsequence), which failed to show any shifted species (datanot shown). This was consistent with our previous observa-tions that the TC1 double-stranded mutant probe was unable to

Figure 1. Ultraviolet cross-linking analysis of a SPy-TC1 double-strandedoligonucleotide complex. (A) The SRC1A promoter region contains twocritical Sp-family member DNA-binding sites, GC1 and GA2, as well asthree TC tracts, TC1, TC2 and TC3. SPy double-stranded binding siteslocated within TC1 and TC2 are shaded gray. The major transcription startsites are located just downstream of TC3 and are marked by an arrow.(B) An EMSA was ®rst performed by incubating wild-type TC1 double-stranded radiolabeled oligonucleotides with SW620 nuclear extract followedby exposure of the gel to UV light. The shifted species was isolated byexcision, and the radiolabeled SPy-DNA complex either eluted out of theexcised gel slice and subjected to analysis by SDS±PAGE (lane A), orelectrophoresed out of the gel slice following insertion into the well of anSDS±polyacrylamide gel (lane B). The gels were then visualized byautoradiography.



Figure 2. EMSA analysis of ion-exchange and DNA-af®nity chromatography fractions. (A) EMSA analysis of fractions from ®rst round step-wise elution ofion-exchange column using a TC1 double-stranded radiolabeled oligonucleotide. (B) EMSA of fractions from second round linear elution ion-exchangecolumn using a TC1 double-stranded radiolabeled oligonucleotide. Fraction numbers from each column are shown along the tops of each panel, including thesalt concentration gradients used for each elution. (C) EMSA analysis of fractions eluted from the SPy DNA af®nity column using a radiolabeled TC1single-stranded pyrimidine oligonucleotide. DNA af®nity fraction 17 was resolved by SDS±PAGE and ¯uorescently stained with SYPRO Ruby-Red.



bind SPy (16), suggesting that the complex observed inFigure 2B was indeed SPy. The DNA af®nity column wasconstructed as described in Materials and Methods, andthe pooled SPy-containing fractions from the second ion-exchange column loaded. The DNA af®nity column waswashed extensively, eluted with a linear gradient of 0±1.0 MKCl and fractions assayed for SPy activity by EMSA (Fig. 2C).Since the af®nity column was constructed with double-stranded TC1 oligonucleotides, assaying the eluted fractionswith a radiolabeled double-stranded TC1 oligonucleotidewould likely have been unsuccessful, particularly withfractions containing high salt concentrations. Therefore, tocircumvent these potential detection problems, the fractionswere assayed using radiolabeled TC1 pyrimidine single-stranded oligonucleotides, which resulted in a single fraction(no. 17) showing SPy-binding (Fig. 2C). Fraction 17 was thenconcentrated by lyophilization and analyzed by SDS±PAGE,which revealed the presence of one prominent polypeptidewith an apparent molecular weight of ~60 kDa (Fig. 2C,bottom panel).

Identi®cation of SPy by MALDI-TOF massspectrometry

To reveal the identity of SPy, in-gel tryptic digestion wasperformed followed by analysis of the tryptic peptides byMALDI-TOF mass spectrometry. The 60 kDa polypeptidewas excised from the Ruby-Red-stained SDS±PAGE gel(Fig. 2C) along with several protein-free gel pieces asbackground controls. The gel fragments were then digested

