mbinfo MBInfo http://www.mechanobio.info/ http://www.mechanobio.info/ edwards-1999-1 Activation of LIM-kinase by Pak1 couples Rac/Cdc42 GTPase signalling to actin cytoskeletal dynamics. in vitro In vitro protein kinase assay protein kinase assay autoradiography autoradiography Edwards D.C., Sanders L.C., Bokoch G.M., Gill G.N. To test the activation effect of Pak1 on LIMK1, the rate of cofilin phosphorylation by LIMK1 was calculated by measuring the incorporation of radiolabelled-P into Ser3 of cofilin. Inferred from the paper, all human LIMK1 proteins were purified from HEK293 cells in Fig.1a, b & Fig. 2Ab; whereas the kinase domains of the human LIMK1 in Fig. 1c & Fig. 2Aa, B were purified from the baculovirus-infected SF-21 cells. Inferred from the reference paper (PMID: 7618083), all PAK1 proteins were purified from E.coli cells. bokoch@scripps.edu;ggill@ucsd.edu Nat. Cell Biol. (1465-7392) 1999 Only protein-protein interactions imex curation edwards-1999-2 Activation of LIM-kinase by Pak1 couples Rac/Cdc42 GTPase signalling to actin cytoskeletal dynamics. human-a_431 Homo sapiens epidermoid carcinoma a_431 Human epidermoid carcinoma anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Edwards D.C., Sanders L.C., Bokoch G.M., Gill G.N. To test the activation effect of Pak1 on LIMK1, the rate of cofilin phosphorylation by LIMK1 was calculated by measuring the incorporation of radiolabelled-P into Ser3 of cofilin. Inferred from the paper, all human LIMK1 proteins were purified from HEK293 cells in Fig.1a, b & Fig. 2Ab; whereas the kinase domains of the human LIMK1 in Fig. 1c & Fig. 2Aa, B were purified from the baculovirus-infected SF-21 cells. Inferred from the reference paper (PMID: 7618083), all PAK1 proteins were purified from E.coli cells. bokoch@scripps.edu;ggill@ucsd.edu Nat. Cell Biol. (1465-7392) 1999 Only protein-protein interactions imex curation edwards-1999-3 Activation of LIM-kinase by Pak1 couples Rac/Cdc42 GTPase signalling to actin cytoskeletal dynamics. human-kidney Human kidney kidney kidney [Renal] anti bait coip anti bait coimmunoprecipitation anti bait coip anti tag western anti tag western blot anti tag western Edwards D.C., Sanders L.C., Bokoch G.M., Gill G.N. To test the activation effect of Pak1 on LIMK1, the rate of cofilin phosphorylation by LIMK1 was calculated by measuring the incorporation of radiolabelled-P into Ser3 of cofilin. Inferred from the paper, all human LIMK1 proteins were purified from HEK293 cells in Fig.1a, b & Fig. 2Ab; whereas the kinase domains of the human LIMK1 in Fig. 1c & Fig. 2Aa, B were purified from the baculovirus-infected SF-21 cells. Inferred from the reference paper (PMID: 7618083), all PAK1 proteins were purified from E.coli cells. bokoch@scripps.edu;ggill@ucsd.edu Nat. Cell Biol. (1465-7392) 1999 Only protein-protein interactions imex curation edwards-1999-4 Activation of LIM-kinase by Pak1 couples Rac/Cdc42 GTPase signalling to actin cytoskeletal dynamics. in vitro In vitro pull down pull down autoradiography autoradiography Edwards D.C., Sanders L.C., Bokoch G.M., Gill G.N. To test the activation effect of Pak1 on LIMK1, the rate of cofilin phosphorylation by LIMK1 was calculated by measuring the incorporation of radiolabelled-P into Ser3 of cofilin. Inferred from the paper, all human LIMK1 proteins were purified from HEK293 cells in Fig.1a, b & Fig. 2Ab; whereas the kinase domains of the human LIMK1 in Fig. 1c & Fig. 2Aa, B were purified from the baculovirus-infected SF-21 cells. Inferred from the reference paper (PMID: 7618083), all PAK1 proteins were purified from E.coli cells. bokoch@scripps.edu;ggill@ucsd.edu Nat. Cell Biol. (1465-7392) 1999 Only protein-protein interactions imex curation edwards-1999-5 Activation of LIM-kinase by Pak1 couples Rac/Cdc42 GTPase signalling to actin cytoskeletal dynamics. chlae-cos_7 Cercopithecus aethiops simian cells transformed with SV40 Grivet cos_7 cercopithecus aethiops, by transformation with SV40 fluorescence imaging fluorescence microscopy fluorescence imaging immunostaining immunostaining Edwards D.C., Sanders L.C., Bokoch G.M., Gill G.N. To test the activation effect of Pak1 on LIMK1, the rate of cofilin phosphorylation by LIMK1 was calculated by measuring the incorporation of radiolabelled-P into Ser3 of cofilin. Inferred from the paper, all human LIMK1 proteins were purified from HEK293 cells in Fig.1a, b & Fig. 2Ab; whereas the kinase domains of the human LIMK1 in Fig. 1c & Fig. 2Aa, B were purified from the baculovirus-infected SF-21 cells. Inferred from the reference paper (PMID: 7618083), all PAK1 proteins were purified from E.coli cells. bokoch@scripps.edu;ggill@ucsd.edu Nat. Cell Biol. (1465-7392) 1999 Only protein-protein interactions imex curation limk1_human LIM domain