The neuronal-specific SGK1.1 (SGK1_v2) kinase as a transcriptional modulator of BAG4, Brox, and PPP1CB genes expression

Abstract

The Serum- and Glucocorticoid-induced Kinase 1, SGK1, exhibits a broad range of cellular functions that include regulation of the number of ion channels in plasma membrane and modulation of signaling pathways of cell survival. This diversity of functions is made possible by various regulatory processes acting upon the SGK1 gene, giving rise to various isoforms: SGK1_v1-5, each with distinct properties and distinct aminotermini that serve to target proteins to different subcellular compartments. Among cellular effects of SGK1 expression is to indirectly modulate gene transcription by phosphorylating transcriptional factors of the FOXO family. Here we examined if SGK1.1 (SGK1_v2; NM_001143676), which associates primarily to the plasma membrane, is also able to regulate gene expression. Using a differential gene expression approach we identified six genes upregulated by SGK1.1 in HeLa cells. Further analysis of transcript and protein levels validated two genes: BCL2-associated athanogene 4 (BAG-4) and Brox. The results indicate that SGK1.1 regulates gene transcription upon a different set of genes some of which participate in cell survival pathways (BAG-4) and others in intracellular vesicular traffic (Brox).

Figure 1

Western blot of HeLa cells lysates and purified recombinant Brox protein (Brox) probed with pre-immune serum (PRE-Ab), Brox serum and purified Brox specific antibodies (AbBrox).

Figure 2

Western blots (A,C), quantification of mRNA relative expression (B) and bar chart of protein expression (D) of BAG-4, Brox, LYCAT, PPP1CB and RDX of same eight HeLa cultures of transfected cells used for (D) data. (A) Examples of SGK1.1 expression in transfected and no transfected HeLa cells and tubulin reference; (B) Mean ± standard deviation for BAG-4, Brox, PPP1CB and RDX gene expression as ×10⁵ relative to β-actin; (C) Examples of quantitated proteins and tubulin reference; (D) BAG-4, Brox, PPP1CB and RDX proteins expression levels relative to β-tubulin shown as mean ± standard deviation.

Figure 3

Subcellular localization of BAG4, Brox and PPP1CB in non-transfected HeLa cells. (A) Studied proteins using specifics antibodies are shown in green; (B) merge of studied proteins (green) and nucleus (blue) labeled with DAPI; (C) merge of studied proteins (green), nucleus (blue) and β tubulin (red).

Figure 4

Immunolocalization of BAG-4, Brox and PPP1CB in non-transfected HeLa cells and SGK1.1S515D or SGK1.1K220A transfected HeLa cells. Negative control for secondary antibody at the same exposure time of the corresponding experiment.