Increased apoptosis in differentiating p27-deficient mouse embryonic stem cells

Bryja, Vı́tězslav; Pachernı́k, Jiřı́; Souček, Karel; Horváth, Viktor; Dvořák, Petr; Hampl, Aleš

doi:10.1007/s00018-004-4081-4

Cited by 27 publications

(40 citation statements)

References 41 publications

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“…Expanded protocol can be found in SI Appendix. Immunoblotting and sample preparation was performed as previously described (44) and developed using either light-sensitive films (Agfa, GE Healthcare) or chemiluminescence documentation system FusionSL (Vilber-Lourmat). MS for posttranslational modifications was performed as previously described (27), for full experimental details, see SI Appendix.…”

Section: Methodsmentioning

confidence: 99%

Dishevelled is a NEK2 kinase substrate controlling dynamics of centrosomal linker proteins

Červenka

Valnohová

Bernatík

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Dishevelled (DVL) is a key scaffolding protein and a branching point in Wnt signaling pathways. Here, we present conclusive evidence that DVL regulates the centrosomal cycle. We demonstrate that DVL dishevelled and axin (DIX) domain, but not DIX domain-mediated multimerization, is essential for DVL's centrosomal localization. DVL accumulates during the cell cycle and associates with NIMA-related kinase 2 (NEK2), which is able to phosphorylate DVL at a multitude of residues, as detected by a set of novel phospho-specific antibodies. This creates interfaces for efficient binding to CDK5 regulatory subunitassociated protein 2 (CDK5RAP2) and centrosomal Nek2-associated protein 1 (C-NAP1), two proteins of the centrosomal linker. Displacement of DVL from the centrosome and its release into the cytoplasm on NEK2 phosphorylation is coupled to the removal of linker proteins, an event necessary for centrosomal separation and proper formation of the mitotic spindle. Lack of DVL prevents NEK2-controlled dissolution of loose centrosomal linker and subsequent centrosomal separation. Increased DVL levels, in contrast, sequester centrosomal NEK2 and mimic monopolar spindle defects induced by a dominant negative version of this kinase. Our study thus uncovers molecular crosstalk between centrosome and Wnt signaling.Wnt signaling | centrosome | Dishevelled | NEK2 | linker proteins

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Section: Methodsmentioning

confidence: 99%

Dishevelled is a NEK2 kinase substrate controlling dynamics of centrosomal linker proteins

Červenka

Valnohová

Bernatík

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…However, this observation goes against the hypothesis concerning the role of TNAP in fast proliferating cells. RA induces a decrease in the proliferation and accumulation of cells in the G1 phase of the cell cycle in both ES and EC cells [80–82]. …”

Section: Alkaline Phosphatase and Stem Cellsmentioning

confidence: 99%

Alkaline Phosphatase in Stem Cells

Štefková

Procházková

Pachernı́k

2015

Stem Cells International

166

107

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Alkaline phosphatase is an enzyme commonly expressed in almost all living organisms. In humans and other mammals, determinations of the expression and activity of alkaline phosphatase have frequently been used for cell determination in developmental studies and/or within clinical trials. Alkaline phosphatase also seems to be one of the key markers in the identification of pluripotent embryonic stem as well as related cells. However, alkaline phosphatases exist in some isoenzymes and isoforms, which have tissue specific expressions and functions. Here, the role of alkaline phosphatase as a stem cell marker is discussed in detail. First, we briefly summarize contemporary knowledge of mammalian alkaline phosphatases in general. Second, we focus on the known facts of its role in and potential significance for the identification of stem cells.

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“…Western blot analysis and protein samples were prepared as described previously (Bryja et al, 2004). The antibodies used were: anti-Dvl2 (sc-13974), anti-Dvl3 (sc-8027) and anti-c-Myc (sc-40) (Santa Cruz Biotechnology); anti-␤-catenin (BD Bioscences); anti-active-␤-catenin (anti-ABC, Upstate Biotechnology); antiphospho-␤-catenin (S45, Biosource) anti-phospho-serin (AB1603, Chemicon International) and anti-MBP (Dako).…”

Section: Western Blottingmentioning

confidence: 99%

Wnt-5a induces Dishevelled phosphorylation and dopaminergic differentiation via a CK1-dependent mechanism

Bryja

Schulte

Rawal

et al. 2007

Journal of Cell Science

159

170

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Previously, we have shown that Wnt-5a strongly regulates dopaminergic neuron differentiation by inducing phosphorylation of Dishevelled (Dvl). Here, we identify additional components of the Wnt-5a-Dvl pathway in dopaminergic cells. Using in vitro gain-of-function and loss-of-function approaches, we reveal that casein kinase 1 (CK1) δ and CK1ϵ are crucial for Dvl phosphorylation by non-canonical Wnts. We show that in response to Wnt-5a, CK1ϵ binds Dvl and is subsequently phosphorylated. Moreover, in response to Wnt-5a or CK1ϵ, the distribution of Dvl changed from punctate to an even appearance within the cytoplasm. The opposite effect was induced by a CK1ϵ kinase-dead mutant or by CK1 inhibitors. As expected, Wnt-5a blocked the Wnt-3a-induced activation of β-catenin. However, both Wnt-3a and Wnt-5a activated Dvl2 by a CK1-dependent mechanism in a cooperative manner. Finally, we show that CK1 kinase activity is necessary for Wnt-5a-induced differentiation of primary dopaminergic precursors. Thus, our data identify CK1 as a component of Wnt-5a-induced signalling machinery that regulates dopaminergic differentiation, and suggest that CK1δ/ϵ-mediated phosphorylation of Dvl is a common step in both canonical and non-canonical Wnt signalling.

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Increased apoptosis in differentiating p27-deficient mouse embryonic stem cells

Cited by 27 publications

References 41 publications

Dishevelled is a NEK2 kinase substrate controlling dynamics of centrosomal linker proteins

Dishevelled is a NEK2 kinase substrate controlling dynamics of centrosomal linker proteins

Alkaline Phosphatase in Stem Cells

Wnt-5a induces Dishevelled phosphorylation and dopaminergic differentiation via a CK1-dependent mechanism

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