with trypsin and their masses determined by MALDI-TOFmass spectrometry. As shown in Figure 3A, the gel slicecontaining the 60 kDa SPy polypeptide had a spectrumcontaining many more peptides than the background spec-trum, which showed peaks for the trypsin enzyme only(Fig. 3B). The resulting experimental peptide masses gener-ated from the gel slice containing the 60 kDa polypeptide werethen searched against predicted tryptic peptide masses for allthe proteins contained within the NCBI protein database,using the MS-Fit sequence database search program availableat the ProteinProspector website (http://prospector.ucsf.edu).As shown in Figure 4A and B, 14 (out of 32 submitted) massesmatched precisely to the Homo sapiens pyrimidine bindingprotein hnRNP K. The correlation of the matches wassigni®cant, with a maximum difference in mass of±21.2 p.p.m. between the submitted and matched peptides,with a majority of the differences within 10 p.p.m. Submittedpeptide masses that fall within 50 p.p.m. of the matchedmasses are considered signi®cant, and protein identi®cationunambiguous, if >15% of the matched protein sequence iscovered by these peptides (32). The locations of the experi-mentally derived tryptic peptides according to the amino acidsequence of hnRNP K are shown in Figure 4B.

EMSA and western analysis con®rm SPy identity ashnRNP K

The precise match between SPy tryptic peptides and thoseof hnRNP K suggested with a high probability that SPywas hnRNP K; however, we proceeded to con®rm our

Figure 3. MALDI-TOF mass spectrometric analysis of SPy tryptic peptides. The polypeptide present in af®nity fraction 17 was resolved by SDS±PAGE,stained with Ruby-Red, excised from the gel and subject to tryptic digestion. MALDI-TOF mass spectrometric analysis of the subsequent tryptic peptidesproduced the spectrum shown in (A). The spectrum in (B) represents the masses of tryptic peptides present in a background piece from the SDS±PAGE.



identi®cation using hnRNP K antibodies in EMSAs andwestern blot analyses. EMSAs using both polyclonal andmonoclonal anti-hnRNP K antibodies produced supershiftedSPy complexes (Fig. 5A, polyclonal and B, monoclonal).The EMSA in Figure 5A was performed with an hnRNP Kpolyclonal antibody (30) and produced a faint supershiftedspecies, while the hnRNP K monoclonal antibody 3C2 (29),as shown in Figure 5B, produced a robust supershiftedspecies and an associated reduction in intensity of the SPycomplex. Furthermore, western blot analysis of fractions16±18 of the DNA af®nity puri®cation using the anti-hnRNPK monoclonal antibody (3C2) showed immunoreactivity witha 60 kDa species in fraction 17 only. This ®nding wasconsistent with the identi®cation of SPy as hnRNP K asrevealed by the mass spectrometry data, and stronglysuggested that the factor previously referred to as SPy wasindeed hnRNP K.

SRC1A promoter activity is dependent on hnRNP Kbinding

Previously we reported that SPy/hnRNP K bound speci®callyto double-stranded polypurine:polypyrimidine sequences lo-cated within the SRC1A promoter region (Fig. 1A) (16). Pointmutations in two of these tracts, TC1 and TC2, abolished theability of SPy/hnRNP K to bind its double-stranded target andresulted in reduced promoter activity, especially in concertwith mutations in critical Sp1 binding sites (16). However, ourwork also showed that even these mutated pyrimidinesequences, when in single-stranded form, were still able tobind SPy/hnRNP K with a high af®nity. Thus, some of theobserved residual promoter activity might have been the resultof such single-stranded binding. Because of this relaxed SPy/hnRNP K single-stranded binding speci®city, we hypothe-sized that more drastic mutations disrupting the pyrimidine

Figure 4. ProteinProspector search results of SPy tryptic peptide masses. The peptide masses resulting from MALDI-TOF analysis of SPy tryptic peptideswere searched using the ProteinProspector website against the masses of virtually derived tryptic peptides present in the NCBI protein database. Fourteen of32 submitted peptide masses (A) were found to match precisely with virtually derived tryptic peptide masses of hnRNP K. The table shows submitted andmatched masses of the corresponding peptides, including the difference in mass between the two (delta p.p.m.). Matches to within 50 p.p.m. are consideredsigni®cant (32). (B) Amino acid sequence of hnRNP K showing the regions that matched to SPy tryptic peptides (bold and underlined). Numbered regionscorrespond to the locations within hnRNP K of identi®ed tryptic peptides from (A). These peptides cover 32% of the matched protein sequence, providingunambiguous identi®cation.



