Lineage specific composition of cyclin D-CDK4/CDK6-p27 complexes reveals distinct functions of CDK4, CDK6 and individual D-type cyclins in differentiating cells of embryonic origin.

OBJECTIVES: This article is to study the role of G(1)/S regulators in differentiation of pluripotent embryonic cells. MATERIALS AND METHODS: We established a P19 embryonal carcinoma cell-based experimental system, which profits from two similar differentiation protocols producing endodermal or neuroectodermal lineages. The levels, mutual interactions, activities, and localization of G(1)/S regulators were analysed with respect to growth and differentiation parameters of the cells. RESULTS AND CONCLUSIONS: We demonstrate that proliferation parameters of differentiating cells correlate with the activity and structure of cyclin A/E-CDK2 but not of cyclin D-CDK4/6-p27 complexes. In an exponentially growing P19 cell population, the cyclin D1-CDK4 complex is detected, which is replaced by cyclin D2/3-CDK4/6-p27 complex following density arrest. During endodermal differentiation kinase-inactive cyclin D2/D3-CDK4-p27 complexes are formed. Neural differentiation specifically induces cyclin D1 at the expense of cyclin D3 and results in predominant formation of cyclin D1/D2-CDK4-p27 complexes. Differentiation is accompanied by cytoplasmic accumulation of cyclin Ds and CDK4/6, which in neural cells are associated with neural outgrowths. Most phenomena found here can be reproduced in mouse embryonic stem cells. In summary, our data demonstrate (i) that individual cyclin D isoforms are utilized in cells lineage specifically, (ii) that fundamental difference in the function of CDK4 and CDK6 exists, and (iii) that cyclin D-CDK4/6 complexes function in the cytoplasm of differentiated cells. Our study unravels another level of complexity in G(1)/S transition-regulating machinery in early embryonic cells.

Inhibition of endocytosis blocks Wnt signalling to beta-catenin by promoting dishevelled degradation.

AIM: The Wnt/Frizzled signalling pathway is highly conserved through evolution. Frizzled, the receptors for Wnts, have the topology of seven transmembrane spanning domain receptors. An important means of regulation of these receptors is internalization and desensitization through clathrin-mediated endocytosis. Therefore, we investigated the effects of endocytosis inhibition on Frizzled4-green fluorescent protein (FZD(4)-GFP) localization, dishevelled levels and Wnt-3a signalling to beta-catenin. METHODS: Experiments were performed in the mouse neuronal cell line SN4741 that has previously proven to be valuable for the investigation of Wnt/Frizzled signalling. FZD(4)-GFP distribution has been examined using confocal laser scanning microscopy. Dishevelled protein expression levels and the activation of beta-catenin upon treatment with endocytosis inhibitors (hyperosmolaric sucrose and K(+) depletion), kinase inhibitors and Wnt-3a were analysed by immunoblotting. RESULTS: Hyperosmotic sucrose and K(+) depletion increased the membrane localization of FZD(4)-GFP, and in parallel triggered fast (1-2 h) and almost complete (approx. 95%) degradation of endogenous dishevelled, which was independent of Wnt-induced, CK1-mediated phosphorylation of dishevelled. In addition, dishevelled depletion induced by endocytosis inhibition completely prevented canonical signalling by Wnt-3a to beta-catenin even when osmotic conditions and endocytosis were reverted to normal. CONCLUSIONS: The data provide evidence for a molecular mechanism that could be a basis for a novel negative feedback loop within the Wnt/Frizzled pathway depending on dishevelled degradation. The identification of molecular details of regulatory mechanisms for the Wnt/Frizzled signalling pathway increases our understanding of pathway regulation, which might be of special physiological significance for embryonic development, cancer and neurological disorders.