Src tyrosine kinase phosphorylation of nuclearreceptor HNF4 correlates with isoform-specificloss of HNF4 in human colon cancerKarthikeyani Chellappaa, Lucy Jankovab, Jake M. Schnabla, Songqin Panc, Yann Brelivetd, Caroline L-S. Funge,Charles Chane, Owen F. Dentf, Stephen J. Clarkeg, Graham R. Robertsonb, and Frances M. Sladeka,1

aDepartment of Cell Biology and Neuroscience, University of California, Riverside, CA 92521; bCancer Pharmacology Unit, ANZAC Research Institute,University of Sydney, Sydney, New South Wales, Australia; c W. M. Keck Proteomics Laboratory, Institute for Integrative Genome Biology, University ofCalifornia, Riverside, CA 92521; dDpartement de Biologie et Gnomique Structurales, Institut de Gntique et de Biologie Molculaire et Cellulaire,67404 Illkirch, France; eDepartment of Anatomical Pathology, fDepartment of Colorectal Surgery, Concord Hospital; and gNorthern Clinical School,University of Sydney, Sydney, New South Wales, Australia

Edited by Tony Hunter, The Salk Institute for Biological Studies, La Jolla, CA, and approved December 15, 2011 (received for review April 28, 2011)

Src tyrosine kinase has long been implicated in colon cancer butmuch remains to be learned about its substrates. The nuclear re-ceptor hepatocyte nuclear factor 4 (HNF4) has just recently beenimplicated in colon cancer but its role is poorly defined. Here weshow that c-Src phosphorylates human HNF4 on three tyrosinesin an interdependent and isoform-specific fashion. The initial phos-phorylation site is a Tyr residue (Y14) present in the N-terminal A/Bdomain of P1- but not P2-driven HNF4. Phospho-Y14 interacts withthe Src SH2 domain, leading to the phosphorylation of two addi-tional tyrosines in the ligand binding domain (LBD) in P1-HNF4.Phosphomimetic mutants in the LBD decrease P1-HNF4 proteinstability, nuclear localization and transactivation function. Immuno-histochemical analysis of approximately 450 human colon cancerspecimens (Stage III) reveals that P1-HNF4 is either lost or localizedin the cytoplasm in approximately 80% of tumors, and that stainingfor active Src correlates with those events in a subset of samples.Finally, three SNPs in the human HNF4 protein, two of whichare in the HNF4 F domain that interacts with the Src SH3 domain,increase phosphorylation by Src and decrease HNF4 protein stabi-lity and function, suggesting that individuals with those variantsmay be more susceptible to Src-mediated effects. This newly iden-tified interaction between Src kinase and HNF4 has importantimplications for colon and other cancers.

HNF4 isoforms SH2 SH3 domain SNP Src kinase tyrosinephosphorylation

Colon cancer, the third most common malignancy in the Uni-ted States, is a multifactorial disease that is influenced byboth genetics and the environment (1, 2). c-Src is a nonreceptortyrosine kinase that is strongly implicated in the development,growth, progression, and metastasis of several human cancers (3).In colon cancer, Src activation is associated with the early stages(4, 5) as well as progression and metastasis (68). Despite thislong association with colon cancer, much remains to be learnedabout Src substrates (9).

Hepatocyte nuclear factor 4alpha (HNF4) (NR2A1) is ahighly conserved member of the nuclear receptor superfamilywith a recently identified endogenous ligand (linoleic acid) thatbinds in a reversible fashion (10, 11). HNF4 is best known for itsrole as a master regulator of liver-specific gene expression andas a key player in beta cells of the pancreas where it is mutatedin an inherited form of type 2 diabetes (1214). HNF4 is alsoexpressed in kidney, stomach, and intestine; several recent papersalso show an important role for HNF4 in the colon (1520).There are two different promoters (P1 and P2) of HNF4Athat are utilized in a temporal and tissue-specific fashion (11)(Fig. S1). While only P1-driven HNF4 (P1-HNF4) is expressedin the adult liver, both P1- and P2-driven HNF4 (P2-HNF4)are expressed in the adult intestine and colon (21, 22). Expression

of P1-HNF4 is decreased in several human cancers includinghepatocellular, gastric, renal, and colorectal carcinomas, whilethe expression of P2-HNF4 is either unchanged or upregulated(22, 23). However, the mechanism responsible for the differentialdysregulation of P1- and P2-HNF4 isoforms is not known. Herewe show that Src kinase preferentially phosphorylates P1-HNF4in vitro and in vivo on multiple residues in a complex fashion,resulting in a loss of function and protein stability of P1- but notP2-HNF4. We also show that the phosphorylation is influencedby the SH2 and SH3 domains of Src and by SNPs in HNF4.Finally, we show that increased staining for active Src is asso-ciated with a loss of nuclear P1-HNF4 in a sizeable cohort ofhuman colorectal tumors. These findings suggest a unique linkbetween an oncogenic kinase, a potent differentiation factor andhuman colon cancer.

