bhf-ucl Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates cardiac sodium channel NaV1.5 gating by multiple phosphorylation sites. Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates cardiac sodium channel NaV1.5 gating by multiple phosphorylation sites. J. Biol. Chem. (0021-9258) 2012 imex curation Ashpole N.M., Herren A.W., Ginsburg K.S., Brogan J.D., Johnson D.E., Cummins T.R., Bers D.M., Hudmon A. Cardiac - Interactions involving cardiac related proteins Methods state: GST-tagged intracellular regions of NaV1.5 (N terminus, L1–L3, and C terminus). Therefore assumed L1 (loop1) is 416 – 711aa based on uniprot record cytoplasmic domain, and this has 594T within L1. ahudmon@iupui.edu 18-Sep-2014: Contacted by IntAct-Help. Only protein-protein interactions in vitro In vitro protein kinase assay protein kinase assay tag visualisation tag visualisation Ashpole N.M., Herren A.W., Ginsburg K.S., Brogan J.D., Johnson D.E., Cummins T.R., Bers D.M., Hudmon A. Cardiac - Interactions involving cardiac related proteins Methods state: GST-tagged intracellular regions of NaV1.5 (N terminus, L1–L3, and C terminus). Therefore assumed L1 (loop1) is 416 – 711aa based on uniprot record cytoplasmic domain, and this has 594T within L1. ahudmon@iupui.edu Accepted 2014-JUL-25 by ORCHARD J. Biol. Chem. (0021-9258) 2012 Only protein-protein interactions imex curation Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates cardiac sodium channel NaV1.5 gating by multiple phosphorylation sites. Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates cardiac sodium channel NaV1.5 gating by multiple phosphorylation sites. J. Biol. Chem. (0021-9258) 2012 imex curation Ashpole N.M., Herren A.W., Ginsburg K.S., Brogan J.D., Johnson D.E., Cummins T.R., Bers D.M., Hudmon A. Cardiac - Interactions involving cardiac related proteins Methods state: GST-tagged intracellular regions of NaV1.5 (N terminus, L1–L3, and C terminus). Therefore assumed L1 (loop1) is 416 – 711aa based on uniprot record cytoplasmic domain, and this has 594T within L1. ahudmon@iupui.edu 18-Sep-2014: Contacted by IntAct-Help. Only protein-protein interactions human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. anti bait coip anti bait coimmunoprecipitation anti bait coip monoclonal western monoclonal antibody western blot monoclonal western Ashpole N.M., Herren A.W., Ginsburg K.S., Brogan J.D., Johnson D.E., Cummins T.R., Bers D.M., Hudmon A. Cardiac - Interactions involving cardiac related proteins Methods state: GST-tagged intracellular regions of NaV1.5 (N terminus, L1–L3, and C terminus). Therefore assumed L1 (loop1) is 416 – 711aa based on uniprot record cytoplasmic domain, and this has 594T within L1. ahudmon@iupui.edu Accepted 2014-JUL-25 by ORCHARD J. Biol. Chem. (0021-9258) 2012 Only protein-protein interactions imex curation Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates cardiac sodium channel NaV1.5 gating by multiple phosphorylation sites. Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates cardiac sodium channel NaV1.5 gating by multiple phosphorylation sites. J. Biol. Chem. (0021-9258) 2012 imex curation Ashpole N.M., Herren A.W., Ginsburg K.S., Brogan J.D., Johnson D.E., Cummins T.R., Bers D.M., Hudmon A. Cardiac - Interactions involving cardiac related proteins Methods state: GST-tagged intracellular regions of NaV1.5 (N terminus, L1–L3, and C terminus). Therefore assumed L1 (loop1) is 416 – 711aa based on uniprot record cytoplasmic domain, and this has 594T within L1. ahudmon@iupui.edu 18-Sep-2014: Contacted by IntAct-Help. Only protein-protein interactions in vitro In vitro protein kinase assay protein kinase assay weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Ashpole N.M., Herren A.W., Ginsburg K.S., Brogan J.D., Johnson D.E., Cummins T.R., Bers D.M., Hudmon A. Cardiac - Interactions involving cardiac related proteins Methods state: GST-tagged intracellular regions of NaV1.5 (N terminus, L1–L3, and C terminus). Therefore assumed L1 (loop1) is 416 – 711aa based on uniprot record cytoplasmic domain, and this has 594T within L1. ahudmon@iupui.edu Accepted 2014-JUL-25 by ORCHARD J. Biol. Chem. (0021-9258) 2012 Only protein-protein interactions imex curation Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates cardiac sodium channel NaV1.5 gating by multiple phosphorylation sites. Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates cardiac sodium channel NaV1.5 gating by multiple phosphorylation sites. J. Biol. Chem. (0021-9258) 2012 imex curation Ashpole N.M., Herren A.W., Ginsburg K.S., Brogan J.D., Johnson D.E., Cummins T.R., Bers D.M., Hudmon A. Cardiac - Interactions involving cardiac related proteins Methods state: GST-tagged intracellular regions of NaV1.5 (N terminus, L1–L3, and C terminus). Therefore assumed L1 (loop1) is 416 – 711aa based on uniprot record cytoplasmic domain, and this has 594T within L1. ahudmon@iupui.edu 18-Sep-2014: Contacted by IntAct-Help. Only protein-protein interactions human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. pull down pull down monoclonal western monoclonal antibody western blot monoclonal western Ashpole N.M., Herren A.W., Ginsburg K.S., Brogan J.D., Johnson D.E., Cummins T.R., Bers D.M., Hudmon A. Cardiac - Interactions involving cardiac related proteins Methods state: GST-tagged intracellular regions of NaV1.5 (N terminus, L1–L3, and C terminus). Therefore assumed L1 (loop1) is 416 – 711aa based on uniprot record cytoplasmic domain, and this has 594T within L1. ahudmon@iupui.edu Accepted 2014-JUL-25 by ORCHARD J. Biol. Chem. (0021-9258) 2012 Only protein-protein interactions imex curation Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates cardiac sodium channel NaV1.5 gating by multiple phosphorylation sites. Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates cardiac sodium channel NaV1.5 gating by multiple phosphorylation sites. J. Biol. Chem. (0021-9258) 2012 imex curation Ashpole N.M., Herren A.W., Ginsburg K.S., Brogan J.D., Johnson D.E., Cummins T.R., Bers D.M., Hudmon A. Cardiac - Interactions involving cardiac related proteins Methods state: GST-tagged intracellular regions of NaV1.5 (N terminus, L1–L3, and C terminus). Therefore assumed L1 (loop1) is 416 – 711aa based on uniprot record cytoplasmic domain, and this has 594T within L1. ahudmon@iupui.edu 18-Sep-2014: Contacted by IntAct-Help. Only protein-protein interactions in vitro In vitro pull down pull down tag fluorescence tag visualisation by fluorescence tag fluorescence Ashpole N.M., Herren A.W., Ginsburg K.S., Brogan J.D., Johnson D.E., Cummins T.R., Bers D.M., Hudmon A. Cardiac - Interactions involving cardiac related proteins Methods state: GST-tagged intracellular regions of NaV1.5 (N terminus, L1–L3, and C terminus). Therefore assumed L1 (loop1) is 416 – 711aa based on uniprot record cytoplasmic domain, and this has 594T within L1. ahudmon@iupui.edu Accepted 2014-JUL-25 by ORCHARD J. Biol. Chem. (0021-9258) 2012 Only protein-protein interactions imex curation Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates cardiac sodium channel NaV1.5 gating by multiple phosphorylation sites. Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates cardiac sodium channel NaV1.5 gating by multiple phosphorylation sites. J. Biol. Chem. (0021-9258) 2012 imex curation Ashpole N.M., Herren A.W., Ginsburg K.S., Brogan J.D., Johnson D.E., Cummins T.R., Bers D.M., Hudmon A. Cardiac - Interactions involving cardiac related proteins Methods state: GST-tagged intracellular regions of NaV1.5 (N terminus, L1–L3, and C terminus). Therefore assumed L1 (loop1) is 416 – 711aa based on uniprot record cytoplasmic domain, and this has 594T within L1. ahudmon@iupui.edu 18-Sep-2014: Contacted by IntAct-Help. Only protein-protein interactions in vitro In vitro protein kinase assay protein kinase assay autoradiography autoradiography Ashpole N.M., Herren A.W., Ginsburg K.S., Brogan J.D., Johnson D.E., Cummins T.R., Bers D.M., Hudmon A. Cardiac - Interactions involving cardiac related proteins Methods state: GST-tagged intracellular regions of NaV1.5 (N terminus, L1–L3, and C terminus). Therefore assumed L1 (loop1) is 416 – 711aa based on uniprot record cytoplasmic domain, and this has 594T within L1. ahudmon@iupui.edu Accepted 2014-JUL-25 by ORCHARD J. Biol. Chem. (0021-9258) 2012 Only protein-protein interactions imex curation kcc2d_human Calcium/calmodulin-dependent protein kinase type II subunit delta CAMK2D CAMKD protein protein