MINT MINT, Dpt of Biology, University of Rome Tor Vergata MINT http://mint.bio.uniroma2.it/mint url>http://mint.bio.uniroma2.it/mint/ https://mint.bio.uniroma2.it MINT-[0-9]+ https://mint.bio.uniroma2.it/index.php/results-interactions/?id=${ac} The Molecular INTeraction database (MINT) is a relational database designed to store interactions between biological molecules. http://mint.bio.uniroma2.it Tyrosine phosphorylation controls cortactin binding to two enterohaemorrhagic Escherichia coli effectors: Tir and EspFu/TccP. Tyrosine phosphorylation controls cortactin binding to two enterohaemorrhagic Escherichia coli effectors: Tir and EspFu/TccP. Cellular microbiology 2007 imex curation Cantarelli VV., Kodama T., Nijstad N., Abolghait SK., Nada S., Okada M., Iida T., Honda T. vcantarelli@weinmann.com.br 04-05-20: Contacted by IntAct-Help. Only protein-protein interactions imex curation mouse-fibroblast Mus musculus fibroblast Fibroblast fluorescence imaging fluorescence microscopy fluorescence imaging immunostaining immunostaining Cantarelli VV., Kodama T., Nijstad N., Abolghait SK., Nada S., Okada M., Iida T., Honda T. vcantarelli@weinmann.com.br Accepted 2020-JAN-15 AT 17:35 GMT AT 17:35 GMT by LUANA Cellular microbiology 2007 Only protein-protein interactions imex curation imex curation Tyrosine phosphorylation controls cortactin binding to two enterohaemorrhagic Escherichia coli effectors: Tir and EspFu/TccP. Tyrosine phosphorylation controls cortactin binding to two enterohaemorrhagic Escherichia coli effectors: Tir and EspFu/TccP. Cellular microbiology 2007 imex curation Cantarelli VV., Kodama T., Nijstad N., Abolghait SK., Nada S., Okada M., Iida T., Honda T. vcantarelli@weinmann.com.br 04-05-20: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro pull down pull down anti tag western anti tag western blot anti tag western Cantarelli VV., Kodama T., Nijstad N., Abolghait SK., Nada S., Okada M., Iida T., Honda T. vcantarelli@weinmann.com.br Accepted 2020-JAN-15 AT 17:35 GMT AT 17:35 GMT by LUANA Cellular microbiology 2007 Only protein-protein interactions imex curation imex curation Tyrosine phosphorylation controls cortactin binding to two enterohaemorrhagic Escherichia coli effectors: Tir and EspFu/TccP. Tyrosine phosphorylation controls cortactin binding to two enterohaemorrhagic Escherichia coli effectors: Tir and EspFu/TccP. Cellular microbiology 2007 imex curation Cantarelli VV., Kodama T., Nijstad N., Abolghait SK., Nada S., Okada M., Iida T., Honda T. vcantarelli@weinmann.com.br 04-05-20: Contacted by IntAct-Help. Only protein-protein interactions imex curation yeast Saccharomyces cerevisiae Baker's yeast 2 hybrid two hybrid 2H 2h Gal4 transcription regeneration two-hybrid yeast two hybrid 2-hybrid classical two hybrid classical two hybrid 2H two-hybrid 2-hybrid Gal4 transcription regeneration 2h yeast two hybrid Y2H Y-2H 2 hybrid predetermined predetermined participant predetermined Cantarelli VV., Kodama T., Nijstad N., Abolghait SK., Nada S., Okada M., Iida T., Honda T. vcantarelli@weinmann.com.br Accepted 2020-JAN-15 AT 17:35 GMT AT 17:35 GMT by LUANA Cellular microbiology 2007 Only protein-protein interactions imex curation imex curation Tyrosine phosphorylation controls cortactin binding to two enterohaemorrhagic Escherichia coli effectors: Tir and EspFu/TccP. Tyrosine phosphorylation controls cortactin binding to two enterohaemorrhagic Escherichia coli effectors: Tir and EspFu/TccP. Cellular microbiology 2007 imex curation Cantarelli VV., Kodama T., Nijstad N., Abolghait SK., Nada S., Okada M., Iida T., Honda T. vcantarelli@weinmann.com.br 04-05-20: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro filter binding filter binding Filter overlay assay Filter overlay assay anti tag western anti tag western blot anti tag western Cantarelli VV., Kodama T., Nijstad N., Abolghait SK., Nada S., Okada M., Iida T., Honda T. vcantarelli@weinmann.com.br Accepted 2020-JAN-15 AT 17:35 GMT AT 17:35 GMT