ResultsSrc Preferentially Phosphorylates P1-HNF4 Both In Vitro and In Vivo.An in vitro kinase assay showed that Src phosphorylates full lengthhuman P1-HNF42 as well as a truncated fragment that corre-sponds to the N-terminal portion [A/B and DNA binding domain(DBD)] (Fig. S2 A and B). Since P1- and P2-HNF4 differ byapproximately 29 amino acids in the A/B domain (Fig. S1C), werepeated the kinase assay and found that Src does not appreciablyphosphorylate P2-HNF48 in vitro (Fig. 1A and Fig. S2G).Cotransfection of HEK293 cells with constitutively active c-Src(c-Src Y530F) and HNF42 also showed in vivo tryosine phos-phorylation of P1-HNF42 using the phospho-Tyr (pY) specificAb 4G10; the signal was greatly reduced in cells pretreated withSrc inhibitor PP1 (Fig. S2H). In contrast, the in vivo tyrosine phos-phorylation signal of HNF48 was also much less than that ofHNF42, even in the presence of the tyrosine phosphatase inhibi-tor pervanadate (PV) (Fig. 1B).

To map Src phosphosites we mutated the two tyrosines in theHNF42 A/B domain to Phe (Y6F and Y14F) and found that onlytheY14Fmutation abolished the in vitro phosphorylation (Fig. S2C).Mass spectrometric analysis also identified a doubly phosphory-lated peptide containing Y277 and Y279 in the ligand bindingdomain (LBD) when P1-HNF42 was ectopically coexpressedwith c-Src Y530F in HEK293 cells (Fig. 1C); P2-HNF48 yielded

Author contributions: K.C., G.R.R., and F.M.S. designed research; K.C., L.J., S.P., and G.R.R.performed research; Y.B. performed computational modeling; K.C., J.M.S., and S.J.C.contributed new reagents/analytic tools; K.C., L.J., S.P., C.L.-S.F., C.C., O.F.D., G.R.R., andF.M.S. analyzed data; and K.C. and F.M.S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. E-mail: frances.sladek@ucr.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1106799109/-/DCSupplemental.

23022307 PNAS February 14, 2012 vol. 109 no. 7 www.pnas.org/cgi/doi/10.1073/pnas.1106799109

6-fold less signal, despite equivalent recovery of the unphosphory-lated peptide (Fig. S2I). AY277/Y279 peptide containing a singlephospho group was also detected; sequencing of the peptide inthree independent experiments indicated that the phosphoryla-tion was on Y277, consistent with Y277 fitting the Src consensussite (DEEIYEEFF) better than Y279 (DNEY277AY279LK) (24).Y14 and Y277 were further validated as Src sites in in vivo and invitro assays using site-directed mutants and an Ab raised againstphospho-Y14 (pY14) (Figs. 1D and Fig. S2D and E). Importantly,the phosphorylation signal is almost completely lost in all Y14Fmutants, confirming that Y14 is critical for subsequent Src-mediated phosphorylation (Fig. 1D and Fig. S2F). Interestingly,a Y279F mutant consistently yielded increased total pYas well aspY14, but only in the presence of PV (Fig. 1D, and Fig. S2J). In-deed, in the absence of PV, the Y279F mutant resulted in reducedtotal pY and pY14 (Fig. S2J), suggesting a dynamic relationshipbetween Y279 and Y14 that may involve a phosphatase.

Phosphomimetic Mutants of P1-HNF4 Negatively Affect ProteinFunction and Stability. Modeling of the HNF4 LBD shows thatY277 flanks a salt bridge between E150 (helix 1) and K281 (helix8) (Fig. 2A, Top) that is part of a communication pathway linkingthe cofactor binding site to the dimer interface (25). In contrast,Y279 is near a hydrophobic cavity at the dimer interface, pointingtowards the interface of helix 9 and helix 1 (Fig. 2A, Bottom).Negatively charged groups at Y277 and Y279 are therefore pre-dicted to adversely affect HNF4 protein dimerization and/ortransactivation. The model also shows that Y277 and Y279 aretoo far apart (15.4 ) for one kinase molecule to phosphorylateboth in a single event (Fig. S3A). This suggests that the phos-phorylation of these residues may occur in a sequential fashion,a notion that is supported by the singly phosphorylated Y277peptide detected by mass spectrometry.

To decipher the role of the three phosphorylated tyrosines,we examined the activity of single and double phosphomimeticmutants of Y14 (Y14E) and Y277/Y279 (Y277E/Y279E), respec-tively, and found that while Y14E had only a marginal effect onHNF42 transactivation (Fig. S3B), the Y277E/Y279E doublemutant completely abolished it (Fig. 2B). Since the DNA binding

activity of Y277E/Y279E was only marginally reduced comparedto that of WT HNF42 (Fig. S3C), we examined the intracellularlocalization of the double mutant and found a substantial cyto-plasmic localization (Fig. 2C, and Fig. S3D) that was reversedin the presence of nuclear export inhibitor leptomycin B (Fig. 2D).However, neither leptomycin B nor increased amounts of expres-sion vector (Fig. 2E), or addition of the potent HNF4 coactiva-tor PGC1 (Fig. 2F), recovered the transactivation function ofY277E/Y279E. Lastly, we examined the protein stability ofY277E/Y279E and found it greatly reduced compared to WTorY277F/Y279F (Fig. 2G and Fig. S3E). Taken together, theseresults indicate that Y277E/Y279E has reduced nuclear localiza-tion and protein stability. It also appears to have a reduced abilityto recruit coactivators and decreased transactivation function,consistent with active Src decreasing the ability of WT HNF42to activate transcription (Fig. 5E, and Fig. S5D and Fig. S6C).

