Evidence for AKT-independent regulation of FOXO1 and FOXO3 in haematopoietic stem and progenitor cells

ABSTRACT

Transcription factors FOXOs (1, 3, 4) are essential for the maintenance of haematopoietic stem cells. FOXOs are evolutionary conserved substrates of the AKT serine threonine protein kinase that are also phosphorylated by several kinases other than AKT. Specifically, phosphorylation by AKT is known to result in the cytosolic localization of FOXO and subsequent inhibition of FOXO transcriptional activity. In addition to phosphorylation, FOXOs are regulated by a number of other post-translational modifications including acetylation, methylation, redox modulation, and ubiquitination that altogether determine these factors' output. Cumulating evidence raises the possibility that in stem cells, including in haematopoietic stem cells, AKT may not be the dominant regulator of FOXO. To address this question in more detail, we examined gene expression, subcellular localization, and response to AKT inhibition of FOXO1 and FOXO3, the main FOXO expressed in HSPCs (haematopoietic stem and progenitor cells). Here we show that while FOXO1 and FOXO3 transcripts are expressed at similar levels, endogenous FOXO3 protein is mostly nuclear compared to the cytoplasmic localization of FOXO1 in HSPCs. Furthermore, inhibition of AKT does not enhance nuclear localization of FOXO1 nor FOXO3. Nonetheless AKT inhibition in the context of loss of NAD-dependent SIRT1 deacetylase modulates FOXO3 localization in HSPCs. Together, these data suggest that FOXO3 is more active than FOXO1 in primitive haematopoietic stem and multipotent progenitor cells. In addition, they indicate that upstream regulators other than AKT, such as SIRT1, maintain nuclear FOXO localization and activity in HSPCs.

KEYWORDS:

• acetylation
• AKT
• deacetylation
• FOXO
• FOXO1
• FOXO3
• hematopoietic stem and progenitor cells
• SIRT1
• Sirtuins

This article is referred to by:

FOXO3a & haematopoietic stem cells: Goodbye PI3K, hello SIRT1?

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors thank the flow cytometry core at the Icahn School of Medicine at Mount Sinai for technical help.

Funding

C.L.B. was partially supported by a Roche TCRC - Young Investigator and R.L. by NIH T32 GM08553-13 and T32 HD075735. This work was supported by funds from RO1 HL116365, the ASH Bridge Award, and Tisch Cancer Institute to SG.