Coa LL et al. (2019), AKT/protein kinase B associates with β-actin in...

ECB-ART-46879

Biosci Rep 2019 Jan 30;391:. doi: 10.1042/BSR20181312.

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AKT/protein kinase B associates with β-actin in the nucleus of melanoma cells.

Coa LL , Abreu TF , Tashima AK , Green J , Pascon RC , Vallim MA , Machado-Jr J .

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The serine-threonine kinase AKT/PKB is a critical regulator of various essential cellular processes, and dysregulation of AKT has been implicated in many diseases, including cancer. Despite AKT action is known to function mainly in the cytoplasm, AKT has been reported to translocate to the nucleus. However, very little is known about the mechanism required for the nuclear import of AKT as well as its function in this cellular compartment. In the present study, we characterized the presence of endogenous nuclear AKT in human melanoma cells and addressed the possible role of AKT by exploring its potential association with key interaction nuclear partners. Confocal and Western blot analyses showed that both phosphorylated and non-phosphorylated forms of AKT are present in melanoma cells nuclei. Using mass spectrometry in combination with protein-crosslinking and co-immunoprecipitation, we identified a series of putative protein partners of nuclear AKT, including heterogeneous nuclear ribonucleoprotein (hnRNP), cytoskeleton proteins β-actin, γ-actin, β-actin-like 2 and vimentin. Confocal microscopy and biochemical analyses validated β-actin as a new nuclear AKT-interacting partner. Cofilin and active RNA Polymerase II, two proteins that have been described to interact and work in concert with nuclear actin in transcription regulation, were also found associated with nuclear AKT. Overall, the present study uncovered a yet unrecognized nuclear coupling of AKT and provides insights into the involvement of AKT in the interaction network of nuclear actin.

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Genes referenced: btaf1 creb1 ctdp1 LOC115919910 LOC115929578 LOC576396 LOC582192 LOC586660 LOC586734 LOC586799 LOC588766 LOC590297 LOC594261 polr3a tubgcp2

