Cell density and phosphorylation control the subcellular localization of adenomatous polyposis coli protein

Loss of functional adenomatous polyposis coli protein (APC) leads to uncont rolled proliferation of colonic epithelial cells, as evidenced by polyp for mation, a prelude to carcinogenesis. As a tumor suppressor, APC targets the oncogene beta -catenin for proteasome-mediated cytoplasmic degradation. Re cently, it was demonstrated that APC also interacts with nuclear beta -cate nin, thereby reducing beta -catenin's activity as a transcription cofactor and enhancing its nuclear export. The first objective of this study was to analyze how cellular context affected APC distribution. We determined that cell density but not cell cycle influenced APC's subcellular distribution, with predominantly nuclear APC found in subconfluent MDCK and intestinal ep ithelial cells but both cytoplasmic and nuclear APC in superconfluent cells . Redistribution of APC protein did not depend on continual nuclear export. Focusing on the two defined nuclear localization signals in the C-terminal third of APC (NLS1(APC) and NLS2(APC)), we found that phosphorylation at t he CK2 site increased and phosphorylation at the PKA site decreased NLS2(AP C)-mediated nuclear translocation. Cell density-mediated redistribution of beta -galactosidase was achieved by fusion to NLS2(APC) but not to NLS1(APC ). Both the CK2 and PKA sites were important for this density-mediated redi stribution, and pharmacological agents that target CK2 and PKA instigated r elocalization of endogenous APC. Our data provide evidence that physiologic al signals such as cell density regulate APC's nuclear distribution, with p hosphorylation sites near NLS2(APC) being critical for this regulation.