content of the single-stranded targets would be required tocompletely prevent SPy/hnRNP K single-stranded binding.Single-stranded pyrimidine oligonucleotides, based on theTC1 and TC2 sequences and harboring four pyrimidine topurine substitutions (Fig. 6A), were subsequently designedand shown by EMSA to completely abolish SPy/hnRNP K'ssingle- and double-stranded binding ability (data not shown).These TC1 and TC2 single-stranded SPy/hnRNP K-bindingmutations were then introduced both individually and collect-ively into the 0.38SRC-CAT reporter construct by site-directed mutagenesis. Surprisingly, when assayed by transienttransfection into SW480 cells, the constructs containingindividual mutations abolishing SPy/hnRNP K single-stranded binding at either TC1 or TC2 resulted in CAT levels

similar to those observed with the previously reportedindividual double-stranded mutations (Fig. 6A). However,there was a signi®cant difference observed between the single-stranded SPy/hnRNP K mutations in TC1 and TC2 togethercompared to the TC1 and TC2 double-stranded mutationstogether. The TC1-TC2 single-stranded mutations togetherreduced CAT levels to 30% of wild-type while the TC1-TC2double-stranded mutant reduced CAT levels to just <70%(Fig. 6A). These ®ndings suggest that ultimately it may be theability of SPy/hnRNP K to bind these TC tracts in single-stranded form that is critical for maximum SRC1A promoteractivity.

To further examine the effects of SPy/hnRNP K'sinteraction with the SRC1A promoter, we co-transfected the

Figure 5. Antibodies speci®c for hnRNP K recognize SPy. EMSA analysis of a SPy-TC1 double-stranded DNA complex in the presence of increasingamounts of anti-hnRNP K (A) polyclonal or (B) monoclonal 3C2 antibody. Addition of Ets antibody to the EMSA reactions in both (A) and (B) is shown as acontrol. (C) Western analysis of DNA af®nity fractions 16±18 (of Fig. 2C) using anti-hnRNP K monoclonal antibody 3C2.



wild-type 0.38SRC-CAT vector with increasing concentra-tions of wild-type TC1 single-stranded oligonucleotides. Theobjective here was to titrate out endogenous SPy/hnRNP K,which is present at relatively high levels in SW480 cells. Asshown in Figure 6B, addition of wild-type TC1 oligonucle-otides reduced CAT levels in a dose-dependent manner, by~50%. However, the addition of TC1ss mut oligonucleotide,which is unable to bind SPy/hnRNP K in vitro (sequenceshown in Fig. 6A), showed no effect on 0.38SRC-CATreporter levels, even at the highest concentration (Fig. 6C).Lastly, we repeated these experiments using the 0.38SRC-CAT vector containing both TC1 and TC2 single-strandedmutations. In this case, co-transfection of either wild-type ormutated versions of the TC1 oligonucleotide had no effect(Fig. 6D and E, respectively). These results demonstrate thatthe in vitro binding properties of SPy/hnRNP K correlatestrongly with the activity of the SRC1A promoter in vivo. Inconjunction with the mutational analysis we conclude thatSPy/hnRNP K is integrally involved in regulating SRC1Apromoter activity.

DISCUSSION

In this paper we further dissect the DNA binding character-istics of SPy and its effect on SRC1A promoter activity. Usinga combination of DNA af®nity chromatography and MALDI-TOF mass spectrometry we also report the identi®cation ofthis factor as hnRNP K. The relatively rapid migration of theSPy complex on EMSA gels, coupled with our UV-cross-linking experiments and subsequent puri®cation of SPy as asingle 60 kDa protein, were entirely consistent with thehypothesis that the SPy complex observed on EMSA gelsrepresented a single protein species. This notion was furtherstrengthened by the robust supershift generated in EMSA gelswith the hnRNP K monoclonal antibody, which also con-®rmed the identity of SPy as hnRNP K. Interestingly, whilethe calculated mass of hnRNP K is 50±51 kDa, it is known tomigrate as a 60±65 kDa species on SDS±polyacrylamide gels(17).

