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 The membrane localization of Ras2p and the association between Cdc25p and Ras2-GTP are regulated by protein kinase A (PKA) in the yeast Saccharomyces cerevisiae. The membrane localization of Ras2p and the association between Cdc25p and Ras2-GTP are regulated by protein kinase A (PKA) in the yeast Saccharomyces cerevisiae. FEBS Lett. (0014-5793) 2011 Dong J., Bai X. suggested by lperfetto dongjianbio@tju.edu.cn in vitro In vitro pull down pull down predetermined predetermined participant predetermined Dong J., Bai X. suggested by lperfetto dongjianbio@tju.edu.cn FEBS Lett. (0014-5793) 2011 cdc25_yeast Cell division control protein 25 CDC25 CTN1 YLR310C L2142.6 protein protein yeast Saccharomyces cerevisiae Baker's yeast MSDTNTSIPNTSSAREAGNASQTPSISSSSNTSTTTNTESSSASLSSSPSTSELTSIRPIGIVVAAYDFNYPIKKDSSSQLLSVQQGETIYILNKNSSGWWDGLVIDDSNGKVNRGWFPQNFGRPLRDSHLRKHSHPMKKYSSSKSSRRSSLNSLGNSAYLHVPRNPSKSRRGSSTLSASLSNAHNAETSSGHNNTVSMNNSPFSAPNDASHITPQSSNFNSNASLSQDMTKSADGSSEMNTNAIMNNNETNLQTSGEKAGPPLVAEETIKILPLEEIEMIINGIRSNIASTWSPIPLITKTSDYKLVYYNKDLDIYCSELPLISNSIMESDDICDSEPKFPPNDHLVNLYTRDLRKNANIEDSSTRSKQSESEQNRSSLLMEKQDSKETDGNNNSINDDDNNNENNKNEFNEAGPSSLNSLSAPDLTQNIQSRVVAPSRSSILAKSDIFYHYSRDIKLWTELQDLTVYYTKTAHKMFLKENRLNFTKYFDLISDSIVFTQLGCRLMQHEIKAKSCSKEIKKIFKGLISSLSRISINSHLYFDSAFHRKKMDTMNDKDNDNQENNCSRTEGDDGKIEVDSVHDLVSVPLSGKRNVSTSTTDTLTPMRSSFSTVNENDMENFSVLGPRNSVNSVVTPRTSIQNSTLEDFSPSNKNFKSAKSIYEMVDVEFSKFLRHVQLLYFVLQSSVFSDDNTLPQLLPRFFKGSFSGGSWTNPFSTFITDEFGNATKNKAVTSNEVTASSSKNSSISRIPPKMADAIASASGYSANSETNSQIDLKASSAASGSVFTPFNRPSHNRTFSRARVSKRKKKYPLTVDTLNTMKKKSSQIFEKLNNATGEHLKIISKPKSRIRNLEINSSTYEQINQNVLLLEILENLDLSIFINLKNLIKTPSILLDLESEEFLVHAMSSVSSVLTEFFDIKQAFHDIVIRLIMTTQQTTLDDPYLFSSMRSNFPVGHHEPFKNISNTPLVKGPFHKKNEQLALSLFHVLVSQDVEFNNLEFLNNSDDFKDACEKYVEISNLACIIVDQLIEERENLLNYAARMMKNNLTAELLKGEQEKWFDIYSEDYSDDDSENDEAIIDDELGSEDYIERKAANIEKNLPWFLTSDYETSLVYDSRGKIRGGTKEALIEHLTSHELVDAAFNVTMLITFRSILTTREFFYALIYRYNLYPPEGLSYDDYNIWIEKKSNPIKCRVVNIMRTFLTQYWTRNYYEPGIPLILNFAKMVVSEKIPGAEDLLQKINEKLINENEKEPVDPKQQDSVSAVVQTTKRDNKSPIHMSSSSLPSSASSAFFRLKKLKLLDIDPYTYATQLTVLEHDLYLRITMFECLDRAWGTKYCNMGGSPNITKFIANANTLTNFVSHTIVKQADVKTRSKLTQYFVTVAQHCKELNNFSSMTAIVSALYSSPIYRLKKTWDLVSTESKDLLKNLNNLMDSKRNFVKYRELLRSVTDVACVPFFGVYLSDLTFTFVGNPDFLHNSTNIINFSKRTKIANIVEEIISFKRFHYKLKRLDDIQTVIEASLENVPHIEKQYQLSLQVEPRSGNTKGSTHASSASGTKTAKFLSEFTDDKNGNFLKLGKKKPPSRLFR CA90053435C85465 CA90053435C85465 raf1_human RAF proto-oncogene serine/threonine-protein kinase RAF1 RAF Proto-oncogene c-RAF Raf-1 protein protein human Homo sapiens Human MEHIQGAWKTISNGFGFKDAVFDGSSCISPTIVQQFGYQRRASDDGKLTDPSKTSNTIRVFLPNKQRTVVNVRNGMSLHDCLMKALKVRGLQPECCAVFRLLHEHKGKKARLDWNTDAASLIGEELQVDFLDHVPLTTHNFARKTFLKLAFCDICQKFLLNGFRCQTCGYKFHEHCSTKVPTMCVDWSNIRQLLLFPNSTIGDSGVPALPSLTMRRMRESVSRMPVSSQHRYSTPHAFTFNTSSPSSEGSLSQRQRSTSTPNVHMVSTTLPVDSRMIEDAIRSHSESASPSALSSSPNNLSPTGWSQPKTPVPAQRERAPVSGTQEKNKIRPRGQRDSSYYWEIEASEVMLSTRIGSGSFGTVYKGKWHGDVAVKILKVVDPTPEQFQAFRNEVAVLRKTRHVNILLFMGYMTKDNLAIVTQWCEGSSLYKHLHVQETKFQMFQLIDIARQTAQGMDYLHAKNIIHRDMKSNNIFLHEGLTVKIGDFGLATVKSRWSGSQQVEQPTGSVLWMAPEVIRMQDNNPFSFQSDVYSYGIVLYELMTGELPYSHINNRDQIIFMVGRGYASPDLSKLYKNCPKAMKRLVADCVKKVKEERPLFPQILSSIELLQHSLPKINRSASEPSLHRAAHTEDINACTLTTSPRLPVF DIP protein P04049 original sequence version: 117 DIP protein P04049 original sequence version: 106 DIP protein P04049 original sequence version: 114 DIP protein P04049 original sequence version: 141 DIP protein P04049 original sequence version: 142 DIP protein P04049 original sequence version: 147 DIP protein P04049-1 original sequence