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Normal human breast epithelial (HBE) cells which reached confluence ceased growth and tightly adhered to each other, forming a monolayer. In quiescent cells thus arrested by density, E-cadherin colocalized and coimmunoprecipitated with alpha- and beta-catenins in the boundary region between adjacent cells. By contrast, immunocytostaining and Western blot analyses revealed that E-cadherin colocalized and coprecipitated with beta-catenin but not with alpha-catenin in exponentially growing cells at low density. As a comparable amount of alpha-catenin was detected in the total cell lysate of cells at different densities, it is suggested that alpha-catenin is present but dissociates from the E-cadherin-beta-catenin complex in growing cells. beta-Catenin was tyrosine phosphorylated in growing cells at low density but not in quiescent cells at confluence. Tyrosine phosphorylation of beta-catenin was concomitantly induced with association of beta-catenin with EGF receptor (EGFR) when quiescent cells at confluence were dissociated into single cells by tryptic digestion, being accompanied by dissociation of alpha-catenin from E-cadherin. Both tyrosine phosphorylation and association of beta-catenin with EGFR were inhibited by tyrphostin, a specific inhibitor of the EGFR tyrosine kinase, whereas dissociation of alpha-catenin from E-cadherin was not. The results suggest that tyrosine phosphorylation of beta-catenin is achieved by EGFR upon tryptic digestion of cells and concurrent with but independent of dissociation of alpha-catenin from E-cadherin. beta-Catenin thus phosphorylated at tyrosine is suggested to play the role in preventing alpha-catenin once dissociated from reassociating with E-cadherin until cells reach confluence.

Cell shape and adhesion of cultured mammalian cells change dramatically during mitosis, however, how cell cycle-dependent alterations in cell adhesion are regulated remain to be elucidated. We show here that normal human mammary epithelial (HME) cells which became less adhesive and adopted the rounded morphology during the G(2)/M phase of the cell cycle significantly reduced their dependence on beta1 integrin-mediated adhesion to laminin, by using function blocking antibody to beta1 integrin. In G(2)/M cells, both total and cell surface expressions of beta1 integrin were comparable with those in G(1) cells but it was phosphorylated at threonines 788-789 within its cytoplasmic domain and coimmunoprecipitated Ca(2+)/calmodulin-dependent protein kinase (CaMK) II. The threonine phosphorylated beta1 integrin significantly reduced its intracellular linkage with actin, with no significant reduction in the actin expression. In contrast, beta1 integrin in G(1) cells was not threonine phosphorylated but formed a link with actin and coimmunoprecipitated the core enzyme of the serine/threonine protein phosphatase (PP) 2A. The results suggest that reduced beta1 integrin-mediated cell adhesion of HME cells to the substratum during mitosis may be induced by beta1 integrin phosphorylation at threonines 788-789 and its reduced ability to link with the actin cytoskeleton.

Hoe 642 enhanced myocardial tolerance against infarction, and this enhanced tolerance was not influenced by anesthetic agents commonly used for infarct size studies. Infarct size limitation by Hoe 642 was not inhibited by polyB, suggesting that cardioprotection by Na(+)-H+ exchange inhibition is not PKC mediated and thus may be unrelated to preconditioning.

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