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 DNA damage-induced CHK1 autophosphorylation at Ser296 is regulated by an intramolecular mechanism. DNA damage-induced CHK1 autophosphorylation at Ser296 is regulated by an intramolecular mechanism. FEBS Lett. (0014-5793) 2012 Okita N., Minato S., Ohmi E., Tanuma S., Higami Y. nokita7@rs.noda.tus.ac.jp in vitro In vitro protein kinase assay protein kinase assay western blot western blot Immuno blot Immuno blot Okita N., Minato S., Ohmi E., Tanuma S., Higami Y. nokita7@rs.noda.tus.ac.jp FEBS Lett. (0014-5793) 2012 chk1_human Serine/threonine-protein kinase Chk1 CHEK1 CHK1 CHK1 checkpoint homolog Checkpoint kinase-1 Cell cycle checkpoint kinase protein protein human Homo sapiens Human MAVPFVEDWDLVQTLGEGAYGEVQLAVNRVTEEAVAVKIVDMKRAVDCPENIKKEICINKMLNHENVVKFYGHRREGNIQYLFLEYCSGGELFDRIEPDIGMPEPDAQRFFHQLMAGVVYLHGIGITHRDIKPENLLLDERDNLKISDFGLATVFRYNNRERLLNKMCGTLPYVAPELLKRREFHAEPVDVWSCGIVLTAMLAGELPWDQPSDSCQEYSDWKEKKTYLNPWKKIDSAPLALLHKILVENPSARITIPDIKKDRWYNKPLKKGAKRPRVTSGGVSESPSGFSKHIQSNLDFSPVNSASSEENVKYSSSQPEPRTGLSLWDTSPSYIDKLVQGISFSQPTCPDHMLLNSQLLGTPGSSQNPWQRLVKRMTRFFTKLDADKSYQCLKETCEKLGYQWKKSCMNQVTISTTDRRNNKLIFKVNLLEMDDKILVDFRLSKGDGLEFKRHFLKIKGKLIDIVSSQKIWLPAT DIP protein O14757 original sequence version: 102 0ABD0FAB67E60F67 chek1-6 1 CHK1 2 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined self self self self in vitro translated in vitro translated protein in vitro translated phosphorylated phosphorylated residue phosres phosphorylated residue PhosRes Phospho Phosphorylation Phosphorylation (O of Serine, Threonine, Tyrosine and Aspartate, N epsilon of Lysine) western blot western blot Immuno blot Immuno blot certain certain sequence position certain certain certain sequence position certain resulting-ptm tag tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined O14757:p.Lys38Ala mutation disrupting interaction mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain K A Tue Jul 12 10:23:49 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'mutation disrupting' Wed May 02 19:04:52 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys38ala' Tue Jun 30 18:06:54 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys38Ala' phosphorylation phosphorylation reaction phosphorylation true mint f2c f2d As shown in Figure 2C, only CHK1(WT)-6His was autophoshorylated during incubation with ATP. After incubation without ATP, a basal level of Ser296 phosphorylation was only observed in CHK1(WT)-6His, and not in the other mutants. This observation implies that the basal phosphorylation of recombinant CHK1 has already occurred in an in vitro transcription/translation system. Next, to confirm whether Ser296 autophosphorylation is regulated by an intramolecular mechanism as suggested in Figure 2A, we performed a CHK1 autophosphorylation assay using the combinations shown in (Figure 2D). In the reaction combination of 3F-CHK1(WT)-3M and CHK1(WT)-6His, both Ser296 phosphorylation levels were increased by the addition of ATP. However, in the reaction combinations of 3F-CHK1(WT)-3M and CHK1(K38A)-6His or 3F-CHK1(WT)-3M and CHK1(D130A)-6His, only 3F-CHK1(WT)-3M was efficiently autophosphorylated at Ser296. To check for any unexpected artifacts produced by recombinant preparation methods, we also used a combination of 3F-CHK1(D130A)-3M and CHK1(WT)-6His, which gave a similar result. These results clearly show that Ser296 autophosphorylation of CHK1 is mainly regulated by an intramolecular mechanism. true chek1-6 1 CHK1 2 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined self self self self in vitro translated in vitro translated protein in vitro translated phosphorylated phosphorylated residue phosres phosphorylated residue PhosRes Phospho Phosphorylation Phosphorylation (O of Serine, Threonine, Tyrosine and Aspartate, N epsilon of Lysine) western blot western blot Immuno blot Immuno blot certain certain sequence position certain