Active Src Decreases P1- But Not P2-HNF4 Protein Stability In Vivo.c-Src Y530F also negatively affected HNF42 protein stability

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Fig. 1. Src preferentially phosphorylates P1-HNF42 on Tyr14, Tyr 277, andTyr279. (A) In vitro Src kinase assay with HNF4 IPd from bacterial lysatecontaining full-length GST-tagged P1- or P2-HNF4 (2 and 8, respectively),recombinant Src kinase and 32P--ATP. Shown is an autoradiograph of a blotfollowing SDS-PAGE. Bottom, IB with HNF4 Ab. (B) In vivo Src kinase assay ofCOS-7 cells cotransfected with HNF42 and c-Src WT or Y530F and treated100 M PV for different time points as indicated. IPd HNF4 was probedwith anti-pY (4G10) or HNF4 Ab. (C) Mass spectrometry (nano-LC/MS/MS)analysis of HNF4 IPd from HEK293 cells cotransfected with c-Src Y530F andHNF42 or HNF48 after a 10-min PV treatment. A peptide containing Y277/Y279 was detected atmz 1471.2, +2, corresponding to a doubly phosphory-lated fragment. The identity of the 1471.2, +2 peptide was verified using asynthetic peptide corresponding to the peptide sequence shown. (D) In vivoSrc kinase assay as in (B) but with WT or Y to F mutants of HNF42 cotrans-fected with c-Src Y530F into HEK293 cells and treated with 100 M PV for10 min prior to lysis. pY14, phospho-specific Ab to pY14 HNF4.

Fig. 2. Tyr277 and Tyr279 are key residues for Src-mediated effects onHNF4. (A) Three-dimensional model of human HNF4 LBD in the region ofY277 and Y279. The two subunits of the homodimer are shown in cyan andgreen. Key residues and helices are indicated. (B) Luciferase activity (relativelight units, RLU) from transiently transfected Cos-7 cells with WT or doublemutants of HNF42 and an HNF4-responsive luciferase construct (ApoB.-85-47.E4.Luc). Data are the means of triplicate samples from one experiment;error bars show s.d. *P < 0.002 WT vs. Y277E/Y279E mutant. (C) Subcellularlocalization of WT and double mutants of Y277 and Y279 in HNF42 ex-pressed in COS-7 cells. Immunolabeled cells (445 Ab) were visualized usinga Zeiss 510 confocal microscope (40) and digitally magnified. (D) Subcellularlocalization as in (C) but after leptomycin B (LMB) or vehicle treatment for8 h of HNF42 Y277E/ Y279E mutant. Immunolabeled cells (445 Ab) werevisualized with a Nikon Eclipse Ti inverted microscope (20) and digitallymagnified. (E) Fold difference (normalized RLU in the presence or absenceof HNF42) of COS-7 cells transfected with WT or Y277E/Y279E mutant ofHNF42 and ApoAI-4.Luc reporter treated with LMB or vehicle for 9 h. Dataare the means of triplicate samples from one experiment; error bars shows.d. *P < 0.05 vehicle vs. LMB for WT, n.s., no significant change. (F) Folddifference (normalized RLU in the presence or absence of PGC1) of WT orY277E/Y279E mutant of HNF42 cotransfected in HEK293 cells with ApoAI-4.Luc reporter and CMV.-gal. *P < 0.0005 fold difference in mock-trans-fected vs. WT HNF42. (G) Quantification of protein stability of WT anddouble mutants of Y277 and Y279 in HNF42 expressed in Cos-7 cells in thepresence of 50 MCHX at the indicated time points. Experiments in BGwereperformed two to three times with similar results.

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in an HEK293 cotransfection assay, while Src inhibitor PP2increased HNF42 protein levels (Fig. 3A and Fig. S4A, Right).Proteasome inhibitors (MG132 and ALLN) (Fig. 4A, Right andFig. S4A, Left) substantially increased HNF42 protein levels, butlysosomal inhibitors (chloroquine and ammonium chloride) hadonly a marginal effect (Fig. S4A), suggesting that Src decreasesHNF42 protein stability mainly via the proteasome pathway. Toexamine the effect of endogenous Src activation on endogenousHNF4, we treated human colorectal cancer cells CaCo2, whichexpress both P1- and P2-HNF4 isoforms, with EGF to induceSrc activity (Figs. S4 B and C). The results show a loss ofP1- but not P2-HNF4 upon EGF treatment at later time points(Figs. 3B and Fig. S4D). We also show that endogenous P1- butnot P2-HNF4 is phosphorylated in CaCo2 cells by both IP/IB(Fig. 3C) and mass spectrometry; the latter showed that as muchas 40% of the P1-HNF4 was doubly phosphorylated at Y277/Y279 in CaCo2 cells (Fig. 3D). Finally, a 24-h treatment ofCaCo2 cells with Src inhibitor PP2 significantly decreased endo-genous P1-HNF4 phosphorylation (Fig. 3E) and increased thelevel of P1-HNF4 protein (Fig. 3F). In contrast, there was amarked decrease in P2-HNF4 protein upon PP2 treatment,which could be due to the fact that P1-HNF4 is known to de-crease transcription of the P2 promoter (21). All told, selectivephosphorylation of endogenous P1-HNF4 in a colon cancer cellline correlates with decreased protein stability and is consistentwith the experiments using ectopic expression and phosphomi-metic mutants.