kinase 1 LIMK1 LIMK protein protein human Homo sapiens Human MRLTLLCCTWREERMGEEGSELPVCASCGQRIYDGQYLQALNADWHADCFRCCDCSASLSHQYYEKDGQLFCKKDYWARYGESCHGCSEQITKGLVMVAGELKYHPECFICLTCGTFIGDGDTYTLVEHSKLYCGHCYYQTVVTPVIEQILPDSPGSHLPHTVTLVSIPASSHGKRGLSVSIDPPHGPPGCGTEHSHTVRVQGVDPGCMSPDVKNSIHVGDRILEINGTPIRNVPLDEIDLLIQETSRLLQLTLEHDPHDTLGHGLGPETSPLSSPAYTPSGEAGSSARQKPVLRSCSIDRSPGAGSLGSPASQRKDLGRSESLRVVCRPHRIFRPSDLIHGEVLGKGCFGQAIKVTHRETGEVMVMKELIRFDEETQRTFLKEVKVMRCLEHPNVLKFIGVLYKDKRLNFITEYIKGGTLRGIIKSMDSQYPWSQRVSFAKDIASGMAYLHSMNIIHRDLNSHNCLVRENKNVVVADFGLARLMVDEKTQPEGLRSLKKPDRKKRYTVVGNPYWMAPEMINGRSYDEKVDVFSFGIVLCEIIGRVNADPDYLPRTMDFGLNVRGFLDRYCPPNCPPSFFPITVRCCDLDPEKRPSFVKLEHWLETLRMHLAGHLPLGPQLEQLDRGFWETYRRGESGLPAHPEVPD pak1_human Serine/threonine-protein kinase PAK 1 PAK1 p21-activated kinase 1 p65-PAK Alpha-PAK protein protein human Homo sapiens Human MSNNGLDIQDKPPAPPMRNTSTMIGAGSKDAGTLNHGSKPLPPNPEEKKKKDRFYRSILPGDKTNKKKEKERPEISLPSDFEHTIHVGFDAVTGEFTGMPEQWARLLQTSNITKSEQKKNPQAVLDVLEFYNSKKTSNSQKYMSFTDKSAEDYNSSNALNVKAVSETPAVPPVSEDEDDDDDDATPPPVIAPRPEHTKSVYTRSVIEPLPVTPTRDVATSPISPTENNTTPPDALTRNTEKQKKKPKMSDEEILEKLRSIVSVGDPKKKYTRFEKIGQGASGTVYTAMDVATGQEVAIKQMNLQQQPKKELIINEILVMRENKNPNIVNYLDSYLVGDELWVVMEYLAGGSLTDVVTETCMDEGQIAAVCRECLQALEFLHSNQVIHRDIKSDNILLGMDGSVKLTDFGFCAQITPEQSKRSTMVGTPYWMAPEVVTRKAYGPKVDIWSLGIMAIEMIEGEPPYLNENPLRALYLIATNGTPELQNPEKLSAIFRDFLNRCLEMDVEKRGSAKELLQHQFLKIAKPLSSLTPLIAAAKEATKNNH limk1-pak1 1310455 n/a 1310460 enzyme target enzyme target substrate substrate unspecified role unspecified role kinase domain activation domain activation domain tag activation domain certain certain sequence position certain certain certain sequence position certain false spofr-sf_21 Spodoptera frugiperda insect cells sf_21 Insect cells derived from immature ovaries of fall armyworm Spodoptera frugiperda pupae. full name: IPLB-SF-21-AE cells are susceptible to Baculovirus infection. n/a Pak1 1310461 enzyme enzyme unspecified role unspecified role Q13153:p.Thr423Glu mutation increasing mutation increasing interaction mutation increasing certain certain sequence position certain certain certain sequence position certain false The mutation for the constitutively activated form of Pak1 protein is inferred from the reference paper. (PMID: 10551809) 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) phosphorylation phosphorylation reaction phosphorylation false false false Fig1. c limk1-limk1 1310455 n/a 1310460 putative self putative self putative self putative self human-kidney Human kidney kidney kidney [Renal] phosphorylation phosphorylation reaction phosphorylation false true false Fig. 2Ab pak1-pak1 1310455 n/a 1310461 putative self putative self putative self putative self ecoli Escherichia coli (strain K12) phosphorylation phosphorylation reaction phosphorylation false true false Fig. 2Ab pak1-limk1 1310456 n/a 1310461 unspecified role unspecified role bait bait n/a 1310460 unspecified role unspecified role prey prey physical association physical association false false false Fig. 3a limk1-pak1 1310457 n/a 1310461 unspecified role unspecified role prey prey region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false n/a 1310460 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false physical association physical association false false false Fig. 3b Constitutively activated Rac(Q61L) enhanced complex formation between Pak and LIMK1, compared with the complex formation induced by inactive Rac(T17N). pak1-limk1 1310458 n/a 1310461 unspecified role unspecified role bait bait region binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false 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 region binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false LIMK1 binds to the region between amino acids 67 to 150 in the Pak1 N terminus, which contains the p21-binding domain (amino acids 67-86) and the autoinhibitory regulatory domain (amino acids 83-149). ecoli Escherichia coli (strain K12) n/a 1310460 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 autoradiography autoradiography undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false in vitro In vitro physical association physical association false false false Fig. 3c LIMK1 binds to a region of Pak1 that includes its autoinhibitory domain, and this interaction is increased by Rac or Cdc42 binding to the p21-binding domain of Pak1. limk1-pak1-1 1310459 n/a 1310461 unspecified role unspecified role neutral component neutral component nucl transfection nucleic acid transfection nucl transfection region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false n/a 1310460 unspecified role unspecified role neutral component neutral component nucl transfection nucleic acid transfection nucl transfection colocalization colocalization false false false Fig. 7