Glycogen synthase kinase 3 beta regulates cyclin D1 proteolysis and subcellular localization

The activities of cyclin D-dependent kinases serve to integrate extracellul ar signaling during G(1) phase with the fell-cycle engine that regulates DN A replication and mitosis. Induction of D-type cyclins and their assembly i nto holoenzyme complexes depend on mitogen stimulation. Conversely, the fac t that D-type cyclins are labile proteins guarantees that the subunit pool shrinks rapidly when cells are deprived of mitogens. Phosphorylation of cyc lin D1 on a single threonine residue near the carboxyl terminus (Thr-286) p ositively regulates proteasomal degradation of D1. Now, we demonstrate that glycogen synthase kinase-3 beta (GSK-3 beta) phosphorylates cyclin D1 spec ifically on Thr-286, thereby triggering rapid cyclin D1 turnover. Because t he activity of GSK-3 beta can be inhibited by signaling through a pathway t hat sequentially involves Ras, phosphatidylinositol-3-OH kinase (PI3K), and protein kinase B (Akt), the turnover of cyclin D1, like its assembly, is a lso Ras dependent and, hence, mitogen regulated. In contrast, Ras mutants d efective in PI3K signaling, or constitutively active mitogen-activated prot ein kinase-kinase (MEK1) mutants that act downstream of Ras to activate ext racellular signal-regulated protein kinases (ERKs), cannot stabilize cyclin D1. In direct contrast to cyclin D1, which accumulates in the nucleus duri ng G(1) phase and exits into the cytoplasm during S phase, GSK-3 beta is pr edominantly cytoplasmic during G(1) phase, but a significant fraction enter s the nucleus during S phase. A highly stable D1 mutant in which an alanine is substituted for the threonine at position 286 and that is refractory to phosphorylation by GSK-3 beta remained in the nucleus throughout the cell cycle. Overexpression of an active, but not a kinase-defective, form of GSK -3 beta in mouse fibroblasts caused a redistribution of cyclin D1 from the cell nucleus to the cytoplasm. Therefore, phosphorylation and proteolytic t urnover of cyclin D1 and its subcellular localization during the cell divis ion cycle are linked through the action of GSK-3 beta.