LIP1, a cytoplasmic protein functionally linked to the Peutz-Jeghers syndrome kinase LKB1Darrin P. Smith1, Sydonia I. Rayter1, Christiane Niederlander2, James Spicer1, C. Mike Jones2 and Alan Ashworth1,2,*
1The Breakthrough Toby Robins Breast Cancer Research Centre and 2Section of Gene Function and Regulation, Chester Beatty Laboratories, Institute of Cancer Research, Fulham Road, London SW3 6JB, UK
Received July 20, 2001; Revised and Accepted October 8, 2001
LKB1 is a serine/threonine kinase which is inactivatedby mutation in the Peutz-Jeghers polyposis andcancer predisposition syndrome (PJS). We haveidentified a novel leucine-rich repeat containingprotein, LIP1, that interacts with LKB1. The LIP1 geneconsists of 25 exons, maps to human chromosome2q36 and encodes a protein of 121 kDa. LIP1 appearsto be a cytoplasmically located protein whereas weand others have shown previously that LKB1 ispredominantly nuclear, with only a small proportionof cells showing strong cytoplasmic expression.However, when LKB1 and LIP1 are co-expressed, theproportion of cytoplasmic LKB1 dramaticallyincreases, suggesting that LIP1 may regulate LKB1function by controlling its subcellular localization.Ectopic expression of both LKB1 and LIP1 inXenopus embryos induces a secondary body axis,providing further evidence for a functional linkbetween the two proteins. This phenotype resemblesthe effects of ectopic expression of TGFβ super-family members and their downstream effectors. Apossible role for LIP1 and LKB1 in TGFβ signalling issupported by the observation that LIP1 interacts withthe TGFβ-regulated transcription factor SMAD4,forming a LKB1–LIP1–SMAD4 ternary complex.SMAD4 mutations give rise to juvenile polyposissyndrome, which is clinically similar to PJS. Our datasuggest an unsuspected mechanistic link betweenthese two syndromes.
INTRODUCTION
Peutz-Jeghers syndrome (PJS) is a dominantly inherited conditioncharacterized by multiple gastrointestinal hamartomatouspolyps and mucocutaneous pigmented spots on the lips, digitsand buccal mucosa (1,2). Patients with PJS are at least 10 timesmore likely to develop cancer than the general population (3).Malignant neoplasms may occur in a variety of tissuesincluding colon (possibly by malignant transformation of thehamartomas), small intestine, breast, cervix, ovary andpancreas. The gene for PJS was mapped to chromosome 19p13
by linkage analysis and comparative genomic hybridization.Loss of the presumptive wild-type allele in the epithelialcomponent of the gastrointestinal hamartomas suggested thatthe PJS gene was a tumour suppressor and that there may be ahamartoma–adenoma–carcinoma sequence in neoplastictransformation (4). Multiple independent mutations werefound in the gene LKB1 (STK11) in affected members of PJSfamilies (5,6). LKB1 is only rarely mutated in the sporadiccounterpart of the tumours found in PJS (7). However,epigenetic inactivation of LKB1 may be important, particularlyin the papillary subtype of breast carcinoma associated withPJS (8).
LKB1 encodes a serine/threonine kinase and is the humanorthologue of the Xenopus gene XEEK1 (9). LKB1 hasautocatalytic kinase activity and PJS mutations have beenshown to cause loss or severe abrogation of the autokinaseactivity (10,11). Expression of exogenous wild-type LKB1,but not LKB1 with PJS-associated mutations, in some LKB1deficient cancer cell lines causes growth suppression byblocking the cell-cycle at the G1/S transition (12) supportingits proposed role as a tumour suppressor gene. Exogenoushuman LKB1 and mouse Lkb1 are predominantly nuclearproteins in transfected cells but a significant minority of cellsshow strong cytoplasmic expression (12,13 and unpublisheddata). A nuclear localization signal of the single basic type islocated towards the N-terminus of the protein (13). LKB1 has aputative CAAX box prenylation signal at the C-terminus, andtruncated LKB1 may be prenylated and localized to the plasmamembrane (14); however, membrane localization of full-length LKB1 has not been reported. As yet, no substrates ofLKB1 have been identified and nothing is known about how itsactivity is regulated.
