Identification of the Axin and Frat Binding Region of Glycogen Synthase Kinase-3

Fraser, Elizabeth; Young, Neville; Dajani, Rana; Franca-Koh, Jonathan; Ryves, Jonathan; Williams, Robin S. B.; Yeo, Margaret; Webster, Marie-Thérèse; Richardson, Chris; Smalley, Matthew J.; Pearl, Laurence H.; Harwood, Adrian J.; Dale, Trevor

doi:10.1074/jbc.m109462200

Cited by 112 publications

(115 citation statements)

References 51 publications

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“…1A), suggesting that BIS1 specifically interacted with BIN2. Consistent with a recent result indicating that kinase activity is not required for a GSK3/substrate interaction in animals (Fraser et al, 2002), the kinase-dead BIN2 protein containing a Lys-69-Arg mutation was still able to interact with BIS1 (Fig. 1A).…”

Section: Bes1 and Bzr1 Interact Specifically With Bin2 In Yeast Cellssupporting

confidence: 78%

Two Putative BIN2 Substrates Are Nuclear Components of Brassinosteroid Signaling

et al. 2002

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GSK3 is a highly conserved kinase that negatively regulates many cellular processes by phosphorylating a variety of protein substrates. BIN2 is a GSK3-like kinase in Arabidopsis that functions as a negative regulator of brassinosteroid (BR) signaling. It was proposed that BR signals, perceived by a membrane BR receptor complex that contains the leucine (Leu)-rich repeat receptor-like kinase BRI1, inactivate BIN2 to relieve its inhibitory effect on unknown downstream BR-signaling components. Using a yeast (Saccharomyces cerevisiae) two-hybrid approach, we discovered a potential BIN2 substrate that is identical to a recently identified BR-signaling protein, BES1. BES1 and its closest homolog, BZR1, which was also uncovered as a potential BR-signaling protein, display specific interactions with BIN2 in yeast. Both BES1 and BZR1 contain many copies of a conserved GSK3 phosphorylation site and can be phosphorylated by BIN2 in vitro via a novel GSK3 phosphorylation mechanism that is independent of a priming phosphorylation or a scaffold protein. Five independent bes1 alleles containing the same proline-233-Leu mutation were identified as semidominant suppressors of two different bri1 mutations. Overexpression of the wild-type BZR1 gene partially complemented bin2/ϩ mutants and resulted in a BRI1 overexpression phenotype in a BIN2 ؉ background, whereas overexpression of a mutated BZR1 gene containing the corresponding proline-234-Leu mutation rescued a weak bri1 mutation and led to a bes1-like phenotype. Confocal microscopic analysis indicated that both BES1 and BZR1 proteins were mainly localized in the nucleus. We propose that BES1/BZR1 are two nuclear components of BR signaling that are negatively regulated by BIN2 through a phosphorylation-initiated process.BRs are a special class of plant polyhydroxysteroids that have wide distribution throughout the plant kingdom and play many important roles throughout plant development that include seed germination, stem elongation, pollen tube growth, vascular differentiation, skotomorphogenesis, and stress resistance (Clouse and Sasse, 1998;Steber and McCourt, 2001). It was well documented that gene regulation is critical for many BR-elicited physiological responses (Clouse and Feldmann, 1999), however, the signaling mechanism from BR perception to gene regulation remains largely unknown. Extensive genetic screens for BR-insensitive-signaling mutants in Arabidopsis have so far identified only two genes, BRI1 and BIN2 (Clouse et al., 1996;Kauschmann et al., 1996;Li and Chory, 1997;Noguchi et al., 1999;Li et al., 2001;Li and Nam, 2002).BRI1 encodes a Leu-rich repeat receptor-like kinase that is composed of an extracellular domain, a singlepass transmembrane segment, and an intracellular kinase domain (Li and Chory, 1997). BRI1 is a plasma membrane-localized protein and can function as a Ser/Thr kinase when expressed in Escherichia coli or animal cell culture (Friedrichsen et al., 2000;Oh et al., 2000). The extracellular domain of BRI1 can confer BR responsiveness to the kinase ...

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Section: Bes1 and Bzr1 Interact Specifically With Bin2 In Yeast Cellssupporting

confidence: 78%

Two Putative BIN2 Substrates Are Nuclear Components of Brassinosteroid Signaling

et al. 2002

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“…Lithium is commonly used to inhibit GSK3␤, but it is known to exhibit other effects like activating Akt (28). Moreover, the overexpression of a dominant negative mutant of GSK3 (27) caused a greater HIF-1 transcriptional activity, suggesting that GSK3 inhibition leading to the accumulation of HIF-1␣ is correlated with a greater transcriptional activity. Finally, hypoxia-induced increase in VEGF secretion and in Glut-1 expression was also enhanced by GSK3 inhibition in the presence of lithium.…”

Section: Fig 7 Effect Of Pi3k Inhibition On Hif-1␣ Expressionmentioning

confidence: 99%

“…This vector was co-transfected with 100 ng of the control vector pRL-SV40 (Promega, Madison, WI) and with or without 1 g of expression vector. Regarding the dominant negative mutant, a vector expressing a kinase-inactive mutant of GSK3 was used (27). pCMV-Myc was used as the empty vector.…”

Section: Hepg2mentioning

confidence: 99%

Regulation of Hypoxia-inducible Factor-1α Protein Level during Hypoxic Conditions by the Phosphatidylinositol 3-Kinase/Akt/Glycogen Synthase Kinase 3β Pathway in HepG2 Cells