hnRNP K was ®rst identi®ed as one of more than 20proteins found in ribonucleoprotein particles where it bindsRNA (19). Its highly modular structure allows a number ofdiverse interactions with an unusually large range of mol-ecules. This includes single-, double- and triple-stranded DNAas well as various proteins including transcriptional regulatorssuch as TATA binding protein (TBP) (23) and Zik-1 (33),inducible serine threonine kinases (34), the oncoprotein Vav(20) and members of the c-Src family of tyrosine kinases (35).This has led to proposals that hnRNP K acts as some form ofplatform or scaffold to facilitate diverse molecular interactions(35,36). Mirroring this diversity, hnRNP K to date has beendirectly implicated in numerous cellular processes includingtranscription, mRNA transport, splicing and translationalregulation (22±25). In addition to its many partners androles, hnRNP K is also modi®ed by a number of kinases,including casein kinase II (37), PKC (38) and tyrosine kinasessuch as c-Src and Lck (21,39). While the effect of suchphosphorylations is not well understood, they have beenshown to modulate DNA and RNA binding ability (36).

hnRNP K has been implicated in the transcriptionalregulation of several genes (23,24,40). The work described

Figure 6. Effect of SPy/hnRNP K DNA-binding mutations and titration ofSPy/hnRNP K protein on SRC1A promoter activity. (A) Mutations thatabolish SPy single-stranded binding (i.e. four incorporated purines withineither the pyrimidine strand of TC1, TC2 or both together) were introducedinto 0.38SRC-CAT and reporter levels assayed by transient transfection inSW480 cells. Solid black bars represent SPy double-stranded mutations;gray bars represent SPy single-stranded mutations. CAT levels are expressedrelative to wild-type 0.38SRC-CAT (open bar). Sequences of the mutationsare shown below. (B) Single-stranded oligonucleotides capable of SPy/hnRNP K binding (TC1 CT) as well as (C), the mutant form, incapable ofSPy/hnRNP K binding (TC1ss mut CT) were co-transfected with 0.38SRC-CAT into SW480 cells. 0.38SRC-CAT harboring TC1+TC2ss mutationswas cotransfected with both TC1 CT (D) and TC1ss mut CT (E), single-stranded oligonucleotides into SW480 cells. The black triangle representsincreasing concentrations (see Materials and Methods) of the competitoroligonucleotide in the presence of consistent quantities of reporter plasmid.CAT levels were standardized to total protein and b-galactosidase expres-sion, and are the average of three duplicate experiments.



here shows that SPy/hnRNP K interacts in vitro withfunctionally important SRC1A promoter elements and there-fore likely regulates SRC expression in vivo too. Our reasonsfor this conclusion can be summarized as follows. (i) Singlepoint mutations within either TC1 or TC2, which disrupted theability of hnRNP K to bind double-stranded targets in vitro,affected promoter activity in vivo. (ii) More drastic mutationsthat destroyed the ability of SPy/hnRNP K to bind single-stranded targets in vitro had an even more pronounced effectin vivo. (iii) Titration of endogenous hnRNP K with single-stranded oligonucleotides shown to bind SPy/hnRNP Kstrongly in vitro reduced SRC1A promoter activity in vivo,while mutated versions of these oligonucleotides had no effect.

