A healthy human colon is in a continuous state of cellular renewal, and consequently, epithelial cells lining the colon undergo a highly regulated series of cell divisions. Since loss of APC function initiates colorectal tumor formation, characterized by unregulated cellular proliferation, we hypothesized that APC normally functions to maintain normal colon cell proliferation. Furthermore, in serving this function, APC might display a distinct subcellular localization in proliferating cells compared to quiescent cells. We found that the subcellular localization of APC was stable as MDCK epithelial cells passed through the cell cycle (Fig. ). APC was predominantly nuclear, with some cytoplasmic concentration near the cell's edge, consistent with previous reports using asynchronous MDCK cells (6
). However, when epithelial cells from kidney or intestine became superconfluent, presumably on entering G0
, APC was evenly distributed between the cytoplasm and nucleus (Fig. ). This is the first demonstration that cellular context influences the subcellular localization of APC.
Our observation that blocking Crm-1-mediated nuclear export with LMB did not result in a nuclear accumulation of APC in superconfluent epithelial cells (Fig. ) suggests that only a small fraction of the total APC population shuttles between the nucleus and cytoplasm in quiescent cells. Alternatively, it is possible that most APC molecules shuttle between the nucleus and cytoplasm in quiescent cells, but only rarely. After showing that the increased cytoplasmic APC in quiescent cells was not dependent on continual nuclear export, we focused on the role of nuclear import signals. We demonstrated that a stretch of 32 amino acids containing NLS2APC was sufficient to confer nuclear localization to the otherwise cytoplasmic protein β-Gal, in a manner dependent on cell density (Fig. ). NLS1APC, although adequate to drive the nuclear localization of β-Gal, was not markedly regulated by phosphorylation (Fig. ) and was not influenced by cell density (Fig. ). Additionally, we showed that two intrinsic phosphorylation sites modulated nuclear localization driven by NLS2APC and were necessary for the differential localization of β-Gal-NLS2APC in quiescent versus proliferating cells (Fig. and ).
We hypothesize, based on the similarity between NLS2APC and the NLS of SV40 T-ag, that NLS2APC mediates nuclear import of APC. Demonstration that fusion of either NLS1APC or NLS2APC with the large cytoplasmic protein β-Gal resulted in its nuclear localization further implicated NLS1APC and NLS2APC as mediators of nuclear import. The steady-state localization of APC might also be influenced by nuclear and/or cytoplasmic retention. Since the APC sequence analyzed in this study extends beyond the monopartite basic stretch of amino acids that we classified as NLS2APC, it is possible that other elements in this region affect nuclear or cytoplasmic retention and ultimately, APC's subcellular localization.
Apart from containing two potential phosphorylation sites, the NLS2APC
peptide used in our experiments encompasses the third axin-binding motif of APC, SAMP#3 (3
). It is possible that binding of the NLS2APC
region to axin participates in the differential localization of NLS2APC
-β-Gal in quiescent cells. Altering both phosphorylation sites in NLS2APC
so as to promote nuclear import resulted in the loss of the cytoplasmic β-Gal in quiescent cells (Fig. ). Therefore, if axin-APC interactions are responsible for cytoplasmic retention of APC, the single amino acid substitutions that promoted nuclear localization must have disrupted axin and APC binding. Conversely, if axin-APC interactions are responsible for nuclear retention of APC, the single amino acid substitutions that promoted nuclear localization of β-Gal-NLS2APC
in quiescent cells must have enhanced axin and APC binding. The structure of a portion of axin, crystallized with APC-SAMP#3, predicted contact between the potential CK2 site, Ser2034
, and axin, with no contact at the potential PKA site, Ser2054
). Then again, the equivalent position of Ser2034
is an aspartic acid in human, murine, and Xenopus
SAMP#1 and an alanine in Drosophila melanogaster
SAMP#2, suggesting tolerance for both amino acid substitutions used in this study. Detailed analyses of axin's subcellular localization as well as the binding kinetics of endogenous axin with mutant APC are necessary to further examine this intriguing possibility.
Using various β-Gal chimeras, we demonstrated that potential phosphorylation sites near NLS2APC are critical modulators of nuclear localization, responsible for differences in β-Gal-NLS2APC localization in proliferating versus quiescent cells. We predicted that phosphorylated serine residues, Ser2034APC and Ser2054APC, function in a similar manner in full-length APC in vivo. Indeed, a brief activation of CK2 and inhibition of PKA in quiescent MDCK or IEC-6 cells resulted in a dramatic relocalization of endogenous APC from the cytoplasm to the nucleus (Fig. ). Similarly, simultaneous inhibition of CK2 and activation of PKA in proliferating MDCK or IEC-6 cells resulted in a shift of endogenous APC from the nucleus to the cytoplasm. In the future it will be interesting to determine whether Ser2034 and Ser2054 are differentially phosphorylated within the context of endogenous APC protein in cells grown under different conditions. So far, our attempts to use matrix-assisted laser desorption ionization–time of flight analysis of purified APC protein digested with trypsin to determine the phosphorylation status of specific APC residues have been impeded by the relatively low abundance and large size of APC protein (K. Neufeld, personal communication).