Src SH2 and SH3 Domains Interact with P1-HNF4. Since Src SH2 andSH3 domains (bind pYand proline-rich regions, respectively) areknown to play a role in regulating Src activity and processivephosphorylation of its substrates, we examined their ability tointeract with HNF4. Not only did WT HNF42 phosphorylatedby active Src bind the Src SH2 domain in vitro, but Y14 wasrequired for maximal interaction (Fig. 4A, and Fig. S5B). In con-trast, P2-HNF48 did not bind Src SH2 appreciably (Fig. 4B). Toverify the specificity of the interaction, we showed that the SrcSH2 mutant R178A does not interact with phospho-HNF42(Fig. S5B), and that the pY14 peptide binds directly to the WTSrc SH2 domain, but not the R178A mutant (Fig. 4C). We alsofound that the pY14 but not the nonphosphorylated (Y14) pep-

tide can effectively compete with phospho-HNF42 for bindingthe Src SH2 domain (Fig. 4D). Finally, using an in vivo kinaseassay, we show that a WT Src SH2 domain is required for totaltyrosine phosphorylation of HNF42 but is less important forY14 phosphorylation (Fig. 4E). These results are consistent withthe notion that phosphorylation of Y14 is the first event and thatthe Src SH2 domain subsequently binds pY14. The results are

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Fig. 3. Active Src decreases P1- but not P2-HNF4 protein stability; tyrosine phosphorylation of endogenous P1-HNF4. (A) Left, Protein stability ofWT HNF42expressed in HEK293 cells in the presence or absence of c-Src Y530F. Graph represents HNF4 protein level normalized to Coomassie stain. Data are means oftriplicates from one of at least 10 representative experiments; error bars show s.e.m. *P < 0.05.Right top, Right, IB of HNF42 and -actin from whole cellextracts of HEK293 treated with either DMSO or 50 M MG132 for 8 h. Shown are representative blots from three independent experiments. (B) Proteinstability of P1- and P2-HNF4 in CaCo2 cells harvested at the indicated time points after EGF + CHX treatment. Shown is one representative quantificationof EGF-treated versus untreated controls from five independent experiments. (C) In vivo phosphorylation of endogenous HNF4 from CaCo2 cells. Top, IPdphospho-Tyr proteins (4G10) were IBd with either P1- or P2-HNF4 Abs. Bottom, IPd HNF4 (445) was probed with pY14 or P1-HNF4 Ab. (D) Mass spectro-metry analysis as in Fig. 1C but of P1-HNF4 IPd (N1.14 Ab) from CaCo2 cells. Signals corresponding to the nonphosphorylated Y277/Y279 (mz 1391.2, +2) andpY277pY279 (mz 1471.2, +2) peptides were detected. Ratio of the amount of pY277/pY279 to Y277/Y279 is indicated. (E) As in (C, Top) but from CaCo2 cellstreated with ethanol or 10 MPP2 for different time points. Values corresponding to normalized phospho P1-HNF4 (IPd P1-HNF4 divided by input P1-HNF4)are shown. (F) IB of P1-HNF4 and P2-HNF4 from whole cell extracts of CaCo2 cells after 24 h or 48 h treatment with 10 M PP2 or DMSO. Shown are triplicatesfrom one of three independent experiments.

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Fig. 4. Src SH2 domain interacts with pY14 and is critical for phosphorylationof Y277 and Y279. (A) Pulldown assay with GST or GST.Src-SH2 proteins andwhole cell extracts from HEK293 cells cotransfected with c-Src Y530F andWT or the indicated Y to F mutants of HNF42. IBs with 445 show theamount of HNF4 in the Input and after the Pulldown. (B) As in (A) but withhuman HNF42 and HNF48. (C) Pulldown assays as in (A) but with WT orR178A mutants of Src SH2 and 5 g of pY14 peptide (Fig. S2D). Shown is anIB of bound peptide after Pulldown spotted onto PVDF membrane and de-tected by an Ab that recognizes the pY14 peptide. (D) Pulldown as in (A)but the interaction between WT HNF42 and GST.Src-SH2 was competedwith either the pY14 or the Y14 peptide. Shown is the IB with the HNF4antibody from one representative experiment and quantification of boundHNF4 relative to no peptide from 5 and 2 independent experiments forpY14 and Y14 competition, respectively. Error bar represents s.e.m. *P < 0.01relative to no peptide competition. (E) In vivo Src kinase assay in HEK293 cellscotransfected in 100-mm plates with HNF42WT (5 g) and either c-Src Y530For Src SH2 mutant (c-Src R178A/Y530F) at different ratios of transfectedDNA (5 g to 0.5 g). IPd HNF4was probed with 4G10, pY14 or P1/P2 HNF4Ab. Indicated is the fold difference in the phosphorylation signal with theSrc SH2 mutant relative to c-Src Y530F at each ratio. Fold difference forthe 10.2 ratio was calculated using a darker exposure of the 4G10 blot. NA,not applicable as no signal was observed.