human Homo sapiens Human MASTTTCTRFTDEYQLFEELGKGAFSVVRRCMKIPTGQEYAAKIINTKKLSARDHQKLEREARICRLLKHPNIVRLHDSISEEGFHYLVFDLVTGGELFEDIVAREYYSEADASHCIQQILESVNHCHLNGIVHRDLKPENLLLASKSKGAAVKLADFGLAIEVQGDQQAWFGFAGTPGYLSPEVLRKDPYGKPVDMWACGVILYILLVGYPPFWDEDQHRLYQQIKAGAYDFPSPEWDTVTPEAKDLINKMLTINPAKRITASEALKHPWICQRSTVASMMHRQETVDCLKKFNARRKLKGAILTTMLATRNFSAAKSLLKKPDGVKESTESSNTTIEDEDVKARKQEIIKVTEQLIEAINNGDFEAYTKICDPGLTAFEPEALGNLVEGMDFHRFYFENALSKSNKPIHTIILNPHVHLVGDDAACIAYIRLTQYMDGSGMPKTMQSEETRVWHRRDGKWQNVHFHRSGSPTVPIKPPCIPNGKENFSGGTSLWQNI DIP protein Q13557 original sequence version: 123 DIP protein Q13557 original sequence version: 113 62999FBAB98120CE syntide_2_peptide Syntide-2 synthetic peptide peptide peptide polypeptide oligopeptide oligopeptide polypeptide chemical synthesis Chemical synthesis (Chemical synthesis) PLARTLSVAGLPGKK An amino acid sequence which is not represented in UniProt The native peptide PLARTLSVAGLPGKK is a selective Substrate for CaM kinase II and PKC. it is also a poor substrate for phosphorylase kinase and is not phosphorylated by myosin light chain kinase. The sequence of PLARTLSVAGLPGKK is homologous to phosphorylation site 2 in glycogen synthase. CBB20424AD7344D2 scn5a_human Sodium channel protein type 5 subunit alpha Sodium channel protein type V subunit alpha Voltage-gated sodium channel subunit alpha Nav1.5 Sodium channel protein cardiac muscle subunit alpha SCN5A hH1 protein protein human Homo sapiens Human MANFLLPRGTSSFRRFTRESLAAIEKRMAEKQARGSTTLQESREGLPEEEAPRPQLDLQASKKLPDLYGNPPQELIGEPLEDLDPFYSTQKTFIVLNKGKTIFRFSATNALYVLSPFHPIRRAAVKILVHSLFNMLIMCTILTNCVFMAQHDPPPWTKYVEYTFTAIYTFESLVKILARGFCLHAFTFLRDPWNWLDFSVIIMAYTTEFVDLGNVSALRTFRVLRALKTISVISGLKTIVGALIQSVKKLADVMVLTVFCLSVFALIGLQLFMGNLRHKCVRNFTALNGTNGSVEADGLVWESLDLYLSDPENYLLKNGTSDVLLCGNSSDAGTCPEGYRCLKAGENPDHGYTSFDSFAWAFLALFRLMTQDCWERLYQQTLRSAGKIYMIFFMLVIFLGSFYLVNLILAVVAMAYEEQNQATIAETEEKEKRFQEAMEMLKKEHEALTIRGVDTVSRSSLEMSPLAPVNSHERRSKRRKRMSSGTEECGEDRLPKSDSEDGPRAMNHLSLTRGLSRTSMKPRSSRGSIFTFRRRDLGSEADFADDENSTAGESESHHTSLLVPWPLRRTSAQGQPSPGTSAPGHALHGKKNSTVDCNGVVSLLGAGDPEATSPGSHLLRPVMLEHPPDTTTPSEEPGGPQMLTSQAPCVDGFEEPGARQRALSAVSVLTSALEELEESRHKCPPCWNRLAQRYLIWECCPLWMSIKQGVKLVVMDPFTDLTITMCIVLNTLFMALEHYNMTSEFEEMLQVGNLVFTGIFTAEMTFKIIALDPYYYFQQGWNIFDSIIVILSLMELGLSRMSNLSVLRSFRLLRVFKLAKSWPTLNTLIKIIGNSVGALGNLTLVLAIIVFIFAVVGMQLFGKNYSELRDSDSGLLPRWHMMDFFHAFLIIFRILCGEWIETMWDCMEVSGQSLCLLVFLLVMVIGNLVVLNLFLALLLSSFSADNLTAPDEDREMNNLQLALARIQRGLRFVKRTTWDFCCGLLRQRPQKPAALAAQGQLPSCIATPYSPPPPETEKVPPTRKETRFEEGEQPGQGTPGDPEPVCVPIAVAESDTDDQEEDEENSLGTEEESSKQQESQPVSGGPEAPPDSRTWSQVSATASSEAEASASQADWRQQWKAEPQAPGCGETPEDSCSEGSTADMTNTAELLEQIPDLGQDVKDPEDCFTEGCVRRCPCCAVDTTQAPGKVWWRLRKTCYHIVEHSWFETFIIFMILLSSGALAFEDIYLEERKTIKVLLEYADKMFTYVFVLEMLLKWVAYGFKKYFTNAWCWLDFLIVDVSLVSLVANTLGFAEMGPIKSLRTLRALRPLRALSRFEGMRVVVNALVGAIPSIMNVLLVCLIFWLIFSIMGVNLFAGKFGRCINQTEGDLPLNYTIVNNKSQCESLNLTGELYWTKVKVNFDNVGAGYLALLQVATFKGWMDIMYAAVDSRGYEEQPQWEYNLYMYIYFVIFIIFGSFFTLNLFIGVIIDNFNQQKKKLGGQDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPLNKYQGFIFDIVTKQAFDVTIMFLICLNMVTMMVETDDQSPEKINILAKINLLFVAIFTGECIVKLAALRHYYFTNSWNIFDFVVVILSIVGTVLSDIIQKYFFSPTLFRVIRLARIGRILRLIRGAKGIRTLLFALMMSLPALFNIGLLLFLVMFIYSIFGMANFAYVKWEAGIDDMFNFQTFANSMLCLFQITTSAGWDGLLSPILNTGPPYCDPTLPNSNGSRGDCGSPAVGILFFTTYIIISFLIVVNMYIAIILENFSVATEESTEPLSEDDFDMFYEIWEKFDPEATQFIEYSVLSDFADALSEPLRIAKPNQISLINMDLPMVSGDRIHCMDILFAFTKRVLGESGEMDALKIQMEEKFMAANPSKISYEPITTTLRRKHEEVSAMVIQRAFRRHLLQRSLKHASFLFRQQAGSGLSEEDAPEREGLIAYVMSENFSRPLGPPSSSSISSTSFPPSYDSVTRATSDNLQVRGSDYSHSEDLADFPPSPDRDRESIV DIP protein Q14524 original sequence version: 163 DIP protein Q14524 original sequence version: 119 DIP protein Q14524 original sequence version: 138 This sequence has been withdrawn from Uniprot. 