SUPPLEMENTARY INFORMATION

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1 doi: /nature05765 SUPPLEMENTARY INFORMATION Helicobacter pylori targets PAR1/MARK kinase to disrupt epithelial cell polarity Iraj Saadat 1, Hideaki Higashi 1, Chikashi Obuse 2, Mayumi Umeda 1, Naoko Murata-Kamiya 1, Yasuhiro Saito 1, Huaisheng Lu 1, Naomi Ohnishi 1, Takeshi Azuma 3, Atsushi Suzuki 4, Shigeo Ohno 4 & Masanori Hatakeyama 1 1 Division of Molecular Oncology, Institute for Genetic Medicine and Division of Chemistry, Graduate School of Science, Hokkaido University, Sapporo , Japan. 2 Faculty of Advanced Life Science, Hokkaido University, Sapporo , Japan. 3 Internal Center for Medical Research and Treatment, Kobe University Graduate School of Medicine, Kobe , Japan. 4 Department of Molecular Biology, Yokohama City University Graduate School of Medical Science, Yokohama , Japan. CONTENT Supplementary Figures (13) Supplementary table (1) References to supplementary information 1

2 Supplementary Figure 1 a Merge ZO-1 x-y (Apical) x-y (Middle) 3D (Downward) 3D (Upward) Merge ZO-1 b y-z x-z caga-positive H. pylori c Merge E- adherin Merge gp135 phalloidin Supplementary Figure 1 Mislocalization of ZO-1, E-cadherin and gp135 by. a, Confocal views of ZO-1 localization in MDCK cells expressing at 24 h after transfection of a vector. b, Compared with control, gastric mucosa infected with caga-positive H. pylori showed reduced ZO-1 staining at the tight junctions (ZO-1 ring), which was concomitantly associated with diffuse staining of ZO-1 along the lateral membranes. Informed consent was obtained from each patient after description of the nature and protocol of the study. c, Confocal x-z views of E-cadherin (upper) or gp135 (lower) localization in MDCK cells expressing at 24 h after transfection of a vector. Scale bar, 10 µm. 2

3 Supplementary Figure 2 -: PR Supplementary Figure 2 PR is not phosphorylated in MDCK cells. MDCK cells were transiently transfected with an expression vector for - or PR - in which all of the tyrosine residues that constitute EPIYA motifs were replaced by alanine residues 1. Cell lysates prepared were immunoblotted with an anti- or anti-phosphotyrosine antibody. 3

4 Supplementary Figure 3 ABCCC Supplementary Figure 3 Subcellular localization of ABCCC in non-polarized MDCK cells. Non-polarized MDCK cells were transfected with an expression vector for - or ABCCC - and were stained with an anti- antibody. Confocal x-y, x-z and y-z sectional views are shown. Scale bar, 10 µm. 4

5 Supplementary Figure 4 Supplementary Figure 4 Extrusion of -positive cells from polarized MDCK monolayer. Polarized MDCK cells were transfected with an expression vector for - and were stained with an anti- antibody (green). Confocal x-z views of stained cells are shown. -positive cells with bowlshaped morphology, which showed ZO-1, E-cadherin and gp135 mislocalizations, were extruded from the MDCK monolayer and were localized on top of the monolayer without showing any sign of apoptosis. Nuclei were stained with DAPI. Scale bar, 10 µm. 5

6 Supplementary Figure 5 MSSARTPLPT LNERDTEQPT LGHLDSKPSS KSNMIRGRNS ATSADEQPHI 50 GNYRLLKTIG KGNFAKVKLA RHILTGKEVA VKIIDKTQLN SSSLQKLFRE 100 VRIMKVLNHP NIVKLFEVIE TEKTLYLVME YASGGEVFDY LVAHGRMKEK 150 EARAKFRQIV SAVQYCHQKF IVHRDLKAEN LLLDADMNIK IADFGFSNEF 200 TFGNKLDTFC GSPPYAAPEL FQGKKYDGPE VDVWSLGVIL YTLVSGSLPF 250 DGQNLKELRE RVLRGKYRIP FYMSTDCENL LKKFLILNPS KRGTLEQIMK 300 DRWMNVGHED DELKPYVEPL PDYKDPRRTE LMVSMGYTRE EIQDSLVGQR 350 YNEVMATYLL LGYKSSELEG DTITLKPRPS ADLTNSSAPS PSHKVQRSVS 400 ANPKQRRFSD QAAGPAIPTS NSYSKKTQSN NAENKRPEED RESGRKASST 450 AKVPASPLPG LERKKTTPTP STNSVLSTST NRSRNSPLLE RASLGQASIQ 500 NGKDSLTMPG SRASTASASA AVSAARPRQH QKSMSASVHP NKASGLPPTE 550 SNCEVPRPST APQRVPVASP SAHNISSSGG APDRTNFPRG VSSRSTFG 600 QLRQVRDQQN LPYGVTPASP SGHSQGRRGA SGSIFSKFTS KFVRRNLSFR 650 FARRNLNEPE SKDRVETLRP HVVGSGGNDK EKEEFREAKP RSLRFTWSMK 700 TTSSMEPNEM MREIRKVLDA NSCQSELHEK YMLLCMHGTP GHEDFVQWEM 750 EVCKLPRLSL NGVRFKRISG TSMAFKNIAS KIANELKL 788 Supplementary Figure 5 Identification of PAR1b as a human protein that specifically binds to ABCCs but not ABCCC by mass spectrometric analysis. LC/MS/MS spectra of the tryptic peptides identified (lower panels) are shown in red characters along with the full amino-acid sequences of human PAR1b (ID number Q7KZI7: long splicing form consisting of 788 amino-acid residues), showing all peptides that were sequenced from PAR1b. It should be noted that PAR1b cdna (ID number: NM_017490) used in the present study encodes a short splicing form (745 amino acids). The short splicing form starts from residue 34 and contains two internal deletions, lack of residue 412 and deletion of residues , of the long splicing form. The sequences underlined with blue and green lines are identical to those of human PAR1c/MARK1 and PAR1a/MARK3, respectively. 6

