IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ Structural and Functional Analysis of the GADD34:PP1 eIF2α Phosphatase. Structural and Functional Analysis of the GADD34:PP1 eIF2α Phosphatase. Cell reports 2015 imex curation Choy MS., Yusoff P., Lee IC., Newton JC., Goh CW., Page R., Shenolikar S., Peti W. shirish.shenolikar@duke-nus.edu.sg;wolfgang_peti@brown.edu 24-May-2021: Contacted by IntAct-Help. Only protein-protein interactions imex curation chlae-cos_7 Cercopithecus aethiops simian cells transformed with SV40 Grivet cos_7 cercopithecus aethiops, by transformation with SV40 bifc bimolecular fluorescence complementation bifc western blot western blot Immuno blot Immuno blot Choy MS., Yusoff P., Lee IC., Newton JC., Goh CW., Page R., Shenolikar S., Peti W. shirish.shenolikar@duke-nus.edu.sg;wolfgang_peti@brown.edu Accepted 2021-APR-27 AT 09:24 BST AT 09:24 BST by PPORRAS Cell reports 2015 Only protein-protein interactions imex curation imex curation Structural and Functional Analysis of the GADD34:PP1 eIF2α Phosphatase. Structural and Functional Analysis of the GADD34:PP1 eIF2α Phosphatase. Cell reports 2015 imex curation Choy MS., Yusoff P., Lee IC., Newton JC., Goh CW., Page R., Shenolikar S., Peti W. shirish.shenolikar@duke-nus.edu.sg;wolfgang_peti@brown.edu 24-May-2021: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro fps fluorescence polarization spectroscopy Fluorescence anisotropy FPS FPS Fluorescence anisotropy fps predetermined predetermined participant predetermined Choy MS., Yusoff P., Lee IC., Newton JC., Goh CW., Page R., Shenolikar S., Peti W. shirish.shenolikar@duke-nus.edu.sg;wolfgang_peti@brown.edu Accepted 2021-APR-27 AT 09:24 BST AT 09:24 BST by PPORRAS Cell reports 2015 Only protein-protein interactions imex curation imex curation Structural and Functional Analysis of the GADD34:PP1 eIF2α Phosphatase. Structural and Functional Analysis of the GADD34:PP1 eIF2α Phosphatase. Cell reports 2015 imex curation Choy MS., Yusoff P., Lee IC., Newton JC., Goh CW., Page R., Shenolikar S., Peti W. shirish.shenolikar@duke-nus.edu.sg;wolfgang_peti@brown.edu 24-May-2021: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro itc isothermal titration calorimetry ITC ITC itc predetermined predetermined participant predetermined Choy MS., Yusoff P., Lee IC., Newton JC., Goh CW., Page R., Shenolikar S., Peti W. shirish.shenolikar@duke-nus.edu.sg;wolfgang_peti@brown.edu Accepted 2021-APR-27 AT 09:24 BST AT 09:24 BST by PPORRAS Cell reports 2015 Only protein-protein interactions imex curation imex curation Structural and Functional Analysis of the GADD34:PP1 eIF2α Phosphatase. Structural and Functional Analysis of the GADD34:PP1 eIF2α Phosphatase. Cell reports 2015 imex curation Choy MS., Yusoff P., Lee IC., Newton JC., Goh CW., Page R., Shenolikar S., Peti W. shirish.shenolikar@duke-nus.edu.sg;wolfgang_peti@brown.edu 24-May-2021: Contacted by IntAct-Help. Only protein-protein interactions imex curation human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Choy MS., Yusoff P., Lee IC., Newton JC., Goh CW., Page R., Shenolikar S., Peti W. shirish.shenolikar@duke-nus.edu.sg;wolfgang_peti@brown.edu Accepted 2021-APR-27 AT 09:24 BST AT 09:24 BST by PPORRAS Cell reports 2015 Only protein-protein interactions imex curation imex curation Structural and Functional Analysis of the GADD34:PP1 eIF2α Phosphatase. Structural and Functional Analysis of the GADD34:PP1 eIF2α Phosphatase. Cell reports 2015 imex curation Choy MS., Yusoff P., Lee IC., Newton JC., Goh CW., Page R., Shenolikar S., Peti W. shirish.shenolikar@duke-nus.edu.sg;wolfgang_peti@brown.edu 24-May-2021: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro