Human Molecular Genetics, 2001, Vol. 10, No. 25 2869-2877
© 2001 Oxford University Press
LIP1, a cytoplasmic protein functionally linked to the Peutz-Jeghers syndrome kinase LKB1
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.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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LKB1 is a serine/threonine kinase which is inactivated by mutation in the Peutz-Jeghers polyposis and cancer predisposition syndrome (PJS). We have identified a novel leucine-rich repeat containing protein, LIP1, that interacts with LKB1. The LIP1 gene consists of 25 exons, maps to human chromosome 2q36 and encodes a protein of 121 kDa. LIP1 appears to be a cytoplasmically located protein whereas we and others have shown previously that LKB1 is predominantly nuclear, with only a small proportion of cells showing strong cytoplasmic expression. However, when LKB1 and LIP1 are co-expressed, the proportion of cytoplasmic LKB1 dramatically increases, suggesting that LIP1 may regulate LKB1 function by controlling its subcellular localization. Ectopic expression of both LKB1 and LIP1 in Xenopus embryos induces a secondary body axis, providing further evidence for a functional link between the two proteins. This phenotype resembles the effects of ectopic expression of TGFß superfamily members and their downstream effectors. A possible role for LIP1 and LKB1 in TGFß signalling is supported by the observation that LIP1 interacts with the TGFß-regulated transcription factor SMAD4, forming a LKB1–LIP1–SMAD4 ternary complex. SMAD4 mutations give rise to juvenile polyposis syndrome, which is clinically similar to PJS. Our data suggest an unsuspected mechanistic link between these two syndromes.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Peutz-Jeghers syndrome (PJS) is a dominantly inherited condition characterized by multiple gastrointestinal hamartomatous polyps and mucocutaneous pigmented spots on the lips, digits and buccal mucosa (1,2). Patients with PJS are at least 10 times more likely to develop cancer than the general population (3). Malignant neoplasms may occur in a variety of tissues including colon (possibly by malignant transformation of the hamartomas), small intestine, breast, cervix, ovary and pancreas. 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 epithelial component of the gastrointestinal hamartomas suggested that the PJS gene was a tumour suppressor and that there may be a hamartoma–adenoma–carcinoma sequence in neoplastic transformation (4). Multiple independent mutations were found in the gene LKB1 (STK11) in affected members of PJS families (5,6). LKB1 is only rarely mutated in the sporadic counterpart of the tumours found in PJS (7). However, epigenetic inactivation of LKB1 may be important, particularly in the papillary subtype of breast carcinoma associated with PJS (8).
LKB1 encodes a serine/threonine kinase and is the human orthologue of the Xenopus gene XEEK1 (9). LKB1 has autocatalytic kinase activity and PJS mutations have been shown to cause loss or severe abrogation of the autokinase activity (10,11). Expression of exogenous wild-type LKB1, but not LKB1 with PJS-associated mutations, in some LKB1 deficient cancer cell lines causes growth suppression by blocking the cell-cycle at the G1/S transition (12) supporting its proposed role as a tumour suppressor gene. Exogenous human LKB1 and mouse Lkb1 are predominantly nuclear proteins in transfected cells but a significant minority of cells show strong cytoplasmic expression (12,13 and unpublished data). A nuclear localization signal of the single basic type is located towards the N-terminus of the protein (13). LKB1 has a putative CAAX box prenylation signal at the C-terminus, and truncated LKB1 may be prenylated and localized to the plasma membrane (14); however, membrane localization of full-length LKB1 has not been reported. As yet, no substrates of LKB1 have been identified and nothing is known about how its activity is regulated.