2304 www.pnas.org/cgi/doi/10.1073/pnas.1106799109 Chellappa et al.

also of interest since Y14 (PApY14TTL) only partially fits theknown consensus for a Src kinase substrate (pY(E/D/T)(E/N/Y)(L/I) ) and SH2 domain ligand [(P/H)(I/P)pY(E/D/V/I)(L/I/E)(I/L/V)] (24). Finally, to show that pY14 is required for phosphor-ylation of the LBD, we examined an N-terminally truncatedHNF42 construct (AB) and found that it was neither phos-phorylated in vivo by active Src (Fig. S6A) nor greatly repressedin transactivation function by Src compared to WT HNF42(Fig. S6C). These results confirm that the P1-driven A/B domainis actively required for the phosphorylation of the LBD.

Since the F domain of HNF4 is proline rich, we predicted andobserved an interaction between WT HNF42 and the Src SH3domain in a GST pulldown assay (Fig. 5A). Interestingly, like theSH2 mutant, an SH3 mutant W121A decreased total HNF42phosphorylation but had little effect on pY14 (Fig. 5B). Theseresults suggest that the Src SH3 domain, like the SH2 domain,is not required for Y14 phosphorylation but that it is required forphosphorylation of the LBD. Consistent with this notion, massspectrometry did not show any phosphorylation on Y277 whenHNF42 was cotransfected with the Src SH3 mutant (Fig. S5C).Finally, a luciferase assay showed that while both the WT andc-Src Y530F decreased HNF42 transcriptional activity, the SH3mutant W121A and, to a lesser extent the SH2 mutant R178A,exhibited less repression (Fig. S5D). Taken together, these resultsshow that HNF42 binds both the Src SH2 and SH3 domains andthat those interactions play an important role in the isoform-specific phosphorylation of HNF4.

SNPs in HNF4 Affect Src Phosphorylation and Protein Stability.Thereare three SNPs in human HNF4 that might be relevant tothe interaction with Src (dbSNP, http://www.ncbi.nlm.nih.gov/projects/SNP/). Introduction of mutations corresponding totwo SNPs in the F domain (P421L, rs6031602, and P436S,rs1063239) dramatically decreased binding to the Src SH3 do-

main (Fig. 5A). Rather unexpectedly, in the presence of cotrans-fected active Src, both variants (P421L and P436S) yielded agreater pY14 signal compared to WT HNF42 (Fig. 5C), espe-cially P421L. Another SNP adjacent to Y279 (L280F, rs6093980)also yielded a greater pY14 signal (Fig. 5C). Importantly, all threeSNPs (L280F, P421L, P436S) exhibited decreased protein stabi-lity compared to WT HNF42, but only in the presence of activeSrc (Fig. 5D and Fig. S6D). Finally, two of the SNP variants(L280F and P421L) exhibited lower levels of transactivation thanWT HNF42 in the absence of active Src, while the third variantP436S activated transcription as well as WT (Fig. 5E). Impor-tantly, all three variants were more susceptible to Src-mediatedrepression of transactivation than WT HNF42, even after nor-malizing to the amount of HNF4 protein (Fig. 5E). This in-creased sensitivity of the SNP variants to Src is consistent with theincreased phosphorylation observed in Fig. 5C.

The results thus far show that the proline-rich F domain ofWT HNF42 interacts with the Src SH3 domain and that this in-teraction is required for full phosphorylation. However, the SNPvariants show that the WT F domain may inhibit Src-mediatedphosphorylation and repression of HNF42 transactivation. Toaddress this apparent discrepancy, we examined the in vivophosphorylation of HNF42 truncated in the F domain (F). Theresults show that HNF4 F is indeed phosphorylated by Src(Fig. S6A). In addition, mass spectrometry of cotransfectedHNF4 and active Src showed that the amount of the singly phos-phorylated pY277 peptide (1431.2) was greater in F than in WTHNF42 (41% vs 3.6%, respectively) (Fig. S6B). Consistent withthe notion that the F domain inhibits Src-mediated phosphoryla-tion of HNF42, the F truncation also exhibited a greater re-duction in transactivation in the presence of active Src than didWTHNF42 (Fig. S6C). Therefore, the F domain appears to playa modulatory role in Src-mediated effects on HNF42 via bindingthe SH3 domain.