phosphatase assay phosphatase assay western blot western blot Immuno blot Immuno blot Choy MS., Yusoff P., Lee IC., Newton JC., Goh CW., Page R., Shenolikar S., Peti W. shirish.shenolikar@duke-nus.edu.sg;wolfgang_peti@brown.edu Accepted 2021-APR-27 AT 09:24 BST AT 09:24 BST by PPORRAS Cell reports 2015 Only protein-protein interactions imex curation imex curation Structural and Functional Analysis of the GADD34:PP1 eIF2α Phosphatase. Structural and Functional Analysis of the GADD34:PP1 eIF2α Phosphatase. Cell reports 2015 imex curation Choy MS., Yusoff P., Lee IC., Newton JC., Goh CW., Page R., Shenolikar S., Peti W. shirish.shenolikar@duke-nus.edu.sg;wolfgang_peti@brown.edu 24-May-2021: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro x-ray diffraction x-ray crystallography X-ray X-ray x-ray diffraction predetermined predetermined participant predetermined Choy MS., Yusoff P., Lee IC., Newton JC., Goh CW., Page R., Shenolikar S., Peti W. shirish.shenolikar@duke-nus.edu.sg;wolfgang_peti@brown.edu Accepted 2021-APR-27 AT 09:24 BST AT 09:24 BST by PPORRAS Cell reports 2015 Only protein-protein interactions imex curation imex curation Structural and Functional Analysis of the GADD34:PP1 eIF2α Phosphatase. Structural and Functional Analysis of the GADD34:PP1 eIF2α Phosphatase. Cell reports 2015 imex curation Choy MS., Yusoff P., Lee IC., Newton JC., Goh CW., Page R., Shenolikar S., Peti W. shirish.shenolikar@duke-nus.edu.sg;wolfgang_peti@brown.edu 24-May-2021: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Choy MS., Yusoff P., Lee IC., Newton JC., Goh CW., Page R., Shenolikar S., Peti W. shirish.shenolikar@duke-nus.edu.sg;wolfgang_peti@brown.edu Accepted 2021-APR-27 AT 09:24 BST AT 09:24 BST by PPORRAS Cell reports 2015 Only protein-protein interactions imex curation imex curation if2a_human Eukaryotic translation initiation factor 2 subunit 1 EIF2S1 EIF2A Eukaryotic translation initiation factor 2 subunit alpha protein protein human Homo sapiens Human MPGLSCRFYQHKFPEVEDVVMVNVRSIAEMGAYVSLLEYNNIEGMILLSELSRRRIRSINKLIRIGRNECVVVIRVDKEKGYIDLSKRRVSPEEAIKCEDKFTKSKTVYSILRHVAEVLEYTKDEQLESLFQRTAWVFDDKYKRPGYGAYDAFKHAVSDPSILDSLDLNEDEREVLINNINRRLTPQAVKIRADIEVACYGYEGIDAVKEALRAGLNCSTENMPIKINLIAPPRYVMTTTTLERTEGLSVLSQAMAVIKEKIEEKRGVFNVQMEPKVVTDTDETELARQMERLERENAEVDGDDDAEEMEAKAED DIP protein P05198 original sequence version: 149 FF3E75E3816E6B1E pp1a_human Serine/threonine-protein phosphatase PP1-alpha catalytic subunit PPP1CA PPP1A protein protein human Homo sapiens Human MSDSEKLNLDSIIGRLLEVQGSRPGKNVQLTENEIRGLCLKSREIFLSQPILLELEAPLKICGDIHGQYYDLLRLFEYGGFPPESNYLFLGDYVDRGKQSLETICLLLAYKIKYPENFFLLRGNHECASINRIYGFYDECKRRYNIKLWKTFTDCFNCLPIAAIVDEKIFCCHGGLSPDLQSMEQIRRIMRPTDVPDQGLLCDLLWSDPDKDVQGWGENDRGVSFTFGAEVVAKFLHKHDLDLICRAHQVVEDGYEFFAKRQLVTLFSAPNYCGEFDNAGAMMSVDETLMCSFQILKPADKNKGKYGQFSGLNPGGRPITPPRNSAKAKK DIP protein P62136 original sequence version: 72 DIP protein P62136 original sequence version: 76 DIP protein P62136 original sequence version: 123 DIP protein P62136 original sequence version: 126 DIP protein P62136-1 original sequence version: 98 60C37E1AD9831DAC pr15a_human Protein phosphatase 1 regulatory subunit 15A Myeloid differentiation primary response protein MyD116 homolog PPP1R15A GADD34 Growth arrest and DNA damage-inducible protein GADD34 protein protein human Homo sapiens Human