In an attempt to find substrates or regulators of LKB1 wescreened a yeast-two-hybrid library with Lkb1 as bait. AnLKB1 interacting protein, LIP1, was identified. We show thatthis protein may be functionally linked to LKB1 as it alters thesubcellular location of Lkb1 when co-expressed, and bothLkb1 and LIP1 cause axis duplication when ectopicallyexpressed in Xenopus embryos. Axis duplication suggests arole for LKB1 and LIP1 in TGFβ signalling which is furthersupported by the interaction of LIP1 with the TGFβ-regulatedtranscription factor SMAD4.
*To whom correspondence should be addressed. Tel: +44 20 7970 6058; Fax: +44 20 7878 3858; Email [email protected]
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RESULTS
LIP1 encodes a novel Lkb1-interacting protein containing six leucine-rich repeat motifs
Yeast-two-hybrid screening of a mouse embryonic cDNAlibrary (15) with mouse Lkb1 (13) (note we have used LKB1 toindicate the human protein and Lkb1 the mouse orthologue)and an autokinase inactive variant (D194A) resulted in theisolation of the same cDNA clone (Y2H-21). The interactionbetween Lkb1 and Y2H-21 was specific as Y2H-21 failed tointeract with a range of control proteins, and required the C-terminal159 residues of Lkb1. Although Y2H-21 was not identical to knowngenes in GenBank, various multiple human ESTs were present inthe sequence database, including those contained in the UniGenecluster Hs.22410 of 71 ESTs (http://www.ncbi.nlm.nih.gov/UniGene). Sequencing of members of this EST cluster resultedin the identification of EST AA134795 which was found tocontain a complete ORF with an in frame stop codon N-terminalto the first Met codon, a polyA addition signal and polyA tail.We have designated the gene encoded by this clone LIP1 (forLkb1 interacting protein 1; GenBank accession no. AF450267);
the amino acid sequence of the protein encoded by LIP1 isshown in Figure 1 aligned with the ORF of the mouse Y2H-21clone.
LIP1 consists of 1099 amino acid residues with a predictedmolecular weight of 121.4 kDa. LIP1 is a widely or ubiquitouslyexpressed gene as human ESTs have been isolated from manytissues including colon, testis, ovary, breast, cervix, aorta,brain, kidney, lung, uterus, oesophagus, placenta, skin andpancreas. Furthermore, the sequence of the probable mouseLIP1 orthologue has recently been reported (16) (GenBankaccession nos AK004757 and BAB23538). Mouse Lip1 is99.4% identical to the ORF in the Y2H-21 clone in the regionof overlap, and 74% identical to human LIP1. cDNA micro-array analysis of 47 adult, neonatal and embryonic tissues (17)(http://www.genome.gsc.riken.go.jp/READ/) shows that Lip1is expressed in all tissues examined, with expression levels inadult tissues varying only 3.4-fold between the highest andlowest expressing tissues.
The LIP1 gene (within BAC clone AC009955) consists of25 exons and at least two alternatively spliced transcripts areexpressed (Fig. 1). One, represented by EST AI149114,contains a 46 amino acid insert and the other (EST
Figure 1. Structure of the LKB1 interacting protein LIP1. (A) The amino acid sequences of human LIP1 and the mouse Y2H-21 clone isolated from the yeast-two-hybridscreen are shown aligned. Vertical lines indicate identical residues, dots conserved residues and dashes are gaps introduced into the sequence for maximum alignment. Thesix LRRs are in bold and underlined, the glutamic acid-rich region is in italics and underlined, and the sequences and positions of alternately spliced exons areindicated. (B) Alignment of the LIP1 LRRs. Identical residues are shaded in black and conserved residues in grey. (C) Location of LIP1 on human chromosome 2.Other genes mapped to the same genetic interval are indicated in the right column. (D) Exon structure of LIP1 cDNA. Met is the initiating methionine codon andTer the termination codon. (E) Schematic alignment of LIP1 and the related protein Nischarin (NIS) indicating the percentage amino acid identity between variousregions of the proteins.