In an attempt to find substrates or regulators of LKB1 we screened a yeast-two-hybrid library with Lkb1 as bait. An LKB1 interacting protein, LIP1, was identified. We show that this protein may be functionally linked to LKB1 as it alters the subcellular location of Lkb1 when co-expressed, and both Lkb1 and LIP1 cause axis duplication when ectopically expressed in Xenopus embryos. Axis duplication suggests a role for LKB1 and LIP1 in TGFß signalling which is further supported by the interaction of LIP1 with the TGFß-regulated transcription factor SMAD4.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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LIP1 encodes a novel Lkb1-interacting protein containing six leucine-rich repeat motifs
Yeast-two-hybrid screening of a mouse embryonic cDNA library (15) with mouse Lkb1 (13) (note we have used LKB1 to indicate the human protein and Lkb1 the mouse orthologue) and an autokinase inactive variant (D194A) resulted in the isolation of the same cDNA clone (Y2H-21). The interaction between Lkb1 and Y2H-21 was specific as Y2H-21 failed to interact with a range of control proteins, and required the C-terminal 159 residues of Lkb1. Although Y2H-21 was not identical to known genes in GenBank, various multiple human ESTs were present in the sequence database, including those contained in the UniGene cluster Hs.22410 of 71 ESTs (http://www.ncbi.nlm.nih.gov/UniGene). Sequencing of members of this EST cluster resulted in the identification of EST AA134795 which was found to contain a complete ORF with an in frame stop codon N-terminal to the first Met codon, a polyA addition signal and polyA tail. We have designated the gene encoded by this clone LIP1 (for Lkb1 interacting protein 1; GenBank accession no. AF450267); the amino acid sequence of the protein encoded by LIP1 is shown in Figure 1 aligned with the ORF of the mouse Y2H-21 clone.
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LIP1 consists of 1099 amino acid residues with a predicted molecular weight of 121.4 kDa. LIP1 is a widely or ubiquitously expressed gene as human ESTs have been isolated from many tissues including colon, testis, ovary, breast, cervix, aorta, brain, kidney, lung, uterus, oesophagus, placenta, skin and pancreas. Furthermore, the sequence of the probable mouse LIP1 orthologue has recently been reported (16) (GenBank accession nos AK004757 and BAB23538). Mouse Lip1 is 99.4% identical to the ORF in the Y2H-21 clone in the region of overlap, and 74% identical to human LIP1. cDNA microarray analysis of 47 adult, neonatal and embryonic tissues (17) (http://www.genome.gsc.riken.go.jp/READ/) shows that Lip1 is expressed in all tissues examined, with expression levels in adult tissues varying only 3.4-fold between the highest and lowest expressing tissues.
The LIP1 gene (within BAC clone AC009955) consists of 25 exons and at least two alternatively spliced transcripts are expressed (Fig. 1). One, represented by EST AI149114, contains a 46 amino acid insert and the other (EST AW957418) encodes an alternate C-terminus. Radiation hybrid mapping (18) (data not shown) localized LIP1 to the human chromosome 2 reference interval D2S164 to D2S163 (http://www.ncbi.nlm.nih.gov/genemap99) and SCL4A3 which is next to LIP1 and has been mapped to human 2q36 (19).
