Dual alterations in casein kinase I-ε and GSK-3β modulate β-catenin stability in hyperproliferating colonic epithelia

Abstract

Casein kinase I (CKI)-ε and GSK-3β phosphorylate β-catenin at Ser⁴⁵(β-cat⁴⁵) and Thr⁴¹/Ser^37,33(β-cat^33,37,41) residues, thereby facilitating its ubiquitination and proteasomal degradation. We used a Citrobacter rodentium-induced transmissible murine colonic hyperplasia (TMCH) model to determine Ser/Thr phosphorylation and biological function of β-catenin during crypt hyperproliferation. TMCH was associated with 3-fold and 3.3-fold increases in CKI-ε cellular abundance and 2-fold and 1.8-fold increase in its activity at 6 and 12 days after infection, respectively. β-Catenin coimmunoprecipitated with both cellular and nuclear CKI-ε and cellular axin at these time points. Cellular β-catenin was constitutively phosphorylated at Ser⁴⁵and underwent subcellular redistribution to cytoskeletal and nuclear fractions at days 6 and 12 of TMCH, respectively. β-cat^33,37,41, however, exhibited only subtle changes in either phosphorylation status or subcellular distribution even after blocking proteasomal degradation in vivo. Interestingly, GSK-3β underwent increased phosphorylation at Ser⁹, leading to 40% and 70% decreases in its activity at these time points, respectively. Coimmunoprecipitation studies exhibited strong association of GSK-3β with PKC-ζ at either time point. Cellular β-cat⁴⁵stabilized and, along with unphosphorylated β-catenin, underwent nuclear translocation, associated with nuclear accumulated Tcf-4 and cAMP response element binding protein binding protein, and was significantly acetylated, leading to increases in DNA binding. Priming of β-catenin at Ser⁴⁵exists in vivo. However, β-cat⁴⁵does not necessarily enter the degradation pathway. Impairment in linking β-cat⁴⁵to subsequent GSK-3β-mediated phosphorylation and degradation may account for increased steady-state levels of both unphosphorylated as well as Ser⁴⁵-phosphorylated β-catenin, which may be causally linked to increases in cell census during TMCH.