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 Akt augments the oncogenic potential of the HBx protein of hepatitis B virus by phosphorylation. Akt augments the oncogenic potential of the HBx protein of hepatitis B virus by phosphorylation. FEBS J. (1742-464X) 2012 Khattar E., Mukherji A., Kumar V. human-hep_g2 Homo sapiens hepatocellular carcinoma (HCC) cells (p53-wt). hep_g2 human hepatocellular carcinoma (HCC) cells (p53-wt). anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Khattar E., Mukherji A., Kumar V. FEBS J. (1742-464X) 2012 Akt augments the oncogenic potential of the HBx protein of hepatitis B virus by phosphorylation. Akt augments the oncogenic potential of the HBx protein of hepatitis B virus by phosphorylation. FEBS J. (1742-464X) 2012 Khattar E., Mukherji A., Kumar V. in vitro In vitro protein kinase assay protein kinase assay predetermined predetermined participant predetermined Khattar E., Mukherji A., Kumar V. FEBS J. (1742-464X) 2012 Akt augments the oncogenic potential of the HBx protein of hepatitis B virus by phosphorylation. Akt augments the oncogenic potential of the HBx protein of hepatitis B virus by phosphorylation. FEBS J. (1742-464X) 2012 Khattar E., Mukherji A., Kumar V. human-huh7 Homo sapiens hepatoma cell line established from a hepatocellular carcinoma huh7 Human hepatoma cell line established from a hepatocellular carcinoma anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Khattar E., Mukherji A., Kumar V. FEBS J. (1742-464X) 2012 x_hbvd3 Protein X X HBx Peptide X pX protein protein hbvd3 Hepatitis B virus genotype D subtype ayw (isolate France/Tiollais/1979) MAARLCCQLDPARDVLCLRPVGAESRGRPFSGSLGTLSSPSPSAVPTDHGAHLSLRGLPVCAFSSAGPCALRFTSARRMETTVNAHQILPKVLHKRTLGLSAMSTTDLEAYFKDCLFKDWEELGEEIRLKVFVLGGCRHKLVCAPAPCNFFTSA mint 29FD1CC9E09A34B5 akt1_mouse RAC-alpha serine/threonine-protein kinase Akt1 Akt Rac RAC-PK-alpha AKT1 kinase Proto-oncogene c-Akt Protein kinase B Thymoma viral proto-oncogene Protein kinase B alpha protein protein mouse Mus musculus Mouse MNDVAIVKEGWLHKRGEYIKTWRPRYFLLKNDGTFIGYKERPQDVDQRESPLNNFSVAQCQLMKTERPRPNTFIIRCLQWTTVIERTFHVETPEEREEWATAIQTVADGLKRQEEETMDFRSGSPSDNSGAEEMEVSLAKPKHRVTMNEFEYLKLLGKGTFGKVILVKEKATGRYYAMKILKKEVIVAKDEVAHTLTENRVLQNSRHPFLTALKYSFQTHDRLCFVMEYANGGELFFHLSRERVFSEDRARFYGAEIVSALDYLHSEKNVVYRDLKLENLMLDKDGHIKITDFGLCKEGIKDGATMKTFCGTPEYLAPEVLEDNDYGRAVDWWGLGVVMYEMMCGRLPFYNQDHEKLFELILMEEIRFPRTLGPEAKSLLSGLLKKDPTQRLGGGSEDAKEIMQHRFFANIVWQDVYEKKLSPPFKPQVTSETDTRYFDEEFTAQMITITPPDQDDSMECVDSERRPHFPQFSYSASGTA DIP protein P31750 original sequence version: 125 DIP protein P31750 original sequence version: 168 DIP protein P31750 original sequence version: 88 98DF28E5EFE03730 1433t_human 14-3-3 protein theta YWHAQ 14-3-3 protein tau 14-3-3 protein T-cell Protein HS1 protein protein human Homo sapiens Human MEKTELIQKAKLAEQAERYDDMATCMKAVTEQGAELSNEERNLLSVAYKNVVGGRRSAWRVISSIEQKTDTSDKKLQLIKDYREKVESELRSICTTVLELLDKYLIANATNPESKVFYLKMKGDYFRYLAEVACGDDRKQTIDNSQGAYQEAFDISKKEMQPTHPIRLGLALNFSVFYYEILNNPELACTLAKTAFDEAIAELDTLNEDSYKDSTLIMQLLRDNLTLWTSDSAGEECDAAEGAEN DIP protein P27348 original sequence version: 164 DIP protein P27348 original sequence version: 173 175534325E9E37C4 creb1_human Cyclic AMP-responsive element-binding protein 1 CREB1 protein protein human Homo sapiens Human MTMESGAENQQSGDAAVTEAENQQMTVQAQPQIATLAQVSMPAAHATSSAPTVTLVQLPNGQTVQVHGVIQAAQPSVIQSPQVQTVQISTIAESEDSQESVDSVTDSQKRREILSRRPSYRKILNDLSSDAPGVPRIEEEKSEEETSAPAITTVTVPTPIYQTSSGQYIAITQGGAIQLANNGTDGVQGLQTLTMTNAAATQPGTTILQYAQTTDGQQILVPSNQVVVQAASGDVQTYQIRTAPTSTIAPGVVMASSPALPTQPAEEAARKREVRLMKNREAARECRRKKKEYVKCLENRVAVLENQNKTLIEELKALKDLYCHKSD DIP protein P16220 original sequence version: 169 DIP protein P16220 original sequence version: 158 F5BA8200EE5184B7 them4_human Acyl-coenzyme A thioesterase THEM4 THEM4 CTMP Carboxyl-terminal modulator protein Thioesterase superfamily member 4 protein protein human Homo sapiens Human