Dictyostelium differentiation-inducing factor-3 activates glycogen synthase kinase-3β and degrades cyclin D1 in mammalian cells

In search of chemical substances applicable for the treatment of cancer and other proliferative disorders, we studied the signal transduction of Dictyostelium differentiation-inducing factors (DIFs) in mammalian cells mainly using HeLa cells. Although DIF-1 and DIF-3 both strongly inhibited cell proliferation by inducing G(0)/G(1) arrest, DIF-3 was more effective than DIF-1. DIF-3 suppressed cyclin D1 expression at both mRNA and protein levels, whereas the overexpression of cyclin D1 overrode DIF-3-induced cell cycle arrest. The DIF-3-induced decrease in the amount of cyclin D1 protein preceded the reduction in the level of cyclin D1 mRNA. The decrease in cyclin D1 protein seemed to be caused by accelerated proteolysis, since it was abrogated by N-acetyl-Leu-Leu-norleucinal, a proteasome inhibitor. DIF-3-induced degradation of cyclin D1 was also prevented by treatment with lithium chloride, an inhibitor of glycogen synthase kinase-3beta (GSK-3beta), suggesting that DIF-3 induced cyclin D1 proteolysis through the activation of GSK-3beta. Indeed, DIF-3 dephosphorylated Ser(9) and phosphorylated tyrosine on GSK-3beta, and it stimulated GSK-3beta activity in an in vitro kinase assay. Moreover, DIF-3 was revealed to induce the nuclear translocation of GSK-3beta by immunofluorescent microscopy and immunoblotting of subcellular protein fractions. These results suggested that DIF-3 activates GSK-3beta to accelerate the proteolysis of cyclin D1 and that this mechanism is involved in the DIF-3-induced G(0)/G(1) arrest in mammalian cells.