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Fig. 1. Expression of mouse E-cadherin affects levels and subcellular localization of Arm and -catenin in cl-8 cells. (A) Whole cell lysates from cl-8 cells and cl-8 cells stably transfected with a construct driving expression of mouse E-cadherin under control of the metallothioneine promoter (cl8mEcad), were analyzed by western blotting with antibodies against mouse E-cadherin, Arm and -catenin. cl8mEcad cells expressed a baseline level of E-cadherin due to the leakiness of the metallothioneine promoter; this level of expression increased eightfold following addition of Cu²⁺ (+) to the culture medium. Two forms of E-cadherin were detected; a slower migrating, 135 kDa precursor and faster migrating, mature 120 kDa protein (Shore and Nelson, 1991). In cl8mEcad cells, levels of both phosphorylated (slower migrating) and hypophosphorylated (faster migrating) Arm were strongly elevated (13-fold in the absence of Cu²⁺, 40-fold after induction with Cu²⁺). -Catenin levels were also increased in cl8mEcad cells (twofold). (B) Northern blot of total RNAs from cl-8 and cl8mEcad cells, probed for Arm. Expression of E-cadherin does not alter the steady state levels of Arm mRNA. (C) Hypotonic lysates of cl-8 and cl8mEcad cells were separated into cytosolic (S) and pellet fractions by centrifugation at 100,000 g; the pellet was re-extracted in buffer containing Triton X-100 and centrifuged again, giving rise to a Triton X-100-soluble membrane fraction (P) and the insoluble pellet (I), containing proteins bound to the cytoskeleton. Fractions were analyzed by western blot with antibodies against Arm and -catenin. In cl-8 cells, Arm was present in approximately equal amounts in the cytosol (S) and membrane fraction (P). -catenin was found predominantly in the cytosolic fraction (S). In cl8mEcad cells, both Arm and -catenin were present mostly in the membrane fraction (P). Upon induction of high E-cadherin levels with Cu²⁺, significant amounts of both proteins were also found in the Triton X-100-insoluble pellet fraction (I).

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