mbinfo MBInfo http://www.mechanobio.info/ http://www.mechanobio.info/ zhang-1998-4 Negative regulation of Rho family GTPases Cdc42 and Rac2 by homodimer formation. in vitro In vitro gtpase assay gtpase assay GTPase gtp hydrolisis gtp hydrolisis GTPase predetermined predetermined participant predetermined Zhang B., Zheng Y. Interaction detection: GST pull down. The source of GST-Cdc42 specified by the author and the source of endogenous Cdc42 inferred from the cells (NIH3T3) used. yzheng@utmem1.utmem.edu J. Biol. Chem. (0021-9258) 1998 Only protein-protein interactions imex curation zhang-1998-3 Negative regulation of Rho family GTPases Cdc42 and Rac2 by homodimer formation. in vitro In vitro chromatography chromatography technology column chromatography chromatography weight identificat confirmation by molecular weight weight identificat Zhang B., Zheng Y. Interaction detection: GST pull down. The source of GST-Cdc42 specified by the author and the source of endogenous Cdc42 inferred from the cells (NIH3T3) used. yzheng@utmem1.utmem.edu J. Biol. Chem. (0021-9258) 1998 Only protein-protein interactions imex curation zhang-1998-1 Negative regulation of Rho family GTPases Cdc42 and Rac2 by homodimer formation. in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Zhang B., Zheng Y. Interaction detection: GST pull down. The source of GST-Cdc42 specified by the author and the source of endogenous Cdc42 inferred from the cells (NIH3T3) used. yzheng@utmem1.utmem.edu J. Biol. Chem. (0021-9258) 1998 Only protein-protein interactions imex curation zhang-1998-2 Negative regulation of Rho family GTPases Cdc42 and Rac2 by homodimer formation. in vitro In vitro pull down pull down weight by comassie molecular weight estimation by coomasie staining weight by comassie Zhang B., Zheng Y. Interaction detection: GST pull down. The source of GST-Cdc42 specified by the author and the source of endogenous Cdc42 inferred from the cells (NIH3T3) used. yzheng@utmem1.utmem.edu J. Biol. Chem. (0021-9258) 1998 Only protein-protein interactions imex curation gtp guanosine triphosphate - GTP 5'-GTP GTP Guanosine 5'-triphosphate guanosine 5'-triphosphoric acid guanosine triphosphate GUANOSINE-5'-TRIPHOSPHATE H4gtp guanosine 5'-(tetrahydrogen triphosphate) small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) InChI=1/C10H16N5O14P3/c11-10-13-7-4(8(18)14-10)12-2-15(7)9-6(17)5(16)3(27-9)1-26-31(22,23)29-32(24,25)28-30(19,20)21/h2-3,5-6,9,16-17H,1H2,(H,22,23)(H,24,25)(H2,19,20,21)(H3,11,13,14,18)/t3-,5-,6-,9-/m1/s1/f/h14,19-20,22,24H,11H2 cdc42_human Cell division control protein 42 homolog CDC42 G25K GTP-binding protein protein protein human Homo sapiens Human MQTIKCVVVGDGAVGKTCLLISYTTNKFPSEYVPTVFDNYAVTVMIGGEPYTLGLFDTAGQEDYDRLRPLSYPQTDVFLVCFSVVSPSSFENVKEKWVPEITHHCPKTPFLLVGTQIDLRDDPSTIEKLAKNKQKPITPETAEKLARDLKAVKYVECSALTQKGLKNVFDEAILAALEPPEPKKSRRCVLL rac2_human Ras-related C3 botulinum toxin substrate 2 p21-Rac2 Small G protein GX RAC2 protein protein human Homo sapiens Human MQAIKCVVVGDGAVGKTCLLISYTTNAFPGEYIPTVFDNYSANVMVDSKPVNLGLWDTAGQEDYDRLRPLSYPQTDVFLICFSLVSPASYENVRAKWFPEVRHHCPSTPIILVGTKLDLRDDKDTIEKLKEKKLAPITYPQGLALAKEIDSVKYLECSALTQRGLKTVFDEAIRAVLCPQPTRQQKRACSLL rhoa_human Transforming protein RhoA Rho cDNA clone 12 ARHA RHO12 RHOA ARH12 protein protein human Homo sapiens Human MAAIRKKLVIVGDGACGKTCLLIVFSKDQFPEVYVPTVFENYVADIEVDGKQVELALWDTAGQEDYDRLRPLSYPDTDVILMCFSIDSPDSLENIPEKWTPEVKHFCPNVPIILVGNKKDLRNDEHTRRELAKMKQEPVKPEEGRDMANRIGAFGYMECSAKTKDGVREVFEMATRAALQARRGKKKSGCLVL rasn_human GTPase NRas Transforming protein N-Ras NRAS HRAS1 protein protein human Homo sapiens Human MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNSKSFADINLYREQIKRVKDSDDVPMVLVGNKCDLPTRTVDTKQAHELAKSYGIPFIETSAKTRQGVEDAFYTLVREIRQYRMKKLNSSDDGTQGCMGLPCVVM cdc42_mouse Cell division control protein 42 homolog Cdc42 G25K GTP-binding protein protein protein mouse Mus musculus Mouse MQTIKCVVVGDGAVGKTCLLISYTTNKFPSEYVPTVFDNYAVTVMIGGEPYTLGLFDTAGQEDYDRLRPLSYPQTDVFLVCFSVVSPSSFENVKEKWVPEITHHCPKTPFLLVGTQIDLRDDPSTIEKLAKNKQKPITPETAEKLARDLKAVKYVECSALTQKGLKNVFDEAILAALEPPEPKKSRRCVLL cdc42-gtp-2 1316690 n/a 1316694 enzyme target enzyme target substrate substrate neutral component neutral component n/a 1316695 enzyme