version: 136 DIP protein P04049 original sequence version: 136 DIP protein P04049 original sequence version: 153 DIP protein P04049 original sequence version: 150 DIP protein P04049 original sequence version: 164 DIP protein P04049 original sequence version: 172 DIP protein P04049-1 original sequence version: 172 DIP protein P04049 original sequence version: 184 DIP protein P04049 original sequence version: 188 DIP protein P04049 original sequence version: 195 EF821B5349711BC3 ras2_yeast Ras-like protein 2 RAS2 ASC1 CTN5 GLC5 YNL098C N2198 protein protein yeast Saccharomyces cerevisiae Baker's yeast MPLNKSNIREYKLVVVGGGGVGKSALTIQLTQSHFVDEYDPTIEDSYRKQVVIDDEVSILDILDTAGQEEYSAMREQYMRNGEGFLLVYSITSKSSLDELMTYYQQILRVKDTDYVPIVVVGNKSDLENEKQVSYQDGLNMAKQMNAPFLETSAKQAINVEEAFYTLARLVRDEGGKYNKTLTENDNSKQTSQDTKGSGANSVPRNSGGHRKMSNAANGKNVNSSTTVVNARNASIESKTGLAGNQATNGKTQTDRTNIDNSTGQAGQANAQSANTVNNRVNNNSKAGQVSNAKQARKQQAAPGGNTSEASKSGSGGCCIIS 6CAAAC531EEAE92F 6CAAAC531EEAE92F raf1-ras2 1 Ras2p 4 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey cell lysate cell lysate physiological level physiological level endogenous level endogenous endogenous endogenous level RAF1-RBD 3 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role bait bait purified purified binding site binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain 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 physical association physical association f2 domino mint To further verify the down-regulation of Ras2p activity by PKA, we adopted the Ras2-GTP/RBD pull-down assay to examine the intracellular Ras2-GTP level in PKA mutants. Cells were incubated on glucose medium to an A600nm of 1. As shown in Fig. 2A, the intracellular level of Ras2-GTP is elevated in PKA-deleted cells (DJ301), whereas it is remarkably diminished in PKA-activated cells (DJ296-3). Though its high PKA activity, mutant carrying RAS2val19 allele (PMA203) exhibits an astonishingly high level of Ras2-GTP 6 due to its impaired GTPase activity [25].The different levels of Ras2-GTP in three mutants of elevated and constitutive PKA (DJ296-2, DJ296-3 and DJ296-4) might be due to the different activity of the three catalytic subunits (Fig. 2B). Above all, these data reveal a down-regulation of intracellular level of Ras2-GTP by PKA activity raf1-cdc25-1 1 RAF1-RBD 3 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role bait bait 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 Cdc25p 2 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 cell lysate cell lysate ha tag ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined Ras2p 4 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey cell lysate cell lysate physiological level physiological level endogenous level endogenous endogenous endogenous level association association homomint domino mint f4a f4b f4c f4d Quantitative analysis (Fig. 4D) of western blot (Fig. 4B,C) shows that the ratios of Ras2-GTP/Cdc25p are increased in PKA-deleted mutants (DJ402 and DJ424), whereas they are decreased in PKA-activated mutants (DJ304 and DJ301), revealing that the intracellular association between Cdc25p and Ras2-GTP is down-regulated by PKA. In mutant ZHS804 that expressing ras2val19 allele, though the extremely high level of Ras2-GTP (the sixth lane of Fig. 4B), the Cdc25p bound to Ras2-GTP is not accordingly increased compared with DJ304 and DJ301 (the sixth lane of Fig. 4C). This is consistent with the low GTPase of Ras2val19 suppressing the Cdc25p requirement for activation of adenylate cyclase [25].