7 Supplementary Figure 6 a c -: PAR1b-T7: ABCCC ABC PR IP: anti- ABCCC ABCC-s EPIYA segments A B C C C A B C PR ABCCC ABCC-s 1009 FPLKRHDKVDDLSKVGRSVSPEPIYATIDDLGGP---FPLKRHDKVDDLSKVGLSRNQELAQKIDNLSQAVSEAKAGFFSN AINRKIDRINKIASAGKGVGGFSGAGRSASPEPIYATIDFDEANQAGFPLRRSAAVNDLSKVGLSREQELTRRIGDLSQAVSEAKTGHFGN 1012 b d ABD IP: anti- -: PAR1b-T7: ABD EPIYA segments A B D A C C C ABD Number of CM ABD A B D ABCC-s : EPIYA motif : CM sequence ABCC e ABD ABD-16AA -: PAR1b-T7: IP: anti- ABD EPIYA segments ABD-16AA A B D : EPIYA motif : CM sequence ABD ABD-16AA f Bound PAR1b (-fold) : ABD ABC ABCCC Number of CM: : PAR1b-T7: ABCC-16AA 1025 IP: anti-t7 ABCC-s ABCC ABCC 16AA : EPIYA motif : CM sequence 0 ABCC-s ABCC ABCC 16AA Supplementary Figure 6 Delineation of region required for PAR1b interaction. a, COS-7 cells were transfected with a T7-tagged PAR1b vector together with - (ABCCC-type Western ) or its mutant vector (see also Fig. 1a). b, Schematic of ABD-type East Asian and its mutants used in this work (upper). COS- 7 cells were transfected with a T7-tagged PAR1b vector together with ABD - or its mutant vector. c, Sequence alignment of the residues of ABCCC-type Western and residues of ABD-type East Asian that are respectively required for the binding of Western and East Asian with PAR1b. The 16-amino-acid sequence boxed in red is the -multimerization (CM) sequence 2. Each of the EPIYA-C segments in Western contains a single CM sequence. Hence, ABC, a major form of Western, possesses two CM sequences, one within the EPIYA-C segment and the other immediately downstream of the EPIYA-C segment, and ABCCC possesses four CM sequences. On the other hand, ABD-type East Asian contains only a single CM sequence immediately downstream of the EPIYA-D segment 2. d, COS-7 cells were transfected with a PAR1b-T7 vector together with the expression vector for the Western mutant with various numbers of CM sequences. e, ABD-16AA was made by specifically deleting the 16-amino-acid CM sequence from East Asian ABD. COS-7 cells were transfected with a PAR1b-T7 vector together with ABD -, ABD-16AA - or control empty vector. (a, b, d, e) Total cell lysates () were subjected to immunoprecipitation (IP) with an anti- or anti-t7 antibody. The immunoprecipitates were immunoblotted (IB) with the indicated antibodies. f, COS-7 cells were transfected with a T7-tagged PAR1b vector together with ABCCC -, ABC - or ABD - vector. Cell lysates prepared were immunoprecipitated with an anti-t7 antibody and the immunoprecipitates were immunoblotted with an anti- antibody. Intensities of each protein bands were quantitated by a lumino-image analyzer. Relative amounts of -bound PAR1b were calculated from the immunoblotting data, defining the values in ABD as 1. Error bars are ± s.d. (n=3). 7