What then might be the role of hnRNP K in SRC1Aregulation? Previous studies have shown that hnRNP K can

activate the c-myc promoter through interactions with asimilar polypurine:polypyrimidine tract (29). Michelotti et al.(23) reported that hnRNP K can interact with a CT elementin vitro and induce a single-stranded conformation whenpresent in a supercoiled plasmid. They proposed a modelwhereby hnRNP K could induce a single-stranded `bubble' inthe c-myc promoter by virtue of its high af®nity forpolypyrimidine sequences, thereby facilitating the entry ofother components of the basal transcriptional machinery. Ourprevious characterization of SPy/hnRNP K DNA-bindingspeci®city leads us to propose a similar model for SRC (Fig. 7).However, we have shown for the ®rst time that SPy/hnRNP Kactually has a distinct double-stranded DNA sequence bindingrequirement, namely the CTTCC motif present in TC1 andTC2. We therefore propose that this could be an important

Figure 7. Possible model for SRC1A transcriptional regulation by Sp1 and hnRNP K. Our data suggest a model by which hnRNP K could recognize and bindspeci®cally to double-stranded sequences within TC1 and TC2 followed by strand separation that would be facilitated by the increased af®nity of hnRNP Kfor single-stranded DNA. The resulting single-stranded `bubble' could encompass the entire TC-tract region or more discrete regions therein. For example,the ability of hnRNP K to bind TBP could recruit TFIID to the TC3 region and aid in the assembly of a pre-initiation complex. Transactivation would beaccomplished by Sp1, most notably in this model, from binding to the GC1 site. Such a structure would be in agreement with the location of the preferredtranscription start sites, which are located ~20 bp downstream of TC3 and would also account for other minor sites, which are located throughout the TC-tractregion.



factor determining promoter selectivity by SPy/hnRNP K,which would be dependent on the presence of this motif.Interestingly, the c-myc CT element previously mentionedcontains exactly such a motif (TCCTCCCCACCTTCC-CCACCCTCCCCACCCTCCCCA). In addition, hnRNP Khas been shown to interact with a similar sequence (ACC-CTCCCCTCCCCTGTAACTCCACCCCTTCCCCA) foundin the neuronal nicotinic acetylcholine receptor promoter,which also contains the same motif (41).

Thus, we propose that following the binding of SPy/hnRNP K to its double-stranded target, melting of the SRC1Apromoter region would be facilitated by the higher af®nityof SPy/hnRNP K for the single-stranded pyrimidine strands.SPy/hnRNP K could then recruit other elements of the basaltranscriptional machinery, namely TFIID, through the knowninteraction between hnRNP K and TBP (23). Our mutationand transfection experiments (Fig. 6) also support such amodel, since to signi®cantly disrupt SRC1A promoter activity,the ability of SPy/hnRNP K to bind to both TC1 and TC2 insingle-stranded form must be destroyed. However, it should benoted that these constructs still contain an intact TC3 tract,which is also capable of binding SPy/hnRNP K with highaf®nity in single-stranded form. We are currently assessing thecontribution of TC3 to the remaining promoter activity.

It is interesting to note that despite the long-term observa-tion that hnRNP K has remarkable nucleic acid bindingabilities there has not been a systematic study to examineDNA-binding speci®city. This is especially true of thedouble-stranded DNA-binding ability of hnRNP K. Indeed,where binding studies have been carried out the source ofhnRNP K tends to be recombinant hnRNP K isolated fromprokaryotic sources. Given the results of our studies and thelikelihood that phosphorylation could in¯uence bindingability, we feel such binding studies are a priority and shouldhelp identify other promoters potentially regulated byhnRNP K. Polypurine:polypyrimidine tracts are not uncom-mon in eukaryotic promoter regions and the models describedhere could represent a method by which hnRNP K could targetthe basal transcriptional machinery to speci®c promoters,especially ones with high GC content which often lack TATAboxes.