841E3A365931190B kcc2a_rat Calcium/calmodulin-dependent protein kinase type II subunit alpha Camk2a protein protein rat Rattus norvegicus (Rat) Rat MATITCTRFTEEYQLFEELGKGAFSVVRRCVKVLAGQEYAAKIINTKKLSARDHQKLEREARICRLLKHPNIVRLHDSISEEGHHYLIFDLVTGGELFEDIVAREYYSEADASHCIQQILEAVLHCHQMGVVHRDLKPENLLLASKLKGAAVKLADFGLAIEVEGEQQAWFGFAGTPGYLSPEVLRKDPYGKPVDLWACGVILYILLVGYPPFWDEDQHRLYQQIKAGAYDFPSPEWDTVTPEAKDLINKMLTINPSKRITAAEALKHPWISHRSTVASCMHRQETVDCLKKFNARRKLKGAILTTMLATRNFSGGKSGGNKKNDGVKESSESTNTTIEDEDTKVRKQEIIKVTEQLIEAISNGDFESYTKMCDPGMTAFEPEALGNLVEGLDFHRFYFENLWSRNSKPVHTTILNPHIHLMGDESACIAYIRITQYLDAGGIPRTAQSEETRVWHRRDGKWQIVHFHRSGAPSVLPH DIP protein P11275 original sequence version: 135 DIP protein P11275 original sequence version: 82 DIP protein P11275 original sequence version: 134 DIP protein P11275 original sequence version: 117 306F416CCE9B5F62 camk2g-scn5a 3 NaV1.5 9 enzyme target enzyme target substrate substrate unspecified role unspecified role loop l1 sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region 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 ecoli Escherichia coli (strain K12) deltaC-CaMKII 7 enzyme enzyme unspecified role unspecified role region 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 spofr-sf_9 Spodoptera frugiperda insect cells Fall armyworm sf_9 insect cells Derived from pupal ovarian tissue of the fall armyworm Cloned from the parent cell line, IPLB-SF-21-AE (SF-21) derived from immature ovaries of fall armyworm Spodoptera frugiperda pupae cells are susceptible to Baculovirus infection phosphorylation phosphorylation reaction phosphorylation Fig 1C Species from figure legend: bacterially expressed GST fusion proteins of intracellular hNaV1.5 regions were exposed to activated δC-CaMKII with [γ-32P]ATP and methods: GST-tagged intracellular regions of NaV1.5 (N terminus, L1–L3, and C terminus) as well as GST-tagged mutants of the L1 were expressed in Bl-21 cells (these are ecoli) scn5a-camk2d 5 deltaC-CaMKII 7 unspecified role unspecified role prey prey purified purified required autophosphorylation nphosres N-phosphorylated residue NPhosRes undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined autophosphorylation region fluorescent dye fluorescent dye label fluorescent dye undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region 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 spofr-sf_9 Spodoptera frugiperda insect cells Fall armyworm sf_9 insect cells Derived from pupal ovarian tissue of the fall armyworm Cloned from the parent cell line, IPLB-SF-21-AE (SF-21) derived from immature ovaries of fall armyworm Spodoptera frugiperda pupae cells are susceptible to Baculovirus infection NaV1.5 9 monoclonal western monoclonal antibody western blot monoclonal western tag fluorescence tag visualisation by fluorescence tag fluorescence unspecified role unspecified role bait bait region 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 loop l1 sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) direct interaction direct interaction Fig 2C scn5a-camk2g 4 deltaC-CaMKII 7 anti tag western anti tag western blot anti tag western monoclonal western monoclonal antibody western blot monoclonal western unspecified role unspecified role prey prey region 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 required autophosphorylation nphosres N-phosphorylated residue NPhosRes undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined autophosphorylation NaV1.5 9 unspecified role unspecified role bait bait loop l1 sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region 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 direct interaction direct interaction Fig 2B camk2d-scn5a-1 3 deltaC-CaMKII 7 enzyme enzyme unspecified role unspecified role intein tag tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 562-rosetta ii de3 Escherichia coli rosetta ii de3 rosetta ii de3 NaV1.5 9 weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra enzyme target enzyme target substrate substrate unspecified role unspecified role purified purified Q14524:p.[Ser516Ala;Thr594Ala] mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain S A certain certain sequence position certain certain certain sequence position certain T A Wed May 02 19:51:12 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ser516ala,thr594ala' Tue Jun 30 18:11:53 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Ser516Ala;Thr594Ala]' Q14524:p.Thr594Ala mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain T A Wed May 02 16:55:38 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'thr594ala' Tue Jun 30 17:55:17 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Thr594Ala' region 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 loop l1 sufficient to bind sufficient binding region sufficient to bind autoradiography