by LUANA Cellular microbiology 2007 Only protein-protein interactions imex curation imex curation Tyrosine phosphorylation controls cortactin binding to two enterohaemorrhagic Escherichia coli effectors: Tir and EspFu/TccP. Tyrosine phosphorylation controls cortactin binding to two enterohaemorrhagic Escherichia coli effectors: Tir and EspFu/TccP. Cellular microbiology 2007 imex curation Cantarelli VV., Kodama T., Nijstad N., Abolghait SK., Nada S., Okada M., Iida T., Honda T. vcantarelli@weinmann.com.br 04-05-20: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Cantarelli VV., Kodama T., Nijstad N., Abolghait SK., Nada S., Okada M., Iida T., Honda T. vcantarelli@weinmann.com.br Accepted 2020-JAN-15 AT 17:35 GMT AT 17:35 GMT by LUANA Cellular microbiology 2007 Only protein-protein interactions imex curation imex curation Tyrosine phosphorylation controls cortactin binding to two enterohaemorrhagic Escherichia coli effectors: Tir and EspFu/TccP. Tyrosine phosphorylation controls cortactin binding to two enterohaemorrhagic Escherichia coli effectors: Tir and EspFu/TccP. Cellular microbiology 2007 imex curation Cantarelli VV., Kodama T., Nijstad N., Abolghait SK., Nada S., Okada M., Iida T., Honda T. vcantarelli@weinmann.com.br 04-05-20: Contacted by IntAct-Help. Only protein-protein interactions imex curation human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 fluorescence imaging fluorescence microscopy fluorescence imaging immunostaining immunostaining Cantarelli VV., Kodama T., Nijstad N., Abolghait SK., Nada S., Okada M., Iida T., Honda T. vcantarelli@weinmann.com.br Accepted 2020-JAN-15 AT 17:35 GMT AT 17:35 GMT by LUANA Cellular microbiology 2007 Only protein-protein interactions imex curation imex curation wasl_rat Neural Wiskott-Aldrich syndrome protein Wasl protein protein rat Rattus norvegicus (Rat) Rat MSSGQQPPRRVTNVGSLLLTPQENESLFSFLGKKCVTMSSAVVQLYAADRNCMWSKKCSGVACLVKDNPQRSYFLRIFDIKDGKLLWEQELYNNFVYNSPRGYFHTFAGDTCQVALNFANEEEAKKFRKAVTDLLGRRQRKSEKRRDAPNGPNLPMATVDIKNPEITTNRFYSSQVNNISHTKEKKKGKAKKKRLTKADIGTPSNFQHIGHVGWDPNTGFDLNNLDPELKNLFDMCGISEAQLKDRETSKVIYDFIEKTGGVEAVKNELRRQAPPPPPPSRGGPPPPPPPPHSSGPPPPPARGRGAPPPPPSRAPTAAPPPPPPSRPGVVVPPPPPNRMYPPPPPALPSSAPSGPPPPPPLSMAGSTAPPPPPPPPPPPGPPPPPGLPSDGDHQVPASSGNKAALLDQIREGAQLKKVEQNSRPVSCSGRDALLDQIRQGIQLKSVSDGQESTPPTPAPTSGIVGALMEVMQKRSKAIHSSDEDEDDDDEEDFQDDDEWED DIP protein O08816 original sequence version: 145 DIP protein O08816 original sequence version: 115 480E21F26F7FC77E src8_mouse Src substrate cortactin Cttn Ems1 protein protein mouse Mus musculus Mouse MWKASAGHAVSITQDDGGADDWETDPDFVNDVSEKEQRWGAKTVQGSGHQEHINIHKLRENVFQEHQTLKEKELETGPKASHGYGGKFGVEQDRMDRSAVGHEYQSKLSKHCSQVDSVRGFGGKFGVQMDRVDQSAVGFEYQGKTEKHASQKDYSSGFGGKYGVQADRVDKSAVGFDYQGKTEKHESQKDYSKGFGGKYGIDKDKVDKSAVGFEYQGKTEKHESQKDYVKGFGGKFGVQTDRQDKCALGWDHQEKLQLHESQKDYKTGFGGKFGVQSERQDSSAVGFDYKERLAKHESQQDYAKGFGGKYGVQKDRMDKNASTFEEVVQVPSAYQKTVPIEAVTSKTSNIRANFENLAKEREQEDRRKAEAERAQRMAKERQEQEEARRKLEEQARAKKQTPPASPSPQPIEDRPPSSPIYEDAAPFKAEPSYRGSEPEPEYSIEAAGIPEAGSQQGLTYTSEPVYETTEAPGHYQAEDDTYDGYESDLGITAIALYDYQAAGDDEISFDPDDIITNIEMIDDGWWRGVCKGRYGLFPANYVELRQ DIP protein Q60598 original sequence version: 136 DIP protein Q60598 original sequence version: 78 DIP protein Q60598 original sequence version: 135 8F5CA026ACCD6D4F espfu_eco57 Secreted effector protein EspF(U) EspF-like protein encoded on prophage U Tir-cytoskeleton coupling protein TccP espF(U) tccP Z3072 protein protein eco57 Escherichia coli O157:H7 MINNVSSLFPTVNRNITAVYKKSSFSVSPQKITLNPVKISSPFSPSSSSISATTLFRAPNAHSASFHRQSTAESSLHQQLPNVRQRLIQHLAEHGIXPARSMAEHIPPAPKWPAPPPPVQNEQSRPLPDVAQRLMQHLAEHGIQPARNMAEHIPPAPNWPAPTPPVQNEQSRPLPDVAQRLMQHLAEHGIQPARNMAEHIPPAPXWXAPTPPVQNEQSRPLPDVAQRLMQHLAEHGIZPARSMAEHIPPAPNWPAPPPPVQNEQSRPLPDVAQRLXQHLAEHGIQPARNMAEHIPPAPNWPAPXXPVXNEQSRPLXDVAXRLMQHLAEHGIQPARNMAEHIPPAPNWXAPTPPVQNEQSRPLPDVAQRLMQHLAEHGINTSKRS F8794D8467780135 src8_human Src substrate cortactin CTTN EMS1 Amplaxin Oncogene EMS1 protein protein human Homo sapiens Human