Although the amino acid sequence of LIP1 provides few clues to its possible function, there are six tandem leucine-rich repeats (LRRs) towards the N-terminus (Fig. 1). LRRs are a consensus motif involved in protein–protein interactions that usually occur in tandem copies and are characterized by leucines at invariant positions (20). The LRRs of LIP1 are not closely related to the LRRs of other known proteins except for the presence of the core leucine residues. Other notable features of the LIP1 amino acid sequence are a highly glutamic acid-rich region (30 of 63 residues; Fig. 1), a possible leucine zipper (fitting the consensus L(X)6L(X)6L(X)6L where X is any residue; Fig. 1E), and a proline (9.0%) and leucine (16.1%) rich character. The mammalian proteins for which a function has been proposed most closely related to LIP1 are the human protein IRAS-1 (suggested to be an imidazoline receptor) (21) and its probable mouse orthologue Nischarin, which is believed to be involved in the control of cell migration (22). Nischarin (Fig. 1) and IRAS-1 share low overall sequence identity with LIP1, but have a similar structure with six tandem LRRs towards the N-terminus followed by a glutamic acid-rich region. 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-21 or LIP1 (the splice isoform containing exons XXIIIa and XXVb) were co-transfected into COS cells and co-immunoprecipitation experiments performed (Fig. 2A). MYC–Lkb1 can be co-immunoprecipitated with FLAG–Y2H-21 and FLAG–LIP1 but not with various control proteins demonstrating that Lkb1 interacts specifically with LIP1 in mammalian cells. The control proteins shown are ß-CATENIN, SMAD4 and TCF4 all of which are involved in pathways deregulated in intestinal polyposis syndromes and may therefore have been considered to be candidate Lkb1-interacting proteins. As further specificity controls we have shown that LIP1 does not interact with the serine/threonine kinases B-Raf and mPAR-1 (data not shown). Lkb1 kinase activity is not required for interaction as an inactive mutant of Lkb1 (D194A) interacts with LIP1 equally as well as wild-type Lkb1 (data not shown). The interaction between Lkb1 and LIP1 is not a post-cell-lysis association as Lkb1 cannot be co-immunoprecipitated with LIP1 from an admixture of singly transfected cell lysates (data not shown).
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From yeast-two-hybrid data, a LIP1 interaction site for Lkb1 was localized between residue 278 and the C-terminus (residue 436). Furthermore, yeast-two-hybrid data localized an interaction site for Lkb1 on LIP1 between residues 740 and 915 (the region of LIP1 corresponding to the Y2H-21). Accordingly, the C-terminal region (LIP1-CT; residues 313–1099) of LIP1 co-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-LRRs is somewhat weaker than with full-length LIP1 or LIP1-CT.
Incubation of immunoprecipitated LIP1 in kinase buffer leads to the phosphorylation of LIP1 suggesting the co-immunoprecipitation of an associated kinase. However, mixing immunoprecipitated LIP1 and Lkb1 in kinase buffer under conditions where Lkb1 autophosphorylation is observed (data not shown) (10) does not lead to an increase in LIP1 phosphorylation, suggesting that LIP1 is not a substrate of Lkb1.
LIP1 is a cytoplasmic protein that can anchor Lkb1 in the cytoplasm
We expressed LIP1 in HeLa, COS, NIH-3T3 and MDCK cells by microinjection of expression constructs. In all these cell types, LIP1 was located in the cytoplasm. In HeLa cells 62% of cells (431 cells in total) microinjected showed diffuse expression of LIP1 which fills most of the cytoplasm, and 38% of cells had granular or punctate expression, in which the size of the foci of expression varied. The punctate expression possibly reflects the association of LIP1 with cytoplasmic organelles. However, LIP1 expression does not co-localize with a Golgi marker (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 remains to be determined.
When Lkb1 is expressed alone in HeLa cells the vast majority of cells (99%) show predominantly nuclear expression (Fig. 3; Table 1). Some punctate cytoplasmic expression is seen in 24% of cells in which Lkb1 is predominantly nuclear. Punctate cytoplasmic expression never predominates over nuclear expression, although a very few cells have mainly diffuse cytoplasmic expression. However, when Lkb1 and LIP1 are co-expressed the proportion of microinjected cells in which Lkb1 is predominantly cytoplasmic increases from 0.7 to 30% (P < 0.0001; Fisher’s exact test) (Fig. 3 and Table 1). As controls, we have demonstrated that the subcellular distribution of Lkb1 is not affected by the expression of the proteins EGFP and Smad4 and that LIP1 does not affect the distribution of RAD51 protein. We have observed that LIP1 causes the cytoplasmic accumulation of Lkb1 in COS, NIH-3T3 and MDCK cells in addition to HeLa cells (data not shown).