autoradiography certain certain sequence position certain certain certain sequence position certain Q14524:p.Ser516Ala mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain S A Wed May 02 17:13:47 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ser516ala' Tue Jun 30 18:07:51 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ser516Ala' ecoli Escherichia coli (strain K12) phosphorylation phosphorylation reaction phosphorylation Fig 4A-B Q14524 ser-516 O-phospho-L-serine Q14524 thr-594 O-phospho-L-threonine Note that authors refer to Thr-594 in methods, results and figures, but in results discussing figure 4 they state Ser-594. From the UniProt seq 594 is Thr, and in Fig 4 legend 594 is labelled Thr. There is also a Ser-593. I have assumed that 594 is Thr. camk2d-scn5a-4 1 deltaC-CaMKII 7 enzyme enzyme unspecified role unspecified role intein tag tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 562-rosetta ii de3 Escherichia coli rosetta ii de3 rosetta ii de3 NaV1.5 9 enzyme target enzyme target substrate substrate unspecified role unspecified role region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain Q14524 ser-516 O-phospho-L-serine chemical synthesis Chemical synthesis (Chemical synthesis) phosphorylation phosphorylation reaction phosphorylation Fig 3C column 3 Q14524 ser-516 O-phospho-L-serine From legend: soluble peptide (50 μm) phosphorylation by monomeric δC-CaMKII, methods state soluble peptide S516, NHLSLTRGLSRTSMK which aligns with 507-521 camk2d-scn5a-4 6 deltaC-CaMKII 7 enzyme enzyme unspecified role unspecified role required autophosphorylation nphosres N-phosphorylated residue NPhosRes undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined autophosphorylation region 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 spofr-sf_9 Spodoptera frugiperda insect cells Fall armyworm sf_9 insect cells Derived from pupal ovarian tissue of the fall armyworm Cloned from the parent cell line, IPLB-SF-21-AE (SF-21) derived from immature ovaries of fall armyworm Spodoptera frugiperda pupae cells are susceptible to Baculovirus infection NaV1.5 9 enzyme target enzyme target substrate substrate unspecified role unspecified role region sufficient to bind sufficient binding region sufficient to bind undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined Q14524 ser-483 O-phospho-L-serine Q14524 ser-484 O-phospho-L-serine chemical synthesis Chemical synthesis (Chemical synthesis) phosphorylation phosphorylation reaction phosphorylation Fig 3A-B spot 25-31 Q14524 ser-483 O-phospho-L-serine Q14524 ser-484 O-phospho-L-serine camk2d-scn5a-4 1 NaV1.5 9 enzyme target enzyme target substrate substrate unspecified role unspecified role purified purified region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain Q14524:p.Thr594Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain T A Wed May 02 16:22:16 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'thr594ala' Tue Jun 30 18:42:30 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Thr594Ala' Q14524:p.Ser593Ala mutation increasing mutation increasing interaction mutation increasing certain certain sequence position certain certain certain sequence position certain S A Wed May 02 14:39:08 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ser593ala' Tue Jun 30 18:11:44 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ser593Ala' Q14524:p.Ser593_Thr594delinsAlaAla mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain ST AA Thu Jul 07 12:15:37 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was ser593_594ala Fri Jul 22 11:02:22 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'ser593ala,thr594ala' Wed May 02 18:50:10 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ser_thr593-594ala_ala' Tue Jun 30 18:55:03 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ser593_Thr594delinsAlaAla' chemical synthesis Chemical synthesis (Chemical synthesis) deltaC-CaMKII 7 enzyme enzyme unspecified role unspecified role intein tag tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 562-rosetta ii de3 Escherichia coli rosetta ii de3 rosetta ii de3 phosphorylation phosphorylation reaction phosphorylation Fig 3F Q14524 thr-594 O-phospho-L-threonine From methods: monomeric δC-CaMKII