Novel association of APC with intermediate filaments identified using a new versatile APC antibody.
BACKGROUND
As a key player in suppression of colon tumorigenesis, Adenomatous Polyposis Coli (APC) has been widely studied to determine its cellular functions. However, inconsistencies of commercially available APC antibodies have limited the exploration of APC function. APC is implicated in spindle formation by direct interactions with tubulin and microtubule-binding protein EB1. APC also interacts with the actin cytoskeleton to regulate cell polarity. Until now, interaction of APC with the third cytoskeletal element, intermediate filaments, has remained unexamined.


RESULTS
We generated an APC antibody (APC-M2 pAb) raised against the 15 amino acid repeat region, and verified its reliability in applications including immunoprecipitation, immunoblotting, and immunofluorescence in cultured cells and tissue. Utilizing this APC-M2 pAb, we immunoprecipitated endogenous APC and its binding proteins from colon epithelial cells expressing wild-type APC. Using Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS), we identified 42 proteins in complex with APC, including beta-catenin and intermediate filament (IF) proteins lamin B1 and keratin 81. Association of lamin B1 with APC in cultured cells and human colonic tissue was verified by co-immunoprecipitation and colocalization. APC also colocalized with keratins and remained associated with IF proteins throughout a sequential extraction procedure.


CONCLUSIONS
We introduce a versatile APC antibody that is useful for cell/tissue immunostaining, immunoblotting and immunoprecipitation. We also present evidence for interactions between APC and IFs, independent of actin filaments and microtubules. Our results suggest that APC associates with all three major components of the cytoskeleton, thus expanding potential roles for APC in the regulation of cytoskeletal integrity.
Wang
Yang
Y
Department of Molecular Biosciences, University of Kansas, Lawrence, KS, USA. yang.wang@vanderbilt.edu
Azuma
Yoshiaki
Y
Friedman
David B
DB
Coffey
Robert J
RJ
Neufeld
Kristi L
KL
D011125:Adenomatous Polyposis Coli / Q000378:metabolism*; D000906:Antibodies / Q000276:immunology / Q000378:metabolism*; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003106:Colon / Q000378:metabolism / Q000473:pathology; D004847:Epithelial Cells / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D007381:Intermediate Filament Proteins / Q000032:analysis / Q000378:metabolism*; D007633:Keratins / Q000032:analysis / Q000378:metabolism; D034921:Lamin Type B / Q000032:analysis / Q000378:metabolism; D034622:RNA Interference; D053719:Tandem Mass Spectrometry; D051176:beta Catenin / Q000378:metabolism
D000906:Antibodies; D007381:Intermediate Filament Proteins; D034921:Lamin Type B; D051176:beta Catenin; D007633:Keratins