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In the cells in which Lkb1 is predominantly cytoplasmic the expression is punctate. Figure 3 shows typical immunofluorescence images of the proportion of cytoplasmic and nuclear Lkb1, and demonstrates that they co-localize in discrete punctate structures of varying size. Standard immunofluorescence images are shown to give an impression of the proportion of cytoplasmic and nuclear Lkb1; however, co-localization has been confirmed by confocal immunofluorescence microscopy (not shown). Taken together these results suggest, in agreement with our co-immunoprecipitation experiments, that Lkb1 and LIP1 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 the early blastula embryo and expression levels decline sharply prior to gastrulation (9). This suggests that XEEK1 may function early in embryonic development. We examined the effects of ectopic Lkb1 and LIP1 expression in early Xenopus embryos. Synthetic Lkb1 and LIP1 mRNAs were injected into one ventrovegetal blastomere of eight-cell Xenopus embryos, and the embryos were allowed to develop to tailbud stages (Fig. 4). Both Lkb1 and LIP1 induced secondary body axes that lacked the most anterior structures. The ectopic axes and the somitic muscle they form (Fig. 4C and D) are features similar to those obtained following ectopic expression of dorsalising TGFß family members or their downstream effectors, SMADs (23,24). These results suggest that the activities of both Lkb1 and LIP1 intersect with TGFß-activated signalling pathways, and prompted us to test this directly.
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LIP1 interacts with SMAD4 forming a SMAD4–LIP1–Lkb1 ternary complex
SMAD4 is a TGFß-regulated transcription factor that is mutated in juvenile polyposis syndrome (JPS), a gastrointestinal hamartoma syndrome clinically similar to PJS. First we investigated whether LIP1 affected SMAD4 signalling. TGFß-responsive luciferase reporter constructs [p(CAGA)12-lux containing 12 tandem SMAD3/4 binding sites, and the more complex 3TP-lux containing PMA-responsive elements along with SMAD3/4 binding sites of the PAI-1 promoter (25)] were co-transfected with Lkb1 or LIP1 into TGFß-responsive cell lines (Mv.1.Lu and CCL64). Neither Lkb1 nor LIP1 had any effect on the magnitude of the TGFß response at a range of doses (data not shown).
However, we were able to show that LIP1 and SMAD4 associate in mammalian cells as they can be co-immunoprecipitated from co-transfected COS cells (Fig. 5A). As specificity controls for this interaction, SMAD4 is not co-immunoprecipitated by CDX2 or ß-CATENIN. Furthermore SMAD4 is not co-immunoprecipitated 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 on LIP1 is contained within the C-terminal portion of the protein. Confocal immunofluorescence microscopy (Fig. 5C) shows that SMAD4 and LIP1 co-localize in co-microinjected cells, providing further evidence that they associate in vivo. Lkb1 does not associate directly with SMAD4 when plasmids encoding the two proteins are co-transfected (Figs 2A and 5A). However, both LIP1 and SMAD4 are co-immunoprecipitated with Lkb1 when the three constructs are co-transfected together (Fig. 5B). This demonstrates that a Lkb1–LIP1–SMAD4 ternary complex, in which LIP1 forms a bridge between Lkb1 and SMAD4, exists when the three proteins are co-expressed.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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LIP1 is the first protein described to interact with the Peutz-Jegher’s syndrome tumour suppressor protein LKB1. The existence of PJS genes other than LKB1 has been suggested and LIP1 is a candidate for such a gene. However, these additional PJS loci have so far been mapped to chromosomes 6 and 19q13 (10,26) whereas LIP1 maps to chromosome 2q36. This chromosomal region has not been reported as a major site of allelic losses or gains in the cancers associated with PJS. However, this should not exclude LIP1 as a candidate target for somatic inactivation in PJS-associated tumours as it is possible that epigenetic inactivation, as suggested for LKB1 (8), is important. Further, the glutamic acid-rich coding region of LIP1 which is uninterrupted by introns represents an extensive region of simple repetitive coding DNA. Frameshift mutations in such regions of repetitive coding DNA have been seen in mismatch repair deficient tumours. 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 presence of six tandem copies of a LRR motif towards the N-terminus. LRRs are involved in protein–protein interactions and occur in a wide variety of proteins with diverse cellular locations and functions (20). Most similar to LIP1 are the human protein IRAS-1 and the mouse protein, Nischarin, which is very closely related to IRAS-1 and probably its orthologue. IRAS-1/Nischarin and LIP1 share little primary sequence similarity, but do contain similar motifs including six LRRs. Like LIP1, Nischarin is expressed in a punctate pattern in the cytoplasm of cells (22). IRAS-1 is suggested to encode a receptor for imidazolines (hypotensive agents with binding sites in the brain and a variety of peripheral tissues) (21), but the evidence for this is limited. Nischarin appears to play a negative role in cell migration through an interaction with integrin 
5 and the down-regulation of signalling mediated by the small GTPase Rac; the integrin 5 interaction site on Nischarin is not conserved in LIP1.
