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 http://mint.bio.uniroma2.it/mint anonymous-2014w-2 Gatekeeper tyrosine phosphorylation is autoinhibitory for Symbiosis Receptor Kinase in vitro In vitro molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column predetermined predetermined participant predetermined maitrayee_d@hotmail.com 2014 imex curation Paul A., Samaddar S., Bhattacharya A., Banerjee A., Das A., Chakrabarti S., DasGupta M. FEBS Lett. (0014-5793) Only protein-protein interactions anonymous-2014j-1 Gatekeeper tyrosine phosphorylation is autoinhibitory for Symbiosis Receptor Kinase in vitro In vitro protein kinase assay protein kinase assay autoradiography autoradiography maitrayee_d@hotmail.com 2014 imex curation Paul A., Samaddar S., Bhattacharya A., Banerjee A., Das A., Chakrabarti S., DasGupta M. FEBS Lett. (0014-5793) Only protein-protein interactions e6yc17_arahy protein protein arahy Arachis hypogaea (Peanut) MMDLQDYWILRLVFVVYVLCVYIFIRSASASEGFESIACCADSNYTDPVTTLNYTTDYSSFPDKKSCRHLSETVLHQIRDENFRLFDINEGKRCYNLPTTLNKVYLIRGTFPSENAPGKGSFGVSIGVTVLGTVRSSSQDLRIEGVFRATKNNTDFCLVTEEGNPYISHLELRSVSEEYLQGLNSSVLKLINRSNLGGKEDDIRYPIDQSDRIWKRTTTSPYTPISFNISILDHKSNVTPPLKVLQTALTHPERLEFNNNGLEVKEDYEYLVFLYFLELNNSVREGQRVFDIFVNSEIKEGRFDILNGGSNYRYTLLNVSAKGSLNLTLAKASGSENGPLLNAYEIMQVHPWIEGTNQTDVEVIKKVREQLLVQNQDNKVLKSWSGDPCILSPWHGITCDHSSGPSVITDLDLSSSDLKGPIPSSVTEMTNLRTLNLSHNSFTGEIPSSFPLSSLLISIDVSYNDLEGSLPESISSLPNLKTLYFGCNEHLKEDIPPKLGSSLIQTDGGRCKEEDSRLDQVVVISVVTCGSLLITLVIGVIFVCCYRHKLIPWEGFVGKGYPVTTNLIFSLPSKDDFFIKSVSIQAFTLEYIEEATEKYKTLIGEGGFGPVYRGMLDDGQEVAVKVRSATSTQGTREFDNELNLLSAIQHENLVPLIGYCNEKDQQILVYPFMSNGSLQNRLYGEPAKRKILDWPTRLSIALGAARGLAYLHTFPGRPVIHRDIKSSNILLDHSMCAKVADFGFSKYAPQEGDSNVSLEVRGTAGYLDPEYYTTQQLSEKSDVFSFGVVLLEIVSGREPLDIKRPRNEWSLVEWAKPYIRASKIEEIVDPGIKGGYHAEAMWRVVEVALQCIEPFSAYRPCMDDIVRELEDALIIENNASEYMKSIDSLGGSNRYSFVMDKRVPPSTSSTAESTITSQTLSLPQPR e6yc17-1 1548821 n/a AhSYMRK 1548823 western blot western blot Immuno blot Immuno blot unspecified role unspecified role neutral component neutral component purified purified Stoichiometry: 2.0 direct interaction direct interaction false false false F3A F3B SF1 e6yc17-e6yc17 1548821 n/a AhSYMRK 1548823 western blot western blot Immuno blot Immuno blot unspecified role unspecified role neutral component neutral component purified purified n/a AhSYMRK 1548823 western blot western blot Immuno blot Immuno blot unspecified role unspecified role neutral component neutral component purified purified direct interaction direct interaction false false false F3A F3B SF1 WT AhSYMRK eluted predominantly at around 76 kDa (peak at 16.5 ml) where the catalytically inactive K625E AhSYMRK eluted at around 40 kDa (peak at 17.28 ml) (Fig. 3A and Supplemental Fig. S1), suggesting the active WT polypeptide to be stable in a dimeric form. The monomeric form of K625E polypeptide ruled out the possibility of any influence of the His6-tag on the dimerisation of AhSYMRK kinase domain (Fig. 3A). We noticed that with increasing concentrations around 75 μM, the WT polypeptide showed an increased tendency to oligomerise and form aggregates that eluted within void volume. This indicated that the WT AhSYMRK had a propensity to oligomerise and this propensity was concentration-dependent. Western blot analysis by α-His6 as well as α-SYMRK confirmed that the void volume fraction as well as other fractions eluting at higher molecular weight, consisted of aggregates of the WT AhSYMRK polypeptide (Fig. 3B). e6yc17-1 1548822 n/a AhSYMRK 1548823 autoradiography autoradiography self self self self purified purified 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 false E6YC17:p.Tyr670Glu mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false Stoichiometry: 2.0 phosphorylation phosphorylation reaction phosphorylation false false false F1A F3C As indicated in Fig. 3C, no autophosphorylation was detected in the void fraction (P1) of AhSYMRK. We also tested the fractions eluting in between P1 and P2 containing higher order oligomeric fractions (data not shown), but autophosphorylation was only detectable in the dimeric fraction of AhSYMRK. Immunoblot analysis of P1 and P2 using α-pY670 (665–674) and α-pY indicated that both the oligomeric and dimeric fractions of WT AhSYMRK were autophosphorylated on gatekeeper tyrosine. This indicated that AhSYMRK could actively autophosphorylate on gatekeeper tyrosine before oligomerising into inactive higher order structures. As indicated in Fig. 1A, the wild-type AhSYMRK and the Y670F mutant actively autophosphorylated but the activity of Y670E kinase was severely affected. Thus a phosphomimic (Y670E) but not a structure-mimic (Y670F) substitution in the gatekeeper position has adverse effect on the catalytic activity of AhSYMRK