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Research, Fulham Road, London SW3 6JB, UK. Loss-of-function mutations in the LKB1 (STK11) serine– threonine kinase gene cause Peutz–Jeghers syndrome ...
Oncogene (2003) 22, 4752–4756

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Regulation of the Wnt signalling component PAR1A by the Peutz–Jeghers syndrome kinase LKB1 James Spicer1, Sydonia Rayter1,3, Neville Young1,3, Richard Elliott1, Alan Ashworth*,1 and Darrin Smith1,2 1

Cancer Research UK Gene Function and Regulation Group, The Breakthrough Breast Cancer Research Centre, Institute of Cancer Research, Fulham Road, London SW3 6JB, UK

Loss-of-function mutations in the LKB1 (STK11) serine– threonine kinase gene cause Peutz–Jeghers syndrome, which is associated with inherited susceptibility to colorectal and other cancers. No downstream targets of LKB1 kinase activity have been identified. Here we show that LKB1 can direct the phosphorylation of the serine– threonine kinase PAR1A. The amino-acid residues phosphorylated as a result of LKB1 activity have been identified and phosphorylation at these residues is required for PAR1A kinase activity. PAR1A has previously been implicated as a positive regulator of the Wnt-bcatenin signalling pathway. We show here that LKB1 can modify transcription driven by the Wnt-regulated TCF response element, implicating LKB1 in a pathway known to play a key role in human colorectal tumorigenesis. Oncogene (2003) 22, 4752–4756. doi:10.1038/sj.onc.1206669 Keywords: cancer susceptibility; Peutz–Jeghers syndrome; Wnt signalling; colorectal cancer; protein kinase

Peutz–Jeghers syndrome (PJS) is a dominantly inherited condition associated with intestinal hamartomas and a lifetime risk of colorectal and other cancer at least 10 times greater than that of the general population (Tomlinson and Houlston, 1997). Multiple independent mutations have been found in the gene LKB1 (STK11) in affected members of PJS families (Yoo et al., 2002). Loss of the presumptive wild-type allele in the epithelial component of the gastrointestinal hamartomas suggests that the PJS gene is a tumour suppressor and that there may be a hamartoma–adenoma–carcinoma sequence in neoplastic transformation (Bosman, 1999). However, loss of heterozygosity at LKB1 is not always detected in hamartomas, and some mouse models of PJS have recently suggested that haploinsufficiency might be sufficient to trigger disease progression (Miyoshi et al., 2002; Nakau et al., 2002). LKB1 is mutated in some sporadic tumours (Sanchez-Cespedes et al., 2002), and *Correspondence: A Ashworth; E-mail: [email protected] 2 Current address: Walter & Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Victoria 3050, Australia 3 These authors made an equal contribution to this work Received 22 January 2003; revised 18 March 2003; accepted 21 March 2003

epigenetic inactivation of LKB1 may also occur (Esteller et al., 2000). LKB1 encodes a serine/threonine kinase with autocatalytic activity that is greatly reduced by PJS mutations (Mehenni et al., 1998; Esteller et al., 2000). Two nuclear proteins, p53 (Karuman et al., 2001) and BRG1 (Marignani et al., 2001), and the cytoplasmic protein LIP1 (Smith et al., 2001), have been shown to interact with LKB1. However, none of these has been shown to be an LKB1 substrate in vivo, and as yet no downstream phosphorylation target of LKB1 has been identified. A protein involved in embryonic development in the nematode Caenorhabditis elegans shows sequence similarity to LKB1 (Watts et al., 2000), and has been shown to function together with other gene products including the kinase PAR1. We show here that LKB1 directs the phosphorylation of mammalian PAR1A, regulating its kinase activity. PAR1A has been shown previously to activate Dishevelled (Dvl), a component of the Wnt pathway (Sun et al., 2001), and we therefore investigated the effect of LKB1 activity on the Wnt signalling pathway. Genetic studies in C. elegans have implicated several genes in the control of the polarity of asymmetric cell divisions early in embryogenesis (Jan and Jan, 2000). One of these, PAR4, encodes a protein that shares 42% identity with LKB1 within the kinase domain. Another nematode gene important in polarity determination is PAR1, which also encodes an STK (Guo and Kemphues, 1995). Moreover, the phenotype of mutations in PAR4 is more similar to mutations in PAR1 than to those in other PAR genes (Watts et al., 2000). Given the possibility of functional interaction between PAR1 and PAR4 in C. elegans, we tested whether mammalian proteins similar to C. elegans PAR1 might be signalling partners of LKB1. Several mammalian proteins with similarity to C. elegans PAR1 have been described (Parsa, 1988; Drewes et al., 1997; Peng et al., 1998). One of these, here termed PAR1A, is a human sequence that has been variously described as MARK3 (Drewes et al., 1997), cTAK1 (Peng et al., 1998) and p78 (Parsa, 1988). A cDNA encoding human PAR1A was generated by PCR and cloned into a mammalian expression vector. This plasmid was transfected into HeLa S3 cells in the presence or absence of exogenous LKB1. HeLa S3 cells were chosen for the investigation of PAR1A and LKB1