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AW957418) encodes an alternate C-terminus. Radiationhybrid mapping (18) (data not shown) localized LIP1 to thehuman chromosome 2 reference interval D2S164 to D2S163(http://www.ncbi.nlm.nih.gov/genemap99) and SCL4A3 whichis next to LIP1 and has been mapped to human 2q36 (19).
Although the amino acid sequence of LIP1 provides fewclues to its possible function, there are six tandem leucine-richrepeats (LRRs) towards the N-terminus (Fig. 1). LRRs are aconsensus motif involved in protein–protein interactions thatusually occur in tandem copies and are characterized byleucines at invariant positions (20). The LRRs of LIP1 are notclosely related to the LRRs of other known proteins except forthe presence of the core leucine residues. Other notablefeatures of the LIP1 amino acid sequence are a highly glutamicacid-rich region (30 of 63 residues; Fig. 1), a possible leucinezipper (fitting the consensus L(X)6L(X)6L(X)6L where X isany residue; Fig. 1E), and a proline (9.0%) and leucine (16.1%)rich character. The mammalian proteins for which a functionhas been proposed most closely related to LIP1 are the humanprotein IRAS-1 (suggested to be an imidazoline receptor) (21)and its probable mouse orthologue Nischarin, which isbelieved to be involved in the control of cell migration (22).
Nischarin (Fig. 1) and IRAS-1 share low overall sequenceidentity with LIP1, but have a similar structure with six tandemLRRs towards the N-terminus followed by a glutamic acid-richregion. They are also similarly rich in proline and leucine.
Lkb1 and LIP1 interact in mammalian cells
MYC epitope tagged Lkb1 and FLAG epitope tagged Y2H-21or LIP1 (the splice isoform containing exons XXIIIa andXXVb) were co-transfected into COS cells and co-immuno-precipitation experiments performed (Fig. 2A). MYC–Lkb1can be co-immunoprecipitated with FLAG–Y2H-21 andFLAG–LIP1 but not with various control proteins demonstratingthat Lkb1 interacts specifically with LIP1 in mammalian cells.The control proteins shown are β-CATENIN, SMAD4 andTCF4 all of which are involved in pathways deregulated inintestinal polyposis syndromes and may therefore have beenconsidered to be candidate Lkb1-interacting proteins. Asfurther specificity controls we have shown that LIP1 does notinteract with the serine/threonine kinases B-Raf and mPAR-1(data not shown). Lkb1 kinase activity is not required for inter-action as an inactive mutant of Lkb1 (D194A) interacts with
Figure 2. Co-immunoprecipitation of Lkb1 and LIP1. (A) MYC–Lkb1 was co-transfected with empty expression vector or with expression vectors for FLAG-taggedY2H-21, LIP1, β-CATENIN, SMAD4 or TCF4 into COS cells. Cell lysates were immunoprecipitated with anti-FLAG agarose conjugate and lysates and immunoprecipitatesanalysed by 12% SDS–PAGE and western blotting with anti-MYC or anti-FLAG antibodies. MYC–Lkb1 is comparably expressed in all lysates (centre) and allthe FLAG-tagged constructs are expressed and immunoprecipitated (bottom). MYC–Lkb1 is only co-immunoprecipitated with FLAG–Y2H-21 and FLAG–LIP1(top). (B) MYC–Lkb1 was co-transfected with FLAG-tagged LIP1, LIP1-LRRs (containing residues 2–318 of LIP1), LIP1-CT (containing residues 313–1099 ofLIP1) and cell lysates analysed as in (A). MYC–Lkb1 is comparably expressed in all lysates (centre) and the FLAG-tagged constructs are expressed (bottom).MYC–Lkb1 is co-immunoprecipitated with all three FLAG constructs.
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LIP1 equally as well as wild-type Lkb1 (data not shown). Theinteraction between Lkb1 and LIP1 is not a post-cell-lysisassociation as Lkb1 cannot be co-immunoprecipitated withLIP1 from an admixture of singly transfected cell lysates (datanot shown).