LIP1 is a cytoplasmic protein, and expression is concentrated in punctate structures which may be cellular organelles. In the absence of exogenous LIP1, Lkb1 is nuclear in most cells but in a small proportion it is cytoplasmic and located to punctate structures. Co-expression of LIP1 significantly increases the proportion of punctate cytoplasmic Lkb1, overriding its usual nuclear accumulation. Hence, LIP1 may regulate the function of Lkb1 by controlling its subcellular localization. The cytoplasmic sequestration of nuclear-acting proteins is a common mechanism of their regulation. For example, 14-3-3 proteins sequester and inactivate the phosphatase cdc25C, histone deacetylases and forkhead transcription factors (29–31). However, the observation that Lkb1 and LIP1 have the same phenotypic effect when ectopically expressed in Xenopus embryos suggests that they are functionally linked and also that LIP1 does not serve to oppose Lkb1 action by cytoplasmic sequestration. Lkb1 is a member of the Snf1 family of protein kinases which include mammalian AMP-activated protein kinase (AMPK). AMPK phosphorylates both nuclear and cytoplasmic substrates leading to increased ATP levels. The subcellular distribution of AMPK between nucleus, cytoplasm and particulate cytoplasmic structures is controlled, at least in part, by interaction of the kinase subunit with other subunits (32,33). LIP1 may regulate the subcellular redistribution of Lkb1 allowing it to phosphorylate cytoplasmic as well as nuclear substrates. While this paper was in review Karuman et al. (34) reported that overexpression of LKB1 can induce apoptosis in some cell lines which express wild-type p53 and suggest that the redistribution of cytoplasmic LKB1 to mitochondria during apoptosis may be a component of the apoptotic signal. In HeLa cells p53 is inactivated and so in 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 of apoptosis.
An Lkb1 orthologue, XEEK1, is expressed in Xenopus blastulae (9) suggesting that it functions during early development. This idea was tested by ectopically expressing Lkb1 and LIP1 in early Xenopus embryos. Both induce anteriorly truncated secondary body axes, suggesting that they are indeed functionally linked, and may regulate the same signalling pathway. The cellular localization of SMAD transcription factors is critical in TGFß superfamily signalling. TGFß-related ligands activate heterodimeric cell surface serine/threonine kinase receptors leading to the phosphorylation of R-SMADS (e.g. SMADs 1, 2 or 3). A phosphorylated R-SMAD associates with a co-SMAD (e.g. SMAD4) and the complex translocates from the cytoplasm to the nucleus where in association with co-factors the SMAD complex modulates target gene transcription (35). In Xenopus dorsal mesoderm is induced in the blastula and during gastrulation invaginates and extends to establish the anteroposterior body axis. Some members of the TGFß superfamily are essential for induction of dorsal mesoderm and forcing their expression ventrally (23,24) results in the induction of dorsal mesodermal tissues and formation of secondary body axes. Suppression of dorsal mesoderm formation on the ventral side of the embryo is regulated in part by another class of TGFß ligands, the BMPs and inhibition of their signalling results in the induction of a secondary body axis (36). Alternatively, ventral expression of Wnt family members which regulate the APC/ß-CATENIN complex induces secondary axes, but unlike those seen when TGFß signalling components are tested, Wnt-induced axes develop anterior structures (37). The phenotype of Xenopus embryos ectopically expressing Lkb1 or LIP1 is most similar to that induced by altering TGFß signalling.