8 Supplementary Figure 7 PAR1b-T7 NH2 T7 292 COOH N Catalytic T UBA Spacer Tail PAR1b-T7: -: + IP: anti-t IP: anti-t PAR1b-T7: -: IP: anti-t Supplementary Figure 7 Delineation of PAR1b region required for interaction. COS-7 cells were transfected with the indicated vectors. Numbers shown on each PAR1b mutant represent residue deleted. Total cell lysates () were immunoprecipitated with the respective antibodies and the immunoprecipitates (IP) were immunoblotted (IB) with the indicated antibodies. 8

9 Supplementary Figure 8 a Catalytic domain T domain PAR1b 250 L LNPSKR WMNVGHE ELKP EP PAR1a PAR1c PAR1d LVLNP LVLNP KR KR W W N N GHE E GYE DELKP EELKP EELKP EP EP EP D D ND D RT KR KR KR M M M VM PAR1b PAR1a PAR1c PAR1d UBA domain VSMGYTREEIQDSLVGQRYNEVMATYLLLGYKSSELEG------DTITL- EI YN MATY LLGY E VGMGYSQEEIQESLSKMKYDEITATYLLLGRKSSELDASDSSSSSNLSLA EI KY LLGRK E VTMGFARDEINDALINQKYDEVMATYILLGRKPPEFEGGESLSSGNLCQ- MATY LLGRK E VGMGYTREEIKESLTSQKYNEVTATYLLLGRKTEEGGDRGAPGLALARVR EI KYN LLGRK E PAR1b S Spacer domain KQRR K PAR1a S KQRRY K PAR1c S KQRR K PAR1d S RQRRH K PAR1b PAR1a PAR1c KTQSNNAENKRPE 404 RSQTSTADSDLK- 446 RPQANSVESEQKE 429 b PAR1d -: PAR1a: IP: IB: anti-par1a anti- IB: anti-par1a RSPTSTGEAELK CCC ABCC s IP: anti- -: PAR1c-Myc: IB: anti-myc IB: anti-myc CCC ABCC-s IP: anti- -: PAR1d: IB: anti-par1d IB: anti-par1d CCC ABCC-s Supplementary Figure 8 Interaction of with PAR1 family kinases. a, Sequences are of human origin. Amino-acid sequences that are conserved among the PAR1 family are boxed in gray. Residues in PAR1b are essential for interaction. b, COS-7 cells were transfected with the or mutant - expression vector together with the PAR1a, Myc-tagged PAR1c or PAR1d expression vector. Total cell lysates () were immunoprecipitated with an anti- antibody and the immunoprecipitates (IP) were immunoblotted (IB) with the indicated antibodies. 9

10 Supplementary Figure 9 a PAR1b-T7 PAR1b-T b PAR1b-T7: tau: IB: anti-phosphotau (Ser-262) IB: anti-tau Supplementary Figure 9 Loss of kinase activity in PAR1b mutant lacking the -binding sequence. a, AGS cells were transfected with an expression vector for T7-tagged PAR1b or its mutant, PAR1b-T , that specifically lacks the -binding sequence (residues 250 to 276) and were stained with an anti-t7 antibody. Both and the mutant PAR1b were localized at the membrane. Scale bar, 10 µm. b, AGS cells were transfected with an expression vector for PAR1b-T7 or PAR1b-T together with a tau expression vector, and cell lysates were immunoblotted with an anti-tau or anti-phosphotau (Ser-262) antibody. 10

11 Supplementary Figure 10 a Dominant-negative PAR1b Merge ZO-1 PAR1b Merge E-cadherin PAR1b b ZO-1 mislocalization (%) : ABCC-16AA ABCC ABCCC Number of CM: Supplementary Figure 10 Involvement of -PAR1 interaction in the mislocalization of ZO-1 by. a, Confocal x-z views of ZO-1 and E-cadherin localization in MDCK cells expressing a dominant negative PAR1b, PAR1b MC 3. Scale bar, 10 µm. b, Percent ratio of ZO-1-mislocalized cells to total MDCK cells expressing indicated construct at 24 h after transfection. Error bars are ± s.d. (n=3). Note that ABCC-16AA - was made from ABCC - by specifically deleting the 16- amino-acid -multimerization (CM) sequence to which PAR1b binds (see Supplementary Fig. 6d, upper panel) 11

12 Supplementary Figure 11 a PAR1b apkc PAR1b PAR1b/aPKC PAR1b/aPKC/ PAR1b T595A/aPKC b d IB: anti-par1b IB: anti-apkc -: PAR1b-T7: anti-apkc normal rabbit IgG IP c IP: anti PAR1b-T7 -: apkc-flag IB: anti-flag IP: anti-t apkc-flag: IB: anti-phospho- PAR1b (Thr-595) IB: anti-phospho- PAR1b (Thr-208) IB: anti-flag Supplementary Figure 11 Effect of on the regulation of PAR1b by apkc. a, AGS cells were transfected with the indicated expression vectors and were stained for PAR1b, apkc and. Scale bar, 10 µm. b, Lysates prepared from AGS cells were immunoprecipitated with an anti-apkc antibody or control normal rabbit IgG and the immunoprecipitates were immunoblotted with an anti-par1b or apkc antibody. The result shows physical interaction between endogenous PAR1b and apkc in AGS cells. c, d, Lysates prepared from COS-7 cells transfected with the indicated vectors were immunoprecipitated with the respective antibodies. Total cell lysates () and immunoprecipitates (IP) were subjected to immunoblotting (IB) with the indicated antibody. 12

