MilliporeSigma
  • Kap1 regulates the self-renewal of embryonic stem cells and cellular reprogramming by modulating Oct4 protein stability.

Kap1 regulates the self-renewal of embryonic stem cells and cellular reprogramming by modulating Oct4 protein stability.

Cell death and differentiation (2020-09-09)
Eun Kyoung Do, Hye Ji Moon, Kyung Taek Kang, Jung Won Yoon, Ye Seul Kim, Jeong Kon Seo, Jae Ho Kim
ABSTRACT

Oct4 plays a crucial role in the regulation of self-renewal of embryonic stem cells (ESCs) and reprogramming of somatic cells to induced pluripotent stem cells. However, the molecular mechanisms underlying posttranslational regulation and protein stability of Oct4 remain unclear. Using affinity purification and mass spectrometry analysis, we identified Kap1 as an Oct4-binding protein. Silencing of Kap1 reduced the protein levels of Oct4 in ESCs, whereas the overexpression of Kap1 stimulated the levels of Oct4. In addition, Kap1 overexpression stimulated the self-renewal of ESCs and attenuated the spontaneous differentiation of ESCs in response to LIF withdrawal. Kap1 overexpression increased the stability of Oct4 by inhibiting the Itch-mediated ubiquitination of Oct4. Silencing of Kap1 augmented Itch-mediated ubiquitination and inhibited the stability of Oct4. We identified the lysine 133 (K133) residue in Oct4 as a ubiquitination site responsible for the Kap1-Itch-dependent regulation of Oct4 stability. Preventing ubiquitination at the lysine residue by mutation to arginine augmented the reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells. These results suggest that Kap1 plays a crucial role in the regulation of the pluripotency of ESCs and somatic cell reprogramming by preventing Itch-mediated ubiquitination and the subsequent degradation of Oct4.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Cycloheximide, ≥90% (HPLC)
Sigma-Aldrich
Ponceau S, BioReagent, suitable for electrophoresis
Sigma-Aldrich
Protease Inhibitor Cocktail, for use with mammalian cell and tissue extracts, DMSO solution
Sigma-Aldrich
Z-Leu-Leu-Leu-al, ≥90% (HPLC)
Sigma-Aldrich
Monoclonal ANTI-FLAG® BioM2 antibody produced in mouse, clone M2, purified immunoglobulin, buffered aqueous glycerol solution
Sigma-Aldrich
MISSION® esiRNA, targeting human TRIM28
Sigma-Aldrich
MISSION® pLKO.1-puro Non-Mammalian shRNA Control Plasmid DNA, Targets no known mammalian genes
Sigma-Aldrich
Doxycycline hyclate
Sigma-Aldrich
TRI Reagent®, For processing tissues, cells cultured in monolayer or cell pellets