certain certain sequence position certain resulting-ptm tag tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined O14757:p.Asp130Ala mutation disrupting interaction mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain D A Tue Jul 12 10:24:04 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'mutation disrupting' Wed May 02 15:12:08 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'asp130ala' Tue Jun 30 17:00:48 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Asp130Ala' phosphorylation phosphorylation reaction phosphorylation true mint f2c f2d As shown in Figure 2C, only CHK1(WT)-6His was autophoshorylated during incubation with ATP. After incubation without ATP, a basal level of Ser296 phosphorylation was only observed in CHK1(WT)-6His, and not in the other mutants. This observation implies that the basal phosphorylation of recombinant CHK1 has already occurred in an in vitro transcription/translation system. Next, to confirm whether Ser296 autophosphorylation is regulated by an intramolecular mechanism as suggested in Figure 2A, we performed a CHK1 autophosphorylation assay using the combinations shown in (Figure 2D). In the reaction combination of 3F-CHK1(WT)-3M and CHK1(WT)-6His, both Ser296 phosphorylation levels were increased by the addition of ATP. However, in the reaction combinations of 3F-CHK1(WT)-3M and CHK1(K38A)-6His or 3F-CHK1(WT)-3M and CHK1(D130A)-6His, only 3F-CHK1(WT)-3M was efficiently autophosphorylated at Ser296. To check for any unexpected artifacts produced by recombinant preparation methods, we also used a combination of 3F-CHK1(D130A)-3M and CHK1(WT)-6His, which gave a similar result. These results clearly show that Ser296 autophosphorylation of CHK1 is mainly regulated by an intramolecular mechanism. true chek1-6 1 CHK1 2 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined self self self self in vitro translated in vitro translated protein in vitro translated O14757:p.Ser296Ala 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 10:57:39 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'mutation disrupting' Wed May 02 20:01:33 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ser296ala' Tue Jun 30 18:13:00 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ser296Ala' phosphorylated phosphorylated residue phosres phosphorylated residue PhosRes Phospho Phosphorylation Phosphorylation (O of Serine, Threonine, Tyrosine and Aspartate, N epsilon of Lysine) western blot western blot Immuno blot Immuno blot certain certain sequence position certain certain certain sequence position certain resulting-ptm tag tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined phosphorylation phosphorylation reaction phosphorylation true mint As shown in Figure 2C, only CHK1(WT)-6His was autophoshorylated during incubation with ATP. After incubation without ATP, a basal level of Ser296 phosphorylation was only observed in CHK1(WT)-6His, and not in the other mutants. This observation implies that the basal phosphorylation of recombinant CHK1 has already occurred in an in vitro transcription/translation system. Next, to confirm whether Ser296 autophosphorylation is regulated by an intramolecular mechanism as suggested in Figure 2A, we performed a CHK1 autophosphorylation assay using the combinations shown in (Figure 2D). In the reaction combination of 3F-CHK1(WT)-3M and CHK1(WT)-6His, both Ser296 phosphorylation levels were increased by the addition of ATP. However, in the reaction combinations of 3F-CHK1(WT)-3M and CHK1(K38A)-6His or 3F-CHK1(WT)-3M and CHK1(D130A)-6His, only 3F-CHK1(WT)-3M was efficiently autophosphorylated at Ser296. To check for any unexpected artifacts produced by recombinant preparation methods, we also used a combination of 3F-CHK1(D130A)-3M and CHK1(WT)-6His, which gave a similar result. These results clearly show that Ser296 autophosphorylation of CHK1 is mainly regulated by an intramolecular mechanism. f2c f2d true