In summary, we have identi®ed the SRC pyrimidine-binding protein, SPy, as hnRNP K and suggest that it playsan important role in the regulation of SRC transcription. Ourprevious studies showing that SPy/hnRNP K has distinctdouble-stranded binding requirements also suggest a mech-anism by which hnRNP K could selectively regulate speci®cpromoters. Lastly, we are particularly intrigued by thefrequent observations that hnRNP K and pp60c±src interact,including a recent report showing that such interactions canlead to pp60c±src activation and hnRNP K tyrosine phos-phorylation (39). While no evidence yet exists, it is temptingto speculate that these interactions may be a sign that somepositive or negative feedback regulation of SRC transcriptionexists.

ACKNOWLEDGEMENTS

This work was supported by operating grants from theCanadian Institutes of Health Research (CIHR) as well asthe Saskatchewan Regional Partnership Program.

REFERENCES

1. Abram,C.L. and Courtneidge,S.A. (2000) Src family tyrosine kinases andgrowth factor signaling. Exp. Cell Res., 254, 1±13.

2. Biscardi,J.S., Tice,D.A. and Parsons,S.J. (1999) c-Src, receptor tyrosinekinases and human cancer. Adv. Cancer Res., 76, 61±119.

3. Brown,M.T. and Cooper,J.A. (1996) Regulation, substrates and functionsof src. Biochim. Biophys. Acta, 1287, 121±149.

4. Thomas,S.M. and Brugge,J.S. (1997) Cellular functions regulated by Srcfamily kinases. Annu. Rev. Cell Dev. Biol., 13, 513±609.

5. Bolen,J.B., Veillette,A., Schwartz,A.M., DeSeau,V. and Rosen,N. (1987)Activation of pp60c-src protein kinase activity in human coloncarcinoma. Proc. Natl Acad. Sci. USA, 84, 2251±2255.

6. Cartwright,C.A., Coad,C.A. and Egbert,B.M. (1994) Elevated c-Srctyrosine kinase activity in premalignant epithelia of ulcerative colitis.J. Clin. Invest., 93, 509±515.

7. Cartwright,C.A., Kamps,M.P., Meisler,A.I., Pipas,J.M. and Eckhart,W.(1989) pp60c-src activation in human colon carcinoma. J. Clin. Invest.,83, 2025±2033.

8. Talamonti,M.S., Roh,M.S., Curley,S.A. and Gallick,G.E. (1993) Increasein activity and level of pp60c-src in progressive stages of humancolorectal cancer. J. Clin. Invest., 91, 53±60.

9. Jacobs,C. and Rubsamen,H. (1983) Expression of pp60c-src proteinkinase in adult and fetal human tissue: high activities in some sarcomasand mammary carcinomas. Cancer Res., 43, 1696±1702.

10. Luttrell,D.K., Lee,A., Lansing,T.J., Crosby,R.M., Jung,K.D., Willard,D.,Luther,M., Rodriguez,M., Berman,J. and Gilmer,T.M. (1994)Involvement of pp60c-src with two major signaling pathways in humanbreast cancer. Proc. Natl Acad. Sci. USA, 91, 83±87.

11. Ottenhoff-Kalff,A.E., Rijksen,G., van Beurden,E.A., Hennipman,A.,Michels,A.A. and Staal,G.E. (1992) Characterization of protein tyrosinekinases from human breast cancer: involvement of the c-src oncogeneproduct. Cancer Res., 52, 4773±4778.

12. Verbeek,B.S., Vroom,T.M., Adriaansen-Slot,S.S., Ottenhoff-Kalff,A.E.,Geertzema,J.G., Hennipman,A. and Rijksen,G. (1996) c-Src proteinexpression is increased in human breast cancer. Animmunohistochemical and biochemical analysis. J. Pathol., 180,383±388.

13. Irby,R.B., Mao,W., Coppola,D., Kang,J., Loubeau,J.M., Trudeau,W.,Karl,R., Fujita,D.J., Jove,R. and Yeatman,T.J. (1999) Activating SRCmutation in a subset of advanced human colon cancers. Nature Genet.,21, 187±190.

