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

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 (NLS1(APC)) and 2048-2053 (NLS2(APC)). Both APC NLSs are well conserved among human, mouse, rat, and fly. NLS1(APC) and NLS2(APC) each were sufficient to target the cytoplasmic protein beta-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 NLS2(APC) with the simian virus 40 large T antigen NLS (NLS(SV40 T-ag)) revealed sequence similarity extending to adjacent phosphorylation sites. Changing a serine residue (Ser(2054)) to aspartic acid mutated the potential protein kinase A site adjacent to NLS2(APC), resulting in both inhibition of the NLS2(APC)-mediated nuclear import of a chimeric beta-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.

Figure 1

APC possesses two potential monopartite NLSs. (A) Alignment of NLS1_APC and NLS2_APC with NLS_SV40 T-ag shows that both are classical monopartite NLSs. (B) Comparison of NLS_APC in human, mouse, rat, frog, and fly. Both NLS1_APC and NLS2_APC are highly conserved among the different species.

Figure 2

Either NLS1_APC or NLS2_APC is sufficient to translocate the cytoplasmic protein βGal into the nucleus. βGal-fusion proteins were expressed in mouse L cells and detected by immunofluorescence microscopy. Nuclei were visualized with 4′,6-diamidino-2-phenylindole (DAPI). Areas of overlap between the βGal fusion protein (green) and the nuclei (blue) appear an aqua color. (Bar = 10 μm.) For each construct, 100 cells were scored for βGal localization with conventional fluorescence microscopy. The results of four independent experiments are presented as the incidence of nuclear βGal staining ± SD as follows: βGal, 27(±6)%; βGal-NLS_SV40 T-ag, 100(±0)%; βGal-NLS1_APC, 73(±11)%; βGal-mNLS1_APC, 30(±13)%; βGal-NLS2_APC, 69(±8)%; βGal-mNLS2_APC, 35(±4)%; βGal-NLS1,2_APC, 83(±6)%; and βGal-(NLS2_APC)₂, 91(±4)%.

Figure 3

Both NLS1_APC and NLS2_APC are necessary for optimal nuclear import of full-length APC. The various ectopic flag-tagged APC proteins were localized in Cos7 cells by immunofluorescence microscopy. (Bar = 20 μm.) Cells were scored as above with the results of three independent experiments presented as the incidence of nuclear APC staining ± SD. APC, 18(±1)%; APCmNES, 42(±7)%; APCmNESmNLS1, 17(±10)%; and APCmNESmNLS2, 14(±15)%.

Figure 4

NLS2_APC-mediated nuclear translocation of βGal chimera is regulated by phosphorylation at a PKA site. (A) Alignment of NLS2_APC with NLS_SV40 T-ag reveals similarity of NLS sequence as well as the adjacent putative phosphorylation sites. (B) The NLS2_APC region is highly conserved among different species. NLS2 sequences are bold; the CK2 and PKA sites are underlined. (C) βGal-NLS2_APC localizes to both the cytoplasm and nucleus of L cells. βGal-NLS2_APCmPKA_S/A is predominantly nuclear and βGal-NLS2_APCmPKA_S/D predominantly cytoplasmic. (Bar = 10 μm.) Cells were scored as above, and the incidence of nuclear staining for each βGal fusion is as follows: βGal-NLS2_APC, 69(±8)%; βGal-NLS2_APCmPKA_S/A, 100(±0)%; and βGal-NLS2_APCmPKA_S/D, 29(±6)%. (D) Fractionation and Western immunoblot analysis of 293 cells transiently transfected with various constructs confirmed the subcellular localization of the chimeric proteins. Fractions are labeled as follows: C, cytosol; N, nuclear. The various cell fractions were characterized by striping and reprobing the blot for the marker proteins: lamin as a nuclear marker and rho as a cytoplasmic marker.

Figure 5

Phosphorylation of Ser²⁰⁵⁴ within the PKA site adjacent to NLS2_APC negatively regulates the nuclear import of full-length APC. Cos7 cells expressing APCmNES, APCmNESNLS2mPKA_S/A, or APCmNESNLS2mPKA_S/D were analyzed by immunofluorescence microscopy. (Bar = 20 μm.) Cells were scored, and the incidence of nuclear staining for each construct is shown as follows: APCmNES, 42(±7)%; APCmNESNLS2mPKA_S/A, 42(±11)%; and APCmNESNLS2mPKA_S/D, 19(±14)%. DAPI, 4′,6-diamidino-2-phenylindole.