IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ A keratin cytoskeletal protein regulates protein synthesis and epithelial cell growth. A keratin cytoskeletal protein regulates protein synthesis and epithelial cell growth. Nature (0028-0836) 2006 Kim S., Wong P., Coulombe PA. coulombe@jhmi.edu 13-Nov-2007: Contacted by JYOTI. davet mouse-epithelium Mus musculus epithelium Epithelium [epithelial cells] fluorescence imaging fluorescence microscopy fluorescence imaging immunostaining immunostaining Kim S., Wong P., Coulombe PA. coulombe@jhmi.edu Accepted 2007-NOV-17 by DAVET. 13-Nov-2007: Contacted by JYOTI. Nature (0028-0836) 2006 davet A keratin cytoskeletal protein regulates protein synthesis and epithelial cell growth. A keratin cytoskeletal protein regulates protein synthesis and epithelial cell growth. Nature (0028-0836) 2006 Kim S., Wong P., Coulombe PA. coulombe@jhmi.edu 13-Nov-2007: Contacted by JYOTI. davet mesau-bhk21 Mesocricetus auratus_BHK21 bhk-21 Cells from kidneys of five unsexed, one-day-old hamsters BHK-21/C13 cell coip coimmunoprecipitation co-immunoprecipitation CoIp immunoprecipitation Co-IP co-immunoprecipitation CoIp immunoprecipitation Co-IP coip western blot western blot Immuno blot Immuno blot Kim S., Wong P., Coulombe PA. coulombe@jhmi.edu Accepted 2007-NOV-17 by DAVET. 13-Nov-2007: Contacted by JYOTI. Nature (0028-0836) 2006 davet A keratin cytoskeletal protein regulates protein synthesis and epithelial cell growth. A keratin cytoskeletal protein regulates protein synthesis and epithelial cell growth. Nature (0028-0836) 2006 Kim S., Wong P., Coulombe PA. coulombe@jhmi.edu 13-Nov-2007: Contacted by JYOTI. davet mouse-epithelium Mus musculus epithelium Epithelium [epithelial cells] coip coimmunoprecipitation co-immunoprecipitation CoIp immunoprecipitation Co-IP co-immunoprecipitation CoIp immunoprecipitation Co-IP coip western blot western blot Immuno blot Immuno blot Kim S., Wong P., Coulombe PA. coulombe@jhmi.edu Accepted 2007-NOV-17 by DAVET. Nature (0028-0836) 2006 davet 1433s_mouse 14-3-3 protein sigma Sfn Mkrn3 Stratifin protein protein mouse Mus musculus Mouse MERASLIQKAKLAEQAERYEDMAAFMKSAVEKGEELSCEERNLLSVAYKNVVGGQRAAWRVLSSIEQKSNEEGSEEKGPEVKEYREKVETELRGVCDTVLGLLDSHLIKGAGDAESRVFYLKMKGDYYRYLAEVATGDDKKRIIDSARSAYQEAMDISKKEMPPTNPIRLGLALNFSVFHYEIANSPEEAISLAKTTFDEAMADLHTLSEDSYKDSTLIMQLLRDNLTLWTADSAGEEGGEAPEEPQS C1C40905824B3F48 k1c17_mouse Keratin, type I cytoskeletal 17 Krt17 Krt1-17 Keratin-17 Cytokeratin-17 protein protein mouse Mus musculus Mouse MTTTIRQFTSSSSIKGSSGLGGGSSRTSCRLSGSLGAGSCRLGSASGLGSALGSNSYSSCYSFGTGSGYGGNFGGVDGLLAGGEKATMQNLNDRLASYLDKVRALEEANTELEVKIRDWYQKQAPGPARDYSAYYHTIEDLKNKILVATVDNASILLQIDNARLAADDFRTKFETEQALRMSVEADINGLRRVLDELTLARADLEMQIENLKEELAYLKKNHEEEMNALRGQVGGEINVEMDAAPGVDLSRILSEMRDQYEKMAEKNRKDAEDWFFSKTEELNREVATNSELVQSGKSEISELRRTMQALEIELQSQLSMKASLEGSLAETENRYCVQLSQIQGLIGSVEEQLAQLRCEMEQQNQEYKILLDVKTRLEQEIATYRRLLEGEDAHLTQYKPKEPVTTRQVRTIVEEVQDGKVISSREQVHQTTR DIP protein Q9QWL7 original sequence version: 106 279081DF2ECE105D krt17-sfn 3 5 unspecified role unspecified role bait bait mouse-epithelium Mus musculus epithelium Epithelium [epithelial cells] 4 unspecified role unspecified role prey prey mouse-epithelium Mus musculus epithelium Epithelium [epithelial cells] physical association physical association Supplementary Figures 3a-c. To identify K17-binding proteins that might provide mechanistic insight into this growth defect, we set up a biochemical screen using a reversible chemical crosslinker in cultured skin keratinocytes. We identified 14-3-3sigma (also known as stratifin), an epithelial-specific isoform of 14-3-3 (ref. 16), as a specific K17-binding protein in keratinocytes (Supplementary Fig. 3a). By okadaic acid (a serine/threonine phosphatase inhibitor) and by orthophosphates. Krt17 interacts with 14-3-3sigma in a phosphorylation-dependent manner. krt17-sfn-1 1 4 unspecified role unspecified role neutral component neutral component mouse-epithelium Mus musculus epithelium Epithelium [epithelial cells] 5 unspecified role unspecified role neutral component neutral component mouse-epithelium Mus musculus epithelium Epithelium [epithelial cells] colocalization colocalization Supplementary Figure 3d We find that 14-3-3-sigma colocalizes with K17 in the cytoplasm of skin keratinocytes, where their interaction is phosphorylation-dependant (Supplementary Fig. 3b-d). By wounded or stressed epithelium. The authors noted that ". Like K17 protein , 14-3-3 sigma protein; is clearly induced in the wound edge epithelium after skin injury ". krt17-sfn-2 2 5 unspecified role unspecified role bait bait ser44ala binding region binding-associated region binding region mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain thr9ala binding region binding-associated region binding region mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain mesau-bhk21 Mesocricetus auratus_BHK21 bhk-21 Cells from kidneys of five unsexed, one-day-old hamsters BHK-21/C13 cell 4 unspecified role unspecified role prey prey mesau-bhk21 Mesocricetus auratus_BHK21 bhk-21 Cells from kidneys of five unsexed, one-day-old hamsters BHK-21/C13 cell physical association physical association Supplementary Figures 4a,b. Identifying the amino acid residue(s) mediating 14-3-3 binding to K17 should help to define the role of this interaction with regards to protein synthesis. Two consensus 14-3-3-binding sites, threonine 9 (Thr 9) and serine 44 (Ser 44), are present in the non-helical head domain of K17 (Supplementary Fig. 4a). We mutated these sites to alanine residues and transfected the mutant DNA constructs into BHK fibroblasts. Relative to wild-type K17, T9A and S44A single mutants and the double mutant T9A/S44A show significantly reduced 14-3-3 binding (Supplementary Fig. 4b). The similar loss of binding observed for all mutants implies that Thr 9 and Ser 44 both contribute to binding to 14-3-3 (ref. 25). Relative to wild-type Krt17, its' interaction with 14-3-3sigma, in transfected cells, was significantly reduced in the single mutants T9A and S44A as well as with the double mutant T9A/S44A.