Laforin Negatively Regulates Cell Cycle Progression through Glycogen Synthase Kinase 3β-Dependent Mechanisms

Abstract

Glycogen synthase kinase 3␤ (GSK-3␤) represses cell cycle progression by directly phosphorylating cyclin D1 and indirectly regulating cyclin D1 transcription by inhibiting Wnt signaling. Recently, we reported that the Epm2a-encoded laforin is a GSK-3␤ phosphatase and a tumor suppressor. The cellular mechanism for its tumor suppression remains unknown. Using ex vivo thymocytes and primary embryonic fibroblasts from Epm2a ؊/؊ mice, we show here a general function of laforin in the cell cycle regulation and repression of cyclin D1 expression. Moreover, targeted mutation of Epm2a increased the phosphorylation of Ser9 on GSK-3␤ while having no effect on the phosphorylation of Ser21 on GSK-3␣. In the GSK-3␤ ؉/؉ but not the GSK-3␤ ؊/؊ cells, Epm2a small interfering RNA significantly enhanced cell growth. Consistent with an increased level of cyclin D1, the phosphorylation of retinoblastoma protein (Rb) and the levels of Rb-E2F-regulated genes cyclin A, cyclin E, MCM3, and PCNA are also elevated. Inhibitors of GSK-3␤ selectively increased the cell growth of Epm2a ؉/؉ but not of Epm2a ؊/؊ cells. Taken together, our data demonstrate that laforin is a selective phosphatase for GSK-3␤ and regulates cell cycle progression by GSK-3␤-dependent mechanisms. These data provide a cellular basis for the tumor suppression activity of laforin.Cell cycle progression is facilitated by cyclin-dependent kinases (CDKs) that are activated by cyclins, including cyclin D1, and inhibited by CDK inhibitors (27,29). Cyclin D1 is a critical regulator involved in cell cycle progression through the G 1 phase and into the S phase (18, 33). The active cyclin D1/ CDK4 complex initiates the phosphorylation of retinoblastoma protein (Rb) (9), which disrupts Rb-mediated transcriptional repression of E2F and facilitates cell cycle progression (27,29). However, the activity of cyclin D1/CDK4 is specifically inhibited by four INK4 proteins (p 16INK4a , p 15INK4b , p 18INK4c , and p 19INK4d ) (13,14,34) and by glycogen synthase kinase 3␤ (GSK-3␤) (3, 7). GSK-3␤ phosphorylates cyclin D1 at Thr286 (3, 7) and thereby triggers its nuclear export, ubiquitination, and proteasomal degradation (7).GSK-3␤ is a multifunctional serine/threonine protein kinase (2, 6, 12) and a component of multiple signal transduction pathways, including the insulin, Wnt, and Ras signaling pathways. In the Wnt signaling pathway, GSK-3␤ phosphorylates ␤-catenin for its degradation and is an essential negative regulator of the Wnt/␤-catenin pathway (1, 4). Wnt inhibits the enzymatic activity of GSK-3␤, thereby stabilizing ␤-catenin and enabling its association with T-cell factor (TCF)/lymphoid enhancer-binding factor (LEF) protein complexes to activate the transcription of target genes, including those like c-myc and cyclin D1 (25). However, how GSK-3␤ activity is regulated has been poorly understood.Epm2a encodes a 331-amino-acid dual-specificity phosphatase called laforin (5,10,(20)(21)(22). Loss-of-function mutations in Epm2a result in Lafora disease, an autosomal recessive disorder i...

The dual-specificity phosphatase hYVH1 (DUSP12) is a novel modulator of cellular DNA content

“…22 Downregulation of laforin results in a significant increase in the number of cells at S and G 2 /M phases, thus promoting cell growth. 23 Similarly, downregulation of DUSP11, also results in increased proliferation. 24 However, unlike the above studies, our results suggest that the phosphatase activity seems to be dispensable for the ability of hYVH1 to alter cellular DNA content, as substitution of the nucleophilic Cys115 had no affect on the hYVH1 cell cycle phenotype.…”

STARCH-EXCESS4 Is a Laforin-Like Phosphoglucan Phosphatase Required for Starch Degradation in Arabidopsis thaliana

“…Laforin is widely believed to be a phosphoprotein phosphatase involved in the regulation of glycogen-related enzymes (Liu et al, 2008;Singh et al, 2008;Solaz-Fuster et al, 2008). However, two recent studies showed that laforin is able to dephosphorylate solubilized amylopectin or glycogen in vitro and proposed that laforin is in fact a glucan phosphatase (Worby et al, 2006;Gentry et al, 2007).…”

MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens

“…They include MAP4K3 (FDR = 0.14, ranked 9th out of 7,115), a tumor suppressor kinase in the mitogenactivated protein kinase (MAPK) pathway which induces apoptosis [9][10][11]22], and EPM2A (FDR = 0.14, ranked 10th out of 7,115), another protein phosphatase that negatively regulates cell cycle progression [7,23]. From the ESC dataset, TRP53, a mouse ortholog of the human TP53 tumor suppressor gene [8,24], was ranked first out of the three positively selected genes identified.…”