Mottet¹,

Dumont

Deccache

et al. 2003

Journal of Biological Chemistry

292

230

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Hypoxia initiates an intracellular signaling pathway leading to the activation of the transcription factor hypoxia-inducible factor-1 (HIF-1). HIF-1 activity is regulated through different mechanisms involving stabilization of HIF-1␣, phosphorylations, modifications of redox conditions, and interactions with coactivators. However, it appears that some of these steps can be cell type-specific. Among them, the involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in the regulation of HIF-1 by hypoxia remains controversial. Here, we investigated the activation state of PI3K/Akt/glycogen synthase kinase 3␤ (GSK3␤) in HepG2 cells. Increasing incubation times in hypoxia dramatically decreased both the phosphorylation of Akt and the inhibiting phosphorylation of GSK3␤. The PI3K/Akt pathway was necessary for HIF-1␣ stabilization early during hypoxia. Indeed, its inhibition was sufficient to decrease HIF-1␣ protein level after 5-h incubation in hypoxia. However, longer exposure (16 h) in hypoxia resulted in a decreased HIF-1␣ protein level compared with early exposure (5 h). At that time, Akt was no longer present or active, which resulted in a decrease in the inhibiting phosphorylation of GSK3␤ on Ser-9 and hence in an increased GSK3␤ activity. GSK3 inhibition reverted the effect of prolonged hypoxia on HIF-1␣ protein level; more stabilized HIF-1␣ was observed as well as increased HIF-1 transcriptional activity. Thus, a prolonged hypoxia activates GSK3␤, which results in decreased HIF-1␣ accumulation. In conclusion, hypoxia induced a biphasic effect on HIF-1␣ stabilization with accumulation in early hypoxia, which depends on an active PI3K/Akt pathway and an inactive GSK3␤, whereas prolonged hypoxia results in the inactivation of Akt and activation of GSK3␤, which then down-regulates the HIF-1 activity through down-regulation of HIF-1␣ accumulation.Mammalian cells require a constant supply of oxygen to maintain adequate energy production, which is essential for maintaining normal function and for ensuring cell survival. The cellular response to decreased oxygen levels is regulated by a hypoxia-inducible factor-1 (HIF-1) 1 heterodimeric complex composed of two subunits, HIF-1␣ and arylhydrocarbon receptor nuclear translocator (1).This transcription factor binds to conserved regulatory sequences known as hypoxia-responsive element (HRE) found in the promoter of several target genes such as vascular endothelial growth factor (VEGF), erythropoietin, or glycolytic enzymes (aldolase A, enolase-␣, etc.) and controls their expression in response to hypoxia, leading to the adaptation of cells to decreased oxygen level (2).Only the HIF-1␣ subunit is regulated by a reduced oxygen level, and the regulation occurs in large part at post-translational modifications, resulting in its stabilization, nuclear translocation, DNA binding activity, and proper transcriptional activity. Under normal conditions, HIF-1␣ is hydroxylated at the prolines 564 and 462 residues in the oxygen-dependent degradation domain and, hence, inter...

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“…This phosphorylation is regulated by the physical proximity of GSK3b with its substrate, b-catenin, as axin-GSK3b interaction does not alter GSK3b-specific activity. In the presence of a wnt signal, this complex is destabilized when dishevelled (Dvl) is activated, and along with GSK3b binding protein (GBP), or the mammalian homologue FRAT1, recruit GSK3b away from axin and b-catenin (Li et al 1999;Seidensticker and Behrens 2000;Fraser et al 2002), and thus b-catenin is no longer efficiently phosphorylated by GSK3b. Unphosphorylated b-catenin is more stable and therefore it accumulates and redistributes to the nucleus, where it affects transcription of cell cycle genes such as cyclin D1 and myc (Behrens 2000).…”

mentioning

confidence: 99%

Axin negatively affects tau phosphorylation by glycogen synthase kinase 3β

Stoothoff¹,

Bailey²,

Mi³

et al. 2002

Journal of Neurochemistry

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Glycogen synthase kinase 3b (GSK3b) is an essential protein kinase that regulates numerous functions within the cell. One critically important substrate of GSK3b is the microtubuleassociated protein tau. Phosphorylation of tau by GSK3b decreases tau-microtubule interactions. In addition to phosphorylating tau, GSK3b is a downstream regulator of the wnt signaling pathway, which maintains the levels of b-catenin. Axin plays a central role in regulating b-catenin levels by bringing together GSK3b and b-catenin and facilitating the phosphorylation of b-catenin, targeting it for ubiquitination and degradation by the proteasome. Although axin clearly facilitates the phosphorylation of b-catenin, its effects on the phosphorylation of other GSK3b substrates are unclear.Therefore in this study the effects of axin on GSK3b-mediated tau phosphorylation were examined. The results clearly demonstrate that axin is a negative regulator of tau phosphorylation by GSK3b. This negative regulation of GSK3b-mediated tau phosphorylation is due to the fact that axin efficiently binds GSK3b but not tau and thus sequesters GSK3b away from tau, as an axin mutant that does not bind GSK3b did not inhibit tau phosphorylation by GSK3b. This is the first demonstration that axin negatively affects the phosphorylation of a GSK3b substrate, and provides a novel mechanism by which tau phosphorylation and function can be regulated within the cell.

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Identification of the Axin and Frat Binding Region of Glycogen Synthase Kinase-3

Cited by 112 publications

References 51 publications

Two Putative BIN2 Substrates Are Nuclear Components of Brassinosteroid Signaling

Two Putative BIN2 Substrates Are Nuclear Components of Brassinosteroid Signaling

Regulation of Hypoxia-inducible Factor-1α Protein Level during Hypoxic Conditions by the Phosphatidylinositol 3-Kinase/Akt/Glycogen Synthase Kinase 3β Pathway in HepG2 Cells

Axin negatively affects tau phosphorylation by glycogen synthase kinase 3β

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