enzyme neutral component neutral component ecoli Escherichia coli (strain K12) gtpase reaction gtpase reaction false false false Fig 1B, 1C, 1A. The GTPase activity of Cdc42 was enhanced in the presence of GTPgammaS-bound Cdc42 and GAP, but not in the presence of GDP-bound Cdc42. Isoprenylation of Cdc42 did not affect its GTPase activity. gtp-rac2 1316690 n/a 1316694 enzyme target enzyme target substrate substrate prey prey n/a 1316696 enzyme enzyme neutral component neutral component ecoli Escherichia coli (strain K12) gtpase reaction gtpase reaction false false false Fig 1A, 1C. The GTPase activity of Rac2 was enhanced in the presence of GTPgammaS-loaded Rac2 and GAP, but not in the presence of GDP-loaded Rac2. gtp-rhoa 1316690 n/a 1316697 enzyme enzyme neutral component neutral component ecoli Escherichia coli (strain K12) n/a 1316694 enzyme target enzyme target substrate substrate neutral component neutral component gtpase reaction gtpase reaction false false false Fig 1C, 1A. The GTPase activity of RhoA was low and was enhanced in the presence of GAP, but not in the presence of GTPgammaS-loaded RhoA. gtp-nras 1316690 n/a 1316694 enzyme target enzyme target substrate substrate neutral component neutral component n/a 1316698 enzyme enzyme neutral component neutral component ecoli Escherichia coli (strain K12) gtpase reaction gtpase reaction false false false Fig 1A. The basal N-Ras GTPase activity was low and did not increase with increasing concentrations of NRAS. cdc42-cdc42-1 1316691 n/a 1316695 unspecified role unspecified role neutral component neutral component c-terminal region required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) n/a 1316695 unspecified role unspecified role neutral component neutral component c-terminal region required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) physical association physical association false false false Fig 2A, 3B. Both GTPgammaS and GDP-loaded Cdc42 formed homodimers, but the homodimer formation was slightly more with GDP-loaded Cdc42. rac2-rac2-1 1316691 n/a 1316696 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) n/a 1316696 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) physical association physical association false false false Fig 2A. Both GTPgammaS and GDP-loaded Rac2 formed homodimers, but the homodimer formation was slightly enhanced with GDP-loaded Rac2. rhoa-rhoa-2 1316691 n/a 1316697 unspecified role unspecified role neutral component neutral component c-terminal region required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) n/a 1316697 unspecified role unspecified role neutral component neutral component c-terminal region required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) physical association physical association false false false Fig 2A, 3B. Both GTPgammaS and GDP-bound RhoA formed homodimers, but this was slightly enhanced with GDP-loaded RhoA. cdc42-cdc42-1 1316692 n/a 1316699 unspecified role unspecified role prey prey mouse-nih_3t3 Mus musculus Swiss NIH 3t3 embryo fibroblast cells nih_3t3 Mouse Swiss NIH embryo contact inhibited fibroblast cells n/a 1316695 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 false ecoli Escherichia coli (strain K12) physical association physical association false false false Fig 2B. The GTPgammaS-loaded GST-Cdc42 bound to the GTPgammaS-loaded endogenous Cdc42 and the GDP-loaded GST-Cdc42 bound to the GDP-loaded endogenous Cdc42. Binding between the GST-Cdc42 and endogenous Cdc42 loaded with differing nucleotides was minimal. cdc42-cdc42-1 1316693 n/a 1316695 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 false ecoli Escherichia coli (strain K12) n/a 1316695 unspecified role unspecified role prey prey 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 false ecoli Escherichia coli (strain K12) physical association physical association false false false Fig 4C. GTPgammaS-loaded GST-CDC42 bound to GTPgammaS-loaded His-CDC42 and GDP-loaded GST-CDC42 bound to GDP-loaded His-CDC42. Binding between GST-CDC42 and His-CDC42, each bound to differing nucleotides, was minimal. But the binding between GDP-loaded GST-CDC42 and GTPgammaS-loaded His-CDC42 was enhanced in the presence of AlCl3 and NaF.