13 Supplementary Figure 12 a IP: anti-t7 -: PAR1b-T7: + + IB: anti-shp2 b -Flag: -: PAR1b-T7: IB: anti-flag IB: anti-shp2 IP: anti- Supplementary Figure 12 Effect of PAR1b on the -SHP2 interaction. a, COS-7 cells were cotransfected with - and PAR1b-T7 or control vector. Cell lysates were immunoprecipitated with an anti-t7 antibody and the anti-t7 immunoprecipitates were immunoblotted with the indicated antibodies. b, COS-7 cells were triple transfected with -, -Flag and PAR1b-T7 or control vector. Cell lysates were immunoprecipitated with an anti- antibody and the anti- immunoprecipitates were immunoblotted with the indicated antibodies. 13

14 Supplementary Figure 13 a PAR1 (dimer) -PAR1 interaction phosphorylation -SHP2 interaction Plasma membrane PAR1 PAR1 Src py lateral TJ PAR6 PAR3 Disruption of tight junctions Loss of apical-basolateral polarity PAR6 PAR3 PAR1 multimer apkc ZO-1 PAR1 multimer basal PAR1 P apical py py py SHP2 PAR1 kinase inhibition Impaired tight junctions Disruption of epithelial cell polarity SHP2 activation Hummingbird phenotype Sustained Erk activation b apkc P P SHP2 P P SHP2 ZO-1 P Inhibition of PAR1 kinase Activation of SHP2 Elevated cell motility Hummingbird phenotype in AGS cells TJ Supplementary Figure 13 Biological consequences of -PAR1 interaction. a, A sequential interaction model of with PAR1 and SHP2. PAR1 is present as a multimer, most probably a homodimer, in cells 4. ABC-type Western or ABD-type East Asian binds to the PAR1 dimer via the -multimerization (CM) sequence, which causes dimerization 2. Upon tyrosine phosphorylation by Src family kinases at the EPIYA-C or EPIYA-D segment, the dimerized proteins specifically interact with an SHP2 molecule via the two SH2 domains of SHP2 and thereby aberrantly activate SHP2 phosphatase, a bona-fide human oncoprotein 5. b, Upon complex formation, inhibits PAR1 kinase activity throughout the baso-lateral membrane, thereby inducing loss of apical-basolateral polarity of epithelial cells. Moreover, -associated PAR1 mislocalizes to the tight junctions and apical membrane domain because it does not undergo phosphorylation by apkc. Invasion of PAR1 to the tight junction may also perturb the tight junction function, which further contributes to disruption of epithelial cell polarity. 14

15 Supplementary Table 1 Supplementary Table 1 Proteins that bound to ABCC-s but not ABCCC. no. *IPI no. Mass Total score Peptide matched Annotation 1 IPI Splice Isoform 7 of Serine/threonine-protein kinase PAR1b/MARK2 2 IPI Polyadenylate-binding protein 1 3 IPI ER-resident protein ERdj5 4 IPI ATP-dependent RNA helicase A 5 IPI Splice Isoform 1 of Transcription intermediary factor 1-beta 6 IPI Membrane component, chromosome 11, surface marker 1, isoform 1 7 IPI ELKL motif kinase 2 long form *IPI : International Protein Index 15

16 References to supplementary information 1. Higashi, H. et al. SHP-2 tyrosine phosphatase as an intracellular target of Helicobacter pylori protein. Science 295, (2002). 2. Ren, S., Higashi, H., Lu, H., Azuma, T. & Hatakeyama, M. Structural basis and functional consequence of Helicobacter pylori multimerization in cells. J. Biol. Chem. 281, (2006). 3. Bohm, H., Brinkmann, V., Drab, M., Henske, A. & Kurzchalia, T. V. Mammalian homologues of C. elegans PAR-1 are asymmetrically localized in epithelial cells and may influence their polarity. Curr. Biol. 7, (1997). 4. Panneerselvam, S., Marx, A., Mandelkow, E. M. & Mandelkow, E. Structure of the catalytic and ubiquitin-associated domains of the protein kinase MARK/Par-1. Structure 14, (2006). 5. Hatakeyama, M. Oncogenic mechanisms of the Helicobacter pylori protein, Nat. Rev. Cancer 4, (2004). 16