UniProt UniProt http://www.uniprot.org Hog1 mediates cell-cycle arrest in G1 phase by the dual targeting of Sic1. Hog1 mediates cell-cycle arrest in G1 phase by the dual targeting of Sic1. Nat. Cell Biol. (1465-7392) 2004 Escote X., Zapater M., Clotet J., Posas F. francesc@posas@upf.edu 13-Nov-2007: Contacted by JYOTI. orchard in vitro In vitro protein kinase assay protein kinase assay predetermined predetermined participant predetermined Escote X., Zapater M., Clotet J., Posas F. Kinase assay fig 4e not included. Is performed with CLN2-CDC28 complex. Experimental details not described (origin of CDC28?, immunoprecipitated, purified?). The wild-type or GST-Sic1 mutants were expressed in E.coli, purified and mixed with purified GST-Hog1 or catalytically inactive Hog1. francesc@posas@upf.edu Accepted 2007-NOV-2 by ORCHARD. 13-Nov-2007: Contacted by JYOTI. Nat. Cell Biol. (1465-7392) 2004 orchard Hog1 mediates cell-cycle arrest in G1 phase by the dual targeting of Sic1. Hog1 mediates cell-cycle arrest in G1 phase by the dual targeting of Sic1. Nat. Cell Biol. (1465-7392) 2004 Escote X., Zapater M., Clotet J., Posas F. francesc@posas@upf.edu 13-Nov-2007: Contacted by JYOTI. orchard yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast 2 hybrid two hybrid 2H 2h Gal4 transcription regeneration two-hybrid yeast two hybrid 2-hybrid classical two hybrid classical two hybrid 2H two-hybrid 2-hybrid Gal4 transcription regeneration 2h yeast two hybrid Y2H Y-2H 2 hybrid predetermined predetermined participant predetermined Escote X., Zapater M., Clotet J., Posas F. francesc@posas@upf.edu Accepted 2007-NOV-2 by ORCHARD. Nat. Cell Biol. (1465-7392) 2004 orchard Hog1 mediates cell-cycle arrest in G1 phase by the dual targeting of Sic1. Hog1 mediates cell-cycle arrest in G1 phase by the dual targeting of Sic1. Nat. Cell Biol. (1465-7392) 2004 Escote X., Zapater M., Clotet J., Posas F. francesc@posas@upf.edu 13-Nov-2007: Contacted by JYOTI. orchard in vitro In vitro pull down pull down anti tag western anti tag western blot anti tag western Escote X., Zapater M., Clotet J., Posas F. GST-Sic1 or His-Hog1 were purified from E. coli. GST proteins were incubated with His-Hog1 bound to beads and Sic1 was probed using GST antibody. francesc@posas@upf.edu Accepted 2007-NOV-2 by ORCHARD. 13-Nov-2007: Contacted by JYOTI. Nat. Cell Biol. (1465-7392) 2004 orchard Hog1 mediates cell-cycle arrest in G1 phase by the dual targeting of Sic1. Hog1 mediates cell-cycle arrest in G1 phase by the dual targeting of Sic1. Nat. Cell Biol. (1465-7392) 2004 Escote X., Zapater M., Clotet J., Posas F. francesc@posas@upf.edu 13-Nov-2007: Contacted by JYOTI. orchard yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast anti tag coip anti tag coimmunoprecipitation anti tag coip anti tag western anti tag western blot anti tag western Escote X., Zapater M., Clotet J., Posas F. Yeast extracts containing GST-Hog1 and tagged HA-Sic1 (from its chromosomal locus) were incubated with anti-HA antibodies and precipitated GST proteins were probed. francesc@posas@upf.edu Accepted 2007-NOV-2 by ORCHARD. 