MAPGQAPHQATPWRDAHPFFLLSPVMGLLSRAWSRLRGLGPLEPWLVEAVKGAALVEAGLEGEARTPLAIPHTPWGRRPEEEAEDSGGPGEDRETLGLKTSSSLPEAWGLLDDDDGMYGEREATSVPRGQGSQFADGQRAPLSPSLLIRTLQGSDKNPGEEKAEEEGVAEEEGVNKFSYPPSHRECCPAVEEEDDEEAVKKEAHRTSTSALSPGSKPSTWVSCPGEEENQATEDKRTERSKGARKTSVSPRSSGSDPRSWEYRSGEASEEKEEKAHKETGKGEAAPGPQSSAPAQRPQLKSWWCQPSDEEEGEVKALGAAEKDGEAECPPCIPPPSAFLKAWVYWPGEDTEEEEDEEEDEDSDSGSDEEEGEAEASSSTPATGVFLKSWVYQPGEDTEEEEDEDSDTGSAEDEREAETSASTPPASAFLKAWVYRPGEDTEEEEDEDVDSEDKEDDSEAALGEAESDPHPSHPDQRAHFRGWGYRPGKETEEEEAAEDWGEAEPCPFRVAIYVPGEKPPPPWAPPRLPLRLQRRLKRPETPTHDPDPETPLKARKVRFSEKVTVHFLAVWAGPAQAARQGPWEQLARDRSRFARRITQAQEELSPCLTPAARARAWARLRNPPLAPIPALTQTLPSSSVPSSPVQTTPLSQAVATPSRSSAAAAAALDLSGRRG B257AA17456D1403 ppp1ca-ppp1r15a-1 6 10 unspecified role unspecified role neutral component neutral component purified purified RVxF motif sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain The RVxF motif containing region has been shown by NMR to be intrinsically disordered, hence it only adopts a structure in the bound conformation. ecobd Escherichia coli (strain B / BL21-DE3) 9 unspecified role unspecified role neutral component neutral component purified purified Full length protein sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecobd Escherichia coli (strain B / BL21-DE3) 15 13 direct interaction direct interaction Fig.2A, B and C 2.28A Rwork = 0.161 Rfree = 0.205 ppp1ca-ppp1r15a-2 3 10 unspecified role unspecified role neutral component neutral component purified purified RVxF motif sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecobd Escherichia coli (strain B / BL21-DE3) 9 unspecified role unspecified role neutral component neutral component purified purified Full length protein sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecobd Escherichia coli (strain B / BL21-DE3) 20 18 direct interaction direct interaction Fig.2D ppp1ca-ppp1r15a-3 2 10 competitor competitor competitor unspecified role unspecified role purified purified RVxF motif sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecobd Escherichia coli (strain B / BL21-DE3) 9 unspecified role unspecified role prey prey purified purified Full length protein sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecobd Escherichia coli (strain B / BL21-DE3) 10 unspecified role unspecified role bait bait RVxF motif (extended) sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain DyLight 488 label fluorescent dye fluorescent dye label fluorescent dye certain certain sequence position certain certain certain sequence position certain ecobd Escherichia coli (strain B / BL21-DE3) 25 23 25 27 physical association physical association Fig.S2D ppp1ca-ppp1r15a-4 7 10 unspecified role unspecified role bait bait purified purified RVxF motif sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain His-tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range ecoli Escherichia coli (strain K12) 9 unspecified role unspecified role prey prey cell lysate cell lysate Full length protein sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. 31 34 physical association physical association Fig.S2H and I Addition of NaCl in high concentration (350-500mM) abolishes the binding. ppp1ca-ppp1r15a-5 4 8 unspecified role unspecified role prey prey cell lysate cell lysate full length sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. 9 unspecified role unspecified role prey prey cell lysate cell lysate full length sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. 10 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed O75807:p.