title: Keratin 1 plays a critical role in golgi localization of core 2 N-acetylglucosaminyltransferase M via interaction with its cytoplasmic tail.
abstract: Core 2 N-acetylglucosaminyltransferase 2/M (C2GnT-M) synthesizes all three β6GlcNAc branch structures found in secreted mucins. Loss of C2GnT-M leads to development of colitis and colon cancer. Recently we have shown that C2GnT-M targets the Golgi at the Giantin site and is recycled by binding to non-muscle myosin IIA, a motor protein, via the cytoplasmic tail (CT). But how this enzyme is retained in the Golgi is not known. Proteomics analysis identifies keratin type II cytoskeletal 1 (KRT1) as a protein pulled down with anti-c-Myc antibody or C2GnT-M CT from the lysate of Panc1 cells expressing bC2GnT-M tagged with c-Myc. Yeast two-hybrid analysis shows that the rod domain of KRT1 interacts directly with the WKR(6) motif in the C2GnT-M CT. Knockdown of KRT1 does not affect Golgi morphology but increases the interaction of C2GnT-M with non-muscle myosin IIA and its transportation to the endoplasmic reticulum, ubiquitination, and degradation. During Golgi recovery after brefeldin A treatment, C2GnT-M forms a complex with Giantin before KRT1, demonstrating CT-mediated sequential events of Golgi targeting and retention of C2GnT-M. In HeLa cells transiently expressing C2GnT-M-GFP, knockdown of KRT1 does not affect Golgi morphology but leaves C2GnT-M outside of the Golgi, resulting in the formation of sialyl-T antigen. Interaction of C2GnT-M and KRT1 was also detected in the goblet cells of human colon epithelial tissue and primary culture of colonic epithelial cells. The results indicate that glycosylation and thus the function of glycoconjugates can be regulated by a protein that helps retain a glycosyltransferase in the Golgi.
authors.affiliation: From the VA Nebraska-Western Iowa Health Care System, Department of Research Service, Omaha, Nebraska 68105 and Department of Biochemistry and Molecular Biology, College of Medicine and.
authors.forename: Armen
authors.initials: A
authors.lastname: Petrosyan
authors.affiliation: From the VA Nebraska-Western Iowa Health Care System, Department of Research Service, Omaha, Nebraska 68105 and Department of Biochemistry and Molecular Biology, College of Medicine and.
authors.forename: Mohamed F
authors.initials: MF
authors.lastname: Ali
authors.affiliation: From the VA Nebraska-Western Iowa Health Care System, Department of Research Service, Omaha, Nebraska 68105 and Department of Biochemistry and Molecular Biology, College of Medicine and Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198 pcheng@unmc.edu.
authors.forename: Pi-Wan
authors.initials: PW
authors.lastname: Cheng
mesh_terms: D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism / Q000648:ultrastructure; D006056:Golgi Apparatus / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D000077614:Golgi Matrix Proteins; D006367:HeLa Cells; D006801:Humans; D053556:Keratin-1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry / Q000378:metabolism*; D021381:Protein Transport
chemical_list: D000077614:Golgi Matrix Proteins; C491616:KRT1 protein, human; D053556:Keratin-1; D008565:Membrane Proteins; C087574:macrogolgin; D020126:Brefeldin A; D017351:N-Acetylglucosaminyltransferases; C053024:beta-1,3-galactosyl-O-glycosyl-glycoprotein beta-1,6-acetylglucosaminyl transferase
keywords: Glycobiology; Glycosyltransferase; Golgi; Keratin; Mucus; Trafficking