A role for Lkb1 and LIP1 in regulating TGFß-superfamily signalling is further supported by our observation of an Lkb1–LIP1–SMAD4 ternary complex, in which LIP1 forms a bridge between Lkb1 and SMAD4. Inherited mutations in SMAD4 account for approximately one-third of cases of JPS (38) which, like PJS, is characterized by multiple gastrointestinal hamartomatous polyps and an increased risk of gastrointestinal cancer. As with LKB1, somatic loss of the wild-type SMAD4 allele in the epithelium of JPS polyps suggests that SMAD4 is a tumour suppressor and that there may be hamartoma–adenoma–carcinoma sequence during neoplastic progression (39). Taken together, our results provide evidence for an unsuspected mechanistic link between the juvenile intestinal hamartomatous polyposis syndromes PJS and JPS.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 frame to the Gal4 DNA binding domain in the vector pGBT9 (Clontech) and the resulting plasmids introduced into the yeast strain PJ69-2A (40). These strains were then transformed with a mouse embryonic cDNA library fused to the VP16 as described by Vojtek and Hollenberg (15), and then selected on media lacking His, Leu and Trp and with 10 mM 3-amino-1,2,4-triazole. An identical plasmid designated Y2H-21 was isolated once from the Lkb1-WT screen and three times from the 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) primers were used to PCR amplify a 149 bp fragment of LIP1 from DNA samples originating from 84 clones of the Genebridge4 radiation hybrid panel (24). Results were analysed using the Sanger Centre radiation hybrid web server (http://www.sanger.ac.uk/software/Rhserver).
Eukaryotic expression constructs
Lkb1 cDNAs (13) were cloned into the expression vectors pEF.m6 and pEF.f6 (gifts of Dr R.Marais) under the control of the EF1 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 were generated from EST clone AA134795. pEF.f6-LIP1 was used as the basis for the deletion constructs FLAG–LIP1-LRRs (containing residues 2–318) and FLAG–LIP1-CT (containing residues 313–1099) (details available on request).
Co-immunoprecipitation of LIP1 with Lkb1 and SMAD4
COS-7 cells were transfected with plasmids using lipofectamine reagent (Life Technologies). Two days after transfection the cell lysates were immunoprecipitated with mouse monoclonal anti-FLAG M2 agarose conjugate (Anachem) and analysed by western blotting as described by Lorenzo et al. (41). Mouse monoclonal 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 with expression constructs at 100–200 µg/ml using a Zeiss-Eppendorf microinjection workstation. Twenty-four hours after microinjection, cells were fixed (at –80°C in 50% (v/v) methanol:50% (v/v) acetone for 30 min), permeabilized (at room temperature in PBS with 0.2% Triton X-100 for 5 min), stained with primary antibodies and then fluorescently conjugated secondary antibodies and visualized with a Zeiss Axioplan 2 epifluorescent microscope or a Biorad 1024 confocal imaging system.
Ectopic expression of Lkb1 and LIP1 in Xenopus embryos
Lkb1 and LIP1 cDNAs with N-terminal FLAG epitope tags were cloned into the vector pSP64TBX. 5'-capped synthetic mRNA was produced with SP6 RNA polymerase from SalI-linearized templates and injected into one ventrovegetal cell of an eight-cell Xenopus laevis embryo as described by Jones et al. (42).
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ACKNOWLEDGEMENTS |
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We thank Breakthrough Breast Cancer and the Cancer Research Campaign for financial support.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +44 20 7970 6058; Fax: +44 20 7878 3858; Email alana@icr.ac.uk
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REFERENCES |
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