Regulation of Akt(ser473) phosphorylation by choline kinase in breast carcinoma cells

Abstract

Background: The serine/threonine kinase PKB/Akt plays essential role in various cellular processes including cell growth and proliferation, metabolism and cell survival. The importance of the Akt pathway is highlighted by the mutation of various components of the pathway such as the PTEN and PI3-kinase (P110alpha) in human cancers. In this paper, we employed an RNA interference library targeting all human kinases to screen for kinases involved in the regulation of Akt activation, in particular serine 473 phosphorylation. Here, we transfected the MDA-MB 468 breast cell line with the human kinome siRNA library and measured Akt activation using an antibody specific for phosphoserine 473 of Akt.

Results: The screen revealed that phosphorylation of Akt(ser473) can be regulated by more than 90 kinases. Interestingly, phosphorylation of Akt(ser473), but not thr308, can be severely reduced by inhibition of Choline kinase activity via siRNA or small molecule inhibitors. We show here that the regulation of Akt phosphorylation by Choline kinase is PI3K-independent. In addition, xenograft tumors treated with Choline kinase inhibitors demonstrated a statistically significant decrease in Akt(ser473) phosphorylation. Importantly, the reduction in phosphorylation correlates with regression of these xenograft tumors in the mouse model.

Conclusion: High Choline kinase expression and activity has previously been implicated in tumor development and metastasis. The mechanism by which Choline kinase is involved in tumor formation is still not fully resolved. From our data, we proposed that Choline kinase plays a key role in regulating Akt(ser473) phosphorylation, thereby promoting cell survival and proliferation.

Figure 1

Silencing of Choline kinase A or B reduced Akt(ser 473) phosphorylation in MDA-MB 468 cells. A, The screen was set up using MDA-MB 468 cells as described in the method. The percentage pAkt(ser473) signal is calculated by obtaining total intensity (from fluorohore-Alexa 488) of the signal divided by total number of cells imaged. The reading is compared to the non-targeting siRNA transfected cells (scr, set to be 100%). B, Cells transfected with 50 nM pool (P) or deconvoluted (D1-4) ChoK A or B siRNA for 3 days and 30 μg whole cell lysate were subjected to western blot with the indicated antibodies. The signals obtained from the western blot analysis were quantified with Image J program and Akt(ser473) phosphorylation were normalized to total Akt. The ratios were compared to scr control (set as 1) indicated by the values below the blots. C, siRNA were transfected to cells as (B) for 2 days. 500 ng total RNA were reverse transcribed to cDNA subjected to real time PCR (ABI7500) with the appropriate primer sets. Transcript levels were normalized to the scrambled control (scr). The data is an average of triplicate experiments.

Figure 2

Choline kinase regulates Akt activity. A, MDA-MB 468 cells were transfected with 50 nM of the indicated pool siRNA. 3 days posttransfection, 50 μM LY294002 was added to scr transfected cells for 30 mins. Whole cell lysate were subjected to western blotting with the indicated antibodies. B, siRNA transfection and western blot were performed as (A). Percentage of pAkt(ser473) signal were calculated by quantifying the pAkt(ser473) normalized to total Akt on the immunoblot and compared to the scr control (set as 100%). C, MDA-MB 231 cells were transfected with 50 nM of indicated pool siRNA for 2 days. Cells were serum-starved overnight and stimulated with IGF for 15 mins. Whole cell lysates were harvested and western blot performed with the indicated antibodies. The values below the blot indicate the ratio of normalized pAkt(ser473) signals quantified using Image J to that of scr control (set as 1). D, MCF7 were transfected with 1 μg pcDNA vector, ChoK A or B plasmids for 24 h using Lipofectamine 2000 (Invitrogen). Cells were harvested and ChoK activity determined as described in the methods. E, MCF7 was transfected as in (D) and western blot with indicated antibodies.

Figure 3

ChoK inhibitors inhibit ChoK activity and Akt phosphorylation. A, MDA-MB 468 cells were treated with 20 μM Mn58b for indicated time. Lysates were harvested and ChoK activity determined as described in methods. B, MDA-MB 468 and C, serum-starved MDA-MB 231 were treated with Mn58b for indicated time with the specified concentration. MDA-MD 231 cells were stimulated with IGF for 15 mins. 30 μg cell lysate were subjected to western blot with the indicated antibodies. D, MDA-MB 468 cells were treated with 20 μM TCD828. ChoK activity was determined as described in methods. E, MDA-MB 468 and F, serum starved MDA-MB 231 were treated with TCD828 for the indicated time with the specified concentration. MDA-MD 231 cells were stimulated with IGF for 15 mins. 30 μg cell lysate were subjected to western blot with the indicated antibodies. pAkt(ser473) signals were quantified using Image J program and normalized to respective total Akt signal. Values below the blots indicate the normalized Akt(ser473) phosphorylation compared to untreated control.

Figure 4

ChoKα regulation on Akt activity is PI3K independent. MDA-MB 231 and A549 cells were transfected with 50 nM of pool siRNA targeted to scr control or ChoK A. 2 days post transfection, cells were transfected with 0.5 μg PH-GFP construct for 24 h followed by 8 h serum starvation and 15 mins of IGF stimulation. 50 μM LY294002 was added to scr transfected cells for 30 mins prior to IGF stimulation. Cells were stained with Hoechst, fixed with paraformaldehyde and mounted for fluorescence microscopy.

Figure 5

Mn58b treatment slowed tumor growth through the inhibition of Akt(ser473) phosphorylation. A, Immunosuppressed mice were injected with MDA-MB 231 cells on each flank and tumors were allowed to grow to 0.1 cm³. At week 3.5, Mn58b or vehicle, were administrated to the mice intraperitoneally and the growth of tumor monitored. B, 11 xenografts excised from the mice treated with vehicle (C1-6) or Mn58b (T7-11) were frozen and lysed for western blot with the indicated antibodies or D, fixed in paraformaldehyde and section for immunohistochemistry staining with anti-total Akt and anti-pAkt(ser473). C, pAkt(ser473) signal was quantified with Image J program and normalized to total Akt. The ratios were input as scatter plot and analyzed using unpaired t-test with Prism (GraphPad). ** p < 0.05.