was substituted for δC-CaMKII in Fig. 3, E and F, to avoid potential contaminating quantification of L1 phosphorylation with autophosphorylated δC-CaMKII. Difficult to decide on methods etc for 3F but decided that 3F has PSL axis similar to 3C, therefore assumed same procedure, and they also state immobilize peptide in Figure legend. camk2d-scn5a-4 6 NaV1.5 9 enzyme target enzyme target substrate substrate unspecified role unspecified role Q14524:p.Ser516Ala mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain S A Wed May 02 18:42:08 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ser516ala' Tue Jun 30 18:15:13 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ser516Ala' Q14524:p.Thr594Ala mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain T A Wed May 02 16:52:47 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'thr594ala' Tue Jun 30 16:53:00 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Thr594Ala' Q14524:p.[Ser516Ala;Thr594Ala] mutation disrupting rate mutation disrupting interaction rate mutation disrupting rate certain certain sequence position certain certain certain sequence position certain S A certain certain sequence position certain certain certain sequence position certain T A Wed May 02 16:06:07 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ser516ala,thr594ala' Tue Jun 30 19:54:44 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Ser516Ala;Thr594Ala]' region 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 loop l1 sufficient to bind sufficient binding region sufficient to bind autoradiography autoradiography certain certain sequence position certain certain certain sequence position certain spofr-sf_9 Spodoptera frugiperda insect cells Fall armyworm sf_9 insect cells Derived from pupal ovarian tissue of the fall armyworm Cloned from the parent cell line, IPLB-SF-21-AE (SF-21) derived from immature ovaries of fall armyworm Spodoptera frugiperda pupae cells are susceptible to Baculovirus infection deltaC-CaMKII 7 enzyme enzyme unspecified role unspecified role intein tag tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 562-rosetta ii de3 Escherichia coli rosetta ii de3 rosetta ii de3 phosphorylation phosphorylation reaction phosphorylation Fig 4A-D Q14524 Ser-516 O-phospho-L-serine Q14524 Thr-594 O-phospho-L-threonine Sorry I don't know if I need to comment on the aa that are changed, because this changes the phosphorylation but I am not sure that this 100% means that this changes the binding. If I do have do do this I guess I use the the features for the protein as feature type “rate decreasing”. Im assuming I can have multiple lines for each mutant in the one protein as these are individual mutation constructs. Rather than creating new interactions to capture this. camk2a-scn5a 3 9 weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra enzyme target enzyme target substrate substrate unspecified role unspecified role purified purified human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. alpha-CaMKII 10 enzyme enzyme unspecified role unspecified role spofr-sf_9 Spodoptera frugiperda insect cells Fall armyworm sf_9 insect cells Derived from pupal ovarian tissue of the fall armyworm Cloned from the parent cell line, IPLB-SF-21-AE (SF-21) derived from immature ovaries of fall armyworm Spodoptera frugiperda pupae cells are susceptible to Baculovirus infection phosphorylation phosphorylation reaction phosphorylation Fig 1B camk2d-scn5a-5 3 NaV1.5 9 enzyme target enzyme target substrate substrate unspecified role unspecified role Q14524:p.Ser516Glu mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain S E Wed May 02 15:56:39 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ser516glu' Tue Jun 30 16:14:57 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ser516Glu' Q14524:p.Thr594Glu mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain T E Wed May 02 15:25:34 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'thr594glu' Tue Jun 30 19:45:03 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Thr594Glu' loop l1 sufficient to bind sufficient binding region sufficient to bind autoradiography autoradiography certain certain sequence position certain certain certain sequence position certain region 