Preferential Loss of Nuclear P1-HNF4 Correlates with Active SrcStaining in Human Colon Cancer. In a pilot study of tumors fromhuman patients with colon cancer, we found that only 26 out of43 tumors were positive for a P1-HNF4 specific Ab (P1) whileall 43 tumors showed staining with the Ab that recognizes bothP1- and P2-HNF4 (P1/P2) (Fig. S7 A and B). This preferentialloss of P1-HNF4 is consistent with previous results on a similar-sized cohort of patients (22, 23). Screening a larger cohort of 450consecutive Stage III colon cancer cases revealed four categoriesof tumors when intensity of P1 staining was consideredloss ofall P1 staining (a, approximately 12%); predominantly cytoplas-mic P1 staining (b, approximately 1824%); nuclear and cytoplas-mic P1 staining (c, approximately 4749%); and only nuclearP1 staining (d, approximately 1823%) (Figs. 6 AB, Fig. S7 Cand D). All told, approximately 80% of tumor samples showeda P1-HNF4 staining that was not exclusively nuclear; in contrast,in normal tissue HNF4 staining was observed only in the nucleus(Figs. S7E and S8B).

To assess the state of Src activation in the tumors, a subset ofthe samples (98 tissue microarray cores from invasive front andcentral regions of tumors) were stained with anti-phospho-Srcantibody (pTyr 419, clone 9A6). The tumors showed a consistentstaining for pSrc in the nucleus, especially when P1-HNF4 waseither completely cytoplasmic or absent (Fig. 6C, sections I, II). Incontrast, when there was appreciable nuclear P1-HNF4 in thetumor area, there was low pSrc staining (sections III, IV). Further-more, the adjacent normal tissue, which had essentially exclusivenuclear P1-HNF4 in nearly all the cells, showed little evidenceof pSrc staining (Fig. S8C). All told, the majority of the samples(Fig. S8C, categories 1 + 2, 61 out of 98 tumor cores; X2 7.772,p 0.005) were consistent with the notion that activation of Srcmay result in cytoplasmic localization and/or complete loss ofP1-HNF4. Here we report that cytoplasmic P1-HNF4 staining

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Fig. 5. Src SH3 domain interacts with proline-rich F domain of HNF4; SNPsin HNF42 LBD and F domain affect Src-mediated phosphorylation. (A) Pull-down assays with GST.Src-SH3 protein and whole cell extract from HEK293cells transfected with WTor the indicated SNP variants of HNF42. (B) In vivoSrc kinase assay of HEK293 cells cotransfected with HNF42 and either c-SrcY530F or Src SH3 mutant (c-Src W121A/Y530F). IPd HNF4 was probed with4G10, pY14, or P1/P2 HNF4 Ab. (C) In vivo Src kinase assay as in Fig. 1B butwith HNF42WTor the indicated SNP variants cotransfected with c-Src Y530Finto HEK293 cells. Right, quantification of the pY14 signal relative to the to-tal HNF4 signal from one of three representative experiments. (D) Proteinstability of WT and SNP variants of HNF42 cotransfected with c-Src Y530Finto HEK293 cells. Shown is the average of triplicates from one of two inde-pendent experiments of the HNF4 IB signal normalized to Coomassie stain.*P < 0.02 SNP variants vs. WT. (E) Transcriptional activity ofWTor SNP variantsof HNF42 either in the presence or absence of c-Src Y530F in HEK293 cellscotransfected with pZL.HIV.LTR.AI-4 reporter and CMV.gal. Shown is theRLU normalized to HNF4 protein and -gal activity for WT (Left) and % ofWT (set to 100%) for SNP variants (Right). Data are the mean s:e:m: oftwo independent experiments performed in triplicates. *P < 0.05 for SNPvariants in the presence vs. the absence of c-Src Y530F.

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in human colon cancer correlates with staining for active Src.While active Src is typically found in the cytoplasm and is teth-ered to the plasma membrane via a fatty acid anchor, there is aprevious report of nuclear localization of pSrc in human coloncancer (26). We have also observed nuclear pSrc in CaCo2 cells,as well as in three other human colon cancer cell lines (RKO,SW620, SW480) (Fig. S8D).

DiscussionIn this study, we identify HNF4 as a new target of Src kinase andin the process elucidate a complex interaction that has importantramifications for human colon cancer. We find that Src firstphosphorylates Y14 in the N-terminal A/B domain of P1-HNF4(Fig. 6D, Step 1). Subsequently, pY14 binds the SH2 domain ofSrc and the proline-rich F domain of HNF4 binds the Src SH3domain, thereby facilitating the phosphorylation of first Y277 andthen Y279 in the HNF4 LBD (Step 2). We propose that thistethering of the Src protein brings its kinase domain in closeproximity to the Y277/279 residues that are normally not veryaccessible. Since we have shown previously that the HNF4 Fdomain functions as a transcriptional repressor in part by inter-acting with the LBD (27), we also propose that by binding the Fdomain, the Src SH3 domain disrupts the interaction with theLBD, thereby making Y277 and Y279 more accessible for phos-phorylation. The net result is a triply phosphorylated HNF4(Step 3) in which pY277 and pY279 evidently disrupt the coacti-vator binding site, resulting in a loss of transactivation, nuclearlocalization, and protein stability. We also show that three SNPsin HNF4 (L280F, P421L, and P436S) increase Src-mediatedphosphorylation, degradation of HNF4 protein and repressionof transcription. Therefore, we propose that while the SNP var-iants in the F domain disrupt the interaction with the Src SH3domain, they also abrogate the interaction of the F domain withthe LBD, thereby at least partially exposing Y277 and Y279(Fig. 6E). What is not clear, however, is exactly how the SNPslead to increased phosphorylation of Y14. Importantly, this entiresequence of events happens only on P1-HNF4 as P2-HNF4does not contain Y14. Since P2-HNF48 has the same F domainas P1-HNF42, this (and other experiments presented in thiswork) indicates that the binding of the Src SH3 domain to HNF4alone is not sufficient to cause substantial phosphorylation of