From yeast-two-hybrid data, a LIP1 interaction site for Lkb1was localized between residue 278 and the C-terminus (residue436). Furthermore, yeast-two-hybrid data localized an inter-action site for Lkb1 on LIP1 between residues 740 and 915 (theregion of LIP1 corresponding to the Y2H-21). Accordingly,the C-terminal region (LIP1-CT; residues 313–1099) of LIP1co-immunoprecipitates Lkb1 from co-transfected COS cells(Fig. 2B). The LRR-containing region of LIP1 (LIP1-LRRs;residues 2–318) also co-immunoprecipitates with Lkb1,although the co-immunoprecipitation of Lkb1 with LIP1-LRRsis somewhat weaker than with full-length LIP1 or LIP1-CT.
Incubation of immunoprecipitated LIP1 in kinase bufferleads to the phosphorylation of LIP1 suggesting the co-immunoprecipitation of an associated kinase. However,mixing immunoprecipitated LIP1 and Lkb1 in kinase bufferunder conditions where Lkb1 autophosphorylation is observed(data not shown) (10) does not lead to an increase in LIP1phosphorylation, suggesting that LIP1 is not a substrate ofLkb1.
LIP1 is a cytoplasmic protein that can anchor Lkb1 in the cytoplasm
We expressed LIP1 in HeLa, COS, NIH-3T3 and MDCK cellsby microinjection of expression constructs. In all these celltypes, LIP1 was located in the cytoplasm. In HeLa cells 62% ofcells (431 cells in total) microinjected showed diffuse expression
of LIP1 which fills most of the cytoplasm, and 38% of cellshad granular or punctate expression, in which the size of thefoci of expression varied. The punctate expression possiblyreflects the association of LIP1 with cytoplasmic organelles.However, LIP1 expression does not co-localize with a Golgimarker (BODIPY TR Ceramide), a mitochondrial marker(pECFP-Mito), a lysosomal marker (LysoTracker Red DND-99)or an early endosomal marker (RhoB-EGFP) (data not shown).Therefore, the exact subcytoplasmic location of LIP1 remainsto be determined.
When Lkb1 is expressed alone in HeLa cells the vastmajority of cells (99%) show predominantly nuclear expression(Fig. 3; Table 1). Some punctate cytoplasmic expression isseen in 24% of cells in which Lkb1 is predominantly nuclear.Punctate cytoplasmic expression never predominates overnuclear expression, although a very few cells have mainlydiffuse cytoplasmic expression. However, when Lkb1 andLIP1 are co-expressed the proportion of microinjected cells inwhich Lkb1 is predominantly cytoplasmic increases from 0.7to 30% (P < 0.0001; Fisher’s exact test) (Fig. 3 and Table 1).As controls, we have demonstrated that the subcellular distribu-tion of Lkb1 is not affected by the expression of the proteinsEGFP and Smad4 and that LIP1 does not affect the distributionof RAD51 protein. We have observed that LIP1 causes thecytoplasmic accumulation of Lkb1 in COS, NIH-3T3 andMDCK cells in addition to HeLa cells (data not shown).
In the cells in which Lkb1 is predominantly cytoplasmic theexpression is punctate. Figure 3 shows typical immunofluorescenceimages of the proportion of cytoplasmic and nuclear Lkb1, anddemonstrates that they co-localize in discrete punctate structuresof varying size. Standard immunofluorescence images are
Figure 3. LIP1 anchors Lkb1 in the cytoplasm of HeLa cells. MYC–Lkb1 and/or FLAG–LIP1 expression constructs were microinjected into HeLa cells, and 24 hlater cells were fixed and stained for Lkb1 with FITC–αMYC (green), for LIP1 with TR–αFLAG (red) and with Hoechst DNA stain (blue). (A) Lkb1 alone ispredominantly nuclear with a few cells showing either some punctate cytoplasmic expression or mainly diffuse cytoplasmic expression. (B) LIP1 alone is exclusivelycytoplasmic, and many cells have punctate foci of expression which vary in size. (C and D) LIP1 and Lkb1 co-expression causes the concentration of Lkb1 incytoplasmic foci. Cytoplasmic Lkb1 co-localizes with LIP1 (see the yellow cytoplasmic stain in the MERGE images where the red and green immunofluorescencehas been overlaid); this is most apparent in the large cytoplasmic foci (arrowheads).