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interaction because, unlike many cell lines, they lack endogenous LKB1 (Figure 1a and Tiainen et al., 1999). Epitope-tagged PAR1A is expressed in the absence of LKB1 as a 78 kDa protein, with a minor lower mobility component appearing at 80 kDa on SDS–PAGE (Figure 1b). The coexpression of LKB1 shifts the electrophoretic mobility of PAR1A (Figure 1b). A

Figure 1 Phosphorylation of mammalian PAR1A is directed by LKB1 and is dependent on LKB1 kinase activity. (a) Lysates from the mammalian cell lines shown were harvested as described (Smith et al., 1999), separated by SDS–PAGE, and LKB1 protein detected by immunoblotting with anti-LKB1 antibody (Upstate Biotechnology). The cell lines 293, COS, 3T3, Mv.1.Lu and MCF-7, but not HeLa S3 or A549, express LKB1 with species-specific variation in mobility. (b) HeLa S3 or COS cells were transfected using Lipofectamine 2000 (Life Technologies) with pcDNA3.1 þ expression constructs encoding FLAG-tagged mouse LKB1 and human PAR1A (GenBank accession number AL043945) as indicated. Lysates were separated by SDS–PAGE and proteins detected by immunoblotting with anti-FLAG antibody (Sigma). PAR1A expressed in HeLa S3 cells migrates predominantly at 78 kDa, but its mobility is decreased in the presence of cotransfected LKB1, a result of PAR1A phosphorylation (pPAR1A and ppPAR1A, left panel – see text). A kinase-inactive LKB1 mutant D194A was generated using the Quickchange protocol (Stratagene). Coexpression in HeLa S3 cells of wild-type PAR1A with D194A does not cause the PAR1A mobility shift seen with wild-type LKB1 (middle panel), suggesting that LKB1, kinase activity is required to cause PAR1A mobility shift. In the absence of LKB1 a small proportion of PAR1A is expressed at lower mobility (left and middle lanes), implying the presence in HeLa S3 cells of a weak endogenous PAR1A kinase activity. PAR1A in COS (right panel) and 293 cells (data not shown), which both express endogenous LKB1, is hyperphosphorylated even in the absence of cotransfected LKB1. This implies that endogenous LKB1 directs phosphorylation of PAR1A in these cell lines. (c) A549 cells, which like HeLa S3 cells do not express endogenous LKB1, were transfected with PAR1A with or without LKB1. Hyperphosphorylation of PAR1A occurs in the presence of LKB1

similar result was obtained in A549 cells (Figure 1c), which also lack endogenous LKB1 (Figure 1a and Sanchez-Cespedes et al., 2002). This mobility change is likely to be due to phosphorylation of PAR1A, as it can be partially reversed by incubating cell lysate with lambda phosphatase prior to gel loading (not shown). There are no changes in the mobility of LKB1 in the presence of PAR1A. Together these data are consistent with the suggestion from the C. elegans model system that the two proteins are components of a linear kinase cascade, with the phosphorylation of PAR1A occurring downstream of LKB1. Recently, genetic evidence has been presented which functionally links these two proteins in Drosophila, and the proposal was made that LKB1 acts downstream of PAR1 (Martin and St Johnson, 2003). However, the genetic evidence presented for Drosophila is equally consistent with the model and data described in our paper. An alternative model consistent with our data is an indirect interaction in which the two kinases compete for binding to a third protein, which in turn is the determinant of PAR1A phosphorylation status. To exclude this possibility, PAR1A was transfected into HeLa S3 cells with wild-type or kinase-inactive LKB1 (Figure 1b). LKB1 was mutated to alanine at the key catalytic aspartate 194; expression of this kinase dead variant of LKB1 did not result in PAR1A phosphorylation, suggesting that PAR1A phosphorylation occurs downstream of LKB1 and requires LKB1 kinase activity (Figure 1b). The subcellular localization of LKB1 is subject to regulation (Smith et al., 1999), but we find that the distribution of kinasedead LKB1 is indistinguishable from wild type in HeLa S3 cells (data not shown). Here we have identified for the first time a phosphorylation event directed by LKB1 in vivo; LKB1 may either directly phosphorylate PAR1A, or act indirectly via activation of a PAR1A kinase. Unlike HeLa S3 cells, many cell lines express levels of endogenous protein detectable by immunoblotting with an anti-LKB1 antibody (Figure 1a). In these cell lines, the phosphorylation state of epitope-tagged PAR1A is different from that seen in LKB1-deficient HeLa S3 and A549 bronchial carcinoma cells (Figure 1b,c). In COS (Figure 1b) or 293 cells (not shown), ectopically expressed PAR1A is shifted on SDS–PAGE (80 and 82 kDa) even in the absence of coexpressed LKB1, implying that PAR1A is phosphorylated by an endogenous kinase. By contrast, in LKB1-deficient HeLa S3 and A549 cells, transfected PAR1A migrates as the 78 kDa hypophosphorylated protein species. Thus, PAR1A is phosphorylated in cell lines that express endogenous LKB1, but not in LKB1-deficient cells, suggesting that endogenous LKB1 directs PAR1A phosphorylation. The sites of PAR1A phosphorylation downstream of LKB1 were mapped using site-directed mutagenesis. The regulation of the PAR1A protein used in these studies has not been well characterized and no phosphorylation sites have previously been identified. However, the STKs MARK1 and MARK2 are closely Oncogene