Our data support the proposal that APC localization is controlled by a balance of nuclear import, mediated by NLSAPC, and nuclear export, mediated by the intrinsic NESAPC. A schematic of NLS2 modulation within the APC protein is depicted in Fig. . In proliferating cells, where APC is primarily nuclear, CK2APC2034 is phosphorylated but PKAAPC2054 is not (Fig. A). As cells become superconfluent and cease to divide, APC is more evenly distributed between the cytoplasm and the nucleus, indicating a shift in the balance of APC phosphorylation (Fig. B). Some of the cellular APC is dephosphorylated at the CK2APC2034 site, phosphorylated at the PKAAPC2054 site or both, thereby inhibiting nuclear import mediated by NLS2APC. Consequently, we suggest that CK2APC and PKAAPC, together with NLS2APC, constitute a phosphorylation-regulated module, providing a mechanism for regulated nuclear localization of APC.
FIG. 10 Model for the cell density-influenced regulation of APC's subcellular distribution. The CK2APC and PKAAPC sites function as switches promoting nuclear localization (on) or impeding nuclear localization (off). The CK2APC switch is on when the CK2APC site (more ...)
Our mutagenesis strategy clearly indicated that Ser2034APC
were critical mediators of NLS2APC
activity. Within APC, these serine residues are surrounded by amino acids that fit the consensus pattern for phosphorylation by CK2 and PKA, respectively, and thus we have referred to them as potential CK2 and PKA sites (22
). Additionally, pharmacological agents that specifically target PKA and CK2 influenced the localization of endogenous APC (Fig. ). CK2 inhibitors changed the predominantly nuclear APC distribution in proliferating cells to one that was more cytoplasmic, suggesting that phosphorylation of NLS2APC
by CK2 is important for nuclear APC localization. Conversely, CK2 activation in quiescent cells resulted in increased nuclear APC. Our observations correlate with reported CK2 activities. Elevated CK2 levels and activity have been documented in actively proliferating cells, including those from human tumors and normal tissue (reviewed in reference 14
). Here we report that CK2 and PKA have opposing effects on APC localization, suggesting greater PKA activity in quiescent than in proliferating cells. A correlation between PKA activity and cell quiescence is not well established; however, activity of one PKA isozyme has been associated with reduced colonic proliferation (1
). It remains possible that APC is also phosphorylated at these serines by alternative kinases. Precise identification of the kinase responsible for phosphorylation of a given APC residue in vivo will be challenging but ultimately will greatly increase our understanding of how APC regulation is affected by cellular context.
What are the consequences of APC redistribution to the cytoplasm of quiescent epithelial cells? Our results are compatible with the observation that overexpression of APC in mouse fibroblast cells blocks cell cycle progression from G0
to S (2
). APC-mediated β-catenin degradation appears to occur exclusively in the cytoplasm and not in the nucleus (11
). Thus, our observation of more cytoplasmic APC in quiescent cells than in proliferating cells suggests that cytoplasmic APC controls β-catenin levels in quiescent cells. Consistent with this theory and not surprising given its well-documented role in cell-cycle progression (41
), cyclin D1
, a gene transcriptionally activated by β-catenin, was expressed in five times more proliferating than quiescent cells (F. Zhang, personal communication). Furthermore, pharmacological agents that caused APC redistribution (Fig. ) affected subsequent changes in cyclin D1 levels. As a general rule, higher levels of cytoplasmic APC correlated with less cyclin D1 expression; lower levels of cytoplasmic APC correlated with increased cyclin D1 expression (data not shown). We have previously observed that nuclear APC can bind to and inactivate nuclear β-catenin (27
). Whereas the present results appear to contradict this previous finding, β-catenin binding by nuclear APC is likely regulated, occurring only under specific conditions. As such, we envision the nuclear pool of APC acting as a sentry, ready to quickly dampen the β-catenin/LEF-1 signal under particular circumstances. In this capacity, nuclear APC would be crucial in proliferating cells, where β-catenin activity might be tightly regulated. Additionally, we predict that nuclear APC has functions distinct from its role in β-catenin regulation.
Several other tumor suppressor proteins, such as p53, BRCA1, and von Hippel-Lindau, shuttle between the nucleus and the cytoplasm (24
). Regulated protein movement in and out of the nucleus provides a simple, reversible, and rapid means to control nuclear activity and coordinate nuclear and cytoplasmic events. Regulated nuclear localization of tumor suppressor proteins likely serves as a general mechanism to control responses to different environmental conditions and stimuli in normal cells; therefore, the control of nuclear localization is critical for tumor suppressor function. Moreover, regulated nuclear localization of APC, through differential phosphorylation, is expected to affect β-catenin target genes and may prove critical for normal colonocytes to respond rapidly and precisely to different physiological conditions and extracellular signals.