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shown to give an impression of the proportion of cytoplasmicand nuclear Lkb1; however, co-localization has beenconfirmed by confocal immunofluorescence microscopy (notshown). Taken together these results suggest, in agreementwith our co-immunoprecipitation experiments, that Lkb1 andLIP1 physically associate in the cytoplasm.
Ectopic expression of Lkb1 or LIP1 in Xenopus embryos induces a secondary body axis
The Xenopus orthologue of Lkb1, XEEK1, is expressed in theearly blastula embryo and expression levels decline sharplyprior to gastrulation (9). This suggests that XEEK1 may functionearly in embryonic development. We examined the effects ofectopic Lkb1 and LIP1 expression in early Xenopus embryos.Synthetic Lkb1 and LIP1 mRNAs were injected into one
ventrovegetal blastomere of eight-cell Xenopus embryos, andthe embryos were allowed to develop to tailbud stages (Fig. 4).Both Lkb1 and LIP1 induced secondary body axes that lackedthe most anterior structures. The ectopic axes and the somiticmuscle they form (Fig. 4C and D) are features similar to thoseobtained following ectopic expression of dorsalising TGFβfamily members or their downstream effectors, SMADs(23,24). These results suggest that the activities of both Lkb1and LIP1 intersect with TGFβ-activated signalling pathways,and prompted us to test this directly.
LIP1 interacts with SMAD4 forming a SMAD4–LIP1–Lkb1 ternary complex
SMAD4 is a TGFβ-regulated transcription factor that ismutated in juvenile polyposis syndrome (JPS), a gastrointestinal
Table 1. Co-expression with LIP1 alters the subcellular distribution of Lkb1
Figure 4. Both ectopic Lkb1 and LIP1 induce a secondary body axis in Xenopus embryos. Lkb1 and LIP1 can induce secondary axes in Xenopus embryos thatresemble those induced by dorsalising TGFβ factors. (A and B) Embryos with secondary axes induced by injection of 500 pg Lkb1 or LIP1 mRNA into one ventro-vegetalcell of eight-cell embryos. (A) Control embryo at stage 32 (top) and embryo injected with Lkb1 mRNA (bottom). This treatment leads to induction of second axesin 54% of embryos (n = 116). (B) Dorsal view of stage 20 embryos injected with LIP1 mRNA. Forty-eight percent of embryos developed second axes (n = 86).Both LIP1 and Lkb1 induced axes that develop without visible anterior structures (arrowheads). (C and D) Sections through Lkb1 induced second axes. (C) Transversesections through the anterior part of the second axis confirm the absence of anterior structures. (D) Sections through more posterior regions show the presence ofsomites. The second axis here is cut sagitally whereas the primary axis is seen transverse.
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hamartoma syndrome clinically similar to PJS. First we investigatedwhether LIP1 affected SMAD4 signalling. TGFβ-responsiveluciferase reporter constructs [p(CAGA)12-lux containing12 tandem SMAD3/4 binding sites, and the more complex3TP-lux containing PMA-responsive elements along withSMAD3/4 binding sites of the PAI-1 promoter (25)] were co-transfected with Lkb1 or LIP1 into TGFβ-responsive cell lines(Mv.1.Lu and CCL64). Neither Lkb1 nor LIP1 had any effecton the magnitude of the TGFβ response at a range of doses(data not shown).
However, we were able to show that LIP1 and SMAD4 associatein mammalian cells as they can be co-immunoprecipitated from co-transfected COS cells (Fig. 5A). As specificity controls for thisinteraction, SMAD4 is not co-immunoprecipitated by CDX2or β-CATENIN. Furthermore SMAD4 is not co-immuno-precipitated by LIP1-LRRs (amino acids 2–318), but is co-immunoprecipitated by LIP1-CT (amino acids 313–1099)(Fig. 5A) suggesting that the interaction site for SMAD4 onLIP1 is contained within the C-terminal portion of the protein.