MWKASAGHAVSIAQDDAGADDWETDPDFVNDVSEKEQRWGAKTVQGSGHQEHINIHKLRENVFQEHQTLKEKELETGPKASHGYGGKFGVEQDRMDKSAVGHEYQSKLSKHCSQVDSVRGFGGKFGVQMDRVDQSAVGFEYQGKTEKHASQKDYSSGFGGKYGVQADRVDKSAVGFDYQGKTEKHESQRDYSKGFGGKYGIDKDKVDKSAVGFEYQGKTEKHESQKDYVKGFGGKFGVQTDRQDKCALGWDHQEKLQLHESQKDYKTGFGGKFGVQSERQDSAAVGFDYKEKLAKHESQQDYSKGFGGKYGVQKDRMDKNASTFEDVTQVSSAYQKTVPVEAVTSKTSNIRANFENLAKEKEQEDRRKAEAERAQRMAKERQEQEEARRKLEEQARAKTQTPPVSPAPQPTEERLPSSPVYEDAASFKAELSYRGPVSGTEPEPVYSMEAADYREASSQQGLAYATEAVYESAEAPGHYPAEDSTYDEYENDLGITAVALYDYQAAGDDEISFDPDDIITNIEMIDDGWWRGVCKGRYGLFPANYVELRQ DIP protein Q14247 original sequence version: 148 7799326C2B4383BB tir_eco57 Translocated intimin receptor Tir tir espE Secreted effector protein Tir Z5112 ECs4561 protein protein eco57 Escherichia coli O157:H7 MPIGNLGHNPNVNNSIPPAPPLPSQTDGAGGRGQLINSTGPLGSRALFTPVRNSMADSGDNRASDVPGLPVNPMRLAASEITLNDGFEVLHDHGPLDTLNRQIGSSVFRVETQEDGKHIAVGQRNGVETSVVLSDQEYARLQSIDPEGKDKFVFTGGRGGAGHAMVTVASDITEARQRILELLEPKGTGESKGAGESKGVGELRESNSGAENTTETQTSTSTSSLRSDPKLWLALGTVATGLIGLAATGIVQALALTPEPDSPTTTDPDAAASATETATRDQLTKEAFQNPDNQKVNIDELGNAIPSGVLKDDVVANIEEQAKAAGEEAKQQAIENNAQAQKKYDEQQAKRQEELKVSSGAGYGLSGALILGGGIGVAVTAALHRKNQPVEQTTTTTTTTTTTSARTVENKPANNTPAQGNVDTPGSEDTMESRRSSMASTSSTFFDTSSIGTVQNPYADVKTSLHDSQVPTSNSNTSVQNMGNTDSVVYSTIQHPPRDTTDNGARLLGNPSAGIQSTYARLALSGGLRHDMGGLTGGSNSAVNTSNNPPAPGSHRFV 99C417222D4B4AA1 cttn-espf(u)-1 4 9 unspecified role unspecified role bait bait purified purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range myc tag myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined eco57 Escherichia coli O157:H7 8 unspecified role unspecified role prey prey purified purified gst tag gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined sh3 domain binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) direct interaction direct interaction fig. 4a left espf(u)-cttn-1 2 9 unspecified role unspecified role prey prey purified purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range myc tag myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined eco57 Escherichia coli O157:H7 8 unspecified role unspecified role bait bait purified purified gst tag gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined sh3 domain binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain Q60598:p.[Tyr421Asp;Tyr466Asp] mutation increasing mutation increasing interaction mutation increasing certain certain sequence position certain certain certain sequence position certain Y D certain certain sequence position certain certain certain sequence position certain Y D Mon Dec 16 11:28:44 GMT 2019 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[tyr421asp;tyr466asp]' Tue Jun 30 19:42:45 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Tyr421Asp;Tyr466Asp]' Q60598:p.[Ser405Asp;Ser418Asp] mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain S D certain certain sequence position certain certain certain sequence position certain S D Mon Dec 16 11:44:16 GMT 2019 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[ser405asp;ser418asp]' Tue Jun 30 19:14:56 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Ser405Asp;Ser418Asp]' ecoli Escherichia coli (strain K12) direct interaction direct interaction fig. 4a centre espf(u)-cttn-2 5 9 unspecified role unspecified role bait bait purified purified myc tag myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range 8 unspecified role unspecified role prey prey cell lysate cell lysate physiological level physiological level endogenous level endogenous endogenous endogenous level physical association physical association fig. 4b To confirm these findings further, we used lysates obtained from HeLa cells and D5 cells supplemented with purified His‐EspFu‐myc. HeLa cell lysates contain very low levels of tyrosine‐phosphorylated cortactin, in contrast to D5 lysates. As shown in Fig. 4B, tyrosine‐phosphorylated cortactin co‐immunoprecipitates with His‐EspFu‐myc from D5 cells lysates, and no cortactin was co‐immunoprecipitated from the HeLa cells