HNF4. As we observed in the human colon cancer samples, thenet result is a preferential loss of P1- but not P2-HNF4 underconditions that activate c-Src, or a Src kinase family member(the pSrc antibody recognizes the pY419 of several different Srcfamily kinase members). Furthermore, there appears to be anegative effect of Src on HNF4-mediated transactivation, evenwhen reduced HNF4 protein levels are taken into account. Thissuggests that any phospho-HNF4 protein remaining in thenucleus may also have reduced transcriptional activity.

While a few other nuclear receptors are known to be phos-phorylated by Src kinase (2830), here we report the isoform-specific phosphorylation of a nuclear receptor by Src kinase. Wealso provide evidence suggesting that SNP variants in a nuclearreceptor may increase ones susceptibility to c-Src, and hence toSrc-mediated cancer. Finally, the interaction between Src kinaseand HNF4 described here may also be relevant in other cancers,such as hepatocellular carcinoma, which has been shown to havehigh levels of active Src (31) and a loss of P1-driven HNF4 (22).

Materials and MethodsSrc Kinase Assays. In vitro kinase assays were performed as per the manufac-turers protocol (Millipore) using recombinant active Src (2550 ng), 32P--ATPand purified GST fusion proteins or human HNF4 [wild type (WT) or mutant]immunoprecipitated (IPd) from transfected HEK293 cells. In vivo kinaseassays used HEK293 or COS-7 cells transfected with HNF4 and c-Src Y530Ftreated with 100 M PV for 330 min before lysing with RIPA buffer asindicated. HNF4 was IPd with the affinity purified Ab 445 and then elutedwith 445 peptide. In vivo phosphorylation assays in CaCo2 cells were per-formed with cells at

each tissue core was assessed independently by L.J. and two experiencedpathologists (C.L-S.F., C.C.) for staining intensity (0 absent, 1 weak,2 intermediate, 3 strong staining) using P1-specific and P1/P2 antibodiesas previously described (34). pSrc (pTyr419) immunoreactivity was similarlydetermined. Approval for this study incorporating informed consent wasobtained from the South Western Sydney Area Health Service.

ACKNOWLEDGMENTS. We thank P. Chapuis and L. Bokey for establishing thetumor bank at Concord Hospital; C. Clarke for TMA construction; D. Morasand B. Fang HNF4 LBD and SH2 structure analysis; and C. Wu for GST.Src.SH3.The clinical component was supported by a Cancer Institute New SouthWalesTranslational Program Grant for Colorectal Cancer. All other work wasfunded by an National Institutes of Health R01 grant to F.M.S. (DK053892).

1. Walther A, et al. (2009) Genetic prognostic and predictive markers in colorectal cancer.Nat Rev Cancer 9:489499.

2. ACS (2008) Colorectal Cancer Facts & Figures 2008-2010 (American Cancer Society,Atlanta).

3. Yeatman TJ (2004) A renaissance for SRC. Nat Rev Cancer 4:470480.4. Iravani S, et al. (1998) Elevated c-Src protein expression is an early event in colonic

neoplasia. Lab Invest 78:365371.5. Cartwright CA, Meisler AI, Eckhart W (1990) Activation of the pp60c-src protein kinase

is an early event in colonic carcinogenesis. Proc Natl Acad Sci USA 87:558562.6. Summy JM, Gallick GE (2003) Src family kinases in tumor progression and metastasis.

Cancer Metastasis Rev 22:337358.7. Talamonti MS, Roh MS, Curley SA, Gallick GE (1993) Increase in activity and level of

pp60c-src in progressive stages of human colorectal cancer. J Clin Invest 91:5360.8. Termuhlen PM, Curley SA, Talamonti MS, Saboorian MH, Gallick GE (1993) Site-specific

differences in pp60c-src activity in human colorectal metastases. J Surg Res 54:293298.9. Amanchy R, et al. (2008) Identification of c-Src tyrosine kinase substrates using mass

spectrometry and peptide microarrays. J Proteome Res 7:39003910.10. Yuan X, et al. (2009) Identification of an endogenous ligand bound to a native orphan

nuclear receptor. PLoS One 4:e5609.11. Bolotin E, Schnabl J, Sladek F (2009) HNF4A (Homo sapiens)., Transcription Factor

Encyclopedia, http://www.cisreg.ca/cgi-bin/tfe/home.pl.12. Bolotin E, et al. (2010) Integrated approach for the identification of human hepato-

cyte nuclear factor 4alpha target genes using protein bindingmicroarrays.Hepatology51:642653.