Confocal immunofluorescence microscopy (Fig. 5C) showsthat SMAD4 and LIP1 co-localize in co-microinjected cells,providing further evidence that they associate in vivo. Lkb1does not associate directly with SMAD4 when plasmidsencoding the two proteins are co-transfected (Figs 2A and 5A).However, both LIP1 and SMAD4 are co-immunoprecipitatedwith Lkb1 when the three constructs are co-transfected together(Fig. 5B). This demonstrates that a Lkb1–LIP1–SMAD4 ternarycomplex, in which LIP1 forms a bridge between Lkb1 andSMAD4, exists when the three proteins are co-expressed.
DISCUSSION
LIP1 is the first protein described to interact with the Peutz-Jegher’s syndrome tumour suppressor protein LKB1. The existenceof PJS genes other than LKB1 has been suggested and LIP1 isa candidate for such a gene. However, these additional PJS locihave so far been mapped to chromosomes 6 and 19q13 (10,26)whereas LIP1 maps to chromosome 2q36. This chromosomal
Figure 5. Co-immunoprecipitation and co-localization of LIP1 and SMAD4. (A) An expression vector containing MYC-tagged SMAD4 was co-transfected withempty FLAG expression vector or with expression vectors containing FLAG-tagged LIP1, LIP1-LRRs (containing residues 2–318 of LIP1), LIP1-CT (containingresidues 313–1099 of LIP1), CDX2, Lkb1 or β-CATENIN, into COS cells. Cell lysates were immunoprecipitated with anti-FLAG agarose conjugate and lysatesand immunoprecipitates analysed by 12% SDS–PAGE and western blotting with anti-MYC or anti-FLAG antibodies. MYC–SMAD4 is comparably expressed inall lysates (centre) and all the FLAG-tagged constructs are expressed (bottom). MYC–SMAD4 is only co-immunoprecipitated with FLAG–LIP1 and FLAG–LIP1-CT(top). (B) FLAG–Lkb1 or FLAG vector was co-transfected along with the two MYC-tagged constructs indicated, and cell lysates analysed as in (A). Wheretransfected, MYC–LIP1 and MYC–SMAD4 (centre) and FLAG–Lkb1 (bottom) are comparably expressed. MYC–SMAD4 is only co-immunoprecipitated withFLAG–Lkb1 when MYC–LIP1 is co-transfected. (C) MYC–LIP1 and FLAG–SMAD4 expression constructs were microinjected into HeLa cells, and 24 h latercells were fixed and stained for LIP1 with FITC–αMYC (green) and for SMAD4 with TR–αFLAG (red). Confocal immunofluorescence micrographs are shown.Both LIP1 and SMAD4 are predominantly cytoplasmic, and overlay of green and red immunofluoresence (MERGE) shows that LIP1 and SMAD4 are co-localized.
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region has not been reported as a major site of allelic losses orgains in the cancers associated with PJS. However, this shouldnot exclude LIP1 as a candidate target for somatic inactivationin PJS-associated tumours as it is possible that epigeneticinactivation, as suggested for LKB1 (8), is important. Further, theglutamic acid-rich coding region of LIP1 which is uninterruptedby introns represents an extensive region of simple repetitivecoding DNA. Frameshift mutations in such regions of repetitivecoding DNA have been seen in mismatch repair deficienttumours. For example, the serine-rich coding region of E2F4(27) and the glutamine-rich coding region of CDX2 (28).
The most notable feature of the LIP1 sequence is the presenceof six tandem copies of a LRR motif towards the N-terminus.LRRs are involved in protein–protein interactions and occur ina wide variety of proteins with diverse cellular locations andfunctions (20). Most similar to LIP1 are the human protein IRAS-1and the mouse protein, Nischarin, which is very closely related toIRAS-1 and probably its orthologue. IRAS-1/Nischarin and LIP1share little primary sequence similarity, but do contain similarmotifs including six LRRs. Like LIP1, Nischarin is expressedin a punctate pattern in the cytoplasm of cells (22). IRAS-1 issuggested to encode a receptor for imidazolines (hypotensiveagents with binding sites in the brain and a variety of peripheraltissues) (21), but the evidence for this is limited. Nischarinappears to play a negative role in cell migration through aninteraction with integrin α5 and the down-regulation of signallingmediated by the small GTPase Rac; the integrin α5 interactionsite on Nischarin is not conserved in LIP1.
