title: The cholangiocyte marker, BD. 1, forms a stable complex with CLIP170 and shares an identity with eIF3a, a multifunctional subunit of the eIF3 initiation complex.
abstract: We have previously described the generation of a monoclonal antibody recognizing a novel cholangiocyte marker, designated BD.1, that is expressed by fetal and adult rat cholangiocytes but not hepatocytes or the hepatic progenitor cells known as oval cells. In the present report, we have undertaken a comprehensive examination of BD.1 expressed by long-term cultures of bile duct epithelial cells (BDEC) and prostate epithelial cells (PEC). We show that with continued passage, the levels of BD.1 expressed by BDEC and PEC drop significantly, a decrease that is temporally associated with transition from a diploid to an aneuploid karyotype. Cell cycle analysis revealed cell cycle dependent expression of BD.1 characterized by decreased BD.1 levels within the first 10 h after release from serum starvation followed by reacquisition as cells entered S phase. MAb BD.1 recognized a 170 kDa protein in Western blots and showed strong reactivity with a 170 kDa band in blots prepared from phosphoproteins isolated by metal affinity chromatography. Analysis by mass spectrometry of tryptic peptides generated from BD.1 purified by continuous elution electrophoresis identified the plus end microtubule-binding protein, CLIP170, in the fraction reactive with MAb BD.1. Double immunofluorescence with MAb BD.1 and a MAb specific for CLIP170 showed that both were reactive with intrahepatic bile ducts. However, overexpression or siRNA knockdown of CLIP170 in 293T cells did not significantly alter BD.1 levels, indicating that CLIP170 and BD.1 were distinct, co-migrating proteins. Immunoprecipitation analysis with MAb BD.1 and anti-CLIP170 antibodies showed that under microtubule depolymerizing conditions the two proteins could be co-precipitated with both antibodies, leading us to conclude they were capable of forming stable complexes. Two different protocols were devised to enrich for the CLIP170 binding protein recognized by MAb BD.1. Analysis of tryptic peptides by LC-ESI-MS/MS identified BD.1 as eIF3a, the largest subunit of the elongation initiation factor 3 (eIF3) complex. This identity was confirmed by the simultaneous knockdown of both BD.1 and eIF3a by eIF3a-specific siRNAs and by the strong reactivity of MAb BD.1 with the 170 kDa protein immunoprecipitated with the anti-eIF3a antibody, 5H10. Based on these findings, we concluded that the BD.1 antigen was identical to eIF3a, a multifunctional subunit of the eIf3 complex shown here to associate with microtubules through its interactions with CLIP170.
authors.affiliation: Department of Medicine, Division of Hematology and Oncology, Rhode Island Hospital/The Warren Alpert Medical School of Brown University, Providence, RI 02903, USA. David_Mills@Brown.edu
authors.forename: David R
authors.initials: DR
authors.lastname: Mills
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Rozich
authors.forename: Donna L
authors.initials: DL
authors.lastname: Flanagan
authors.forename: Kate E
authors.initials: KE
authors.lastname: Brilliant
authors.forename: DongQin
authors.initials: D
authors.lastname: Yang
authors.forename: Douglas C
authors.initials: DC
authors.lastname: Hixson
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D001652:Bile Ducts / Q000166:cytology / Q000378:metabolism*; D015415:Biomarkers / Q000737:chemistry / Q000378:metabolism*; D002453:Cell Cycle; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D039621:Eukaryotic Initiation Factor-3 / Q000235:genetics / Q000378:metabolism*; D005434:Flow Cytometry; D055785:Gene Knockdown Techniques; D008297:Male; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism*; D010449:Peptide Mapping; D011467:Prostate / Q000166:cytology; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011916:Rats, Inbred F344; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D000911:Antibodies, Monoclonal; D015415:Biomarkers; C105988:EIF3A protein, human; D039621:Eukaryotic Initiation Factor-3; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D034741:RNA, Small Interfering; C076744:cytoplasmic linker protein 170