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	Figure 1. AKT is localized in the nucleus of melanoma cellsA2058 cells were double immunostained for AKT and CREB and analyzed by confocal laser-scanning microscopy. Images are projections of one stack from the middle plane of the nucleus. (A) AKT is shown in red, CREB is shown in green, DAPI-stained nuclei are shown in blue and DIC represents differential interference contrast image. (B) Merged image of endogenous AKT and CREB. (C) Merged image of AKT and DAPI. The yellow cross in the merged panel of AKT and DAPI shows the confocal z-series reconstruction and corresponding xz and yz orthogonal planes. Confocal image and orthogonal views show that AKT immunostaining is localized in melanoma cells nuclei. (D) Co-localization map for AKT and DAPI, obtained using the Colocalization Threshold plugin for ImageJ, shows pixels with positive signals for both probes in white; scale bar, 5 μm.
	Figure 2. Both phosphorylated and non-phosphorylated AKT are present in melanoma cells nucleiA2058 cells were serum starved for 24 h and then stimulated (+) or not (−) with 10% serum for 30 min. (A) Nuclear and cytoplasmic fractions were analyzed by Western blot with anti-lamin A/C or anti-β-tubulin to confirm the purity of the nuclear and cytoplasmic fractions, respectively. (B) Nuclear and cytoplasmic fractions were analyzed by Western blot with anti-p-AKT-Ser473, anti-p-AKT-Thr308 and anti-total-AKT. (C) A2058 cells were serum starved for 24 h in the presence (+) or absence (−) of the PI3K inhibitor XI (20 μM) and then stimulated with 10% serum for 30 min (+). Nuclear fractions were analyzed by Western blot with anti-p-AKT-Ser473, anti-p-AKT-Thr308 and anti-total-AKT. (B and C) Results were plotted as the mean +/− S.D., n=3. Fold change in AKT phosphorylation was normalized to the levels of total-AKT.
	Figure 3. Identification of nuclear AKT binding proteins in melanoma cells nucleiA2058 cells were serum starved for 24 h and then stimulated (+) or not (−) with 10% serum for 30 min as indicated. Nuclear extracts were submitted to two-step chemical cross-linking and immunoprecipitations (IP) were performed using anti-total AKT or control IgG antibodies. Co-immunoprecipitated materials were resolved on SDS-PAGE. Coomassie blue staining of the gel revealed co-immunoprecipitated bands of approximately 42 and 200 kDa. The identified bands were excised and subjected to trypsin digestion for LC-MS/MS analysis.
	Figure 4. Nuclear AKT interacts with β-actinA2058 cells were serum starved for 24 h and then stimulated (+) or not (−) with 10% serum for 30 min, as indicated. (A) Nuclear lysates were submitted to immunoprecipitation (IP) with anti-total AKT or control IgG antibodies. Cell lysates prior to immunoprecipitation were used as input. Input and co-immunoprecipitated proteins were immunoblotted (IB) for detection of total AKT and β-actin. (B) A2058 cells were double immunostained for AKT and β-actin and analyzed by confocal laser-scanning microscopy. Confocal images show maximum projection of image stacks. AKT is shown in green, β-actin is shown in red, DAPI-stained nuclei are shown in blue. Merged images are shown at higher magnification in the panels below. Reconstructed orthogonal projection is presented as viewed in the xz plane, showing co-localization of AKT with β-actin in the nucleus (yellow pixels, arrowhead); scale bar, 5 μm.
	Figure 5. AKT/β-actin complex is not disturbed by inhibition of AKT phosphorylation(A) A2058 cells were serum starved for 24 h and then stimulated (+) or not (−) with 10% serum for 30 min as indicated. Nuclear fractions were immunoprecipitated (IP) with anti-total AKT or control IgG antibodies. Cell lysates prior to immunoprecipitation were used as input. Input and co-IP proteins were resolved on SDS-PAGE and immunoblotted for the detection of p-AKT Ser473, total AKT and β-actin (B). The result shown in (A) was validated by reverse IP where anti-β-actin was used for immunoprecipitation instead of anti-total AKT. (C) A2058 cells were serum starved for 24 h in the presence (+) or absence (−) of the PI3K inhibitor XI (20 μM) and then stimulated (+) or not (−) with 10% serum for 30 min. Nuclear fractions were analyzed by Western blot with anti-p-AKT Ser473, anti-total AKT and anti-β-actin. (D) A2058 cells were treated as described in C and nuclear lysates were immunoprecipitated with anti-total AKT or control IgG antibodies. Co-IP proteins were resolved on SDS-PAGE and immunoblotted for detection of p-AKT Ser473, total AKT and β-actin.
	Figure 6. Nuclear AKT interacts with cofilin(A) A2058 cells were serum starved for 24 h and then stimulated (+) or not (−) with 10% serum for 30 min, as indicated. Nuclear fractions were immunoprecipitated (IP) with anti-cofilin or control IgG antibodies. Cell lysates prior to immunoprecipitation were used as input. Input and co-IP proteins were resolved on SDS-PAGE and immunoblotted for the detection of p-AKT473, total AKT, β-actin and cofilin. (B and C) A2058 cells were double immunostained for: (B) AKT (green) and Cofilin (red); (C) cofilin (green) and β-actin (red). DAPI-stained nuclei are shown in blue. Immunostained cells were analyzed by confocal laser-scanning microscopy. Images are projections of one stack from the middle plane of the nucleus. Reconstructed orthogonal projections are presented as viewed in the xz planes, showing co-localized immunostaining in the nucleus (yellow pixels, arrowhead); scale bar, 5 μm.
	Figure 7. Both nuclear AKT and cofilin co-localize with active RNA Pol IIA2058 cells were double immunostained either for (A) AKT or (B) Cofilin with RNA Pol II phosphorylated at Ser2 in the CTD. AKT and Cofilin are shown in green, RNA Pol II is shown in red and DAPI-stained nuclei are shown in blue. Immunostained cells were analyzed by confocal laser-scanning microscopy. Images are projections of one stack from the middle plane of the nucleus. Reconstructed orthogonal projections are presented as viewed in the xz plane, showing co-localized immunostaining in the nucleus (yellow pixels, arrowhead); scale bar, 5 μm.
	Figure 8. Schematic representation summarizing the interaction network of nuclear AKT and its predicted location at functional nuclear regions(1) AKT is translocated from the cytoplasm to the nucleus by an yet unknown mechanism; (2) cofilin bound to monomeric actin mediates the transport of actin to the nucleus; (3) In the nucleus, AKT interacts with actin/cofilin complex; (4) AKT interaction with actin/cofilin complex may be target to active Pol II phosphorylated in the CTD.

References [+] :

Adini, RhoB controls Akt trafficking and stage-specific survival of endothelial cells during vascular development. 2003, Pubmed