13-Nov-2007: Contacted by JYOTI. Nat. Cell Biol. (1465-7392) 2004 orchard hog1_yeast Mitogen-activated protein kinase HOG1 HOG1 SSK3 YLR113W L2931 L9354.2 High osmolarity glycerol response protein 1 protein protein yeast Saccharomyces cerevisiae Baker's yeast MTTNEEFIRTQIFGTVFEITNRYNDLNPVGMGAFGLVCSATDTLTSQPVAIKKIMKPFSTAVLAKRTYRELKLLKHLRHENLICLQDIFLSPLEDIYFVTELQGTDLHRLLQTRPLEKQFVQYFLYQILRGLKYVHSAGVIHRDLKPSNILINENCDLKICDFGLARIQDPQMTGYVSTRYYRAPEIMLTWQKYDVEVDIWSAGCIFAEMIEGKPLFPGKDHVHQFSIITDLLGSPPKDVINTICSENTLKFVTSLPHRDPIPFSERFKTVEPDAVDLLEKMLVFDPKKRITAADALAHPYSAPYHDPTDEPVADAKFDWHFNDADLPVDTWRVMMYSEILDFHKIGGSDGQIDISATFDDQVAAATAAAAQAQAQAQAQVQLNMAAHSHNGAGTTGNDHSDIAGGNKVSDHVAANDTITDYGNQAIQYANEFQQ D95A20242587CE06 D95A20242587CE06 smp1_yeast Transcription factor SMP1 YBR1245 YBR182C SMP1 protein protein yeast Saccharomyces cerevisiae Baker's yeast MGRRKIEIEPIKDDRNRTVTFIKRKAGLFKKAHELSVLCQVDIAVIILGSNNTFYEYSSVDMSNLLNVHQNNTDLPHNIIEPSDYGDYVKKPRVVLNERKRRRRRATVLQPASHSGSCTVSSQDSSSVQNNGNLSAPLASNDAGNAGVSTPLVHCHGAISRSGSNHSDCARNSADYQMLQGGLNSGGSFHANDYKESVDQQHVANEAIHRNFMNKRIRPDTHLLLSESNHSNYHNFYPSPYENLPKPSLPASLVGNIPSFQSQFVQVIPANSNPMGKGFNGTGDSESFEAKQKIHPTVAISNTLEGPAPVQAMVHHLHQLNSNRGKLSGKPYLKLNIPKATNDACQRSPAMYSGTASPKTDVQATPNQMLASNMSSPLSRSKFLGFKNNDMDDLYHNGRCGSTYVNNKTFFLKPPIGRPPKFPKSPSSSIVVFPSSVASSTLKSTSSTNSPD 3D53A859D468227C cdc4_yeast Cell division control protein 4 CDC4 YFL009W F-box protein CDC4 E3 ubiquitin ligase complex SCF subunit CDC4 protein protein yeast Saccharomyces cerevisiae Baker's yeast MGSFPLAEFPLRDIPVPYSYRVSGGIASSGSVTALVTAAGTHRNSSTAKTVETEDGEEDIDEYQRKRAAGSGESTPERSDFKRVKHDNHKTLHPVNLQNTGAASVDNDGLHNLTDISNDAEKLLMSVDDGSAAPSTLSVNMGVASHNVAAPTTVNAATITGSDVSNNVNSATINNPMEEGALPLSPTASSPGTTTPLAKTTKTINNNNNIADLIESKDSIISPEYLSDEIFSAINNNLPHAYFKNLLFRLVANMDRSELSDLGTLIKDNLKRDLITSLPFEISLKIFNYLQFEDIINSLGVSQNWNKIIRKSTSLWKKLLISENFVSPKGFNSLNLKLSQKYPKLSQQDRLRLSFLENIFILKNWYNPKFVPQRTTLRGHMTSVITCLQFEDNYVITGADDKMIRVYDSINKKFLLQLSGHDGGVWALKYAHGGILVSGSTDRTVRVWDIKKGCCTHVFKGHNSTVRCLDIVEYKNIKYIVTGSRDNTLHVWKLPKESSVPDHGEEHDYPLVFHTPEENPYFVGVLRGHMASVRTVSGHGNIVVSGSYDNTLIVWDVAQMKCLYILSGHTDRIYSTIYDHERKRCISASMDTTIRIWDLENIWNNGECSYATNSASPCAKILGAMYTLQGHTALVGLLRLSDKFLVSAAADGSIRGWDANDYSRKFSYHHTNLSAITTFYVSDNILVSGSENQFNIYNLRSGKLVHANILKDADQIWSVNFKGKTLVAAVEKDGQSFLEILDFSKASKINYVSNPVNSSSSSLESISTSLGLTRTTIIP 0348F2F8FA78F3BC 0348F2F8FA78F3BC sic1_yeast Protein SIC1 SIC1 SDB25 YLR079W L9449.8 CDK inhibitor p40 protein protein yeast Saccharomyces cerevisiae Baker's yeast MTPSTPPRSRGTRYLAQPSGNTSSSALMQGQKTPQKPSQNLVPVTPSTTKSFKNAPLLAPPNSNMGMTSPFNGLTSPQRSPFPKSSVKRTLFQFESHDNGTVREEQEPLGRVNRILFPTQQNVDIDAAEEEEEGEVLLPPSRPTSARQLHLSLERDEFDQTHRKKIIKDVPGTPSDKVITFELAKNWNNNSPKNDARSQESEDEEDIIINPVRVGKNPFASDELVTQEIRNERKRAMLRENPDIEDVITYVNKKGEVVEKRRLTDEEKRRFKPKALFQSRDQEH 7913636340678F78 7913636340678F78 sic1-hog1-1 2 5 unspecified