Abstract
Inactivating mutations in the adenomatous polyposis coli (APC) gene correlate with progression of colon cancer and familial adenomatous polyposis. The APC tumor suppressor contributes to chromosome segregation and turnover of the oncogenic transcriptional activator beta-catenin, and these activities are impaired by truncating cancer mutations. APC was recently identified as a shuttling protein whose subcellular distribution is regulated by two nuclear localization signals (NLSs) and multiple nuclear export signals (NESs). Here, we show that mutant disease-linked truncated forms of APC, most of which lack the two central NLSs and certain NES sequences, retain nuclear-cytoplasmic shuttling activity. Nuclear export of truncated APC is mediated by a dominant N-terminal NES. Nuclear import of NLS-deficient APC mutants is facilitated by the N-terminal ARM domain. Furthermore, co-expression of the ARM-binding protein, B56 alpha, increased the nuclear localization of mutant and wild-type APC. The minimal B56 alpha-responsive sequence mapped to APC amino acids 302-625. B56 alpha is a regulatory subunit of protein phosphatase 2A; however, its ability to shift APC to the nucleus was independent of phosphatase activity. We conclude that APC nuclear import is regulated by the ARM domain through its interaction with B56 alpha and postulate that APC/B56 alpha complexes target the dephosphorylation of specific proteins within the nucleus.
Highlights
Mutational inactivation of the APC1 gene is a key event in the development of colon cancer and the intestinal polyp disorder, familial adenomatous polyposis (FAP) (1–3)
We compared the subcellular distribution of different adenomatous polyposis coli (APC) truncation peptides that result from frequent germline mutations associated with FAP (22, 23) or from somatic mutations observed in colon tumors (24, 25)
Many of the deletion mutants were more nuclear in SW480 cells than in HCT116 cells, and it is not yet clear if this may relate to differences in the endogenous form of APC expressed by these cell lines
Summary
Cell Culture and Treatments—NIH 3T3 mouse fibroblasts, human HCT116, and SW480 colon carcinoma cells were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal calf serum and were free of mycoplasma. PRev(1.4)-GFP to test APC sequences for possible nuclear export activity as recently described in detail (19). The expression vectors that encode full-length APC (pCMV-APC) and the C-terminal truncated APC mutants, pCMV-APC-(1–2644), pCMV-APC-(1–1941), and pCMVAPC-(1–1309), were described previously (20, 21). The full-length APC cDNA containing site-directed mutations in NES1 or NES1ϩ2 were described previously (12), and the same NES1 mutation (L75A/ L77A) was inserted into the APC deletion constructs APC-(1–302) and APC-(1– 625) by replacing the SalI/KpnI fragment (N terminus of APC including start site and NESs). The ability to block nuclear import (with actinomycin D) or export (with leptomycin B) allows for accurate comparison of the export activity of test NESs inserted into the control vector. For quantitation of nuclear and cytoplasmic fluorescence intensities, more than 100 confocal cell images were analyzed using the NIH Image software as previously described (12)
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