[Val556Ala;Phe558Ala] mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain V A certain certain sequence position certain certain certain sequence position certain F A Tue Jun 30 18:10:50 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Val556Ala;Phe558Ala]' The mutation abolishes binding to PP1alpha (P62136) but not the other binding partner (eIF2alpha, P05198) FLAG tag flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged n-term range n-terminal range n-term range n-term range n-terminal range n-term range PP1 binding region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain eIF2a binding region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain needed for eIF2a binding required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. The two different eIF2a binding regions in GADD34/PR15A represent two different overlapping constructs that were both tested in the experiment. Both are able to bind eIF2a but neither fully contains the other. 37 44 45 37 39 43 physical association physical association Fig.3 ppp1ca-ppp1r15a-6 1 8 unspecified role unspecified role prey prey nucl transfection nucleic acid transfection nucl transfection over-expressed over expressed level over-expressed living cell living cell YC-tag yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined Full length protein sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain chlae-cos_7 Cercopithecus aethiops simian cells transformed with SV40 Grivet cos_7 cercopithecus aethiops, by transformation with SV40 9 unspecified role unspecified role bait bait nucl transfection nucleic acid transfection nucl transfection over-expressed over expressed level over-expressed living cell living cell YN-tag yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined Full length protein sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain chlae-cos_7 Cercopithecus aethiops simian cells transformed with SV40 Grivet cos_7 cercopithecus aethiops, by transformation with SV40 10 unspecified role unspecified role ancillary ancillary nucl transfection nucleic acid transfection nucl transfection over-expressed over expressed level over-expressed living cell living cell RVxF motif sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain O75807:p.[Val556Ala;Phe558Ala] mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain F A certain certain sequence position certain certain certain sequence position certain V A Tue Jun 30 17:56:34 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Val556Ala;Phe558Ala]' chlae-cos_7 Cercopithecus aethiops simian cells transformed with SV40 Grivet cos_7 cercopithecus aethiops, by transformation with SV40 colocalization colocalization Fig.4, Fig.S4 ppp1r15a-ppp1ca-2 2 9 unspecified role unspecified role prey prey purified purified Full length protein sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecobd Escherichia coli (strain B / BL21-DE3) 10 unspecified role unspecified role bait bait RVxF motif (extended) sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain DyLight 488 label fluorescent dye fluorescent dye label fluorescent dye certain certain sequence position certain certain certain sequence position certain ecobd Escherichia coli (strain B / BL21-DE3) 60 58 physical association physical association Fig.S2C The reported Kd value is an approximate value. eif2s1-ppp1ca-1 5 8 enzyme target enzyme target substrate substrate neutral component neutral component purified purified possible PhosRes phosres phosphorylated residue PhosRes Phospho Phosphorylation Phosphorylation (O of Serine, Threonine, Tyrosine and Aspartate, N epsilon of Lysine) undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined observed-ptm observed-ptm observed ptm observed-ptm Binding region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecobd Escherichia coli (strain B / BL21-DE3) 9 unspecified role unspecified role unspecified role unspecified role purified purified Full length protein sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecobd Escherichia coli (strain B / BL21-DE3) 67 65 dephosphorylation dephosphorylation reaction dephosphorylation Fig.S2B