role unspecified role prey prey 8 unspecified role unspecified role bait bait region binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain physical association physical association 3a Two-hybrid experiments using a Sic1 form that does not contain the CDK inhibitory domain to prevent cell-cycle arrest (Sic1DeltaCDK), showed that Hog1 interacted with Sic1 as efficiently as the Hog1 downstream target Smp1 (ref. 16) (Fig. 3a). smp1-hog1-1 2 6 unspecified role unspecified role bait bait 5 unspecified role unspecified role prey prey physical association physical association 3a Two-hybrid experiments using a Sic1 form that does not contain the CDK inhibitory domain to prevent cell-cycle arrest (Sic1DeltaCDK), showed that Hog1 interacted with Sic1 as efficiently as the Hog1 downstream target Smp1 (ref. 16) (Fig. 3a). sic1-hog1-2 4 5 unspecified role unspecified role prey prey 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 8 unspecified role unspecified role bait bait region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association 3b Correspondingly, in vivo co-immunoprecipitation experiments showed that full-length Sic1 precipitated Hog1 (Fig. 3b). sic1-hog1-3 3 5 unspecified role unspecified role bait bait region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 8 unspecified role unspecified role prey prey 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 ecoli Escherichia coli (strain K12) direct interaction direct interaction 3c Morevoer, in vitro experiments using purified proteins showed that Sic1 binds directly to Hog1 (Fig. 3c). hog1-sic1-2 1 5 enzyme enzyme unspecified role unspecified role 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 ecoli Escherichia coli (strain K12) 8 enzyme target enzyme target substrate substrate unspecified role unspecified role P38634:p.Thr173Glu mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain T E Wed May 02 15:27:14 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'thr173glu' Tue Jun 30 17:31:21 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Thr173Glu' ecoli Escherichia coli (strain K12) phosphorylation phosphorylation reaction phosphorylation SIC1 is phosphorylated on Tyr173 as determined by autoradiography and use of SIC1 mutant constructs. sic1-hog1-4 2 8 unspecified role unspecified role bait bait P38634:p.Thr173Glu mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain T E Wed May 02 18:34:19 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'thr173glu' Tue Jun 30 20:02:59 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Thr173Glu' 7 unspecified role unspecified role prey prey physical association physical association 4f Two-hybrid analysis of Cdc4 fused to the GAL4 activator domain with wild-type Sic1DeltaCDK (SIC1) or Sic1 T173EDeltaCDK (Sic1-TE) fused to the LexA-DNA binding domain were evaluated by the filter and liquid beta-galactosidase assay (U, Miller units from three independent assays).