lysates, strongly suggesting that EspFu specifically binds to the tyrosine‐phosphorylated form of cortactin. cttn-espf(u)-2 6 10 unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level fixed cell fixed cell 9 anti tag immunost anti tag immunostaining anti tag immunost immunostaining immunostaining unspecified role unspecified role neutral component neutral component infection infection fixed cell fixed cell flag tag flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined colocalization colocalization fig. 4c Infection of HeLa cells with DMEM‐activated EHEC expressing FLAG‐tagged EspFu (as in Fig. 2C and D) resulted in a perfect colocalization of EspFu‐FLAG and tyrosine‐phosphorylated cortactin, which was visible 45 min after infection (Fig. 4C). As the infection proceeded (120 min), cortactin was dephosphorylated and only EspFu‐FLAG was detected near the bacteria. cttn-wasl-2 3 8 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed SH3 domain binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain Q60598:p.[Tyr421Asp;Tyr466Asp] mutation increasing mutation increasing interaction mutation increasing certain certain sequence position certain certain certain sequence position certain Y D certain certain sequence position certain certain certain sequence position certain Y D Mon Dec 16 11:10:52 GMT 2019 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[tyr421asp; tyr466asp]' Tue Jun 30 20:19:13 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Tyr421Asp;Tyr466Asp]' Q60598:p.Trp525Lys mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain W K Mon Dec 16 11:21:51 GMT 2019 This feature has been corrected as a result of our quality control procedures. The original label was 'p-trp525lys' Tue Jun 30 16:58:10 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Trp525Lys' Q60598:p.[Ser405Asp;Ser418Asp] mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain S D certain certain sequence position certain certain certain sequence position certain S D Mon Dec 16 11:08:41 GMT 2019 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[ser405asp;ser418asp]' Tue Jun 30 17:53:06 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Ser405Asp;Ser418Asp]' 7 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed physical association physical association fig. 4d To further support the results from our Y2H assay, we decided to test the interaction of our cortactin constructs with N‐WASP. N‐WASP specifically interacted with the C‐terminus, but not with the N‐terminus of cortactin (Fig. 4D, grey bars). More importantly, N‐WASP was able to interact with Cort S/D, mimicking serine phosphorylation, but was almost completely unable to interact with the wild type or the construct mimicking tyrosine phosphorylation (Cort Y/D). cttn-espf(u)-3 3 8 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed SH3 domain binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain Q60598:p.[Tyr421Asp;Tyr466Asp] mutation increasing mutation increasing interaction mutation increasing certain certain sequence position certain certain certain sequence position certain Y D certain certain sequence position certain certain certain sequence position certain Y D Mon Dec 16 11:57:44 GMT 2019 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[tyr421asp; tyr466asp]' Tue Jun 30 16:37:12 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Tyr421Asp;Tyr466Asp]' Q60598:p.