Phosphorylation near nuclear localization signal regulates nuclear import of adenomatous polyposis coli protein

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Abstract
Mutation of the adenomatous polyposis coli (APC) gene is an early step in the development of colorectal carcinomas. APC protein is located in both the cytoplasm and the nucleus. The objective of this study was to define the nuclear localization signals (NLSs) in APC protein. APC contains two potential NLSs comprising amino acids 1767–1772 (NLS1APC) and 2048–2053 (NLS2APC). Both APC NLSs are well conserved among human, mouse, rat, and fly. NLS1APC and NLS2APC each were sufficient to target the cytoplasmic protein β-galactosidase to the nucleus. Mutational analysis of APC demonstrated that both NLSs were necessary for optimal nuclear import of full-length APC protein. Alignment of NLS2APC with the simian virus 40 large T antigen NLS (NLSSV40 T-ag) revealed sequence similarity extending to adjacent phosphorylation sites. Changing a serine residue (Ser2054) to aspartic acid mutated the potential protein kinase A site adjacent to NLS2APC, resulting in both inhibition of the NLS2APC-mediated nuclear import of a chimeric β-galactosidase fusion protein and a reduction of full-length APC nuclear localization. Our data provide evidence that control of APC's nuclear import through phosphorylation is a potential mechanism for regulating APC's nuclear activity.