14. Dehm,S., Senger,M.A. and Bonham,K. (2001) SRC transcriptionalactivation in a subset of human colon cancer cell lines. FEBS Lett., 487,367±371.

15. Bonham,K., Ritchie,S.A., Dehm,S.M., Snyder,K. and Boyd,F.M. (2000)An alternative, human SRC promoter and its regulation by hepaticnuclear factor-1a. J. Biol. Chem., 275, 37604±37611.

16. Ritchie,S., Boyd,F.M., Wong,J. and Bonham,K. (2000) Transcription ofthe human c-Src promoter is dependent on Sp1, a novel pyrimidinebinding factor SPy, and can be inhibited by triplex-formingoligonucleotides. J. Biol. Chem., 275, 847±854.

17. Bomsztyk,K., Van Seuningen,I., Suzuki,H., Denisenko,O. andOstrowski,J. (1997) Diverse molecular interactions of the hnRNP Kprotein. FEBS Lett., 403, 113±115.

18. Guillonneau,F., Guieysse,A.L., Le Caer,J.P., Rossier,J. and Praseuth,D.(2001) Selection and identi®cation of proteins bound to DNA triple-helical structures by combination of 2D-electrophoresis and MALDI-TOF mass spectrometry. Nucleic Acids Res., 29, 2427±2436.

19. Krecic,A.M. and Swanson,M.S. (1999) hnRNP complexes: composition,structure and function. Curr. Opin. Cell Biol., 11, 363±371.

20. Hobert,O., Jallal,B., Schlessinger,J. and Ullrich,A. (1994) Novelsignaling pathway suggested by SH3 domain-mediated p95vav/heterogeneous ribonucleoprotein K interaction. J. Biol. Chem., 269,20225±20228.

21. Van Seuningen,I., Ostrowski,J., Bustelo,X.R., Sleath,P.R. andBomsztyk,K. (1995) The K protein domain that recruits the interleukin 1-responsive K protein kinase lies adjacent to a cluster of c-Src and VavSH3-binding sites. Implications that K protein acts as a docking platform.J. Biol. Chem., 270, 26976±26985.

22. Gorlach,M., Wittekind,M., Beckman,R.A., Mueller,L. and Dreyfuss,G.(1992) Interaction of the RNA-binding domain of the hnRNP C proteinswith RNA. EMBO J., 11, 3289±3295.



23. Michelotti,E.F., Michelotti,G.A., Aronsohn,A.I. and Levens,D. (1996)Heterogeneous nuclear ribonucleoprotein K is a transcription factor. Mol.Cell. Biol., 16, 2350±2360.

24. Tomonaga,T. and Levens,D. (1995) Heterogeneous nuclearribonucleoprotein K is a DNA-binding transactivator. J. Biol. Chem.,270, 4875±4881.

25. Ostareck,D.H., Ostareck-Lederer,A., Wilm,M., Thiele,B.J., Mann,M. andHentze,M.W. (1997) mRNA silencing in erythroid differentiation:hnRNP K and hnRNP E1 regulate 15-lipoxygenase translation from the3¢ end. Cell, 89, 597±606.

26. Kadonaga,J.T. and Tjian,R. (1986) Af®nity puri®cation of sequence-speci®c DNA binding proteins. Proc. Natl Acad. Sci. USA, 83,5889±5893.

27. Hall,C.V., Jacob,P.E., Ringold,G.M. and Lee,F. (1983) Expression andregulation of Escherichia coli lacZ gene fusions in mammalian cells.J. Mol. Appl. Genet., 2, 101±109.

28. Andrews,N.C. and Faller,D.V. (1991) A rapid micropreparationtechnique for extraction of DNA-binding proteins from limiting numbersof mammalian cells. Nucleic Acids Res., 19, 2499.

29. Takimoto,M., Tomonaga,T., Matunis,M., Avigan,M., Krutzsch,H.,Dreyfuss,G. and Levens,D. (1993) Speci®c binding of heterogeneousribonucleoprotein particle protein K to the human c-myc promoter,in vitro. J. Biol. Chem., 268, 18249±18258.