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kinase has partial activity against PAR1A. Cotransfection of LKB1 significantly augments this phosphorylation (Figures 1b and 2b). By contrast, wild-type PAR1A expressed in COS cells does not require coexpression of LKB1 for mobility shift to occur (Figures 1b and 2c). This shift in COS cells is abolished by mutation of T211 and S215 to alanine in PAR1A (Figure 2c), implicating the mutated residues as sites of phosphorylation by endogenous LKB1. Phosphorylation at sites in the activation segment produces a marked increase in the activity of most kinases (Johnson et al., 1996). The effect on PAR1A activity of mutations at the identified phosphorylation sites was therefore investigated. Wild-type PAR1A and the AA mutant were transiently expressed in COS cells; in these cells wild-type PAR1A, but not the AA mutant, is phosphorylated at activation segment sites by an endogenous kinase, probably LKB1 (Figure 2c). PAR1A was immunoprecipitated and recovery of protein assessed by immunoblotting (Figure 3a). Kinase activity was then estimated in an in vitro assay in the presence of 32P-ATP (Figure 3b). The substrate used was

Figure 2 LKB1-directed phosphorylation of PAR1A occurs in the activation segment. (a) PAR1A is aligned with part of the activation segment of the similar proteins MARK1 and MARK2. MARKs 1 and 2 are identical within this sequence and are known to be phosphorylated at amino acids indicated by * (Drewes et al., 1997). Residue numbers refer to PAR1A sequence. (b) PAR1A mutations to alanine were introduced at threonine 211 and serine 215 (AA mutant). Expression in HeLa S3 cells of this mutant, in the presence of LKB1, abolishes the LKB1-induced mobility shift seen with wild-type PAR1A (compare lanes 2 and 4). The mobility shift is also impaired by single mutation at either threonine 211 or serine 215 (not shown). (c) Mutation to alanine at T211 and S215 abolishes mobility shifts in PAR1A expressed alone in COS cells, implying that these are also the sites of PAR1A phosphorylation by endogenous LKB1

related to PAR1A and both are phosphorylated in vivo in the activation segment, and this phosphorylation is required for activity (Drewes et al., 1997). Part of the activation segment of PAR1A shares near identity with MARKs 1 and 2, which are themselves identical over this sequence (Figure 2a). This alignment suggested threonine 211 and serine 215 as likely phosphorylation sites on PAR1A. An expression construct encoding PAR1A with the point mutations T211A and S215A (AA mutant) was transfected into HeLa S3 cells alone or together with LKB1. The PAR1A mobility shift is abolished by alteration of threonine 211 and serine 215 to alanine, either together in the AA mutant (Figure 2b), or separately (not shown). Thus, LKB1-directed phosphorylation occurs at these PAR1A residues. Wild-type PAR1A is phosphorylated to a minor extent in the absence of transfected LKB1 (Figures 1 and 2), implying that an endogenous HeLa S3 cell Oncogene

Figure 3 PAR1A mutations at activation loop phosphorylation sites targeted by LKB1 abolish PAR1A kinase activity. (a) COS cells, in which wild-type PAR1A is phosphorylated by endogenous LKB1, were transfected with wild-type PAR1A or the activation segment mutant AA. Cells were lysed (in 10 mm Tris pH 7.6, 1% (v/ v) igepal CA-630, 150 mm NaCl, 50 mm NaF, 1 mm DTT, 5 mm Na4P2O7, 1 mm EDTA, 1  Complete protease inhibitor mix) and PAR1A protein immunoprecipitated with anti-FLAG M2 agarose beads (Sigma). Recovery of the AA PAR1A mutant, assessed by immunoblotting, was at least as good as that of wild type. (b) The PAR1A immunoprecipitates were incubated with recombinant acid-denatured MAP2 substrate using an in vitro kinase assay buffer (40 mm HEPES pH 7.2, 5 mm MgCl2, 2 mm EGTA, 1 mm DTT, 0.1 mm PMSF, 0.03% (v/v) Brij 35). Immunoprecipitate from cells transfected with vector alone (‘mock’) was included as a control. Incorporation of 32P-phosphate (means7s.e.m. for a representative experiment performed in triplicate) was greatly reduced by PAR1A mutations to alanine at T211 and S215