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 Q14524:p.Ser516Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain S A Wed May 02 15:24:28 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ser516ala' Tue Jun 30 17:52:03 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ser516Ala' Q14524:p.Thr594Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain T A Wed May 02 18:43:31 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'thr594ala' Tue Jun 30 19:42:02 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Thr594Ala' ecoli Escherichia coli (strain K12) deltaC-CaMKII 7 enzyme enzyme unspecified role unspecified role intein tag tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 562-rosetta ii de3 Escherichia coli rosetta ii de3 rosetta ii de3 phosphorylation phosphorylation reaction phosphorylation Fig 4D Q14524 ser-516 O-phospho-L-serine Q14524 thr-594 O-phospho-L-threonine Note that authors refer to Thr-594 in methods, results and figures, but in results discussing figure 4 they state Ser-594. From the UniProt seq 594 is Thr, and in Fig 4 legend 594 is labelled Thr. There is also a Ser-593. I have assumed that 594 is Thr. camk2g-scn5a-3 2 NaV1.5 9 monoclonal western monoclonal antibody western blot monoclonal western monoclonal western monoclonal antibody western blot monoclonal western unspecified role unspecified role prey prey deltaC-CaMKII 7 unspecified role unspecified role bait bait region gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag anti tag western anti tag western blot anti tag western undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association Fig 2A camk2d-scn5a-8 1 deltaC-CaMKII 7 enzyme enzyme unspecified role unspecified role purified purified autophosphorylation allosteric ptm allosteric post-translational modification undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined autophosphorylation region 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 spofr-sf_9 Spodoptera frugiperda insect cells Fall armyworm sf_9 insect cells Derived from pupal ovarian tissue of the fall armyworm Cloned from the parent cell line, IPLB-SF-21-AE (SF-21) derived from immature ovaries of fall armyworm Spodoptera frugiperda pupae cells are susceptible to Baculovirus infection NaV1.5 9 monoclonal western monoclonal antibody western blot monoclonal western tag visualisation tag visualisation ancillary ancillary unspecified role unspecified role loop l1 sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region 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 ecoli Escherichia coli (strain K12) 8 enzyme target enzyme target substrate substrate unspecified role unspecified role purified purified synthetic construct synthetic construct phosphorylation phosphorylation reaction phosphorylation Fig 2D A significant increase in syntide-2 phosphorylation was only observed when autophosphorylated CaMKII was incubated with and allowed to bind SCN5A. camk2d-scn5a-9 1 NaV1.5 9 enzyme target enzyme target substrate substrate unspecified role unspecified role region 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 loop l1 sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) deltaC-CaMKII 7 enzyme enzyme unspecified role unspecified role region 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 spofr-sf_9 Spodoptera frugiperda insect cells Fall armyworm sf_9 insect cells Derived from pupal ovarian tissue of the fall armyworm Cloned from the parent cell line, IPLB-SF-21-AE (SF-21) derived from immature ovaries of fall armyworm Spodoptera frugiperda pupae cells are susceptible to Baculovirus infection phosphorylation phosphorylation reaction phosphorylation Fig 1D species from figure legend: bacterially expressed GST fusion proteins of intracellular hNaV1.5 regions were exposed to activated δC-CaMKII with [γ-32P]ATP and methods: GST-tagged intracellular regions of NaV1.5 (N terminus, L1–L3, and C terminus) as well as GST-tagged mutants of the L1 were expressed in Bl-21 cells (these are ecoli) camk2d-scn5a-10 6 NaV1.5 9 enzyme target enzyme target substrate substrate unspecified role unspecified role region sufficient to bind sufficient binding region sufficient to bind undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined