Abstract
The serum- and glucocorticoid-regulated kinase (SGK1) controls cell transformation and tumor progression. SGK1 affects mitotic stability by regulating the expression of RANBP1/RAN. Here, we demonstrate that SGK1 fluctuations indirectly modify the maturation of pre-miRNAs, by modulating the equilibrium of the RAN/RANBP1/RANGAP1 axis, the main regulator of nucleo-cytoplasmic transport. The levels of pre-miRNAs and mature miRNAs were assessed by qRT-PCR, in total extracts and after differential nuclear/cytoplasmic extraction. RANBP1 expression is the limiting step in the regulation of SGK1-SP1 dependent nuclear export. These results were validated in unrelated tumor models and primary human fibroblasts and corroborated in tumor-engrafted nude mice. The levels of pri-miRNAs, DROSHA, DICER and the compartmental distribution of XPO5 were documented. Experiments using RANGTP conformational antibodies confirmed that SGK1, through RANBP1, decreases the level of the GTP-bound state of RAN. This novel mechanism may play a role in the epigenomic regulation of cell physiology and fate.
Highlights
The serum- and glucocorticoid-regulated kinase 1 (SGK1) is a serine/threonine kinase belonging to the AGK kinase family and shares structural and functional similarities with AKT, PKC and S6K1–3
To verify whether SGK1 modulation affects the nuclear export of short non-coding RNAs within the RAN:GTP:Exportin-5 complex[44] by modulating the activity of the RAN/RANBP1 pathway[10,45], we first used qRT-PCR to examine the levels of 85 microRNAs involved in cancer in a hepatic carcinoma-derived HUH7 cell line treated with either SI113 or vehicle alone (DMSO)
RANBP1 is the principal regulator of the RAN GTPase: it cooperates with RANGAP1 (RAN GTPase-activating protein) to promote GTP hydrolysis by RAN; in addition, RANBP1 has been found to bind to RANGTP, regulating its association or dissociation from effectors of the family of nuclear transport receptors[44,45,55,57]
Summary
The serum- and glucocorticoid-regulated kinase 1 (SGK1) is a serine/threonine kinase belonging to the AGK kinase family and shares structural and functional similarities with AKT, PKC and S6K1–3. We demonstrate for the first time that SGK1, acting through SP1-dependent transcriptional and functional regulation of RANBP1 and RANGAP1, affects the binding status of GTP/GDP of RAN as well as RANBP1/ RANGAP1 protein abundance and the localization of XPO5, enhancing the nuclear export and maturation of pre-miRNAs, without significantly affecting pri-miRNA levels, in a cellular model of HCC and in several neoplastic and non-tumoral primary cell lines. This process is reversed either by SI113-dependent or sh-mediated SGK1 inhibition/depletion. We provided additional data supporting a crucial role of RANBP1 in the mechanism of action of SI113, a novel SGK1 inhibitor showing anticancer properties in several neoplastic models
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