13. Odom DT, et al. (2004) Control of pancreas and liver gene expression by HNF transcrip-tion factors. Science 303:13781381.

14. Gupta RK, Kaestner KH (2004) HNF-4alpha: from MODY to late-onset type 2 diabetes.Trends Mol Med 10:521524.

15. Garrison WD, et al. (2006) Hepatocyte nuclear factor 4alpha is essential for embryonicdevelopment of the mouse colon. Gastroenterology 130:12071220.

16. Darsigny M, et al. (2009) Loss of hepatocyte-nuclear-factor-4alpha affects colonic iontransport and causes chronic inflammation resembling inflammatory bowel disease inmice. PLoS One 4:e7609.

17. Babeu JP, Darsigny M, Lussier CR, Boudreau F (2009) Hepatocyte nuclear factor 4alphacontributes to an intestinal epithelial phenotype in vitro and plays a partial role inmouse intestinal epithelium differentiation. Am J Physiol Gastrointest Liver Physiol297:G124134.

18. Ahn SH, et al. (2008) Hepatocyte nuclear factor 4alpha in the intestinal epithelial cellsprotects against inflammatory bowel disease. Inflamm Bowel Dis 14:908920.

19. Darsigny M, et al. (2010) Hepatocyte nuclear factor-4alpha promotes gut neoplasia inmice and protects against the production of reactive oxygen species. Cancer Res70:94239433.

20. Cattin AL, et al. (2009) Hepatocyte nuclear factor 4alpha, a key factor for homeostasis,cell architecture, and barrier function of the adult intestinal epithelium.Mol Cell Biol29:62946308.

21. Briancon N, et al. (2004) Expression of the alpha7 isoform of hepatocyte nuclear factor(HNF) 4 is activated by HNF6/OC-2 and HNF1 and repressed by HNF4alpha1 in the liver.J Biol Chem 279:3339833408.

22. Tanaka T, et al. (2006) Dysregulated expression of P1 and P2 promoter-drivenhepatocyte nuclear factor-4alpha in the pathogenesis of human cancer. J Pathol208:662672.

23. Oshima T, et al. (2007) Downregulated P1 promoter-driven hepatocyte nuclear factor-4alpha expression in human colorectal carcinoma is a new prognostic factor againstliver metastasis. Pathol Int 57:8290.

24. Huang H, et al. (2008) Defining the specificity space of the human SRC homology 2domain. Mol Cell Proteomics 7:768784.

25. Brelivet Y, Kammerer S, Rochel N, Poch O, Moras D (2004) Signature of the oligomericbehaviour of nuclear receptors at the sequence and structural level. EMBO Rep5:423429.

26. Sakai T, Kawakatsu H, Fujita M, Yano J, OwadaMK (1998) An epitope localized in c-Srcnegative regulatory domain is a potential marker in early stage of colonic neoplasms.Lab Invest 78:219225.

27. Sladek FM, Ruse MD, Jr, Nepomuceno L, Huang SM, Stallcup MR (1999) Modulationof transcriptional activation and coactivator interaction by a splicing variation inthe F domain of nuclear receptor hepatocyte nuclear factor 4alpha1. Mol Cell Biol19:65096522.

28. Kim H, Laing M, Muller W (2005) c-Src-null mice exhibit defects in normal mammarygland development and ERalpha signaling. Oncogene 24:56295636.

29. Buitrago C, Vazquez G, De Boland AR, Boland RL (2000) Activation of Src kinase inskeletal muscle cells by 1, 1,25-(OH(2))-vitamin D(3) correlates with tyrosine phos-phorylation of the vitamin D receptor (VDR) and VDR-Src interaction. J Cell Biochem79:274281.

30. Mahajan NP, et al. (2007) Activated Cdc42-associated kinase Ack1 promotes prostatecancer progression via androgen receptor tyrosine phosphorylation. Proc Natl Acad SciUSA 104:84388443.

31. Masaki T, et al. (1998) pp60c-src activation in hepatocellular carcinoma of humans andLEC rats. Hepatology 27:12571264.

32. Davis NC, Newland RC (1983) The reporting of colorectal cancer: The AustralianClinico-pathological Staging (ACPS) System. Med J Aust 1:282.

33. Fielding LP, et al. (1991) Clinicopathological staging for colorectal cancer: An Interna-tional Documentation System (IDS) and an International Comprehensive AnatomicalTerminology (ICAT). J Gastroenterol Hepatol 6:325344.

34. Kishimoto T, et al. (2008) A case of alpha-fetoprotein-producing pulmonary carcinomawith restricted expression of hepatocyte nuclear factor-4alpha in hepatoid foci: A casereport with studies of previous cases. Hum Pathol 39:11151120.

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