Kap1 Regulates the Stability of Lin28A in Embryonic Stem Cells.

Lin28A is an RNA-binding protein that controls mammalian development and maintenance of the pluripotency of embryonic stem cells (ESCs) via regulating the processing of the microRNA let-7. Lin28A is highly expressed in ESCs, and ectopic expression of this protein facilitates reprogramming of somatic cells to induced pluripotent stem cells. However, the mechanisms underlying the post-translational regulation of Lin28A protein stability in ESCs remain unclear. In the present study, we identified Kap1 (KRAB-associated protein 1) as a novel Lin28A-binding protein using affinity purification and mass spectrometry. Kap1 specifically interacted with the N-terminal region of Lin28A through its coiled-coil domain. Kap1 overexpression significantly attenuated Lin28A ubiquitination and increased its stability. However, small interfering RNA-mediated knockdown of Kap1 promoted the ubiquitination of Lin28A, leading to its proteasomal degradation. Trim71, an E3 ubiquitin ligase, induced Lin28A degradation and Kap1 knockdown accelerated the Trim71-dependent degradation of Lin28A. Mutation of the lysine 177 residue of Lin28A to arginine abrogated the ubiquitination and degradation of Lin28A which were accelerated by Kap1 silencing. Moreover, Kap1 overexpression led to the accumulation of Lin28A in the cytoplasm, but not in the nucleus, and reduced the levels of let-7 subtypes. These results suggest that Kap1 plays a key role in regulation of the stability of Lin28A by modulating the Trim71-mediated ubiquitination and subsequent degradation of Lin28A, thus playing a pivotal role in the regulation of ESC self-renewal and pluripotency.

Regulation of the protein stability and transcriptional activity of OCT4 in stem cells.

OCT4 (also known as Oct3 and Oct3/4), which is encoded by Pou5f1, is expressed in early embryonic cells and plays an important role in early development, pluripotency maintenance, and self-renewal of embryonic stem cells. It also regulates the reprogramming of somatic cells into induced pluripotent stem cells. Several OCT4-binding proteins, including SOX2 and NANOG, reportedly regulate gene transcription in stem cells. An increasing number of evidence suggests that not only gene transcription but also post-translational modifications of OCT4 play a pivotal role in regulating the expression and activity of OCT4. For instance, ubiquitination and sumoylation have been reported to regulate OCT4 protein stability. In addition, the phosphorylation of Ser347 in OCT4 also stabilizes the OCT4 protein level. Recently, we identified KAP1 as an OCT4-binding protein and reported the KAP1-mediated regulation of OCT4 protein stability. KAP1 overexpression led to an increased proliferation of mouse embryonic stem cells and promoted the reprogramming of somatic cells resulting in induced pluripotent stem cells. In this review, we discuss how the protein stability and function of OCT4 are regulated by protein-protein interaction in stem cells.