LIP1 is a cytoplasmic protein, and expression is concentrated inpunctate structures which may be cellular organelles. In theabsence of exogenous LIP1, Lkb1 is nuclear in most cells butin a small proportion it is cytoplasmic and located to punctatestructures. Co-expression of LIP1 significantly increases theproportion of punctate cytoplasmic Lkb1, overriding its usualnuclear accumulation. Hence, LIP1 may regulate the functionof Lkb1 by controlling its subcellular localization. The cytoplasmicsequestration of nuclear-acting proteins is a common mechanismof their regulation. For example, 14-3-3 proteins sequester andinactivate the phosphatase cdc25C, histone deacetylases andforkhead transcription factors (29–31). However, the observationthat Lkb1 and LIP1 have the same phenotypic effect whenectopically expressed in Xenopus embryos suggests that theyare functionally linked and also that LIP1 does not serve tooppose Lkb1 action by cytoplasmic sequestration. Lkb1 is amember of the Snf1 family of protein kinases which includemammalian AMP-activated protein kinase (AMPK). AMPKphosphorylates both nuclear and cytoplasmic substratesleading to increased ATP levels. The subcellular distribution ofAMPK between nucleus, cytoplasm and particulate cytoplasmicstructures is controlled, at least in part, by interaction of thekinase subunit with other subunits (32,33). LIP1 may regulatethe subcellular redistribution of Lkb1 allowing it to phosphorylatecytoplasmic as well as nuclear substrates. While this paper wasin review Karuman et al. (34) reported that overexpression ofLKB1 can induce apoptosis in some cell lines which expresswild-type p53 and suggest that the redistribution of cytoplasmicLKB1 to mitochondria during apoptosis may be a componentof the apoptotic signal. In HeLa cells p53 is inactivated and soin our system apoptosis (and mitochondrial redistribution)would not be observed in response to Lkb1 overexpression.Nevertheless these data suggest that one possible role for
cytoplasmically anchored Lkb1 may be in the promotion ofapoptosis.
An Lkb1 orthologue, XEEK1, is expressed in Xenopus blastulae(9) suggesting that it functions during early development. Thisidea was tested by ectopically expressing Lkb1 and LIP1 inearly Xenopus embryos. Both induce anteriorly truncatedsecondary body axes, suggesting that they are indeed functionallylinked, and may regulate the same signalling pathway. Thecellular localization of SMAD transcription factors is criticalin TGFβ superfamily signalling. TGFβ-related ligands activateheterodimeric cell surface serine/threonine kinase receptorsleading to the phosphorylation of R-SMADS (e.g. SMADs 1, 2or 3). A phosphorylated R-SMAD associates with a co-SMAD(e.g. SMAD4) and the complex translocates from the cyto-plasm to the nucleus where in association with co-factors theSMAD complex modulates target gene transcription (35). InXenopus dorsal mesoderm is induced in the blastula and duringgastrulation invaginates and extends to establish the antero-posterior body axis. Some members of the TGFβ superfamilyare essential for induction of dorsal mesoderm and forcingtheir expression ventrally (23,24) results in the induction ofdorsal mesodermal tissues and formation of secondary bodyaxes. Suppression of dorsal mesoderm formation on the ventralside of the embryo is regulated in part by another class of TGFβligands, the BMPs and inhibition of their signalling results inthe induction of a secondary body axis (36). Alternatively,ventral expression of Wnt family members which regulate theAPC/β-CATENIN complex induces secondary axes, butunlike those seen when TGFβ signalling components aretested, Wnt-induced axes develop anterior structures (37). Thephenotype of Xenopus embryos ectopically expressing Lkb1 orLIP1 is most similar to that induced by altering TGFβ signalling.