Q14524 ser-516 O-phospho-L-serine chemical synthesis Chemical synthesis (Chemical synthesis) deltaC-CaMKII 7 enzyme enzyme unspecified role unspecified role required autophosphorylation nphosres N-phosphorylated residue NPhosRes undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined autophosphorylation region 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 spofr-sf_9 Spodoptera frugiperda insect cells Fall armyworm sf_9 insect cells Derived from pupal ovarian tissue of the fall armyworm Cloned from the parent cell line, IPLB-SF-21-AE (SF-21) derived from immature ovaries of fall armyworm Spodoptera frugiperda pupae cells are susceptible to Baculovirus infection phosphorylation phosphorylation reaction phosphorylation Fig 3A-B spot 45-55 Q14524 ser-516 O-phospho-L-serine camk2d-scn5a-11 6 NaV1.5 9 enzyme target enzyme target substrate substrate unspecified role unspecified role region sufficient to bind sufficient binding region sufficient to bind undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined Q14524 ser-593 O-phospho-L-serine Q14524 thr-594 O-phospho-L-threonine chemical synthesis Chemical synthesis (Chemical synthesis) deltaC-CaMKII 7 enzyme enzyme unspecified role unspecified role region 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 required autophosphorylation nphosres N-phosphorylated residue NPhosRes undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined autophosphorylation spofr-sf_9 Spodoptera frugiperda insect cells Fall armyworm sf_9 insect cells Derived from pupal ovarian tissue of the fall armyworm Cloned from the parent cell line, IPLB-SF-21-AE (SF-21) derived from immature ovaries of fall armyworm Spodoptera frugiperda pupae cells are susceptible to Baculovirus infection phosphorylation phosphorylation reaction phosphorylation Fig 3A-B spot 80-88 Q14524 ser-593 O-phospho-L-serine Q14524 thr-594 O-phospho-L-threonine camk2d-scn5a-12 3 deltaC-CaMKII 7 enzyme enzyme unspecified role unspecified role intein tag tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 562-rosetta ii de3 Escherichia coli rosetta ii de3 rosetta ii de3 NaV1.5 9 weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra enzyme target enzyme target substrate substrate unspecified role unspecified role purified purified loop l1 sufficient to bind sufficient binding region sufficient to bind autoradiography autoradiography certain certain sequence position certain certain certain sequence position certain Q14524:p.Thr594Ala mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain T A Wed May 02 15:30:26 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'thr594ala' Tue Jun 30 17:20:45 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Thr594Ala' region 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 Q14524:p.Ser516Ala mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain S A Wed May 02 16:04:22 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ser516ala' Tue Jun 30 15:50:08 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ser516Ala' ecoli Escherichia coli (strain K12) phosphorylation phosphorylation reaction phosphorylation Fig 4C Q14524 ser-516 O-phospho-L-serine Q14524 thr-594 O-phospho-L-threonine Note that authors refer to Thr-594 in methods, results and figures, but in results discussing figure 4 they state Ser-594. From the UniProt seq 594 is Thr, and in Fig 4 legend 594 is labelled Thr. There is also a Ser-593. I have assumed that 594 is Thr. camk2d-scn5a-13 1 NaV1.5 9 enzyme target enzyme target substrate substrate unspecified role unspecified role region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain Q14524 ser-593 O-phospho-L-serine Q14524 thr-594 O-phospho-L-threonine chemical synthesis Chemical synthesis (Chemical synthesis) deltaC-CaMKII 7 enzyme enzyme unspecified role unspecified role intein tag tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 562-rosetta ii de3 Escherichia coli rosetta ii de3 rosetta ii de3 phosphorylation phosphorylation reaction phosphorylation Fig 3C column 5 Q14524 ser-593 O-phospho-L-serine Q14524 thr-594 O-phospho-L-threonine From legend: soluble peptide (50 μm) phosphorylation by monomeric δC-CaMKII, methods state soluble peptide 593/594, LHGKKNSTVDCNGVV which aligns with 587-601