30. Miau,L.H., Chang,C.J., Shen,B.J., Tsai,W.H. and Lee,S.C. (1998)Identi®cation of heterogeneous nuclear ribonucleoprotein K (hnRNP K)as a repressor of C/EBPb-mediated gene activation. J. Biol. Chem., 273,10784±10791.

31. Bouziane,M., Cherny,D.I., Mouscadet,J.F. and Auclair,C. (1996)Alternate strand DNA triple helix-mediated inhibition of HIV-1 U5 longterminal repeat integration in vitro. J. Biol. Chem., 271, 10359±10364.

32. Jensen,O.N., Wilm,M., Shevchenko,A. and Mann,M. (1999) Samplepreparation methods for mass spectrometric peptide mapping directlyfrom 2-DE gels. Methods Mol. Biol., 112, 513±530.

33. Denisenko,O.N., O'Neill,B., Ostrowski,J., Van Seuningen,I. andBomsztyk,K. (1996) Zik1, a transcriptional repressor that interacts with

the heterogeneous nuclear ribonucleoprotein particle K protein. J. Biol.

Chem., 271, 27701±27706.34. Van Seuningen,I., Ostrowski,J. and Bomsztyk,K. (1995) Description of

an IL-1-responsive kinase that phosphorylates the K protein.

Enhancement of phosphorylation by selective DNA and RNA motifs.

Biochemistry, 34, 5644±5650.35. Ostrowski,J., Schullery,D.S., Denisenko,O.N., Higaki,Y., Watts,J.,

Aebersold,R., Stempka,L., Gschwendt,M. and Bomsztyk,K. (2000) Role

of tyrosine phosphorylation in the regulation of the interaction of

heterogenous nuclear ribonucleoprotein K protein with its protein and

RNA partners. J. Biol. Chem., 275, 3619±3628.36. Ostrowski,J., Kawata,Y., Schullery,D.S., Denisenko,O.N., Higaki,Y.,

Abrass,C.K. and Bomsztyk,K. (2001) Insulin alters heterogeneous

nuclear ribonucleoprotein K protein binding to DNA and RNA. Proc.

Natl Acad. Sci. USA, 98, 9044±9049.37. Wadd,S., Bryant,H., Filhol,O., Scott,J.E., Hsieh,T.Y., Everett,R.D. and

Clements,J.B. (1999) The multifunctional herpes simplex virus IE63

protein interacts with heterogeneous ribonucleoprotein K and with casein

kinase 2. J. Biol. Chem., 274, 28991±28998.38. Schullery,D.S., Ostrowski,J., Denisenko,O.N., Stempka,L., Shnyreva,M.,

Suzuki,H., Gschwendt,M. and Bomsztyk,K. (1999) Regulated interaction

of protein kinase Cdelta with the heterogeneous nuclear

ribonucleoprotein K protein. J. Biol. Chem., 274, 15101±15109.39. Ostareck-Lederer,A., Ostareck,D.H., Cans,C., Neubauer,G.,

Bomsztyk,K., Superti-Furga,G. and Hentze,M.W. (2002) c-Src-mediated

phosphorylation of hnRNP K drives translational activation of

speci®cally silenced mRNAs. Mol. Cell. Biol., 22, 4535±4543.40. Tomonaga,T. and Levens,D. (1996) Activating transcription from single

stranded DNA. Proc. Natl Acad. Sci. USA, 93, 5830±5835.41. Du,Q., Melnikova,I.N. and Gardner,P.D. (1998) Differential effects of

heterogeneous nuclear ribonucleoprotein K on Sp1- and Sp3-mediatedtranscriptional activation of a neuronal nicotinic acetylcholine receptor

promoter. J. Biol. Chem., 273, 19877±19883.



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(SPy) as hnRNP K