Leukemia inhibitory factor regulates cyclin-cdk complexes and duration in mouse embryonic (ES) cells

HAL Id: hal-02831239

https://hal.inrae.fr/hal-02831239

Submitted on 7 Jun 2020

HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Leukemia inhibitory factor regulates cyclin-cdk complexes and duration in mouse embryonic (ES) cells

Caline Sakabedoyan, Marielle Afanassieff, Pierre Savatier

To cite this version:

Caline Sakabedoyan, Marielle Afanassieff, Pierre Savatier. Leukemia inhibitory factor regulates cyclin- cdk complexes and duration in mouse embryonic (ES) cells. 2. Annual Meeting of the International Society for Stem Cell Research (ISSCR), Jun 2004, Boston, United States. 2004. �hal-02831239�

2nd Annual Meeting June 10 – 13, 2004

ABSTRACT – POSTER

LEUKEMIA INHIBITORY FACTOR REGULATES CYCLIN-CDK COMPLEXES IN MOUSE EMBRYONIC STEM (ES) CELLS

Caline SAKABEDOYAN, Marielle AFANASSIEFF, and Pierre SAVATIER Laboratory of Cell Molecular Biology

Ecole Normale Supérieure de Lyon CNRS UMR5665, INRA LA913

LYON, France

The propagation of mouse embryonic stem (ES) cells is dependent on leukemia inhibitory factor (LIF), or cytokines of the interleukine-6 family, which can activate the gp130 receptor.

Despite LIF-gp130 playing a critical role in controlling the self-renewal of the mouse ES cells, little is known on the target genes and the cellular processes that it regulates. Here, we show that LIF- gp130 regulates the formation of, and the kinase activity associated with, Cyclin-Cdk complexes.

A protocol based on LIF starvation for 24 hours followed by re-stimulation for 1 to 8h was used throughout this study. Examination of Cyclin-Cdk kinase activities revealed major variations following LIF-starvation and LIF receptor stimulation. Cyclin E-Cdk2 and Cyclin A-Cdk2 kinases were down-regulated by LIF starvation. Kinase activities and protein complex returned to their original levels within 3h following LIF receptor stimulation. In contrast, Cyclin B1-Cdk1 kinase activity increased upon LIF starvation then returned to its original level between 3 to 8h following LIF receptor stimulation. These data indicate that Cyclin-Cdk complexes are differentially regulated by LIF-receptor activity in self-renewing ES cells. Most importantly, Cyclin E-Cdk2 – which is a critical determinant, and rate-limiting driver, of the G1/S transition in mammalian cells – is under positive control by LIF receptor, whereas Cyclin B1-Cdk1 – a critical determinant of the G2/M transition – is under negative control by LIF receptor.

We next investigated the contribution of Cdc25A/Pim-1 pathway in the regulation of Cyclin E- Cdk2 complex activity in response to LIF receptor stimulation. The Cdc25A phosphatase activates Cyclin E-Cdk2 kinase by removing inhibitory phosphates from Cdk2. The Pim-1 serine/threonine kinase phosphorylates, thereby activates, Cdc25A. We observed that Pim-1 expression and Cdc25A activity are both under positive control by the LIF receptor in ES cells. In parallel, we investigated the role of p27kip1, an inhibitor of Cyclin E-Cdk2 kinase and observed that stimulation of the LIF receptor decreased the level of complexes containing both Cyclin E and p27kip1. We conclude that LiF receptor up-regulates the activity of Cyclin-Cdk2 complexes (i) by stimulating activating dephosphorylation of Cdk subunit by Cdc25A phosphatase and (ii) by preventing kinase inhibition by p27kip1.

Activated gp130 recruits and activates two Src-homology domain-containing proteins, the

STAT3 transcription factor, which is essential to self-renewal, and the SHP-2 phosphatase, which

attenuates the STA3-dependent signal through downstream activation of the p42/p44MAP. To

assess the role of STAT3 and SHP-2 in controlling the activity of Cyclin-Cdk complexes in ES cells,

we made use of an engineered ES cells stably expressing a chimeric receptor (either wild-type or

mutated) consisting of the extracellular domain of the G-CSF receptor fused to the transmembrane

and cytoplasmic region of gp130. Homodimerization of the wild-type chimeric receptor, mediated

by G-CSF, induces both STAT3 and SHP-2 recruitments and promotes self-renewal. Mutated

chimeric receptors harbour point mutations that prevent recruitment of either of them. Using the LIF starvation/restimulation protocol described before, we observed that both STAT3 and SHP-2 were required for full activation of Cyclin E-Cdk2 kinase in the presence of serum, whereas only SHP-2 recruitment to gp130 was critical to Cyclin E-Cdk2 activity in serum-free conditions. How SHP-2 contributes to the regulation of the Cyclin E-Cdk2 kinase activity is under investigation.

Taken together, these data indicate that regulation of Cyclin-Cdk complex activities is part of

the cellular response triggered by LIF receptor activation in mouse ES cells. Whether the

modulation of Cyclin-Cdk activities by LIF impacts on cell-cycle distribution, proliferation and self-

renewal is currently under investigation.