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Figure 4 Lkb1 antagonizes PAR1A activation of Wnt signalling. (a) Canonical Wnt pathway signalling downstream of Dvl was assayed by transfecting constructs expressing TCF4, b-galactosidase and the FLAG-tagged proteins shown, as well as active and mutated TCF4-luciferase reporters TOPFLASH and FOPFLASH, into HeLa S3 cells as described (Smalley et al., 1999). Activation of TCF4-mediated transcription is expressed as a ratio of TOPFLASH/FOPFLASH luciferase activity. Wild-type PAR1A cooperates with Dvl to activate TCF4-mediated transcription. LKB1 antagonises this effect of PAR1A on Wnt signalling. Protein expression was confirmed by immunoblotting as indicated. Luciferase activity data, normalized for b-galactosidase, are means for triplicates of a representative experiment (7s.e.m.). (b) Signals downstream of Wnt ligand and of LKB1 may compete for PAR1A substrate. Wnt signalling leads to the phosphorylation and activation of PAR1A and hence of Dvl (Sun et al., 2001; left), but PAR1 family members are also known to have substrates unrelated to Wnt signalling (Drewes et al., 1997; Peng et al., 1998; right)

recombinant microtubule-associated protein 2 (MAP2), which is known to be phosphorylated by the kinases

MARK1 and MARK2 (Peng et al., 1998). Mutation at T211A and S215A (AA) abolishes PAR1A kinase activity, suggesting that LKB1-directed phosphorylation at these sites is required for PAR1 activity. PAR1 was recently identified as a kinase responsible for phosphorylation of the Wnt pathway component Dvl during Drosophila development (Sun et al., 2001). Having shown that LKB1 activity can lead to the phosphorylation of PAR1A, we wanted to study the effect of LKB1 on Wnt signalling. Reporter genes under the control of TCF promoters have been used to assay the activity of the canonical Wnt pathway (Korinek et al., 1997). Expression of PAR1A with Dvl and TCF activates transcription from a transfected plasmid containing multimeric TCF-binding sites upstream of a luciferase reporter gene (TOPFLASH), but not from a reporter plasmid containing mutated TCF-binding sites (FOPFLASH) (Sun et al., 2001; Figure 4a). The effect of LKB1 expression on Wnt signalling was investigated using this assay. LKB1 suppressed the ability of PAR1A to stimulate Wnt signalling downstream of Dvl (Figure 4a). In the absence of PAR1A, LKB1 did not inhibit Wnt signalling (Figure 4a). The effect of LKB1 was specific, as STK15, an unrelated serine/threonine kinase (Zhou et al., 1998), did not inhibit PAR1A stimulation of Wnt signalling (not shown). Similar results were seen in HeLa S3 cells, a cell line lacking endogenous LKB1. These results are consistent with a model in which the tumour suppressor LKB1 competes with Dvl for PAR1A, redirecting PAR1A from Wnt activation to alternative substrates, so downregulating Wnt signalling (Figure 4b). These alternative PAR1A substrates may include MAPs, which are phosphorylated by other PAR1 family members (Drewes et al., 1997). LKB1 appears to act upstream of PAR1A in a kinase cascade, and we have determined the sites at which PAR1A is phosphorylated downstream of LKB1. These sites are required for PAR1A activity, but LKB1 inhibits PAR1A-mediated stimulation of Wnt signalling. LKB1 may act as a molecular switch, redirecting the activity of PAR1A kinase to alternative substrates. Thus, the pathogenesis of PJS may involve dysregulation of the Wnt signalling pathway, as suggested by upregulation of nuclear bcatenin in PJS tumours (Back et al., 1999).

Abbreviations EST, expressed sequence tag; MAP, microtubule-associated protein; MARK, MAP/microtubule affinity-regulating kinase; PJS, Peutz–Jeghers syndrome; STK, serine/threonine kinase. Acknowledgements JS was the recipient of an Institute of Cancer Research clinical research fellowship. This work was supported by Cancer Research UK and Breakthrough Breast Cancer. We thank Trevor Dale, Institute of Cancer Research, for providing reagents for the reporter assay.

Oncogene

Regulation of the Wnt singnalling component PAR1A J Spicer et al

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