A role for Lkb1 and LIP1 in regulating TGFβ-superfamilysignalling is further supported by our observation of an Lkb1–LIP1–SMAD4 ternary complex, in which LIP1 forms a bridgebetween Lkb1 and SMAD4. Inherited mutations in SMAD4account for approximately one-third of cases of JPS (38)which, like PJS, is characterized by multiple gastrointestinalhamartomatous polyps and an increased risk of gastrointestinalcancer. As with LKB1, somatic loss of the wild-type SMAD4allele in the epithelium of JPS polyps suggests that SMAD4 isa tumour suppressor and that there may be hamartoma–adenoma–carcinoma sequence during neoplastic progression(39). Taken together, our results provide evidence for anunsuspected mechanistic link between the juvenile intestinalhamartomatous polyposis syndromes PJS and JPS.
MATERIALS AND METHODS
Yeast-two-hybrid screening
Codons 2–436 (C-terminus) of mouse wild-type Lkb1 (Lkb1-WT)and kinase inactive (Lkb1-D194A) Lkb1 were cloned in frameto the Gal4 DNA binding domain in the vector pGBT9 (Clontech)and the resulting plasmids introduced into the yeast strainPJ69-2A (40). These strains were then transformed with amouse embryonic cDNA library fused to the VP16 asdescribed by Vojtek and Hollenberg (15), and then selected onmedia lacking His, Leu and Trp and with 10 mM 3-amino-1,2,4-triazole. An identical plasmid designated Y2H-21 was
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isolated once from the Lkb1-WT screen and three times fromthe Lkb1-D194A.
Chromosomal mapping of LIP1
LIP1-specific forward (GAC CTT GGC CCT GAC CTC AGG)and reverse (TCG CCT CTC CCT TAA GCC CTG) primerswere used to PCR amplify a 149 bp fragment of LIP1 from DNAsamples originating from 84 clones of the Genebridge4 radiationhybrid panel (24). Results were analysed using the Sanger Centreradiation hybrid web server (http://www.sanger.ac.uk/software/Rhserver).
Eukaryotic expression constructs
Lkb1 cDNAs (13) were cloned into the expression vectorspEF.m6 and pEF.f6 (gifts of Dr R.Marais) under the control of theEF1α promoter such that MYC or FLAG epitopes, respectively,were fused in frame at the N-terminus. A kinase inactive variant(D194A) of Lkb1 was generated by PCR. LIP1 constructs weregenerated from EST clone AA134795. pEF.f6-LIP1 was used asthe basis for the deletion constructs FLAG–LIP1-LRRs(containing residues 2–318) and FLAG–LIP1-CT (containingresidues 313–1099) (details available on request).
Co-immunoprecipitation of LIP1 with Lkb1 and SMAD4
COS-7 cells were transfected with plasmids using lipofectaminereagent (Life Technologies). Two days after transfection thecell lysates were immunoprecipitated with mouse monoclonalanti-FLAG M2 agarose conjugate (Anachem) and analysed bywestern blotting as described by Lorenzo et al. (41). Mousemonoclonal anti-FLAG M5 (Anachem), rabbit polyclonal anti-MYC (Santa Cruz) and HRP-conjugated secondary antibodies(Pierce) and ECL detection reagents (Amersham) were used.
Co-localization of LIP1 with Lkb1 and SMAD4
HeLa cells were grown on glass coverslips and microinjected withexpression constructs at 100–200 µg/ml using a Zeiss-Eppendorfmicroinjection workstation. Twenty-four hours after micro-injection, cells were fixed (at –80°C in 50% (v/v) methanol:50%(v/v) acetone for 30 min), permeabilized (at room temperaturein PBS with 0.2% Triton X-100 for 5 min), stained withprimary antibodies and then fluorescently conjugatedsecondary antibodies and visualized with a Zeiss Axioplan 2epifluorescent microscope or a Biorad 1024 confocal imagingsystem.
Ectopic expression of Lkb1 and LIP1 in Xenopus embryos
Lkb1 and LIP1 cDNAs with N-terminal FLAG epitope tags werecloned into the vector pSP64TBX. 5′-capped synthetic mRNAwas produced with SP6 RNA polymerase from SalI-linearizedtemplates and injected into one ventrovegetal cell of an eight-cell Xenopus laevis embryo as described by Jones et al. (42).
ACKNOWLEDGEMENTS
We thank Breakthrough Breast Cancer and the CancerResearch Campaign for financial support.
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