Glycogen synthase kinase-3β regulates cyclin D1 proteolysis and subcellular localization

Abstract

The activities of cyclin D-dependent kinases serve to integrate extracellular signaling during G 1 phase with the cell-cycle engine that regulates DNA replication and mitosis. Induction of D-type cyclins and their assembly into holoenzyme complexes depend on mitogen stimulation. Conversely, the fact that D-type cyclins are labile proteins guarantees that the subunit pool shrinks rapidly when cells are deprived of mitogens. Phosphorylation of cyclin D1 on a single threonine residue near the carboxyl terminus (Thr-286) positively regulates proteasomal degradation of D1. Now, we demonstrate that glycogen synthase kinase-3␤ (GSK-3␤) phosphorylates cyclin D1 specifically on Thr-286, thereby triggering rapid cyclin D1 turnover. Because the activity of GSK-3␤ can be inhibited by signaling through a pathway that sequentially involves Ras, phosphatidylinositol-3-OH kinase (PI3K), and protein kinase B (Akt), the turnover of cyclin D1, like its assembly, is also Ras dependent and, hence, mitogen regulated. In contrast, Ras mutants defective in PI3K signaling, or constitutively active mitogen-activated protein kinase-kinase (MEK1) mutants that act downstream of Ras to activate extracellular signal-regulated protein kinases (ERKs), cannot stabilize cyclin D1. In direct contrast to cyclin D1, which accumulates in the nucleus during G 1 phase and exits into the cytoplasm during S phase, GSK-3␤ is predominantly cytoplasmic during G 1 phase, but a significant fraction enters 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␤ remained in the nucleus throughout the cell cycle. Overexpression of an active, but not a kinase-defective, form of GSK-3␤ in mouse fibroblasts caused a redistribution of cyclin D1 from the cell nucleus to the cytoplasm. Therefore, phosphorylation and proteolytic turnover of cyclin D1 and its subcellular localization during the cell division cycle are linked through the action of GSK-3␤.[Key Words: Glycogen synthase kinase-3; cyclin D1; Ras signaling; proteolysis; nuclear transport] Received May 7, 1998; revised version accepted September 24, 1998. A family of cyclin-dependent kinases (CDKs) cooperatively regulates mammalian cell cycle progression (for review, see Sherr 1993). During G 1 phase, D-type cyclins (D1, D2, and D3) are synthesized and assemble with either CDK4 or CDK6 in response to growth factor stimulation, thereby generating active holoenzymes that help inactivate the growth-suppressive function of the retinoblastoma protein (Rb) through its phosphorylation (for review, see Weinberg 1995). Cyclin D holoenzyme complexes also titrate CDK inhibitors, such as p27 Kip1 and p21 Cip1, facilitating the activation of cyclin E-CDK2 and subsequent entry into the DNA synthetic phase of the cell cycle (for review, see Sherr and Roberts 1995).Ras-mediated pathways are important for cyclin D1 induction and its assembly with CDKs. Overexpression of activated oncogenic Ras alleles, but...

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