MLRSCAARLRTLGALCLPPVGRRLPGSEPRPELRSFSSEEVILKDCSVPNPSWNKDLRLLFDQFMKKCEDGSWKRLPSYKRTPTEWIQDFKTHFLDPKLMKEEQMSQAQLFTRSFDDGLGFEYVMFYNDIEKRMVCLFQGGPYLEGPPGFIHGGAIATMIDATVGMCAMMAGGIVMTANLNINYKRPIPLCSVVMINSQLDKVEGRKFFVSCNVQSVDEKTLYSEATSLFIKLNPAKSLT DIP protein Q5T1C6 original sequence version: 84 DIP protein Q5T1C6 original sequence version: 87 mint homomint A01071B07A1B5FB2 akt1_human RAC-alpha serine/threonine-protein kinase AKT1 PKB RAC RAC-PK-alpha Protein kinase B Proto-oncogene c-Akt Protein kinase B alpha protein protein human Homo sapiens Human MSDVAIVKEGWLHKRGEYIKTWRPRYFLLKNDGTFIGYKERPQDVDQREAPLNNFSVAQCQLMKTERPRPNTFIIRCLQWTTVIERTFHVETPEEREEWTTAIQTVADGLKKQEEEEMDFRSGSPSDNSGAEEMEVSLAKPKHRVTMNEFEYLKLLGKGTFGKVILVKEKATGRYYAMKILKKEVIVAKDEVAHTLTENRVLQNSRHPFLTALKYSFQTHDRLCFVMEYANGGELFFHLSRERVFSEDRARFYGAEIVSALDYLHSEKNVVYRDLKLENLMLDKDGHIKITDFGLCKEGIKDGATMKTFCGTPEYLAPEVLEDNDYGRAVDWWGLGVVMYEMMCGRLPFYNQDHEKLFELILMEEIRFPRTLGPEAKSLLSGLLKKDPKQRLGGGSEDAKEIMQHRFFAGIVWQHVYEKKLSPPFKPQVTSETDTRYFDEEFTAQMITITPPDQDDSMECVDSERRPHFPQFSYSASGTA DIP protein P31749 original sequence version: 184 DIP protein P31749 original sequence version: 168 DIP protein P31749 original sequence version: 102 DIP protein P31749 original sequence version: 110 DIP protein P31749 original sequence version: 160 DIP protein P31749 original sequence version: 136 DIP protein P31749 original sequence version: 175 DIP protein P31749 original sequence version: 201 DIP protein P31749 original sequence version: 166 DIP protein P31749 original sequence version: 178 DIP protein P31749-1 original sequence version: 194 DIP protein P31749 original sequence version: 106 DIP protein P31749 original sequence version: 141 6EAFF4F8AD436714 akt1-x-1 2 HBx 4 weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra predetermined predetermined participant predetermined enzyme target enzyme target substrate substrate neutral component neutral component purified purified phosphorylated phosphorylated residue phosres phosphorylated residue PhosRes Phospho Phosphorylation Phosphorylation (O of Serine, Threonine, Tyrosine and Aspartate, N epsilon of Lysine) mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain resulting-ptm 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 Akt1 5 weight by comassie molecular weight estimation by coomasie staining weight by comassie predetermined predetermined participant predetermined enzyme enzyme neutral component neutral component purified purified phosphorylation phosphorylation reaction phosphorylation f1b mint To confirm whether HBx is as a substrate of Akt kinase, we used bacterially-expressed recombinant wild type HBx protein and its single point mutant HBxS31A (serine-31 changed to alanine) for in vitro phosphorylation in the presence of recombinant active Akt1. As shown in Fig. 1B, only the wild-type but not the mutant HBx was phosphorylated in the presence of Akt1 (compare lane 2 and 3) suggesting that serine-31 is the site of phosphorylation. akt1-x-1 2 HBx 4 weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra predetermined predetermined participant predetermined enzyme target enzyme target substrate substrate neutral component neutral component 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 binding site binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain Akt1 5 weight by comassie molecular weight estimation by coomasie staining weight by comassie predetermined predetermined participant predetermined enzyme enzyme neutral component neutral component purified purified phosphorylation phosphorylation reaction phosphorylation f1c mint The specificity of Akt phosphorylation motif on HBx was further confirmed using recombinant HBx-GST protein(XG) that has eleven amino acids (aa 25-35) of HBx fused at the carboxy-terminus of glutathione S-transferase protein. Fig. 1C shows that only XG was specifically phosphorylated by Akt1. As expected, the GST fusion protein with mutant peptide (S31A) sequence was not phosphorylated (data not shown). x-akt1 1 HBx 4 