[Ser405Asp;Ser418Asp] mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain S D certain certain sequence position certain certain certain sequence position certain S D Mon Dec 16 11:25:23 GMT 2019 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[ser405asp;ser418asp]' Tue Jun 30 18:00:35 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Ser405Asp;Ser418Asp]' Q60598:p.Trp525Lys mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain W K Mon Dec 16 11:57:11 GMT 2019 This feature has been corrected as a result of our quality control procedures. The original label was 'p.trp525lys' Tue Jun 30 18:48:01 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Trp525Lys' 9 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed physical association physical association fig. 4d the Y2H assay confirmed the interaction between EspFu and N‐WASP (top, white bar), as well as between EspFu and the C‐terminus of cortactin (black bars). More importantly, in accordance with the previous results, our Y2H assay showed a strong interaction between EspFu and Cort Y/D form, as compared with the wild‐type or Cort S/D mutant. wasl-espf(u)-3 3 7 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed 9 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed physical association physical association fig. 4d the Y2H assay confirmed the interaction between EspFu and N‐WASP (top, white bar), as well as between EspFu and the C‐terminus of cortactin (black bars). More importantly, in accordance with the previous results, our Y2H assay showed a strong interaction between EspFu and Cort Y/D form, as compared with the wild‐type or Cort S/D mutant. espf(u)-cttn-3 4 8 unspecified role unspecified role prey prey purified purified ha tag ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 11 unspecified role unspecified role bait bait purified purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined binding region biological feature biological feature certain certain sequence position certain certain certain sequence position certain eco57 Escherichia coli O157:H7 direct interaction direct interaction fig. 5a 1-138 5 8 unspecified role unspecified role unspecified role unspecified role cell lysate cell lysate physiological level physiological level endogenous level endogenous endogenous endogenous level phosrestyr421 phosres phosphorylated residue PhosRes Phospho Phosphorylation Phosphorylation (O of Serine, Threonine, Tyrosine and Aspartate, N epsilon of Lysine) certain certain sequence position certain certain certain sequence position certain observed-ptm observed-ptm observed ptm observed-ptm 11 unspecified role unspecified role bait bait purified purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined binding region binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain eco57 Escherichia coli O157:H7 physical association physical association fig. 5b espf(u)-cttn-4 4 11 unspecified role unspecified role bait bait purified purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range binding region binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain eco57 Escherichia coli O157:H7 8 unspecified role unspecified role prey prey purified purified gst tag gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined sh3 domain binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain Q60598:p.[Tyr421Asp;Tyr466Asp] mutation increasing mutation increasing interaction mutation increasing certain certain sequence position certain certain certain sequence position certain Y D certain certain sequence position certain certain certain sequence position certain Y D Mon Dec 16 11:18:07 GMT 2019 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[tyr421asp;tyr466asp]' Tue Jun 30 17:55:41 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Tyr421Asp;Tyr466Asp]' Q60598:p.[Ser405Asp;Ser418Asp] mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain S D certain certain sequence position certain certain certain sequence position certain S D Tue Jun 30 20:21:58 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Ser405Asp;Ser418Asp]' ecoli Escherichia coli (strain K12) direct interaction direct interaction fig. 5c we performed a gel overlay assay using several GST‐tagged cortactin constructs immobilized onto PVDF membranes. His‐Tir‐Nt was added as the probe and was found to bind weakly to cortactin wild type or the S/D form (Fig. 5C). Compared with that, the binding of His‐Tir‐Nt to Cort Y/D seemed to be only slightly improved (Fig. 5C, Y/D). Then, we decided to phosphorylate the GST‐Cort wild type in vitro using recombinant c‐Src. As shown in Fig. 5C (wild‐pY), His‐Tir‐Nt reacted strongly with this construct, confirming that tyrosine phosphorylation of cortactin is required to bind the N‐terminus of Tir from EHEC. cttn-1 3 11 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed binding region binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain 8 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed SH3 domain binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain Q60598:p.