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell AKT1 9 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level physical association physical association f3a mint The HBx-Akt interaction was investigated in HepG2.2.15 cells where HBx is expressed at physiological level. Cell were treated with PI3 kinase inhibitor LY294002, immunoprecipitated either with anti-HBx or anti-Akt antibodies and immunoblotted for Akt or HBx binding. Our results in Fig. 3A and 3B clearly indicated that HBx interacted with Akt1 kinase in a cellular milieu. akt1-x-1 1 HBx 4 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell AKT1 9 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role bait bait living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level physical association physical association f3b mint The HBx-Akt interaction was investigated in HepG2.2.15 cells where HBx is expressed at physiological level. Cell were treated with PI3 kinase inhibitor LY294002, immunoprecipitated either with anti-HBx or anti-Akt antibodies and immunoblotted for Akt or HBx binding. Our results in Fig. 3A and 3B clearly indicated that HBx interacted with Akt1 kinase in a cellular milieu. akt1-x-1 3 HBx 4 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell P03165:p.Ser31Ala mutation disrupting interaction mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain S A Tue Jul 12 10:23:45 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'mutation disrupting' Wed May 02 20:20:12 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ser31ala' Tue Jun 30 19:40:51 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ser31Ala' 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 AKT1 9 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role bait bait living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level physical association physical association f3c mint Since HBx expression is associated with sustained activation of Akt kinase, we speculated whether HBx did so by disrupting the Akt interaction with its negative regulator known as Carboxyl Terminal Regulatory Protein (CTMP) [27]. To probe this, we immunoprecipitated Akt from the HBx- and HBxS31Atransfected cells and western blotted for HBx and CTMP. We observed that HBx-Akt association precluded CTMP interaction with Akt1 (Fig. 3C) which may relate to sustained Akt1 activity in the presence of HBx [13]. Further, HBxS31A mutant that does not interact with Akt1 also did not affect the Akt-CTMP interaction (Fig. 3C). akt1-them4 3 CTMP 8 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level AKT1 9 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role bait bait living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level physical association physical association f3c homomint mint Since HBx expression is associated with sustained activation of Akt kinase, we speculated whether HBx did so by disrupting the Akt interaction with its negative regulator known as Carboxyl Terminal Regulatory Protein (CTMP) [27]. To probe this, we immunoprecipitated Akt from the HBx- and HBxS31Atransfected cells and western blotted for HBx and CTMP. We observed that HBx-Akt association precluded CTMP interaction with Akt1 (Fig. 3C) which may relate to sustained Akt1 activity in the presence of HBx [13]. Further, HBxS31A mutant that does not interact with Akt1 also did not affect the Akt-CTMP interaction (Fig. 3C). x-creb1 3 HBx 4 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell 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 CREB 7 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level physical association physical association f3d mint x-ywhaq 3 HBx 4 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell 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 14-3-3 theta 6 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level physical association physical association sf1b domino mint Cells were transfected with HBx, treated with DMSO or LY294002 for 30 minutes prior harvesting