[Tyr421Asp;Tyr466Asp] mutation increasing mutation increasing interaction mutation increasing certain certain sequence position certain certain certain sequence position certain Y D certain certain sequence position certain certain certain sequence position certain Y D Mon Dec 16 10:37:39 GMT 2019 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[tyr421asp; tyr466asp]' Tue Jun 30 19:58:10 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Tyr421Asp;Tyr466Asp]' Q60598:p.[Ser405Asp;Ser418Asp] mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain S D certain certain sequence position certain certain certain sequence position certain S D Mon Dec 16 11:30:17 GMT 2019 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[ser405asp;ser418asp]' Tue Jun 30 18:42:41 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Ser405Asp;Ser418Asp]' physical association physical association fig. 5d the Y2H assay confirmed the interaction between EspFu and N‐WASP (top, white bar), as well as between EspFu and the C‐terminus of cortactin (black bars). More importantly, in accordance with the previous results, our Y2H assay showed a strong interaction between EspFu and Cort Y/D form, as compared with the wild‐type or Cort S/D mutant. cttn-tir-3 6 10 unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level fixed cell fixed cell 11 unspecified role unspecified role neutral component neutral component infection infection fixed cell fixed cell myc tag myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined colocalization colocalization fig. 5e Furthermore, using immunofluorescence, Tir and tyrosine‐phosphorylated cortactin were found to colocalize beneath the attached bacteria in HeLa cells infected with DMEM‐activated EHEC for 45 min (Fig. 5E). Accordingly, due to the cortactin dephosphorylation, only Tir was detected in HeLa cells infected with EHEC for 120 min. Infection of HeLa cells with DMEM‐activated EHEC Δtir resulted in a complete failure to induce tyrosine phosphorylation of cortactin, as well as actin assembly (Fig. 5E, Δtir). cttn-espf(u)-4 1 9 anti tag immunost anti tag immunostaining anti tag immunost immunostaining immunostaining unspecified role unspecified role neutral component neutral component fixed cell fixed cell flag tag flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 8 unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level fixed cell fixed cell colocalization colocalization suppl fig 1b when expressed in non‐infected D5 cells, EspFu‐FLAG was found to accumulate in tyrosine‐phosphorylated cortactin‐rich structures (Fig. S1B), again supporting the idea that EspFu associates with tyrosine‐phosphorylated cortactin inside the host cells. cttn-espf(u)-5 1 8 unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level fixed cell fixed cell 9 anti tag immunost anti tag immunostaining anti tag immunost immunostaining immunostaining unspecified role unspecified role neutral component neutral component infection infection fixed cell fixed cell gfp tag gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined colocalization colocalization supll fig 2b Infection of HeLa cells with DMEM‐activated EHEC expressing FLAG‐tagged EspFu (as in Fig. 2C and D) resulted in a perfect colocalization of EspFu‐FLAG and tyrosine‐phosphorylated cortactin, which was visible 45 min after infection (Fig. 4C). As the infection proceeded (120 min), cortactin was dephosphorylated and only EspFu‐FLAG was detected near the bacteria.